**Male stem Cell Niche and Spermatogenesis in the** *Drosophila* **testis — A Tale of Germline-Soma Communication**

Fani Papagiannouli

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58756

#### **1. Introduction**

A fundamental question in biology is how communication and exchange of short-range signals shape the microenvironment for setting up functional tissues. In all adult tissues and organs harboring stem cells, tissue homeostasis and repair relies on the proper communication of stem cells and their differentiating daughter cells with the local tissue microenvironment that homes them [1, 2]. Stem cell research has made outstanding contributions on the factors that maintain stem cells or drive them to generate differentiated daughter cells. The use of stem cells in the development of cell-based medicine and in repairing malformed, damaged or aging tissues demands a better understanding of stem cells at a molecular level and of how they behave in their physiological context.

The basic principles controlling stem cell self-renewal versus differentiation are strikingly conserved during evolution and their regulatory logic is often very similar among homologous stem cell niches. Since the signaling pathways and their regulatory circuits are highly complex in the mammalian system with significant molecular redundancy, they are often difficult to study. Therefore, using a simpler model system such as the *Drosophila* testis allows us to elucidate the underlying cellular and molecular mechanisms of stem cell maintenance and differentiation in a straightforward way.

The *Drosophila* testis provides an excellent system to study *in vivo* how two closely apposed cell types communicate and coordinate their reciprocal interaction. Recent advances in spermatogenesis have shown that testis morphogenesis is achieved through the physical contact and diffusible signals exchanged between the germline and the somatic cell popula‐ tions [3]. Moreover, the *Drosophila* testis provides a powerful system to study germline-soma

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

communication as it is possible to identify the different cell populations with specific markers, study them within the context of their wild type surrounding and trace them after genetic manipulations [2, 4]. Although several signaling molecules, cytoskeletal and other factors have been so far identified, many aspects of the coordination of these events remain unsolved. Using well-established genetic tools, cell-type specific markers and imaging techniques we can manipulate cell function in a spatio-temporal specific way within the germline-soma micro‐ environment and decode how signal transmission and polarity are established, maintained and coordinated on the mechanistic level. Therefore, elucidating the mechanisms and factors that regulate these processes is crucial for understanding cell communication and coordination *per se,* which is a prerequisite for the therapeutic applications in other stem cell systems and in various tissue contexts.

The first signs of testis organogenesis are detected in 1st instar larvae (L1) and a testis with a mature stem cell niche and all premeiotic stages is detected at 3rd instar larvae (L3). The *Drosophila* testis contains two types of stem cells: the germline stem cells (GSCs) and the somatic cyst stem cells (CySCs). Each GSC is surrounded by two somatic cyst stem cells (CySCs) and both types of stem cells are maintained through their association to the hub cells, a cluster of non-dividing cells forming the niche organizer. Upon asymmetric cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast (Gb), whereas each CySC pair divides to generate two CySCs remaining associated with the hub and two distally located post-mitotic daughter somatic cyst cells (SCCs) [1, 11]. Upon asymmetric stem cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast, whereas each CySC pair divides to generate two CySCs and two somatic cyst cells (SCCs) [1, 12]. GSCs divide asymmetrically with the mitotic spindle orientated perpendicular to the hub [13, 14]. After division the GSC remains in contact with the hub and inherits the mother centriole whereas the gonialblast, inherits the daughter centriole and initiates differentiation [15]. However, upon starvation-or genetically-induced GSC loss, the GSC population can be renewed both by symmetric renewal and de-differentiation of transient amplifying sperma‐ togonia, which repopulate the niche and reestablish contact to the hub [16].The gonialblast divides mitotically four more times to give rise to 16 interconnected spermatogonial cells, forming a cyst surrounded by the two SCCs (Fig.1). As germ cells enter their differentiation program of four transient amplifying divisions followed by pre-meiotic gene expression and meiotic divisions, the SCCs grow enormously in size, elongate and wrap the germ cells creating cysts [17] outside "sealed" by extracellular matrix (ECM) [18]. After the growth phase, the

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spermatocytes undergo meiosis and differentiate into elongated spermatids.

**Figure 1.** Diagram depicting early spermatogenesis in *Drosophila*. GSC: germline stem cell, CySC: somatic cyst stem cell, SCC: somatic cyst cell. For simplicity reasons CySCs and SCCs are collectively called cyst cells. Testicular cysts com‐

Testis organogenesis is completed during pupal stages. For the formation of a mature testis and a functional reproductive tract, the *Drosophila* testis contacts the seminal vesicle growing

prise of a pair of cyst cells flanking the germline (GSCs, spermatogonia or spermatocytes).

The proposed chapter gives an overview of the *Drosophila* male stem cell niche and its importance as a model system for understanding stem cell function. The chapter starts with an introduction to the system, focusing on the importance of soma-germline communication, mutual coordination and progressive co-differentiation. As next, follows the role of the stem cell niche and signaling pathways in balancing stem cell maintenance and differentiation. The specification and positioning of the stem cell niche is discussed, in view of recent data in the field, which put the way we understand stem cell niche establishment and maintenance into a new perspective. Finally, the role of septate junctions and cortical polarity components in the somatic lineage is presented, together with open questions and challenges of the current research in the field.

#### **2. The** *Drosophila* **testis**

Organogenesis of the *Drosophila* testis, a structure first made by the coalesce of germ cells and somatic gonadal cells in late embryogenesis, proceeds continuously throughout embryonic and larval stages, to reach maturation in adult stages. The embryonic gonad results from the coalescence of the germ cells that completed migration and the somatic gonadal precursors (SGPs). SPGs are mesodermal cells specified in bilateral clusters within the *eve* domain of abdominal parasegments [5] 10 to 13 [6-9]. The development of male and female gonads already differs at the time of gonad coalescence. In the male gonads three SGP populations are identifiable by their different gene expression: the posterior-SGPs, the posterior male-specific SGPs which die by apoptosis in females [6] and the anterior-SGPs which will give rise to the hub, the core of the testicular niche which will recruit and organize the anterior-most germ cells to become germline stem cells (GSCs) [10]. Therefore, it becomes evident that the different SGP populations joining the male gonad orchestrate testis morphogenesis since the germ cells represent a uniform population at that time. The SGPs are specified initially through the function of Zinc-finger homeodomain protein 1 (Zfh-1) within the cluster of the lateral mesoderm (PS2-14) which work together the homeobox protein Tinman to promote germ cell migration to the lateral mesoderm. Subsequently, Zfh-1 restriction in PS10-13 correlates with the specification of these cells as SPGs.

The first signs of testis organogenesis are detected in 1st instar larvae (L1) and a testis with a mature stem cell niche and all premeiotic stages is detected at 3rd instar larvae (L3). The *Drosophila* testis contains two types of stem cells: the germline stem cells (GSCs) and the somatic cyst stem cells (CySCs). Each GSC is surrounded by two somatic cyst stem cells (CySCs) and both types of stem cells are maintained through their association to the hub cells, a cluster of non-dividing cells forming the niche organizer. Upon asymmetric cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast (Gb), whereas each CySC pair divides to generate two CySCs remaining associated with the hub and two distally located post-mitotic daughter somatic cyst cells (SCCs) [1, 11]. Upon asymmetric stem cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast, whereas each CySC pair divides to generate two CySCs and two somatic cyst cells (SCCs) [1, 12]. GSCs divide asymmetrically with the mitotic spindle orientated perpendicular to the hub [13, 14]. After division the GSC remains in contact with the hub and inherits the mother centriole whereas the gonialblast, inherits the daughter centriole and initiates differentiation [15]. However, upon starvation-or genetically-induced GSC loss, the GSC population can be renewed both by symmetric renewal and de-differentiation of transient amplifying sperma‐ togonia, which repopulate the niche and reestablish contact to the hub [16].The gonialblast divides mitotically four more times to give rise to 16 interconnected spermatogonial cells, forming a cyst surrounded by the two SCCs (Fig.1). As germ cells enter their differentiation program of four transient amplifying divisions followed by pre-meiotic gene expression and meiotic divisions, the SCCs grow enormously in size, elongate and wrap the germ cells creating cysts [17] outside "sealed" by extracellular matrix (ECM) [18]. After the growth phase, the spermatocytes undergo meiosis and differentiate into elongated spermatids.

communication as it is possible to identify the different cell populations with specific markers, study them within the context of their wild type surrounding and trace them after genetic manipulations [2, 4]. Although several signaling molecules, cytoskeletal and other factors have been so far identified, many aspects of the coordination of these events remain unsolved. Using well-established genetic tools, cell-type specific markers and imaging techniques we can manipulate cell function in a spatio-temporal specific way within the germline-soma micro‐ environment and decode how signal transmission and polarity are established, maintained and coordinated on the mechanistic level. Therefore, elucidating the mechanisms and factors that regulate these processes is crucial for understanding cell communication and coordination *per se,* which is a prerequisite for the therapeutic applications in other stem cell systems and

The proposed chapter gives an overview of the *Drosophila* male stem cell niche and its importance as a model system for understanding stem cell function. The chapter starts with an introduction to the system, focusing on the importance of soma-germline communication, mutual coordination and progressive co-differentiation. As next, follows the role of the stem cell niche and signaling pathways in balancing stem cell maintenance and differentiation. The specification and positioning of the stem cell niche is discussed, in view of recent data in the field, which put the way we understand stem cell niche establishment and maintenance into a new perspective. Finally, the role of septate junctions and cortical polarity components in the somatic lineage is presented, together with open questions and challenges of the current

Organogenesis of the *Drosophila* testis, a structure first made by the coalesce of germ cells and somatic gonadal cells in late embryogenesis, proceeds continuously throughout embryonic and larval stages, to reach maturation in adult stages. The embryonic gonad results from the coalescence of the germ cells that completed migration and the somatic gonadal precursors (SGPs). SPGs are mesodermal cells specified in bilateral clusters within the *eve* domain of abdominal parasegments [5] 10 to 13 [6-9]. The development of male and female gonads already differs at the time of gonad coalescence. In the male gonads three SGP populations are identifiable by their different gene expression: the posterior-SGPs, the posterior male-specific SGPs which die by apoptosis in females [6] and the anterior-SGPs which will give rise to the hub, the core of the testicular niche which will recruit and organize the anterior-most germ cells to become germline stem cells (GSCs) [10]. Therefore, it becomes evident that the different SGP populations joining the male gonad orchestrate testis morphogenesis since the germ cells represent a uniform population at that time. The SGPs are specified initially through the function of Zinc-finger homeodomain protein 1 (Zfh-1) within the cluster of the lateral mesoderm (PS2-14) which work together the homeobox protein Tinman to promote germ cell migration to the lateral mesoderm. Subsequently, Zfh-1 restriction in PS10-13 correlates with

in various tissue contexts.

114 Adult Stem Cell Niches

research in the field.

**2. The** *Drosophila* **testis**

the specification of these cells as SPGs.

**Figure 1.** Diagram depicting early spermatogenesis in *Drosophila*. GSC: germline stem cell, CySC: somatic cyst stem cell, SCC: somatic cyst cell. For simplicity reasons CySCs and SCCs are collectively called cyst cells. Testicular cysts com‐ prise of a pair of cyst cells flanking the germline (GSCs, spermatogonia or spermatocytes).

Testis organogenesis is completed during pupal stages. For the formation of a mature testis and a functional reproductive tract, the *Drosophila* testis contacts the seminal vesicle growing out of the genital disc during metamorphosis. The outer sheath of the male reproductive tract develops from two populations of cells: the pigment cells of the testis and the precursors of smooth muscle cells from the genital disc [19]. First, the muscle progenitor cells of the genital disc contact the basal surface of the pigment cells of the testis. Then, migration of muscle and pigment cells proceeds in opposite directions until the gonad and the seminal vesicle have each acquired an inner layer of muscle tissue and an outer layer of pigment cells [19]. It is the addition of the acto-myosin sheath, which gives to the adult testis its characteristic coiledshape. The pigment cells are responsible for the yellow color of the testis sheath and seminal vesicle [17]. *wnt2*, expressed in the SGPs, is required for the correct development of pigment cells [19], and in *wnt2* mutant embryos pigment cells are not specified and *Sox100B* is not expressed in pigment cell precursors [20, 21].

towards the direction (A→P) of differentiation. The HCC finally is engulfed by cells of the terminal epithelium to allow coiling of the spermatid bundles towards the testis base [27].

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So far the main evidence for cyst cell (CySCs and SCCs) function came from the analysis of individual signal transduction pathways that establish a cross talk between the soma and the germline. In this chapter recent findings critically affecting germline-soma communication and coordination will be highlighted, with emphasis on the role of cytoskeletal and scaffolding components such as integrins and adaptor proteins, ECM and the septate junction components. Interestingly, the *Drosophila* testis cyst cells show striking similarities with the Sertoli cells, the supportive cells of the mammalian germline, in terms of cytoskeletal and scaffolding compo‐ nents [2]. Moreover, the genes presented in this study show high degree of conservation to their vertebrate homologues [18, 23]. Accordingly, although we use *Drosophila* spermatogen‐ esis as a model for its powerful genetic tools, accessible imaging and the wealth of underlying prior knowledge on which to built on, the regulatory mechanisms discovered in the *Drosophi‐ la* testis provide paradigms for regulatory strategies in spermatogenesis and allow us to discern the complexity of niche and testis homeostasis in other organisms and stem cell systems in other tissues, which will eventually advance the basic knowledge required for stem cell

**2.2. Niche Homeostasis: Signaling regulation of stemness vs. differentiation**

Tissue specific stem cells are the lifetime source of many types of differentiated cells. They reside in microenvironments, the stem cells niches that have an important role in stem cell behavior [28]. Gamete development requires a coordinated soma-germ line interaction that keeps the balance between germline stem cell renewal and differentiation. The balance between stem cell identity and differentiation at the *Drosophila* testicular niche results from signals exchanged among the hub, GSCs and CySCs. The Janus-kinase transducer and activator of transcription (JAK-STAT) pathway was the first signaling pathway found to regulate GSC and CySC maintenance in the *Drosophila* testis [29, 30]. The hub cells secrete the ligand Unpaired (Upd), which activates the JAK-STAT pathway in adjacent GSCs and CySCs [29-31]. In the absence of JAK-STAT signaling the GSCs differentiate and are unable to selfrenew, whereas ectopic expression of *upd* in the germline greatly expands the population of GSCs and CySCs in adult as well as in the larval testis [29, 30]. In GSCs, STAT is required so that E-cadherin (E-cad) maintains the connection of the GSC to the hub and ectopic E-cad partially rescues the maintenance of STAT-depleted GSCs [32]. Another STAT target in GSCs is *chickadde*, the homologue of the *Drosophila* profilin. Chic is required cell autonomously to maintain GSCs by facilitating GSC-hub contact possibly via E-cad whereas Chic in the SCCs is affecting germ cell enclosure and restricting trans-amplifying (TA) spermatogonial divisions [33]. When GSCs divide, their daughter cells displaced from the hub are thought to receive lower levels of hub-derived signals and therefore differentiate. In CySCs, STAT is critical for maintaining their stem cell character and the activation of targets essential for their identity such as *zfh-1* and *chinmo* [32, 34]. *zfh-1* is expressed predominantly in CySCs and their imme‐ diate SCCs, and ectopic expression in late SCCs outside the niche leads in accumulation of GSC-and CySCs-like cells which fill in the whole testis. Similarly, *chinmo* is expressed in

applications.

#### **2.1. Cyst cells: The safeguards of the Germline**

Critical for testis differentiation and morphogenesis is the cyst microenvironment created by the cyst cells (CySCs and SCCs) that enclose the germline cells, accompany them throughout their differentiation steps up to sperm individualization and maintain cyst integrity and architecture [22, 23]. Although it is well established that soma-germline physical contact is critical for the cell communication and for promoting their mutual development and differ‐ entiation [3], it remains so far elusive how these tightly packed cysts coordinate adhesion and cell shape changes with signaling and membrane addition on a mechanistic level.

The thin and squamous cyst cells lack the columnar epithelial structure of e.g. the ovarian follicular epithelium, which caught the attention of scientists analyzing apico-basal polarity many years ago. For this reason, several questions concerning cyst cell architecture, apicalbasal polarity and sub-cellular localization of cytoskeletal proteins such as Dlg, Integrin and Talin remained unclear. Preliminary data show that cyst cells are polarized with an innerapical surface phasing the germline (Fig. 2E; arrowheads) [22] and an outer-basal surface surrounded by ECM [18]. Critical cytoskeletal and polarity components localize at cyst cells, such as Rho1, Bazooka (Baz), Fasciclin II (FasII), Integrin-linked kinase (ILK), βPS-Integrin (encoded by the *myospheroid* gene) (Fig. 2B-F'), as well as the septate junction proteins Dlg, Scrib and Lgl (Fig.3 A-D). Moreover, cyst cells are able to extend projections in between the germline spermatogonia (small insets of Fig.3 A-C) and spermatocytes (Fig.2 C-C', E-F'; yellow arrowheads), similar to what was previously observed in the embryonic gonads [24]. On the morphological level, the orientation of the SCCs flanking the germ cells changes in comparison to their mother CySCs via a not yet uncovered mechanism. The two CySCs flanking the same GSC are arranged parallel to the testis anterior-posterior axis (A/P) and attach to the hub whereas their post-mitotic daughter SCCs change their orientation perpendicular to the A/P testis axis (Fig.1). During terminal differentiation, the two cyst cells of the same cyst acquire different identities followed by morphological changes [25]: the forward SCC becomes the "head cyst cell" (HCC) onto which all 64 spermatid heads are anchored shortly after meiosis, and the posterior one becomes the much larger "tail cyst" (TCC) that surrounds the spermatid tails of 1.8 mm length [26]. This results in creating polarized cysts across the testis A/P axis and towards the direction (A→P) of differentiation. The HCC finally is engulfed by cells of the terminal epithelium to allow coiling of the spermatid bundles towards the testis base [27].

out of the genital disc during metamorphosis. The outer sheath of the male reproductive tract develops from two populations of cells: the pigment cells of the testis and the precursors of smooth muscle cells from the genital disc [19]. First, the muscle progenitor cells of the genital disc contact the basal surface of the pigment cells of the testis. Then, migration of muscle and pigment cells proceeds in opposite directions until the gonad and the seminal vesicle have each acquired an inner layer of muscle tissue and an outer layer of pigment cells [19]. It is the addition of the acto-myosin sheath, which gives to the adult testis its characteristic coiledshape. The pigment cells are responsible for the yellow color of the testis sheath and seminal vesicle [17]. *wnt2*, expressed in the SGPs, is required for the correct development of pigment cells [19], and in *wnt2* mutant embryos pigment cells are not specified and *Sox100B* is not

Critical for testis differentiation and morphogenesis is the cyst microenvironment created by the cyst cells (CySCs and SCCs) that enclose the germline cells, accompany them throughout their differentiation steps up to sperm individualization and maintain cyst integrity and architecture [22, 23]. Although it is well established that soma-germline physical contact is critical for the cell communication and for promoting their mutual development and differ‐ entiation [3], it remains so far elusive how these tightly packed cysts coordinate adhesion and

The thin and squamous cyst cells lack the columnar epithelial structure of e.g. the ovarian follicular epithelium, which caught the attention of scientists analyzing apico-basal polarity many years ago. For this reason, several questions concerning cyst cell architecture, apicalbasal polarity and sub-cellular localization of cytoskeletal proteins such as Dlg, Integrin and Talin remained unclear. Preliminary data show that cyst cells are polarized with an innerapical surface phasing the germline (Fig. 2E; arrowheads) [22] and an outer-basal surface surrounded by ECM [18]. Critical cytoskeletal and polarity components localize at cyst cells, such as Rho1, Bazooka (Baz), Fasciclin II (FasII), Integrin-linked kinase (ILK), βPS-Integrin (encoded by the *myospheroid* gene) (Fig. 2B-F'), as well as the septate junction proteins Dlg, Scrib and Lgl (Fig.3 A-D). Moreover, cyst cells are able to extend projections in between the germline spermatogonia (small insets of Fig.3 A-C) and spermatocytes (Fig.2 C-C', E-F'; yellow arrowheads), similar to what was previously observed in the embryonic gonads [24]. On the morphological level, the orientation of the SCCs flanking the germ cells changes in comparison to their mother CySCs via a not yet uncovered mechanism. The two CySCs flanking the same GSC are arranged parallel to the testis anterior-posterior axis (A/P) and attach to the hub whereas their post-mitotic daughter SCCs change their orientation perpendicular to the A/P testis axis (Fig.1). During terminal differentiation, the two cyst cells of the same cyst acquire different identities followed by morphological changes [25]: the forward SCC becomes the "head cyst cell" (HCC) onto which all 64 spermatid heads are anchored shortly after meiosis, and the posterior one becomes the much larger "tail cyst" (TCC) that surrounds the spermatid tails of 1.8 mm length [26]. This results in creating polarized cysts across the testis A/P axis and

cell shape changes with signaling and membrane addition on a mechanistic level.

expressed in pigment cell precursors [20, 21].

116 Adult Stem Cell Niches

**2.1. Cyst cells: The safeguards of the Germline**

So far the main evidence for cyst cell (CySCs and SCCs) function came from the analysis of individual signal transduction pathways that establish a cross talk between the soma and the germline. In this chapter recent findings critically affecting germline-soma communication and coordination will be highlighted, with emphasis on the role of cytoskeletal and scaffolding components such as integrins and adaptor proteins, ECM and the septate junction components. Interestingly, the *Drosophila* testis cyst cells show striking similarities with the Sertoli cells, the supportive cells of the mammalian germline, in terms of cytoskeletal and scaffolding compo‐ nents [2]. Moreover, the genes presented in this study show high degree of conservation to their vertebrate homologues [18, 23]. Accordingly, although we use *Drosophila* spermatogen‐ esis as a model for its powerful genetic tools, accessible imaging and the wealth of underlying prior knowledge on which to built on, the regulatory mechanisms discovered in the *Drosophi‐ la* testis provide paradigms for regulatory strategies in spermatogenesis and allow us to discern the complexity of niche and testis homeostasis in other organisms and stem cell systems in other tissues, which will eventually advance the basic knowledge required for stem cell applications.

#### **2.2. Niche Homeostasis: Signaling regulation of stemness vs. differentiation**

Tissue specific stem cells are the lifetime source of many types of differentiated cells. They reside in microenvironments, the stem cells niches that have an important role in stem cell behavior [28]. Gamete development requires a coordinated soma-germ line interaction that keeps the balance between germline stem cell renewal and differentiation. The balance between stem cell identity and differentiation at the *Drosophila* testicular niche results from signals exchanged among the hub, GSCs and CySCs. The Janus-kinase transducer and activator of transcription (JAK-STAT) pathway was the first signaling pathway found to regulate GSC and CySC maintenance in the *Drosophila* testis [29, 30]. The hub cells secrete the ligand Unpaired (Upd), which activates the JAK-STAT pathway in adjacent GSCs and CySCs [29-31]. In the absence of JAK-STAT signaling the GSCs differentiate and are unable to selfrenew, whereas ectopic expression of *upd* in the germline greatly expands the population of GSCs and CySCs in adult as well as in the larval testis [29, 30]. In GSCs, STAT is required so that E-cadherin (E-cad) maintains the connection of the GSC to the hub and ectopic E-cad partially rescues the maintenance of STAT-depleted GSCs [32]. Another STAT target in GSCs is *chickadde*, the homologue of the *Drosophila* profilin. Chic is required cell autonomously to maintain GSCs by facilitating GSC-hub contact possibly via E-cad whereas Chic in the SCCs is affecting germ cell enclosure and restricting trans-amplifying (TA) spermatogonial divisions [33]. When GSCs divide, their daughter cells displaced from the hub are thought to receive lower levels of hub-derived signals and therefore differentiate. In CySCs, STAT is critical for maintaining their stem cell character and the activation of targets essential for their identity such as *zfh-1* and *chinmo* [32, 34]. *zfh-1* is expressed predominantly in CySCs and their imme‐ diate SCCs, and ectopic expression in late SCCs outside the niche leads in accumulation of GSC-and CySCs-like cells which fill in the whole testis. Similarly, *chinmo* is expressed in comparable levels in CySCs and early SCCs, is required for CySCs and not GSC renewal, and ectopic expression causes accumulation of GSCs-and CySCs-like cells. Furthermore, *zfh-1* and *chinmo* are not expressed in GSCs meaning that STAT can activate distinct downstream cascades in the GSC vs. CySCs. *ken* and *barbie* (*ken*) is another gene necessary and sufficient to promote CySC identity, yet in a STAT independent manner and with similar ectopic pheno‐ types like *zfh-1* and *chinmo* [35]. At the same time, Suppressor of cytokine signaling 36E (Socs36E) suppresses Jak-Stat signaling in the CySCs preventing them from outcompeting the GSCs and thereby maintains the proper balance of GSCs and CySCs, in a manner that depends on the adhesion protein integrin [36].

can be indeed very complex. Finally, antagonistic functions between the *Drosophila* β-catenin Armadillo (Arm) and the microRNAs-(miR-) 310-313 suggest that modulation of the Wingless signaling activity is important to buffer germ cell and somatic differentiation in the *Drosophi‐*

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Critical for germ cell differentiation is the expression of *bag of marbles (bam)* and *benign gonial cell neoplasm (bgcn)* in dividing spermatogonial cells in order to regulate their proliferation [48]. *bam* transcription is negatively regulated by the cooperation of the Glass bottom boat (Gbb) and Decapentaplegic (Dpp) signaling pathways emanating from the hub and CySCs to maintain the GSC identity [42]. Bam is required cell autonomously in TA spermatogonia to stop proliferation and enter the spermatocyte differentiation program [49]. The switch from TA proliferation to differentiation is mediated by translational control: Mei-P26 facilitates the accumulation of Bam in TA cells whereas Bam and Bcgn bind *mei-P26* 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through subsequent regulatory states that is: from a Mei-P26 on/Bam off to

Another signaling pathway restricting GSC proliferation is mediated by Epidermal Growth Factor Receptor (EGFR), whose inactivation in SCCs leads to an expansion of male GSCs [50]. In *Drosophila* testis, the major ligand of the EGFR pathway, Spitz (Spi) is secreted from the germline cells to stimulate the EGFR on cyst cells (CySCs and SCCs) [25]. Removal of either *spi* or *stet* from the germline cells, or removal of the EGFR from the cyst cells resulted in increased division frequencies of GSCs but did not affect the division frequencies of CySCs, suggesting that EGF signaling downregulates GSC divisions. Likewise, Raf, an EGFR downstream component, is required in SCCs to limit GSC expansion [51-53]. In testes mutated for the *rhomboid* homologue *stet*, the germ cells fail to associate with SCCs. Furthermore, germ cells recruit CySCs via the ligand Spitz, which binds to EGFR, and acts through the nucleotide exchange factor Vav to regulate the activity of Rac1, a down‐ stream component of the EGFR pathway. Taken together, EGF signaling from the germ‐ line cells produces differential Rac-and Rho-activities across the cyst cells that leads to a directional growth of the cyst cells around the germline cells [25]. Finally, Zero popula‐ tion growth (Zpg), the *Drosophila* gap junction Innexin 4, is localized to the spermatogo‐ nia surface, primarily on the sides adjacent to SCCs [54] and is required for the survival

and differentiation of early germ cells in both sexes [55, 56].

**3. The male stem cell niche: Specification and positioning**

The somatic cells of the hub form the organizing center, a cluster of non-dividing cells, at the anterior part of the embryonic male gonad originating, as already discussed, from SGPs [10]. However, not only the hub but also the cyst cells are specified from the SGPs and the common origin between hub and CySCs has been shown by lineage tracing experiments [57]. This is further supported by the fact that both cell types can be traced using the same cell markers such as Zfh-1 and Traffic Jam (TJ) [25]. Hub cell fate vs. cyst cell fate is specified prior to gonad

*la* testis [47].

a Bam on/Mei-P26 off state.

Interestingly, very recent findings revealed that the Hedgehog (Hh) ligand secreted from the hub cells activates the Hh signaling in CySCs (and not in the GSCs) with critical function in CySC maintenance [37-40]. Hh overexpression leads in increased number of CySCs, identified as Zfh-1 positive cyst cells outside the niche, which can still proliferate in contrast to the normal post-mitotic SCCs. Furthermore, rescue of STAT depleted testis by Hh signaling activation in the CySCs can rescue the CySCs but GSC and germline maintenance is still impaired, as these Zfh-1 positive CySCs are not able to induce the GSC over-proliferation phenotype observed in SCCs ectopic Zfh-1 activation [38]. This suggests that [1] *zfh-1* expression relies on inputs from both Hh and JAK-STAT signaling pathways and that [2] apart from Zfh-1 other STAT regulated factors are necessary for allowing the CySC-to-GSC communication, which pro‐ motes GSC maintenance.

Notably, BMP seems to be the primary pathway leading to GSC self-renewal in the *Drosophi‐ la* testis [41-44]. BMP ligands and the BMP modulator *magu*, are expressed in the hub and CySCs that serve as the GSC niche and their loss results in reduced GSC numbers and *bam* derepression, whereas the hub and CySCs remain unaffected [42-44]. This could also suggest that expansion of GSC population by the JAK-STAT signaling could be due to its activation in the CySCs that consequently leads to enhanced expression of BMP ligands from CySCs [32] that finally drive GSC expansion. The BMP pathway is also negatively regulated in the course of testis morphogenesis along embryonic-larval-adult stages via Smurf (SMAD ubiquitination regulatory factor) [45]. High BMP levels are required at the initial steps of niche establishment when the hub cells attract the nearby germ cells to become GSCs in late embryogenesis up to early 3rd instar larval stages. Apparently, BMP signaling is spatially and temporally downre‐ gulated in stem cells and early germline cells in late 3rd instar larval and pupal testes through Smurf proteolytic activity. The described BMP downregulation seems to be critical for the normal decrease in stem cell number during pupal development, for restricting TA sperma‐ togonia proliferation and control of the testis size. This dynamic regulation indicates the requirement for fine trimming the BMP signaling intensity during subsequent developmental stages and might even suggest a difference between establishment vs. maintenance of certain cell populations across different stages. Yet, another recent story revealed that GSC charac‐ teristics can be maintained over time even after ablating the CySC and SCCs [46]. Without CySCs and SCCs, early germ cells away from the hub failed to initiate differentiation and maintained their GSC-like characteristics. Therefore, it becomes evident that the interactions between different stem cell populations and how one stem cell population influences the other can be indeed very complex. Finally, antagonistic functions between the *Drosophila* β-catenin Armadillo (Arm) and the microRNAs-(miR-) 310-313 suggest that modulation of the Wingless signaling activity is important to buffer germ cell and somatic differentiation in the *Drosophi‐ la* testis [47].

comparable levels in CySCs and early SCCs, is required for CySCs and not GSC renewal, and ectopic expression causes accumulation of GSCs-and CySCs-like cells. Furthermore, *zfh-1* and *chinmo* are not expressed in GSCs meaning that STAT can activate distinct downstream cascades in the GSC vs. CySCs. *ken* and *barbie* (*ken*) is another gene necessary and sufficient to promote CySC identity, yet in a STAT independent manner and with similar ectopic pheno‐ types like *zfh-1* and *chinmo* [35]. At the same time, Suppressor of cytokine signaling 36E (Socs36E) suppresses Jak-Stat signaling in the CySCs preventing them from outcompeting the GSCs and thereby maintains the proper balance of GSCs and CySCs, in a manner that depends

Interestingly, very recent findings revealed that the Hedgehog (Hh) ligand secreted from the hub cells activates the Hh signaling in CySCs (and not in the GSCs) with critical function in CySC maintenance [37-40]. Hh overexpression leads in increased number of CySCs, identified as Zfh-1 positive cyst cells outside the niche, which can still proliferate in contrast to the normal post-mitotic SCCs. Furthermore, rescue of STAT depleted testis by Hh signaling activation in the CySCs can rescue the CySCs but GSC and germline maintenance is still impaired, as these Zfh-1 positive CySCs are not able to induce the GSC over-proliferation phenotype observed in SCCs ectopic Zfh-1 activation [38]. This suggests that [1] *zfh-1* expression relies on inputs from both Hh and JAK-STAT signaling pathways and that [2] apart from Zfh-1 other STAT regulated factors are necessary for allowing the CySC-to-GSC communication, which pro‐

Notably, BMP seems to be the primary pathway leading to GSC self-renewal in the *Drosophi‐ la* testis [41-44]. BMP ligands and the BMP modulator *magu*, are expressed in the hub and CySCs that serve as the GSC niche and their loss results in reduced GSC numbers and *bam* derepression, whereas the hub and CySCs remain unaffected [42-44]. This could also suggest that expansion of GSC population by the JAK-STAT signaling could be due to its activation in the CySCs that consequently leads to enhanced expression of BMP ligands from CySCs [32] that finally drive GSC expansion. The BMP pathway is also negatively regulated in the course of testis morphogenesis along embryonic-larval-adult stages via Smurf (SMAD ubiquitination regulatory factor) [45]. High BMP levels are required at the initial steps of niche establishment when the hub cells attract the nearby germ cells to become GSCs in late embryogenesis up to early 3rd instar larval stages. Apparently, BMP signaling is spatially and temporally downre‐ gulated in stem cells and early germline cells in late 3rd instar larval and pupal testes through Smurf proteolytic activity. The described BMP downregulation seems to be critical for the normal decrease in stem cell number during pupal development, for restricting TA sperma‐ togonia proliferation and control of the testis size. This dynamic regulation indicates the requirement for fine trimming the BMP signaling intensity during subsequent developmental stages and might even suggest a difference between establishment vs. maintenance of certain cell populations across different stages. Yet, another recent story revealed that GSC charac‐ teristics can be maintained over time even after ablating the CySC and SCCs [46]. Without CySCs and SCCs, early germ cells away from the hub failed to initiate differentiation and maintained their GSC-like characteristics. Therefore, it becomes evident that the interactions between different stem cell populations and how one stem cell population influences the other

on the adhesion protein integrin [36].

118 Adult Stem Cell Niches

motes GSC maintenance.

Critical for germ cell differentiation is the expression of *bag of marbles (bam)* and *benign gonial cell neoplasm (bgcn)* in dividing spermatogonial cells in order to regulate their proliferation [48]. *bam* transcription is negatively regulated by the cooperation of the Glass bottom boat (Gbb) and Decapentaplegic (Dpp) signaling pathways emanating from the hub and CySCs to maintain the GSC identity [42]. Bam is required cell autonomously in TA spermatogonia to stop proliferation and enter the spermatocyte differentiation program [49]. The switch from TA proliferation to differentiation is mediated by translational control: Mei-P26 facilitates the accumulation of Bam in TA cells whereas Bam and Bcgn bind *mei-P26* 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through subsequent regulatory states that is: from a Mei-P26 on/Bam off to a Bam on/Mei-P26 off state.

Another signaling pathway restricting GSC proliferation is mediated by Epidermal Growth Factor Receptor (EGFR), whose inactivation in SCCs leads to an expansion of male GSCs [50]. In *Drosophila* testis, the major ligand of the EGFR pathway, Spitz (Spi) is secreted from the germline cells to stimulate the EGFR on cyst cells (CySCs and SCCs) [25]. Removal of either *spi* or *stet* from the germline cells, or removal of the EGFR from the cyst cells resulted in increased division frequencies of GSCs but did not affect the division frequencies of CySCs, suggesting that EGF signaling downregulates GSC divisions. Likewise, Raf, an EGFR downstream component, is required in SCCs to limit GSC expansion [51-53]. In testes mutated for the *rhomboid* homologue *stet*, the germ cells fail to associate with SCCs. Furthermore, germ cells recruit CySCs via the ligand Spitz, which binds to EGFR, and acts through the nucleotide exchange factor Vav to regulate the activity of Rac1, a down‐ stream component of the EGFR pathway. Taken together, EGF signaling from the germ‐ line cells produces differential Rac-and Rho-activities across the cyst cells that leads to a directional growth of the cyst cells around the germline cells [25]. Finally, Zero popula‐ tion growth (Zpg), the *Drosophila* gap junction Innexin 4, is localized to the spermatogo‐ nia surface, primarily on the sides adjacent to SCCs [54] and is required for the survival and differentiation of early germ cells in both sexes [55, 56].

#### **3. The male stem cell niche: Specification and positioning**

The somatic cells of the hub form the organizing center, a cluster of non-dividing cells, at the anterior part of the embryonic male gonad originating, as already discussed, from SGPs [10]. However, not only the hub but also the cyst cells are specified from the SGPs and the common origin between hub and CySCs has been shown by lineage tracing experiments [57]. This is further supported by the fact that both cell types can be traced using the same cell markers such as Zfh-1 and Traffic Jam (TJ) [25]. Hub cell fate vs. cyst cell fate is specified prior to gonad coalesce in a subset of somatic gonadal precursor cells (SGPs) upon Notch signaling activation [57]. In a next step, the *abdominal A (abd-A)* and *Abdominal B (Abd-B) Hox* genes promote the distinct identities of the SGP clusters: anterior SGP identity (PS10-11) is specified by *Abd-A* and repressed by *Abd-B*, a combination of *Abd-A* and *Abd-B* specifies the posterior SPGs (PS12) and *Abd-B* alone specifies the male-specific [58] SPGs (PS13) [9, 10, 20, 59]. Thus, *Abd-A* and *Abd-B* pattern the A/P axis of the formed gonad. Moreover, *Abd-B* can control the correct hub positioning by upregulating the tyrosine-kinase *sevenless (sev)* in the ms-SGPs. Sev is activated by the Boss ligand emanating from the primordial germ cells to represses ectopic hub differ‐ entiation [60] whereas the Epidermal growth factor receptor (EGFR) signaling represses hub formation in the rest of the SGPs [61]. Specification of CySCs vs. hub cell fate relies as well on the antagonistic function of *lines (lin)* and *brother of odd with entrails limited (bowl).* Bowl is a zinc finger transcription factor required in the hub cells and its antagonist Lin is a cytoplasmic protein with catalytic activity whereas Drumstick (Drm) competes with Lin for binding to Bowl [25, 62]. This regulatory network was supported by analysis of mutant phenotypes: *bowl* mutant gonads had fewer hub cells, *lines* mutant gonads had increased number of hub cells, whereas *lines* depleted CySCs acquired some hub-like properties and markers [57]. Once specified, the hub cells are able to recruit the anterior-most germ cells to become the germline stem cells (GSCs) [63], giving rise to the male stem cell niche [64].

We have discussed how the posteriorly expressed Hox genes *AbdA* and *AbdB* promote the distinct identities of the SGP clusters in the embryonic male gonad and how the diffusible signals and physical contact of germ and somatic cells keep the balance between stem cell renewal and differentiation in the larval and adult testis. However, it is interesting to under‐ stand how the male stem cell niche is maintained from its initial specification up to the adult stages and how this morphogenetic process is coordinated. In order to ensure normal niche function in the *Drosophila* testis, the hub cells not only need to be properly specified but also need to be correctly placed. Integrin-mediated adhesion is important for maintaining the correct position of the embryonic hub cells during gonad morphogenesis. In the absence of integrin-mediated adhesion, the hub cells still form a cluster, but instead of remaining at the anterior part of the gonad they migrate to the middle part of the developing gonad [65]. Disruption of integrin-mediated adhesion in adult testis by knocking down *talin/rhea,* an integrin-binding and essential focal adhesion protein of the Integrin-cytoskeleton link [66, 67], results in GSC loss and gradual hub disappearance, a phenotype, which becomes more severe as adult males age [67]. As in *talin*-depleted adult testis the hub is progressively lost, the signals that normally emanate from the hub to instruct stem cell renewal are absent, driving the balance between stem cell maintenance and differentiation towards more differentiation and progressive stem cell loss [65]. A similar hub displacement phenotype is observed by depleting adult testis of Lasp [68], an actin-binding protein. From the vertebrate system we know that Lasp interacts genetically with Integrin [69] and in blood platelets Lasp requires Integrin for its proper localization to the cytoskeleton [70]. Moreover, expression levels of Integrin and Talin are critical for occupation of the niche as CySCs with enhanced integrin-mediated adhesion are able to compete and displace their neighboring GSCs [36].

**Figure 2.** Somatic cyst cells are thin, elongated cells with apical and basal surfaces surrounded by ECM. (A) Schematic diagram of early *Drosophila* spermatogenesis. Somatic cyst cells (SCC) are thin, squamous cells and wrap the germline creating cysts surrounded by ECM (orange). (B-F') Components of cyst cells (red) co-stained with Dlg (green). Here, only the spermatocyte region is shown. Baz (B, B') and FasII (D, D') co-localize with Dlg. Rho1 and Dlg decorate the SCCs but do not co-localize (C, C'). In SCCs Dlg and Integrin are not co-localizing, with Dlg being apical (inner side; white arrowheads) and PS-Integrin more basal (facing outside; white arrows) (E, E'). ILK decorates the SCC cytoplasm and Dlg decorates SCCs facing the germline (white arrowheads) (F, F'). Yellow arrowheads in (C), (E) and (F) show SCC cellular projections growing in between the germ cells. Testes are oriented anterior left. Scale Bar: 10 mm. (G) Sche‐ matic diagram depicting a close up of a spermatocyte cyst with key players involved in niche positioning (for simplicity only one somatic cyst cell is shown). Within the spermatocytes, red line indicates the spermatocyte nuclear membrane,

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green dots illustrate Abd-B in the nucleolus and blue represents the nucleoplasm.

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coalesce in a subset of somatic gonadal precursor cells (SGPs) upon Notch signaling activation [57]. In a next step, the *abdominal A (abd-A)* and *Abdominal B (Abd-B) Hox* genes promote the distinct identities of the SGP clusters: anterior SGP identity (PS10-11) is specified by *Abd-A* and repressed by *Abd-B*, a combination of *Abd-A* and *Abd-B* specifies the posterior SPGs (PS12) and *Abd-B* alone specifies the male-specific [58] SPGs (PS13) [9, 10, 20, 59]. Thus, *Abd-A* and *Abd-B* pattern the A/P axis of the formed gonad. Moreover, *Abd-B* can control the correct hub positioning by upregulating the tyrosine-kinase *sevenless (sev)* in the ms-SGPs. Sev is activated by the Boss ligand emanating from the primordial germ cells to represses ectopic hub differ‐ entiation [60] whereas the Epidermal growth factor receptor (EGFR) signaling represses hub formation in the rest of the SGPs [61]. Specification of CySCs vs. hub cell fate relies as well on the antagonistic function of *lines (lin)* and *brother of odd with entrails limited (bowl).* Bowl is a zinc finger transcription factor required in the hub cells and its antagonist Lin is a cytoplasmic protein with catalytic activity whereas Drumstick (Drm) competes with Lin for binding to Bowl [25, 62]. This regulatory network was supported by analysis of mutant phenotypes: *bowl* mutant gonads had fewer hub cells, *lines* mutant gonads had increased number of hub cells, whereas *lines* depleted CySCs acquired some hub-like properties and markers [57]. Once specified, the hub cells are able to recruit the anterior-most germ cells to become the germline

We have discussed how the posteriorly expressed Hox genes *AbdA* and *AbdB* promote the distinct identities of the SGP clusters in the embryonic male gonad and how the diffusible signals and physical contact of germ and somatic cells keep the balance between stem cell renewal and differentiation in the larval and adult testis. However, it is interesting to under‐ stand how the male stem cell niche is maintained from its initial specification up to the adult stages and how this morphogenetic process is coordinated. In order to ensure normal niche function in the *Drosophila* testis, the hub cells not only need to be properly specified but also need to be correctly placed. Integrin-mediated adhesion is important for maintaining the correct position of the embryonic hub cells during gonad morphogenesis. In the absence of integrin-mediated adhesion, the hub cells still form a cluster, but instead of remaining at the anterior part of the gonad they migrate to the middle part of the developing gonad [65]. Disruption of integrin-mediated adhesion in adult testis by knocking down *talin/rhea,* an integrin-binding and essential focal adhesion protein of the Integrin-cytoskeleton link [66, 67], results in GSC loss and gradual hub disappearance, a phenotype, which becomes more severe as adult males age [67]. As in *talin*-depleted adult testis the hub is progressively lost, the signals that normally emanate from the hub to instruct stem cell renewal are absent, driving the balance between stem cell maintenance and differentiation towards more differentiation and progressive stem cell loss [65]. A similar hub displacement phenotype is observed by depleting adult testis of Lasp [68], an actin-binding protein. From the vertebrate system we know that Lasp interacts genetically with Integrin [69] and in blood platelets Lasp requires Integrin for its proper localization to the cytoskeleton [70]. Moreover, expression levels of Integrin and Talin are critical for occupation of the niche as CySCs with enhanced integrin-mediated

stem cells (GSCs) [63], giving rise to the male stem cell niche [64].

120 Adult Stem Cell Niches

adhesion are able to compete and displace their neighboring GSCs [36].

**Figure 2.** Somatic cyst cells are thin, elongated cells with apical and basal surfaces surrounded by ECM. (A) Schematic diagram of early *Drosophila* spermatogenesis. Somatic cyst cells (SCC) are thin, squamous cells and wrap the germline creating cysts surrounded by ECM (orange). (B-F') Components of cyst cells (red) co-stained with Dlg (green). Here, only the spermatocyte region is shown. Baz (B, B') and FasII (D, D') co-localize with Dlg. Rho1 and Dlg decorate the SCCs but do not co-localize (C, C'). In SCCs Dlg and Integrin are not co-localizing, with Dlg being apical (inner side; white arrowheads) and PS-Integrin more basal (facing outside; white arrows) (E, E'). ILK decorates the SCC cytoplasm and Dlg decorates SCCs facing the germline (white arrowheads) (F, F'). Yellow arrowheads in (C), (E) and (F) show SCC cellular projections growing in between the germ cells. Testes are oriented anterior left. Scale Bar: 10 mm. (G) Sche‐ matic diagram depicting a close up of a spermatocyte cyst with key players involved in niche positioning (for simplicity only one somatic cyst cell is shown). Within the spermatocytes, red line indicates the spermatocyte nuclear membrane, green dots illustrate Abd-B in the nucleolus and blue represents the nucleoplasm.

#### **3.1. Some function, different mechanisms: How the Boss/Sev-AbdB cross-talk regulates niche positioning and integrity**

its receptor Sev, Boss becomes internalized in the *sev*-expressing cell (Cagan et al., 1992; Kramer, 1993; Kramer et al., 1991] whereas in the fat body, in response to stimulation by glucose, Boss becomes enclosed in internalized vesicles (Kohyama-Koganeya et al., 2008]. In the *Drosophila* testis, Boss is found in the germline spermatocytes, primarily in vesicles (Fig. 3G), whereas Sev localizes in the cyst cells enclosing them. Abd-B performs its function by affecting Boss internalization in the germline, as Boss is lost from internalized vesicles in *Abd-B* depleted testes [18]. Expression of activated Sev in cyst cells of Abd-B depleted testes could fully rescue the phenotype, meaning the Boss exerts its function via Sev activation. Similarly, a partial rescue of hub positioning and integrin localization was observed by expressing the *shibire*(*shi*) gene [72, 73], which is critical for the endocytic uptake of receptors from the plasma membrane [74, 75] in spermatocytes of *Abd-B* depleted testes. This further suggested that Boss

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In order to elucidate how the Hox transcription factor Abd-B affects Boss localization, genes directly regulated by Abd-B in the *Drosophila* testis were identified by mapping Abd-B binding sites *in vivo* using the DNA adenine methyltransferase identification (DamID) technology [76-79]. This analysis resulted in the identification of 1804 Abd-B binding regions in larval testes, which are associated with 2771 genes. To determine over-representation of GO terms, GO terms were grouped using their annotated Biological Process and subsequently the overrepresentation of GO term groups among the identified genes was analyzed [18]. Since Abd-B controls signaling between the germline and somatic lineage by regulating genes required for Boss receptor recycling or trafficking, further analysis focused on genes involved in trafficking processes. Two genes, one encoding the non-receptor tyrosine kinase Src oncogene at 42A (Src42A) and another one encoding the putative signal recognition binding protein Sec63, were identified as potential mediators of Boss function in the larval testis. In support of a direct regulatory interaction between *src42A* and Abd-B in the larval testis, *src42A* mRNA levels [80] were found to be significantly downregulated in spermatocytes of *AbdBRNAi::T100* animals (with *T100-GAL4* driving expression of UAS-AbdBRNAi in germline spermatocytes), and likewise the activity of the protein tyrosine kinase Src42A was dramatically reduced [18]. Importantly, functional analysis revealed that *src42A* depleted testes mimic the loss of *Abd-B* function: in contrast to wild-type testes, Boss protein was not detected in vesicles, the hub was mispositioned and βPS-integrin was not properly localized in somatic cyst cells of *src42A*

functions in a dynamin-dependent way for its endocytic recycling.

depleted testes. Same results were obtained for *sec63*.

**4. Dlg, Scrib & Lgl: New functions in the** *Drosophila* **testis**

The *discs large (dlg), scribble (scrib) and lethal* [2] *giant larvae (lgl)* genes were initially identified in *Drosophila* as tumor suppressor genes (TSGs) whose mutations lead to neoplastic transfor‐ mation, such as imaginal disc overgrowth and brain tumors [81-84]. Mutant flies die after an extended larval life as "giant" larvae without pupariation. In these tumors the overproliferat‐ ing cells lose their typical epithelial apico-basal polarity, fail to organize an epithelial mono‐ layer and terminally differentiate [84-86]. Therefore, all three TSGs are additionally classified as "cell polarity genes" [83, 84, 87-89]. Since their initial discovery, *dlg, scrib* and *lgl* have been

As already mentioned, the Boss/Sev signaling pathway plays an important role in hub positioning in the *Drosophila* embryonic male gonads by preventing ectopic niche differentia‐ tion in the posterior gonadal somatic cells. *Abd-B*, upstream of this cascade, activates *sev* in the posterior SGPs [60] and consistent with the fact that weak *Abd-B* mutant alleles result in hub expansion and integrity defects in embryonic gonads [10]. A very recent study revealed a new role for the posterior *Hox* gene *Abd-B* in the larval and adult testis. Analysis of the role of the Hox protein Abd-B in the *Drosophila* testis revealed that Abd-B present in the germline spermatocytes acts upstream of the Boss/Sev pathway to regulate hub positioning and integrity, which finally leads to loss of Integrin and Actin localization in the neighboring cyst cells [18]. Analysis of the genetic interactions of *Abd-B* with *integrin* and focal adhesion proteins, revealed that male stem cell niche positioning is regulated by a number of factors, which link Integrin to the extracellular matrix (ECM) and actin filaments. Interestingly, the incorrect placement of the niche in *Abd-B* depleted testes, results in cell non-autonomous centrosome mispositioning and reduced GSC divisions, leading to a dramatic reduction of the pre-meiotic stages of the adult testis, a hallmark of aging in testis [14, 71].

Taken together these studies show that the same players, AbdB, Boss, Sev and Integrin, are used in larval stages to preserve hub positioning and integrity after the initial establishment at embryonic stages but using a slightly variable mechanism: (a) In embryonic gonads, *Abd-B* from the male-specific SGPs regulates *sev* expression in the same cells, whereas Boss signals from the germ cells signals to the Sev expressing cells to ensure that the niche develops in the anterior region of the gonad [60]. Integrin is also required in the somatic cells of the embryonic gonads for anterior positioning of the hub [65]. (b) In larval testes, Abd-B regulates the same process from the germline spermatocytes and via the Boss/Sev pathway controls integrin localization in the neighboring SCCs. This expression switch of Abd-B from the somatic to the germline lineage not only highlights that the mechanism of Abd-B dependent hub positioning is different between embryonic and larval stages but also raises the interesting questions of why and how Abd-B changes its expression and thus the mechanism of hub positioning. During adult stages when testis morphogenesis is completed with the addition of the actomyosin sheath originating from the genital disc [19], hub positioning and integrity is regulated by Sev, Boss [60] and Integrin [68] whereas Abd-B regulates hub positioning from a different cell type in comparison to embryonic and larval stages which is this time the cells of the actomyosin sheath, originating from the genital disc. It seems that the occurrence of new cell types and cell interactions in the course of testis organogenesis made it necessary to adapt the whole stem cell system to the new cellular conditions by reusing the same main players of niche positioning in an alternative manner.

#### *3.1.1. Boss mediates, in a Dynamin-and Src-dependent way, germline-soma signaling in larval testis*

*Drosophila* Boss is an atypical G-protein coupled receptor membrane protein that was first identified as a ligand of the Sevenless (Sev) tyrosine kinase involved in eye differentiation. Previous studies in the eye showed that upon binding of the transmembrane protein Boss to its receptor Sev, Boss becomes internalized in the *sev*-expressing cell (Cagan et al., 1992; Kramer, 1993; Kramer et al., 1991] whereas in the fat body, in response to stimulation by glucose, Boss becomes enclosed in internalized vesicles (Kohyama-Koganeya et al., 2008]. In the *Drosophila* testis, Boss is found in the germline spermatocytes, primarily in vesicles (Fig. 3G), whereas Sev localizes in the cyst cells enclosing them. Abd-B performs its function by affecting Boss internalization in the germline, as Boss is lost from internalized vesicles in *Abd-B* depleted testes [18]. Expression of activated Sev in cyst cells of Abd-B depleted testes could fully rescue the phenotype, meaning the Boss exerts its function via Sev activation. Similarly, a partial rescue of hub positioning and integrin localization was observed by expressing the *shibire*(*shi*) gene [72, 73], which is critical for the endocytic uptake of receptors from the plasma membrane [74, 75] in spermatocytes of *Abd-B* depleted testes. This further suggested that Boss functions in a dynamin-dependent way for its endocytic recycling.

**3.1. Some function, different mechanisms: How the Boss/Sev-AbdB cross-talk regulates**

As already mentioned, the Boss/Sev signaling pathway plays an important role in hub positioning in the *Drosophila* embryonic male gonads by preventing ectopic niche differentia‐ tion in the posterior gonadal somatic cells. *Abd-B*, upstream of this cascade, activates *sev* in the posterior SGPs [60] and consistent with the fact that weak *Abd-B* mutant alleles result in hub expansion and integrity defects in embryonic gonads [10]. A very recent study revealed a new role for the posterior *Hox* gene *Abd-B* in the larval and adult testis. Analysis of the role of the Hox protein Abd-B in the *Drosophila* testis revealed that Abd-B present in the germline spermatocytes acts upstream of the Boss/Sev pathway to regulate hub positioning and integrity, which finally leads to loss of Integrin and Actin localization in the neighboring cyst cells [18]. Analysis of the genetic interactions of *Abd-B* with *integrin* and focal adhesion proteins, revealed that male stem cell niche positioning is regulated by a number of factors, which link Integrin to the extracellular matrix (ECM) and actin filaments. Interestingly, the incorrect placement of the niche in *Abd-B* depleted testes, results in cell non-autonomous centrosome mispositioning and reduced GSC divisions, leading to a dramatic reduction of the pre-meiotic

Taken together these studies show that the same players, AbdB, Boss, Sev and Integrin, are used in larval stages to preserve hub positioning and integrity after the initial establishment at embryonic stages but using a slightly variable mechanism: (a) In embryonic gonads, *Abd-B* from the male-specific SGPs regulates *sev* expression in the same cells, whereas Boss signals from the germ cells signals to the Sev expressing cells to ensure that the niche develops in the anterior region of the gonad [60]. Integrin is also required in the somatic cells of the embryonic gonads for anterior positioning of the hub [65]. (b) In larval testes, Abd-B regulates the same process from the germline spermatocytes and via the Boss/Sev pathway controls integrin localization in the neighboring SCCs. This expression switch of Abd-B from the somatic to the germline lineage not only highlights that the mechanism of Abd-B dependent hub positioning is different between embryonic and larval stages but also raises the interesting questions of why and how Abd-B changes its expression and thus the mechanism of hub positioning. During adult stages when testis morphogenesis is completed with the addition of the actomyosin sheath originating from the genital disc [19], hub positioning and integrity is regulated by Sev, Boss [60] and Integrin [68] whereas Abd-B regulates hub positioning from a different cell type in comparison to embryonic and larval stages which is this time the cells of the actomyosin sheath, originating from the genital disc. It seems that the occurrence of new cell types and cell interactions in the course of testis organogenesis made it necessary to adapt the whole stem cell system to the new cellular conditions by reusing the same main players of niche

*3.1.1. Boss mediates, in a Dynamin-and Src-dependent way, germline-soma signaling in larval testis*

*Drosophila* Boss is an atypical G-protein coupled receptor membrane protein that was first identified as a ligand of the Sevenless (Sev) tyrosine kinase involved in eye differentiation. Previous studies in the eye showed that upon binding of the transmembrane protein Boss to

**niche positioning and integrity**

122 Adult Stem Cell Niches

stages of the adult testis, a hallmark of aging in testis [14, 71].

positioning in an alternative manner.

In order to elucidate how the Hox transcription factor Abd-B affects Boss localization, genes directly regulated by Abd-B in the *Drosophila* testis were identified by mapping Abd-B binding sites *in vivo* using the DNA adenine methyltransferase identification (DamID) technology [76-79]. This analysis resulted in the identification of 1804 Abd-B binding regions in larval testes, which are associated with 2771 genes. To determine over-representation of GO terms, GO terms were grouped using their annotated Biological Process and subsequently the overrepresentation of GO term groups among the identified genes was analyzed [18]. Since Abd-B controls signaling between the germline and somatic lineage by regulating genes required for Boss receptor recycling or trafficking, further analysis focused on genes involved in trafficking processes. Two genes, one encoding the non-receptor tyrosine kinase Src oncogene at 42A (Src42A) and another one encoding the putative signal recognition binding protein Sec63, were identified as potential mediators of Boss function in the larval testis. In support of a direct regulatory interaction between *src42A* and Abd-B in the larval testis, *src42A* mRNA levels [80] were found to be significantly downregulated in spermatocytes of *AbdBRNAi::T100* animals (with *T100-GAL4* driving expression of UAS-AbdBRNAi in germline spermatocytes), and likewise the activity of the protein tyrosine kinase Src42A was dramatically reduced [18]. Importantly, functional analysis revealed that *src42A* depleted testes mimic the loss of *Abd-B* function: in contrast to wild-type testes, Boss protein was not detected in vesicles, the hub was mispositioned and βPS-integrin was not properly localized in somatic cyst cells of *src42A* depleted testes. Same results were obtained for *sec63*.

#### **4. Dlg, Scrib & Lgl: New functions in the** *Drosophila* **testis**

The *discs large (dlg), scribble (scrib) and lethal* [2] *giant larvae (lgl)* genes were initially identified in *Drosophila* as tumor suppressor genes (TSGs) whose mutations lead to neoplastic transfor‐ mation, such as imaginal disc overgrowth and brain tumors [81-84]. Mutant flies die after an extended larval life as "giant" larvae without pupariation. In these tumors the overproliferat‐ ing cells lose their typical epithelial apico-basal polarity, fail to organize an epithelial mono‐ layer and terminally differentiate [84-86]. Therefore, all three TSGs are additionally classified as "cell polarity genes" [83, 84, 87-89]. Since their initial discovery, *dlg, scrib* and *lgl* have been recognized as having important roles also in other forms of polarity as well as in regulation of the actin cytoskeleton, cell signaling and vesicular trafficking [86, 90].

*4.1.2. Vesicle and membrane trafficking*

binding to the β–Pix-GIT1 complex [115].

*4.1.3. Gene regulation and signaling output*

109, 111].

chromatin to regulate secondary gene expression [118].

Several pieces of evidence suggest that Dlg, Scrib and Lgl are involved in vesicle and membrane trafficking [86, 102]: i) Dlg and Strabismus (VanGogh) form a complex that allows membrane deposition during cellularization in *Drosophila* embryos [109] ii) Dlg regulates membrane proliferation of the subsynaptic reticulum (SSR) in NMJs by binding the t-SNARE protein Gtaxin [110, 111] iii) Dlg and Lgl genetically interact with Exo84 which is required for mem‐ brane addition [112] iv) the yeast Lgl homologues Sro7p and Sro77p interact directly with Exo84p and Sec9p traggicking components [113], v) mammalian Lgl binds Syntaxin-4 (t-SNARE) to direct protein trafficking [114], and vi) mammalian Scrib regulates exocytosis by

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Recent studies associate Dlg, Scrib and Lgl with transcriptional response and signaling output since they can regulate the shuttling of critical components between junctional complexes and the nucleus. Such a shuttling mechanism has been described for the Dlg and Scrib vertebrate homologues [116, 117]. In *Drosophila* salivary glands, Lgl together with non-muscle myosin regulate in the cytoplasm access to chromatin modifiers, remodeling and transcription factors necessary for salivary gland degeneration [118]. In wild type salivary glands, chromatin remodeling factors are localized in the nucleus to bind chromatin whereas in the absence of Lgl they accumulate in the cytoplasm and the cortical nuclear zone but cannot bind to

Taken together, Dlg, Scrib and Lgl emerge as dynamic cytoskeletal components which affect polarity, cell structure and behavior by directing the trafficking of proteins to proper plasma membrane surfaces of the cell, and by organizing and stabilizing supramolecular adhesion and signaling complexes through their action as scaffolding adaptor molecules [83-86, 89-91,

**4.2. Dlg, Scrib & Lgl in testis somatic cells promote cyst cell function & testis homeostasis**

Septate junctions are primary candidates for cyst integrity and coordination, as apart from acting as sealing junctions in epithelia and neurons by mediating cell-cell adhesion, they act as scaffolding networks together with multiple pathways to promote organ morphogenesis [120]. Although the function of Dlg, Scrib and Lgl as TSGs has been intensively studied, their role in testis development has been largely overlooked, as mutations in their coding genes do not result in testis tumors. Moreover, the fact that testes lack an easy to study columnar epithelium, which facilitates analysis of apicobasal polarity genes, didn't favor the analysis of these genes in this stem cell system for many years. The last years a number of studies addressed the role of *scrib*, *dlg* and *lgl* scaffolding proteins in the *Drosophila* male gonad, testis architecture and homeostasis [22-24, 119, 121]. Prompted by the observation that the septate junction protein Scrib [122] is expressed in the newly formed embryonic *Drosophila* gonads [88], Scrib dynamics in the embryonic gonads was investigated [24]. During gonad formation Scrib forms a polygonal network around the germ cells and is present primarily in the somatic

Dlg belongs to the MAGUK (membrane-associated guanylate kinases) protein family, a class of scaffolding proteins that recruit signaling molecules into localized multimolecular com‐ plexes [83, 91]. Dlg localizes at the cytoplasmic side of septate junctions between adjacent epithelial cells (the equivalent of vertebrate tight junctions), as well as in neuromuscular junctions (NMJs). It contains three PDZ domains involved in protein-protein interactions with membrane or cytoskeletal proteins, an SH3 domain and a GUK domain. Scrib is also a septate junctional protein of the LAP protein family, containing four PDZ domains and leucine-rich repeats (LRRs) [85, 87, 91, 92]. Lgl is a cytosolic protein containing two WD40 motifs, involved in protein-protein interactions [87]. Lgl can bind to non-muscle myosin II and to the cytoske‐ leton matrix, along the baso-lateral portion of the plasma membrane of epithelial cells to affect cell polarization [93]. All three proteins, often referred to as the Dlg-polarity module, are highly conserved in sequence among different species and growing evidence suggests that they are functionally conserved to a large degree since the vertebrate homologues can rescue the polarity defects and tumorous overgrowth of the respective *Drosophila* mutants [94-96].

#### **4.1. Dlg, Scrib & Lgl: Multitasking proteins in common pathways in various tissues**

Research over several years, defined *dlg, scrib* and *lgl* as key players in numerous tissues contents and malignancies at different time points throughout development, and revealed their multitasking role in: polarity and septate junction establishment; nervous system and brain development; organ development; cancer initiation, progression and metastasis; and mechanism of cooperation with various signaling pathways (Ras, Salvador-Warts-Hippo, Dpp, JNK, Wg, EGFR etc) [22, 97-104]. Some of their common modes of action across different tissues and organisms are analyzed below.

#### *4.1.1. Polarity establishment in various cellular contexts*

The Dlg polarity module works in cooperation with the Crumbs-(Crb, Pals1 & Patj) and the Par-(Bazooka/Par3, Par6, αPKC) polarity complexes to control polarity in several tissues. In epithelial cells, polarity is established in a finely balanced process involving cooperative and antagonistic interactions among the apical Par-and Crumbs-complexes and the basolateral Dlg-complex, which restrict the activity of each complex to its specific membrane domain [85, 86]. In neuroblast asymmetric cell division Dlg, Scrib and Lgl cooperate with the Par and Inscutable-Pins complexes whereas microtubules induce Pins & Gαi cortical polarity through Dlg and Khc-73 interactions [86, 105, 106]. In the *Drosophila* ectoderm, phosphorylation of αPKC is required for Lgl to establish the lateral domain and to prevent apical Lgl recruitment. Lgl homologues genetically interact with Par components to regulate apicobasal polarity in *Xenopus* and MDCK epithelial cells, and in partitioning cell fate determinants in *C.elegans* [85, 90, 91, 107]. Finally, the Dlg polarity module has critical functions also in *Drosophila* dorsal closure formation, in patterning anterior and posterior follicle cells, in wound healing proc‐ esses, in planar cell polarity, in formation of synapses and in NMJs together with other polarity, scaffolding and receptor complexes [86, 102, 108].

#### *4.1.2. Vesicle and membrane trafficking*

recognized as having important roles also in other forms of polarity as well as in regulation of

Dlg belongs to the MAGUK (membrane-associated guanylate kinases) protein family, a class of scaffolding proteins that recruit signaling molecules into localized multimolecular com‐ plexes [83, 91]. Dlg localizes at the cytoplasmic side of septate junctions between adjacent epithelial cells (the equivalent of vertebrate tight junctions), as well as in neuromuscular junctions (NMJs). It contains three PDZ domains involved in protein-protein interactions with membrane or cytoskeletal proteins, an SH3 domain and a GUK domain. Scrib is also a septate junctional protein of the LAP protein family, containing four PDZ domains and leucine-rich repeats (LRRs) [85, 87, 91, 92]. Lgl is a cytosolic protein containing two WD40 motifs, involved in protein-protein interactions [87]. Lgl can bind to non-muscle myosin II and to the cytoske‐ leton matrix, along the baso-lateral portion of the plasma membrane of epithelial cells to affect cell polarization [93]. All three proteins, often referred to as the Dlg-polarity module, are highly conserved in sequence among different species and growing evidence suggests that they are functionally conserved to a large degree since the vertebrate homologues can rescue the polarity defects and tumorous overgrowth of the respective *Drosophila* mutants [94-96].

**4.1. Dlg, Scrib & Lgl: Multitasking proteins in common pathways in various tissues**

tissues and organisms are analyzed below.

*4.1.1. Polarity establishment in various cellular contexts*

scaffolding and receptor complexes [86, 102, 108].

Research over several years, defined *dlg, scrib* and *lgl* as key players in numerous tissues contents and malignancies at different time points throughout development, and revealed their multitasking role in: polarity and septate junction establishment; nervous system and brain development; organ development; cancer initiation, progression and metastasis; and mechanism of cooperation with various signaling pathways (Ras, Salvador-Warts-Hippo, Dpp, JNK, Wg, EGFR etc) [22, 97-104]. Some of their common modes of action across different

The Dlg polarity module works in cooperation with the Crumbs-(Crb, Pals1 & Patj) and the Par-(Bazooka/Par3, Par6, αPKC) polarity complexes to control polarity in several tissues. In epithelial cells, polarity is established in a finely balanced process involving cooperative and antagonistic interactions among the apical Par-and Crumbs-complexes and the basolateral Dlg-complex, which restrict the activity of each complex to its specific membrane domain [85, 86]. In neuroblast asymmetric cell division Dlg, Scrib and Lgl cooperate with the Par and Inscutable-Pins complexes whereas microtubules induce Pins & Gαi cortical polarity through Dlg and Khc-73 interactions [86, 105, 106]. In the *Drosophila* ectoderm, phosphorylation of αPKC is required for Lgl to establish the lateral domain and to prevent apical Lgl recruitment. Lgl homologues genetically interact with Par components to regulate apicobasal polarity in *Xenopus* and MDCK epithelial cells, and in partitioning cell fate determinants in *C.elegans* [85, 90, 91, 107]. Finally, the Dlg polarity module has critical functions also in *Drosophila* dorsal closure formation, in patterning anterior and posterior follicle cells, in wound healing proc‐ esses, in planar cell polarity, in formation of synapses and in NMJs together with other polarity,

the actin cytoskeleton, cell signaling and vesicular trafficking [86, 90].

124 Adult Stem Cell Niches

Several pieces of evidence suggest that Dlg, Scrib and Lgl are involved in vesicle and membrane trafficking [86, 102]: i) Dlg and Strabismus (VanGogh) form a complex that allows membrane deposition during cellularization in *Drosophila* embryos [109] ii) Dlg regulates membrane proliferation of the subsynaptic reticulum (SSR) in NMJs by binding the t-SNARE protein Gtaxin [110, 111] iii) Dlg and Lgl genetically interact with Exo84 which is required for mem‐ brane addition [112] iv) the yeast Lgl homologues Sro7p and Sro77p interact directly with Exo84p and Sec9p traggicking components [113], v) mammalian Lgl binds Syntaxin-4 (t-SNARE) to direct protein trafficking [114], and vi) mammalian Scrib regulates exocytosis by binding to the β–Pix-GIT1 complex [115].

#### *4.1.3. Gene regulation and signaling output*

Recent studies associate Dlg, Scrib and Lgl with transcriptional response and signaling output since they can regulate the shuttling of critical components between junctional complexes and the nucleus. Such a shuttling mechanism has been described for the Dlg and Scrib vertebrate homologues [116, 117]. In *Drosophila* salivary glands, Lgl together with non-muscle myosin regulate in the cytoplasm access to chromatin modifiers, remodeling and transcription factors necessary for salivary gland degeneration [118]. In wild type salivary glands, chromatin remodeling factors are localized in the nucleus to bind chromatin whereas in the absence of Lgl they accumulate in the cytoplasm and the cortical nuclear zone but cannot bind to chromatin to regulate secondary gene expression [118].

Taken together, Dlg, Scrib and Lgl emerge as dynamic cytoskeletal components which affect polarity, cell structure and behavior by directing the trafficking of proteins to proper plasma membrane surfaces of the cell, and by organizing and stabilizing supramolecular adhesion and signaling complexes through their action as scaffolding adaptor molecules [83-86, 89-91, 109, 111].

#### **4.2. Dlg, Scrib & Lgl in testis somatic cells promote cyst cell function & testis homeostasis**

Septate junctions are primary candidates for cyst integrity and coordination, as apart from acting as sealing junctions in epithelia and neurons by mediating cell-cell adhesion, they act as scaffolding networks together with multiple pathways to promote organ morphogenesis [120]. Although the function of Dlg, Scrib and Lgl as TSGs has been intensively studied, their role in testis development has been largely overlooked, as mutations in their coding genes do not result in testis tumors. Moreover, the fact that testes lack an easy to study columnar epithelium, which facilitates analysis of apicobasal polarity genes, didn't favor the analysis of these genes in this stem cell system for many years. The last years a number of studies addressed the role of *scrib*, *dlg* and *lgl* scaffolding proteins in the *Drosophila* male gonad, testis architecture and homeostasis [22-24, 119, 121]. Prompted by the observation that the septate junction protein Scrib [122] is expressed in the newly formed embryonic *Drosophila* gonads [88], Scrib dynamics in the embryonic gonads was investigated [24]. During gonad formation Scrib forms a polygonal network around the germ cells and is present primarily in the somatic gonadal cells, the so-called gonadal mesoderm, that surrounds them. Scrib synthesis in the gonadal mesoderm is cell autonomous, since analysis of agametic gonads and pseudo-gonads made of aggregated germ cells revealed that Scrib in the germ cells requires a direct contact to the gonadal mesoderm [24].

As Dlg, Scrib and Lgl act cooperatively in several tissue contexts [23, 84], their function during male gonad and testis development was analyzed in a comparable way [22, 119]. This work revealed that cell autonomous *scrib* and *dlg* expression in the gonadal meso‐ derm affects critically the internal structure of the gonads by establishing the intimate contacts of the germ cells to the gonadal mesoderm [24, 119]. At later stages, *dlg*, *scrib* and *lgl* expression in the hub, CySCs and SCCs (Fig.3 A-C) is indispensable for testis develop‐ ment and homeostasis, as depletion of these genes results in extremely small testes with reduced number of germline stem cells and impaired differentiation (Fig.3 E-H). More‐ over, Dlg localization in CySCs establishes a tight connection between GSCs and CySCs, and thereby preserves the niche architecture. In late SCCs *dlg* expression is critical for their survival, growth, expansion and for maintaining the integrity of the cysts [22]. This is supported by the observation that the Eya-positive SCCs present in the wild-type testes (Fig.3I; arrowheads) are lost in *dlg* testes (Fig.3J) and die due to apoptosis [22]. Similar to *dlg*, *lgl* testes also lose Eya-positive SCCs (Fig.3L), whereas in *scrib* testes late SCCs are still present (Fig.3K; arrowheads) but the size of these Eya-positive nuclei and of overall testis size is significantly reduced [119]. In contrast to the overgrowth phenotypes observed in imaginal discs and brain hemispheres, the extensive defects in *dlg, scrib* and *lgl* mutant testes underline the importance of the somatic lineage in the establishment of a tight somagermline adhesion and cyst integrity, which is a prerequisite for a functional male stem cell niche and proper testis differentiation [2, 23, 119].

Another striking finding was the formation of wavy and ruffled plasma membrane upon *dlg* over-expression in somatic cyst cells capping the spermatocyte cysts. Up to now, there is no mechanism describing how cyst cells in *Drosophila* testis grow enormously, elongate and ensheath the germ cells of spermatogonial and spermatocyte cysts or how spermatid differ‐ entiation and individualization is guided by the polarized head and tail SCC. From other systems we know that Dlg regulates membrane proliferation in a subset of NMJs in a dosedependent fashion [123] and is an important player in the process of polarized membrane insertion during cellularization [109, 124-126].

124-126]. The fact that membrane proliferation is also involved in mechanisms such as tissue spreading and cell surface extensions, including membrane ruffles [127, 128] and com‐ bined with our results on SCCs membrane ruffling upon Dlg overexpression it can be suggested that polarized membrane insertion, mediated by Dlg, might conduct SCCs

**Figure 3.** Dlg, Scrib and Lgl in the somatic lineage have critical functions in niche architecture, testis differentiation and homeostasis. (A-C) Dlg, Scrib and Lgl localize in somatic hub, somatic stem and cyst cells in *Drosophila* testis. (D) Dlg overexpression leads to ruffled membranes of somatic cyst cells, showing that Dlg promotes somatic cyst cell growth and membrane addition. (E-H) *dlg, scrib* and *lgl* mutant testes are extremely small, with reduced number of germline stem cells and impaired differentiation with only few spermatogonial cysts. Traffic Jam (TJ) marks the somat‐ ic stem cell and cyst cell nuclei, Vasa the germline, Armadillo (Arm) the hub and somatic stem and cyst cells, a-Spectrin the fusome growing through the interconnected spermatogonia and spermatocytes. (I-L) In *dlg* and *lgl* testes late so‐ matic cyst cells are lost as no Eyes Absent (Eya)-positive cyst cells are observed and the tight connection between the cyst cells and the germline is lost. In *scrib* testes Eya-positive somatic cyst cells are present, however testes are small and underdeveloped. Arrows point at the somatic cyst cell membrane. Arrowheads point at Eya-positive late somatic

Male stem Cell Niche and Spermatogenesis in the *Drosophila* testis — A Tale of Germline-Soma Communication

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127

Cell polarity and signaling are fundamental biological processes that impact stem cell function, cancer, cell migration, tissue morphogenesis and response to pathogenic infections. Growing

growth, expansion and spreading over the germ cells of testicular cysts.

**5. Conclusions and future perspectives**

cyst cells. Testis hub is oriented towards the left. Scale Bar: 10mm

Another way to interpret this result would be to consider that Dlg regulates the intensity of germ cell encapsulation through the Egfr pathway, which is the major signaling pathway active at the microenvironment of the spermatogonial cysts [50, 51]. Membrane ruffling, detected in somatic cells upon *dlg* over-expression, is highly reminiscent of the formation of lammellipodia-like structures, formed upon up-regulation of Rac1 in SCCs [53]. Rac1 is a downstream component of the Egfr pathway and acts antagonistically to Rho in order to regulate germ cell encapsulation; moreover, Rho activation perturbates TJ function in various experimental systems [129]. It has already been shown that Dlg regulates mem‐ brane proliferation in a subset of NMJs in a dose-dependent fashion [123] and is an important player in the process of polarized membrane insertion during cellularization [109,

Male stem Cell Niche and Spermatogenesis in the *Drosophila* testis — A Tale of Germline-Soma Communication http://dx.doi.org/10.5772/58756 127

**Figure 3.** Dlg, Scrib and Lgl in the somatic lineage have critical functions in niche architecture, testis differentiation and homeostasis. (A-C) Dlg, Scrib and Lgl localize in somatic hub, somatic stem and cyst cells in *Drosophila* testis. (D) Dlg overexpression leads to ruffled membranes of somatic cyst cells, showing that Dlg promotes somatic cyst cell growth and membrane addition. (E-H) *dlg, scrib* and *lgl* mutant testes are extremely small, with reduced number of germline stem cells and impaired differentiation with only few spermatogonial cysts. Traffic Jam (TJ) marks the somat‐ ic stem cell and cyst cell nuclei, Vasa the germline, Armadillo (Arm) the hub and somatic stem and cyst cells, a-Spectrin the fusome growing through the interconnected spermatogonia and spermatocytes. (I-L) In *dlg* and *lgl* testes late so‐ matic cyst cells are lost as no Eyes Absent (Eya)-positive cyst cells are observed and the tight connection between the cyst cells and the germline is lost. In *scrib* testes Eya-positive somatic cyst cells are present, however testes are small and underdeveloped. Arrows point at the somatic cyst cell membrane. Arrowheads point at Eya-positive late somatic cyst cells. Testis hub is oriented towards the left. Scale Bar: 10mm

124-126]. The fact that membrane proliferation is also involved in mechanisms such as tissue spreading and cell surface extensions, including membrane ruffles [127, 128] and com‐ bined with our results on SCCs membrane ruffling upon Dlg overexpression it can be suggested that polarized membrane insertion, mediated by Dlg, might conduct SCCs growth, expansion and spreading over the germ cells of testicular cysts.

#### **5. Conclusions and future perspectives**

gonadal cells, the so-called gonadal mesoderm, that surrounds them. Scrib synthesis in the gonadal mesoderm is cell autonomous, since analysis of agametic gonads and pseudo-gonads made of aggregated germ cells revealed that Scrib in the germ cells requires a direct contact

As Dlg, Scrib and Lgl act cooperatively in several tissue contexts [23, 84], their function during male gonad and testis development was analyzed in a comparable way [22, 119]. This work revealed that cell autonomous *scrib* and *dlg* expression in the gonadal meso‐ derm affects critically the internal structure of the gonads by establishing the intimate contacts of the germ cells to the gonadal mesoderm [24, 119]. At later stages, *dlg*, *scrib* and *lgl* expression in the hub, CySCs and SCCs (Fig.3 A-C) is indispensable for testis develop‐ ment and homeostasis, as depletion of these genes results in extremely small testes with reduced number of germline stem cells and impaired differentiation (Fig.3 E-H). More‐ over, Dlg localization in CySCs establishes a tight connection between GSCs and CySCs, and thereby preserves the niche architecture. In late SCCs *dlg* expression is critical for their survival, growth, expansion and for maintaining the integrity of the cysts [22]. This is supported by the observation that the Eya-positive SCCs present in the wild-type testes (Fig.3I; arrowheads) are lost in *dlg* testes (Fig.3J) and die due to apoptosis [22]. Similar to *dlg*, *lgl* testes also lose Eya-positive SCCs (Fig.3L), whereas in *scrib* testes late SCCs are still present (Fig.3K; arrowheads) but the size of these Eya-positive nuclei and of overall testis size is significantly reduced [119]. In contrast to the overgrowth phenotypes observed in imaginal discs and brain hemispheres, the extensive defects in *dlg, scrib* and *lgl* mutant testes underline the importance of the somatic lineage in the establishment of a tight somagermline adhesion and cyst integrity, which is a prerequisite for a functional male stem cell

Another striking finding was the formation of wavy and ruffled plasma membrane upon *dlg* over-expression in somatic cyst cells capping the spermatocyte cysts. Up to now, there is no mechanism describing how cyst cells in *Drosophila* testis grow enormously, elongate and ensheath the germ cells of spermatogonial and spermatocyte cysts or how spermatid differ‐ entiation and individualization is guided by the polarized head and tail SCC. From other systems we know that Dlg regulates membrane proliferation in a subset of NMJs in a dosedependent fashion [123] and is an important player in the process of polarized membrane

Another way to interpret this result would be to consider that Dlg regulates the intensity of germ cell encapsulation through the Egfr pathway, which is the major signaling pathway active at the microenvironment of the spermatogonial cysts [50, 51]. Membrane ruffling, detected in somatic cells upon *dlg* over-expression, is highly reminiscent of the formation of lammellipodia-like structures, formed upon up-regulation of Rac1 in SCCs [53]. Rac1 is a downstream component of the Egfr pathway and acts antagonistically to Rho in order to regulate germ cell encapsulation; moreover, Rho activation perturbates TJ function in various experimental systems [129]. It has already been shown that Dlg regulates mem‐ brane proliferation in a subset of NMJs in a dose-dependent fashion [123] and is an important player in the process of polarized membrane insertion during cellularization [109,

to the gonadal mesoderm [24].

126 Adult Stem Cell Niches

niche and proper testis differentiation [2, 23, 119].

insertion during cellularization [109, 124-126].

Cell polarity and signaling are fundamental biological processes that impact stem cell function, cancer, cell migration, tissue morphogenesis and response to pathogenic infections. Growing scientific evidence suggests that these processes are intimately linked. Moreover, shuttling of signaling complexes into specific intracellular regions happens via their recruitment in subcellular domains guided by polarity scaffolds. The microenvironment of the male testis cysts, built by the cyst cell-germline intimate connection, provides an ideal model system to inves‐ tigate how soma-germline adhesion and cell morphological changes are coordinated with cell communication and exchange of short-range signals.

[3] Walker MR, Patel KK, Stappenbeck TS. The stem cell niche. J Pathol. 2009;217(2):

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So far the main evidence for cyst cell (CySCs and SCCs) function came from the analysis of individual signal transduction pathways that establish a cross-talk between the soma and the germline. Now we know that cyst cells are crucially important for soma-germline cyst integrity, overall rigidity and for setting up a functional cyst microenvironment. To this end, it is important (a) to investigate the requirement of the somatic lineage, the cyst cells, as safeguard of germline function, and (b) to characterize the local soma-germline communica‐ tion within the cysts with focus on how polarity scaffolds and signaling platforms promote this. Resolving the basic features of cyst's microenvironment and soma-germline coordination will allow the study of more complex questions in the future such as long-range signaling at the level of cyst-cyst communication. Moreover, the use of a combination of genetic, genomic and high-resolution microscopy techniques to approach these questions will enable us to adapt tools, already successfully established in other tissues and model systems (such as FRAP, FRET and organ cultures) to the *Drosophila* testis.

#### **Acknowledgements**

The author wishes to thank the *Drosophila* community for providing generously fly stocks and antibodies and apologizes to all whose work has not been sited due to space limitations.

## **Author details**

Fani Papagiannouli\*

Centre for Organismal Studies (COS), University of Heidelberg, Germany

#### **References**


[3] Walker MR, Patel KK, Stappenbeck TS. The stem cell niche. J Pathol. 2009;217(2): 169-80.

scientific evidence suggests that these processes are intimately linked. Moreover, shuttling of signaling complexes into specific intracellular regions happens via their recruitment in subcellular domains guided by polarity scaffolds. The microenvironment of the male testis cysts, built by the cyst cell-germline intimate connection, provides an ideal model system to inves‐ tigate how soma-germline adhesion and cell morphological changes are coordinated with cell

So far the main evidence for cyst cell (CySCs and SCCs) function came from the analysis of individual signal transduction pathways that establish a cross-talk between the soma and the germline. Now we know that cyst cells are crucially important for soma-germline cyst integrity, overall rigidity and for setting up a functional cyst microenvironment. To this end, it is important (a) to investigate the requirement of the somatic lineage, the cyst cells, as safeguard of germline function, and (b) to characterize the local soma-germline communica‐ tion within the cysts with focus on how polarity scaffolds and signaling platforms promote this. Resolving the basic features of cyst's microenvironment and soma-germline coordination will allow the study of more complex questions in the future such as long-range signaling at the level of cyst-cyst communication. Moreover, the use of a combination of genetic, genomic and high-resolution microscopy techniques to approach these questions will enable us to adapt tools, already successfully established in other tissues and model systems (such as FRAP, FRET

The author wishes to thank the *Drosophila* community for providing generously fly stocks and antibodies and apologizes to all whose work has not been sited due to space limitations.

[1] Fuller MT, Spradling AC. Male and female Drosophila germline stem cells: two ver‐

[2] Papagiannouli F, Lohmann I. Shaping the niche: Lessons from the Drosophila testis

Centre for Organismal Studies (COS), University of Heidelberg, Germany

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communication and exchange of short-range signals.

and organ cultures) to the *Drosophila* testis.

**Acknowledgements**

128 Adult Stem Cell Niches

**Author details**

Fani Papagiannouli\*

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**Chapter 6**

**Adult Stem Cell Niches — Stem Cells in the Female**

The female genital tract is a complex and physiologically dynamic system of organs which undergo continuous and profound changes during the reproductive years. Each of its components – ovaries, fallopian tubes and uterus - has unique and indispensable roles in reproduction. Each month, under stimulation from the pituitary gland, the ovaries produce and release a mature oocyte, which moves into the neighboring fallopian tube. Concep‐ tion takes place in the lumen of the tube and the mucosal epithelium lining plays a critical part in the transport of the gametes and the successful transfer of the zygote to the uterus, where it implants 6-12 days after fertilization. Considering the importance of successful reproduction for species survival, there is strong evolutionary pressure to make the process robust and to respond quickly to any cellular damage with effective repair mechanisms. Also, the inner layer of the uterus, the endometrium, is subjected to monthly shedding and regeneration in order to sustain a suitable environment for implantation of a potential embryo. Similar to other tissues like intestine and hair, which continue to undergo rapid cellular turnover throughout adult life, there is an increasing number of studies describ‐ ing the existence of adult stem cells in the genital tract that ensure tissue renewal through‐

Historically, adult stem cells have been described in vivo as rare, slow-cycling cells that maintain self-renewal by asymmetric division and can differentiate into different proge‐ nies. The traditional method of identification has been BrdU labeling, and designated stem cells have been described as label retaining cells (LRC), although there has been some doubt concerning the accuracy of the underlying premise that stem cells efficiently incorporate BrdU [1]. Since BrdU is a mutagen and toxic, this prevents successful recovery and in vitro analysis of labeled cells. However, both old and new experimental approaches recently

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mirjana Kessler, Rike Zietlow and Thomas F. Meyer

Additional information is available at the end of the chapter

**Reproductive System**

http://dx.doi.org/10.5772/58842

**1. Introduction**

out life.

[129] Fischer A, Stuckas H, Gluth M, Russell TD, Rudolph MC, Beeman NE, et al. Impaired tight junction sealing and precocious involution in mammary glands of PKN1 trans‐ genic mice. J Cell Sci. 2007;120(Pt 13):2272-83.

## **Adult Stem Cell Niches — Stem Cells in the Female Reproductive System**

Mirjana Kessler, Rike Zietlow and Thomas F. Meyer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58842

#### **1. Introduction**

[128] Albertson R, Riggs B, Sullivan W. Membrane traffic: a driving force in cytokinesis.

[129] Fischer A, Stuckas H, Gluth M, Russell TD, Rudolph MC, Beeman NE, et al. Impaired tight junction sealing and precocious involution in mammary glands of PKN1 trans‐

Trends Cell Biol. 2005;15(2):92-101.

138 Adult Stem Cell Niches

genic mice. J Cell Sci. 2007;120(Pt 13):2272-83.

The female genital tract is a complex and physiologically dynamic system of organs which undergo continuous and profound changes during the reproductive years. Each of its components – ovaries, fallopian tubes and uterus - has unique and indispensable roles in reproduction. Each month, under stimulation from the pituitary gland, the ovaries produce and release a mature oocyte, which moves into the neighboring fallopian tube. Concep‐ tion takes place in the lumen of the tube and the mucosal epithelium lining plays a critical part in the transport of the gametes and the successful transfer of the zygote to the uterus, where it implants 6-12 days after fertilization. Considering the importance of successful reproduction for species survival, there is strong evolutionary pressure to make the process robust and to respond quickly to any cellular damage with effective repair mechanisms. Also, the inner layer of the uterus, the endometrium, is subjected to monthly shedding and regeneration in order to sustain a suitable environment for implantation of a potential embryo. Similar to other tissues like intestine and hair, which continue to undergo rapid cellular turnover throughout adult life, there is an increasing number of studies describ‐ ing the existence of adult stem cells in the genital tract that ensure tissue renewal through‐ out life.

Historically, adult stem cells have been described in vivo as rare, slow-cycling cells that maintain self-renewal by asymmetric division and can differentiate into different proge‐ nies. The traditional method of identification has been BrdU labeling, and designated stem cells have been described as label retaining cells (LRC), although there has been some doubt concerning the accuracy of the underlying premise that stem cells efficiently incorporate BrdU [1]. Since BrdU is a mutagen and toxic, this prevents successful recovery and in vitro analysis of labeled cells. However, both old and new experimental approaches recently

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

confirmed the existence of adult stem cells in the female reproductive system using the mouse model system with BrdU pulse labeling [2,3], as well as with transgenic animals with fluorescently labeled histone 2B [4], although it is not yet clear if the two methods do in fact label the same cells.

**2. Anatomy and histopathology of cervix, uterine endometrium and**

Distinct portions of the mucosa along the tract are morphologically and functionally special‐ ized to facilitate the successful completion of the reproductive process: from the oocyte maturation (ovary) through the transport of gametes (fallopian tube) to the implantation of the embryo in the endometrium and establishment of a viable pregnancy (uterus). The ovarian surface epithelium (OSE) is built of simple, flat, cuboidal cells, without mature adhesion

Adult Stem Cell Niches — Stem Cells in the Female Reproductive System

http://dx.doi.org/10.5772/58842

141

**fallopian tube and ovary**

junctions or prominent polarity (Fig. 1A).

**Figure 1.** Overview of the histology of the female genital tract

Deregulation of the adult stem cell niche, which is the main custodian of homeostasis in healthy tissues, is considered a potentially significant step in the etiology of cancer as well as other proliferative disorders, such as endometriosis. Therefore, basic research into the biology of adult stem cells is a new and promising field in the search for novel therapeu‐ tic strategies for these diseases. This chapter will provide an overview of the current understanding of adult stem cell niches in the female genital tract, and how new evi‐ dence regarding its molecular regulation changes our perspective of analyzing and treating some of its most common pathologies: endometriosis as well as endometrial and ovarian cancer. Based on the available evidence, it is safe to conclude that the female reproduc‐ tive tract harbors adult stem cells. However, in contrast to an already very comprehen‐ sive and detailed insight into the structure and regulation of the adult stem cell niche in the gastro-intestinal tract, hair follicle or hematopoetic tissue, details of the niche organiza‐ tion in the genital tract mucosa remain at best sketchy. Experimental data on adult stem cells from the genital tract almost exclusively originate from in vitro studies of primary culture isolates and so-called "functional assays" describing clonality assays, sphere formation and differentiation capacity for the small population of presumptive stem cell candidates. Still, many questions remain unanswered regarding the molecular mecha‐ nisms of epithelial renewal in a system which during the average reproductive period undergoes more than 400 cycles of phenotypical changes in response to shifts in hormo‐ nal stimulation. Of particular importance is the relationship between adult stem cells in the healthy tissue and so-called cancer stem cells and we will address the most important developments in this area of research.

We will also briefly review contentious recent evidence for a putative reserve of germline stem cells (GSCs) in the ovary, which would represent a further population of adult stem cells, akin to spermatogonia in men. One of the pillars of reproductive medicine and infertility treatments is the dogma that women are born with all potential oocytes in place, arrested in the first meiotic prophase. Thus, the available "ovarian reserve" is a limiting factor in infertility treatments, and egg donation remains the only option for patients who show diminished parameters for remaining primordial follicles. Following on from the postulation that somatic, mitotically active cells in Drosophila melanogaster can act as GSCs [5] several groups have provided evidence for the existence of GSCs in mouse [6,7] as well as human ovaries [8]. However, they are yet to find final acceptance in the scientific community. If they do indeed exist, they would without doubt revolutionize the field of reproductive medicine.

Before we review the current "state of the art" of adult stem cell research in the female genital tract, it is useful to summarize its basic anatomy and histopathology in order to understand the environment in which stem cells function.

## **2. Anatomy and histopathology of cervix, uterine endometrium and fallopian tube and ovary**

confirmed the existence of adult stem cells in the female reproductive system using the mouse model system with BrdU pulse labeling [2,3], as well as with transgenic animals with fluorescently labeled histone 2B [4], although it is not yet clear if the two methods do

Deregulation of the adult stem cell niche, which is the main custodian of homeostasis in healthy tissues, is considered a potentially significant step in the etiology of cancer as well as other proliferative disorders, such as endometriosis. Therefore, basic research into the biology of adult stem cells is a new and promising field in the search for novel therapeu‐ tic strategies for these diseases. This chapter will provide an overview of the current understanding of adult stem cell niches in the female genital tract, and how new evi‐ dence regarding its molecular regulation changes our perspective of analyzing and treating some of its most common pathologies: endometriosis as well as endometrial and ovarian cancer. Based on the available evidence, it is safe to conclude that the female reproduc‐ tive tract harbors adult stem cells. However, in contrast to an already very comprehen‐ sive and detailed insight into the structure and regulation of the adult stem cell niche in the gastro-intestinal tract, hair follicle or hematopoetic tissue, details of the niche organiza‐ tion in the genital tract mucosa remain at best sketchy. Experimental data on adult stem cells from the genital tract almost exclusively originate from in vitro studies of primary culture isolates and so-called "functional assays" describing clonality assays, sphere formation and differentiation capacity for the small population of presumptive stem cell candidates. Still, many questions remain unanswered regarding the molecular mecha‐ nisms of epithelial renewal in a system which during the average reproductive period undergoes more than 400 cycles of phenotypical changes in response to shifts in hormo‐ nal stimulation. Of particular importance is the relationship between adult stem cells in the healthy tissue and so-called cancer stem cells and we will address the most important

We will also briefly review contentious recent evidence for a putative reserve of germline stem cells (GSCs) in the ovary, which would represent a further population of adult stem cells, akin to spermatogonia in men. One of the pillars of reproductive medicine and infertility treatments is the dogma that women are born with all potential oocytes in place, arrested in the first meiotic prophase. Thus, the available "ovarian reserve" is a limiting factor in infertility treatments, and egg donation remains the only option for patients who show diminished parameters for remaining primordial follicles. Following on from the postulation that somatic, mitotically active cells in Drosophila melanogaster can act as GSCs [5] several groups have provided evidence for the existence of GSCs in mouse [6,7] as well as human ovaries [8]. However, they are yet to find final acceptance in the scientific community. If they do indeed exist, they would without doubt revolutionize the field of

Before we review the current "state of the art" of adult stem cell research in the female genital tract, it is useful to summarize its basic anatomy and histopathology in order to understand

in fact label the same cells.

140 Adult Stem Cell Niches

developments in this area of research.

reproductive medicine.

the environment in which stem cells function.

Distinct portions of the mucosa along the tract are morphologically and functionally special‐ ized to facilitate the successful completion of the reproductive process: from the oocyte maturation (ovary) through the transport of gametes (fallopian tube) to the implantation of the embryo in the endometrium and establishment of a viable pregnancy (uterus). The ovarian surface epithelium (OSE) is built of simple, flat, cuboidal cells, without mature adhesion junctions or prominent polarity (Fig. 1A).

**Figure 1.** Overview of the histology of the female genital tract

The stroma of the ovary is filled with luteinized stromal cells, decidual cells, neuroendocrine cells, fat and muscle cells and a population of endometrial-like stromal cells. In addition, the ovary contains the pool of primordial follicles, which consist of immature primary oocytes, arrested at birth in the prophase of meiosis 1, surrounded by a densely packed shell of somatic granulosa cells, which are of key importance for successful growth and maturation of the ovum. The developing oocyte and neighboring granulosa cells represent a perfect example of the "niche" where cell-cell interaction and signaling from surrounding tissue compartments determine cell fate and differentiation (Fig. 1B). The process of ovulation, or specifically follicule rupture, creates a pro-inflammatory environment high in reactive oxygen species [9, 10], that requires extensive repair mechanisms and tissue remodeling to avoid permanent damage to the ovary. Indeed OSE cells, though simple in their cellular phenotype, are uniquely adapted to not only respond to stress conditions, but also to actively participate in the tissue breakdown and remodeling that enables rupture of the follicle through the ovarian surface [11]. They express proteolytic enzymes such are metalloprotease 2 and 9 [12], and can undergo epithelial mesenchymal transition (EMT) [13], which may facilitate efficient repair of the injured ovarian surface during the post-ovulatory phase of the menstrual cycle. The ovaries also produce hormones that control the cellular changes associated with the menstrual cycle. The granulosa cells of growing follicles secrete exponentially increasing amounts of estradiol and progesterone. After oocyte release, the remaining follicular cells undergo transformation into the corpus luteum, which immediately starts producing progesterone under the control of pituitary LH pulses. These two main phases of the menstrual cycle – estradiol-driven follicular phase and progesterone-driven luteal phase – determine cyclical homeostasis of all mucosal surfaces in the lower genital tract including fallopian tube, uterus and cervix.

key step in the development of premalignant lesions. As well as the potentially genotoxic effects of follicular fluid, their connection to the uterus also renders the fallopian tubes vulnerable to ascending infections by sexually transmitted pathogens. Chlamydia trachomatis and Neisseria gonorrhea are major causes of the inflammatory disease salpingitis, which is marked by scarring and tissue injury and dramatically increases the risk for tubal occlusion and infertility. In addition, there is increasing evidence that at least some STDs may also have

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The uterus is a muscular organ with great capacity for growth and physiological transforma‐ tion, which is necessary to support pregnancy. It consists of three layers: the outer serosal layer, or perimetrium, the myometrium, which contributes the most to the volume and mass of the organ, and the inner mucosal layer, or endometrium, which is vital for the initiation of pregnancy. The structure of the endometrial layer has traditionally been divided into the stratum basalis and the stratum functionalis, which is subjected to monthly cyclical renewal, differentiation and shedding. The stratum functionalis changes greatly during each menstrual cycle and is further divided into stratum compactum and stratum spongiosum. The stratum functionalis consists of glandular epithelium (Fig. 1C) residing on the supportive connective tissue and blood vessels which supply nutrients. It is at its thinnest at the beginning of the follicular phase, and proliferates strongly under stimulation by estradiol. The estradiol peak coincides with the maximum follicle diameter prior to ovulation and with maximal thickness of the proliferative endometrium. Following ovulation, rising progesterone levels, trigger differentiation of the stratum functionalis into "secretory" endometrium. At the cellular level, endometrial glands, which resemble narrow straight tubes during the follicular phase, begin to swell as progesterone stimulates the columnar epithelial cells to produce and secrete glycogen granules. It is assumed that this glycogen-rich environment serves as an energy depot for the implanting blastocyst. In addition, stromal cells undergo a profound change, converting from a fibroblast-like phenotype into rounded cells that produce prolactin and insulin growth factor binding protein [21,22]. This complex transformation of the functional layer of the endometrium, called decidualization, is a prerequisite for successful attachment and invasion of the trophoblast and thereby initiation of the pregnancy. Progesterone secretion by the corpus luteum is limited to around two weeks, after which it disintegrates if no chorionic gonadotro‐ pin from an implanted embryo is present in the circulation to rescue its function. In the absence of progesterone, the endometrial stratum functionalis, epithelium and supportive connective

The uterus is separated from the vaginal canal by the narrow muscular cervix. Due to its physiological elasticity, the cervix tightens under the influence of progesterone, and essentially seals the uterus from the outside environment during the second part of the cycle. This protective barrier is enhanced in the case of pregnancy by the formation of a mucus plug that fills the endocervical canal. The entrance to the cervix is called external orifice of the uterus, or external os. The endocervix is lined by simple columnar epithelium, with a similar structure to the endometrial monolayer, while the outer part is lined by stratified squamous epithelium. The segment in between – the squamo-columnar junction (SCJ) – is a dynamic zone, which does not have a fixed location but migrates under the influence of major hormonal changes,

a pro-malignant effect on host cells [18-20].

tissue is shed and expelled by menstrual bleeding.

The fallopian tube, or salpinx, is ~ 10 cm long and anatomically divided into three segments: the isthmus, which connects to the uterus, the ampulla, which constitutes the middle part, and the infundibulum, proximal to the ovary. The infundibulum terminates in relatively large opening, the ostium, which has many fine projections, or fimbriae, that capture the oocyte upon follicule rupture and guide it towards the ampulla, where fertilization takes place. The histology of the tube, starkly different from the ovarian surface, is characterized by the presence of an epithelial monolayer of highly differentiated columnar cells with two distinct cell types: secretory – producing tubular fluid – and ciliated – enabling transport along the lumen (Fig. 1A). Numerous mucosal folds in the distal and ampullar regions provide a suitable environment for the early stages of blastocyst development. Contractility of the tube is ensured by the muscular layer surrounding the epithelium. Although, unlike uterine endometrium, fallopian epithelium does not undergo extensive monthly shedding, it responds to the follicular phase hormonal environment by proliferation [14]. Changes in homeostasis are supported by global gene expression data from fallopian tube mucosal samples, which show marked differences between follicular and luteal phase [15]. The tubal epithelium is also exposed to the pro-inflammatory environment associated with ovulation, leading for instance to an increase in double stranded DNA breaks marked by phospho-γH2A.X in a mouse model in vivo [16]. Exposing human fallopian tube isolates to follicular fluid ex vivo increases expression of inflammation-related genes and DNA repair components [17]. In particular, there is a noticeable accumulation of p53 it the nuclei of tubal cells, which is thought to be a key step in the development of premalignant lesions. As well as the potentially genotoxic effects of follicular fluid, their connection to the uterus also renders the fallopian tubes vulnerable to ascending infections by sexually transmitted pathogens. Chlamydia trachomatis and Neisseria gonorrhea are major causes of the inflammatory disease salpingitis, which is marked by scarring and tissue injury and dramatically increases the risk for tubal occlusion and infertility. In addition, there is increasing evidence that at least some STDs may also have a pro-malignant effect on host cells [18-20].

The stroma of the ovary is filled with luteinized stromal cells, decidual cells, neuroendocrine cells, fat and muscle cells and a population of endometrial-like stromal cells. In addition, the ovary contains the pool of primordial follicles, which consist of immature primary oocytes, arrested at birth in the prophase of meiosis 1, surrounded by a densely packed shell of somatic granulosa cells, which are of key importance for successful growth and maturation of the ovum. The developing oocyte and neighboring granulosa cells represent a perfect example of the "niche" where cell-cell interaction and signaling from surrounding tissue compartments determine cell fate and differentiation (Fig. 1B). The process of ovulation, or specifically follicule rupture, creates a pro-inflammatory environment high in reactive oxygen species [9, 10], that requires extensive repair mechanisms and tissue remodeling to avoid permanent damage to the ovary. Indeed OSE cells, though simple in their cellular phenotype, are uniquely adapted to not only respond to stress conditions, but also to actively participate in the tissue breakdown and remodeling that enables rupture of the follicle through the ovarian surface [11]. They express proteolytic enzymes such are metalloprotease 2 and 9 [12], and can undergo epithelial mesenchymal transition (EMT) [13], which may facilitate efficient repair of the injured ovarian surface during the post-ovulatory phase of the menstrual cycle. The ovaries also produce hormones that control the cellular changes associated with the menstrual cycle. The granulosa cells of growing follicles secrete exponentially increasing amounts of estradiol and progesterone. After oocyte release, the remaining follicular cells undergo transformation into the corpus luteum, which immediately starts producing progesterone under the control of pituitary LH pulses. These two main phases of the menstrual cycle – estradiol-driven follicular phase and progesterone-driven luteal phase – determine cyclical homeostasis of all mucosal surfaces in the lower genital tract including fallopian tube, uterus and cervix.

142 Adult Stem Cell Niches

The fallopian tube, or salpinx, is ~ 10 cm long and anatomically divided into three segments: the isthmus, which connects to the uterus, the ampulla, which constitutes the middle part, and the infundibulum, proximal to the ovary. The infundibulum terminates in relatively large opening, the ostium, which has many fine projections, or fimbriae, that capture the oocyte upon follicule rupture and guide it towards the ampulla, where fertilization takes place. The histology of the tube, starkly different from the ovarian surface, is characterized by the presence of an epithelial monolayer of highly differentiated columnar cells with two distinct cell types: secretory – producing tubular fluid – and ciliated – enabling transport along the lumen (Fig. 1A). Numerous mucosal folds in the distal and ampullar regions provide a suitable environment for the early stages of blastocyst development. Contractility of the tube is ensured by the muscular layer surrounding the epithelium. Although, unlike uterine endometrium, fallopian epithelium does not undergo extensive monthly shedding, it responds to the follicular phase hormonal environment by proliferation [14]. Changes in homeostasis are supported by global gene expression data from fallopian tube mucosal samples, which show marked differences between follicular and luteal phase [15]. The tubal epithelium is also exposed to the pro-inflammatory environment associated with ovulation, leading for instance to an increase in double stranded DNA breaks marked by phospho-γH2A.X in a mouse model in vivo [16]. Exposing human fallopian tube isolates to follicular fluid ex vivo increases expression of inflammation-related genes and DNA repair components [17]. In particular, there is a noticeable accumulation of p53 it the nuclei of tubal cells, which is thought to be a The uterus is a muscular organ with great capacity for growth and physiological transforma‐ tion, which is necessary to support pregnancy. It consists of three layers: the outer serosal layer, or perimetrium, the myometrium, which contributes the most to the volume and mass of the organ, and the inner mucosal layer, or endometrium, which is vital for the initiation of pregnancy. The structure of the endometrial layer has traditionally been divided into the stratum basalis and the stratum functionalis, which is subjected to monthly cyclical renewal, differentiation and shedding. The stratum functionalis changes greatly during each menstrual cycle and is further divided into stratum compactum and stratum spongiosum. The stratum functionalis consists of glandular epithelium (Fig. 1C) residing on the supportive connective tissue and blood vessels which supply nutrients. It is at its thinnest at the beginning of the follicular phase, and proliferates strongly under stimulation by estradiol. The estradiol peak coincides with the maximum follicle diameter prior to ovulation and with maximal thickness of the proliferative endometrium. Following ovulation, rising progesterone levels, trigger differentiation of the stratum functionalis into "secretory" endometrium. At the cellular level, endometrial glands, which resemble narrow straight tubes during the follicular phase, begin to swell as progesterone stimulates the columnar epithelial cells to produce and secrete glycogen granules. It is assumed that this glycogen-rich environment serves as an energy depot for the implanting blastocyst. In addition, stromal cells undergo a profound change, converting from a fibroblast-like phenotype into rounded cells that produce prolactin and insulin growth factor binding protein [21,22]. This complex transformation of the functional layer of the endometrium, called decidualization, is a prerequisite for successful attachment and invasion of the trophoblast and thereby initiation of the pregnancy. Progesterone secretion by the corpus luteum is limited to around two weeks, after which it disintegrates if no chorionic gonadotro‐ pin from an implanted embryo is present in the circulation to rescue its function. In the absence of progesterone, the endometrial stratum functionalis, epithelium and supportive connective tissue is shed and expelled by menstrual bleeding.

The uterus is separated from the vaginal canal by the narrow muscular cervix. Due to its physiological elasticity, the cervix tightens under the influence of progesterone, and essentially seals the uterus from the outside environment during the second part of the cycle. This protective barrier is enhanced in the case of pregnancy by the formation of a mucus plug that fills the endocervical canal. The entrance to the cervix is called external orifice of the uterus, or external os. The endocervix is lined by simple columnar epithelium, with a similar structure to the endometrial monolayer, while the outer part is lined by stratified squamous epithelium. The segment in between – the squamo-columnar junction (SCJ) – is a dynamic zone, which does not have a fixed location but migrates under the influence of major hormonal changes, such as puberty, pregnancy and menopause. Prior to puberty, squamous epithelium covers the outer segment of the cervix and the lower part of the canal. During puberty and first pregnancy, the columnar epithelium of the endocervix expands and covers the outer rim of the external os. Through contact with the low pH of the vagina, the columnar epithelium undergoes metaplasia over time and converts back towards a squamous phenotype (squamous metaplasia). It is widely accepted that over 90% of malignancies of the cervix originate from cellular changes which are initiated in this region of intense tissue remodeling-thus the SCJ is frequently labelled "transformation zone" (TZ).

Putative GSCs have been identified by co-labelling with proliferative and germ cell markers and described either as small cells within the ovarian cortex [6,26-28] or clusters of cells which also include somatic cells [29]. Other studies supported the discovery of ovarian GSCs [26,30] but the considerable differences in marker expression, in vitro phenotype and differentiation potential observed between these studies make the case somewhat controversial. In addition, other groups have failed to repeat these findings and challenged their validity [24,31,32]. Kerr et al described the expulsion of oocytes through the OSE into the peritoneum during the postnatal phase of oocyte reduction, which could potentially be interpreted as GSCs [32]. By using tamoxifen-induced random labelling of cells, Lei and Spradling traced the numbers of follicles over time and argue that the follicle pool is in fact highly stable with a half-life of 10 – 11 months, which would make the follicle pool at birth large enough to support the ~500 ovulations required during the life time of a mouse [33]. They also failed to observe the generation of new follicles even after depletion with busulphan toxin, thus putting into question that there is in fact a need to explain regeneration of follicle numbers, a finding that is further supported by mathematical modelling [34]. Similarly other groups, failed to observe replenishment of the follicle pool by donor bone marrow-derived cells [35] or after chemical depletion [36].

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It has been argued that some of these contradicting findings could be explained by the use of Ddx4 –which was previously thought to be a cytoplasmic germ cell marker, but was used by Zou et al [7] and White et al [8] to isolate cells based on their finding that the protein also contains a transmembrane domain in GSCs. They suggest that the reason many other labora‐ tories have failed to identify GSCs in ovaries using Ddx4 [24] is due to the fact that cytoplasmic Ddx4 expression in oocytes masks the presence of rare GSCs. Similarly, SSEA-1, which was used by Johnson's group to identify ovarian GSCs [25], was reported by Bristol-Gould et al [34] to overlap only with cells from the HSC lineage. Widespread acceptance of adult ovarian GSCs will no doubt depend on the identification of a marker signature or more robust methods which

The difficulty in pinpointing putative ovarian GSCs or their niche in vivo, while at the same time apparently being expandable and giving rise to the appropriate differentiated tissue in vitro, mirrors the experience for stem cells of somatic adult tissues, as we shall explore below. However, the unique properties and complex embryonic development of germ cells do in fact

In the current model, primordial germ cells (PGCs) derive from a small number of epiblast cells which are specified before differentiation into the different germ layers begins. PGCs subsequently undergo a complex migration through the allantois, along the developing hindgut, finally entering the dorsal mesentery and the developing gonads. Despite the obvious importance of these early developmental processes for future fertility, they remain little understood. Mikedis and Downs have suggested that PGCs temporarily reside in an "allantoic core domain" (ACD) which they propose has similar functions to the Spemann organiser, consisting of a stem cell pool which extends the body axis in a posterior direction – contributing not only to the germ cell lineage but also the three germ layers – effectively creating a strong interface between the future umbilical cord and the developing embryo [37]. The stem cells in the ACD express Oct4, Blimp1, Stella and Fragilis – markers thought to be specific for PGCs –

allows identification of these cells by all laboratories with the relevant expertise.

give rise to some important differences.

#### **3. Putative female GSCs**

Before devoting our attention to the somatic adult stem cells of the female genital tract, we will review the rather contentions field of presumptive GSCs in the adult ovary. Since the 1950s, the accepted dogma in reproductive biology has been that in mammals primordial germ cell (PGC)-derived oogonia cease proliferation shortly after birth and differentiate into primary oocytes, which arrest in prophase of meiosis I until fertilization triggers the completion of meiosis [23]. As a consequence, the pool of available oocytes is finite – and exhaustion of the pool of resulting follicles is believed to be responsible for menopause. In the last decade, however, this assumption has been challenged by a series of papers reporting the existence of GSCs in the ovaries of mice and humans. First hints came from observations by Tilly's group [6], who tried to assess the dynamics of germ cell loss during adult life. Their estimation that up to a third of immature follicles in mice are degenerating at any given time led them to postulate that this unexpectedly high rate is incompatible with the slow rate of decline observed in the follicle reserve. In trying to resolve this contradiction, they identified cells on the ovarian surface of young mice that express the meiotic entry marker SCP3, as well as BrdUincorporating cells that simultaneously express the germ cell marker Ddx4, suggesting that these cells may be proliferating GSCs responsible for replenishing the follicle pool.

Further evidence for ovarian GSCs that are capable of proliferation followed by differentiation into oocytes came from the observation that immunomagnetically isolated Ddx4+ cells from both mouse and human ovaries can be expanded in vitro and give rise to oocytes following transplantation into donor ovarian tissue [7,8]. In the mouse, GFP-labelled putative Ddx4+GSCs were able to give rise to offspring following transplantation, with transmission through the germline to subsequent generations [7]. Using transplantation of premeiotic female PGCs Zhang et al [24] further provided proof of principle that the adult ovary is able to support oogenesis. Tilly's group subsequently showed that following extensive doxyrubininduced loss, follicle numbers recover within 36 h. Reasoning that the small numbers of presumed GSCs they had previously identified in the adult ovary would not be sufficient to support this rapid recovery, they transplanted bone marrow from mice expressing GFP under the Oct 4 promoter into germ cell-deficient Atm-/-mice and identified GFP-labelled primordial follicles [25]. They concluded that a subpopulation of circulating bone marrow stem cells that express PGC marker genes are responsible for the observed oocyte replenishment.

Putative GSCs have been identified by co-labelling with proliferative and germ cell markers and described either as small cells within the ovarian cortex [6,26-28] or clusters of cells which also include somatic cells [29]. Other studies supported the discovery of ovarian GSCs [26,30] but the considerable differences in marker expression, in vitro phenotype and differentiation potential observed between these studies make the case somewhat controversial. In addition, other groups have failed to repeat these findings and challenged their validity [24,31,32]. Kerr et al described the expulsion of oocytes through the OSE into the peritoneum during the postnatal phase of oocyte reduction, which could potentially be interpreted as GSCs [32]. By using tamoxifen-induced random labelling of cells, Lei and Spradling traced the numbers of follicles over time and argue that the follicle pool is in fact highly stable with a half-life of 10 – 11 months, which would make the follicle pool at birth large enough to support the ~500 ovulations required during the life time of a mouse [33]. They also failed to observe the generation of new follicles even after depletion with busulphan toxin, thus putting into question that there is in fact a need to explain regeneration of follicle numbers, a finding that is further supported by mathematical modelling [34]. Similarly other groups, failed to observe replenishment of the follicle pool by donor bone marrow-derived cells [35] or after chemical depletion [36].

such as puberty, pregnancy and menopause. Prior to puberty, squamous epithelium covers the outer segment of the cervix and the lower part of the canal. During puberty and first pregnancy, the columnar epithelium of the endocervix expands and covers the outer rim of the external os. Through contact with the low pH of the vagina, the columnar epithelium undergoes metaplasia over time and converts back towards a squamous phenotype (squamous metaplasia). It is widely accepted that over 90% of malignancies of the cervix originate from cellular changes which are initiated in this region of intense tissue remodeling-thus the SCJ is

Before devoting our attention to the somatic adult stem cells of the female genital tract, we will review the rather contentions field of presumptive GSCs in the adult ovary. Since the 1950s, the accepted dogma in reproductive biology has been that in mammals primordial germ cell (PGC)-derived oogonia cease proliferation shortly after birth and differentiate into primary oocytes, which arrest in prophase of meiosis I until fertilization triggers the completion of meiosis [23]. As a consequence, the pool of available oocytes is finite – and exhaustion of the pool of resulting follicles is believed to be responsible for menopause. In the last decade, however, this assumption has been challenged by a series of papers reporting the existence of GSCs in the ovaries of mice and humans. First hints came from observations by Tilly's group [6], who tried to assess the dynamics of germ cell loss during adult life. Their estimation that up to a third of immature follicles in mice are degenerating at any given time led them to postulate that this unexpectedly high rate is incompatible with the slow rate of decline observed in the follicle reserve. In trying to resolve this contradiction, they identified cells on the ovarian surface of young mice that express the meiotic entry marker SCP3, as well as BrdUincorporating cells that simultaneously express the germ cell marker Ddx4, suggesting that

these cells may be proliferating GSCs responsible for replenishing the follicle pool.

into oocytes came from the observation that immunomagnetically isolated Ddx4+

express PGC marker genes are responsible for the observed oocyte replenishment.

Further evidence for ovarian GSCs that are capable of proliferation followed by differentiation

both mouse and human ovaries can be expanded in vitro and give rise to oocytes following transplantation into donor ovarian tissue [7,8]. In the mouse, GFP-labelled putative Ddx4+GSCs were able to give rise to offspring following transplantation, with transmission through the germline to subsequent generations [7]. Using transplantation of premeiotic female PGCs Zhang et al [24] further provided proof of principle that the adult ovary is able to support oogenesis. Tilly's group subsequently showed that following extensive doxyrubininduced loss, follicle numbers recover within 36 h. Reasoning that the small numbers of presumed GSCs they had previously identified in the adult ovary would not be sufficient to support this rapid recovery, they transplanted bone marrow from mice expressing GFP under the Oct 4 promoter into germ cell-deficient Atm-/-mice and identified GFP-labelled primordial follicles [25]. They concluded that a subpopulation of circulating bone marrow stem cells that

cells from

frequently labelled "transformation zone" (TZ).

**3. Putative female GSCs**

144 Adult Stem Cell Niches

It has been argued that some of these contradicting findings could be explained by the use of Ddx4 –which was previously thought to be a cytoplasmic germ cell marker, but was used by Zou et al [7] and White et al [8] to isolate cells based on their finding that the protein also contains a transmembrane domain in GSCs. They suggest that the reason many other labora‐ tories have failed to identify GSCs in ovaries using Ddx4 [24] is due to the fact that cytoplasmic Ddx4 expression in oocytes masks the presence of rare GSCs. Similarly, SSEA-1, which was used by Johnson's group to identify ovarian GSCs [25], was reported by Bristol-Gould et al [34] to overlap only with cells from the HSC lineage. Widespread acceptance of adult ovarian GSCs will no doubt depend on the identification of a marker signature or more robust methods which allows identification of these cells by all laboratories with the relevant expertise.

The difficulty in pinpointing putative ovarian GSCs or their niche in vivo, while at the same time apparently being expandable and giving rise to the appropriate differentiated tissue in vitro, mirrors the experience for stem cells of somatic adult tissues, as we shall explore below. However, the unique properties and complex embryonic development of germ cells do in fact give rise to some important differences.

In the current model, primordial germ cells (PGCs) derive from a small number of epiblast cells which are specified before differentiation into the different germ layers begins. PGCs subsequently undergo a complex migration through the allantois, along the developing hindgut, finally entering the dorsal mesentery and the developing gonads. Despite the obvious importance of these early developmental processes for future fertility, they remain little understood. Mikedis and Downs have suggested that PGCs temporarily reside in an "allantoic core domain" (ACD) which they propose has similar functions to the Spemann organiser, consisting of a stem cell pool which extends the body axis in a posterior direction – contributing not only to the germ cell lineage but also the three germ layers – effectively creating a strong interface between the future umbilical cord and the developing embryo [37]. The stem cells in the ACD express Oct4, Blimp1, Stella and Fragilis – markers thought to be specific for PGCs – but appear to contribute also to other tissues [38]. These observations, as well as the fact that hematopoietic stem cells also migrate from the proximal epiblast to the embryonic aortagonad-mesonephric region during the same period of development, imply that it is theoreti‐ cally possible that there may be "intermixing" or indeed that there is a common precursor pool for PGCs and a subpopulation of bone marrow stem cells.

**4. Uterine endometrial stem cells**

From the volume of work, it is fair to say that human endometrium has been the most intensively studied portion of the female genital tract in the field of adult stem cell research. This is partly due to the fact that the uterus is by far the most accessible portion of the tract, where sample collection and analysis is much less invasive compared to investigating ovary or fallopian tubes, and partly to the logic assumption that such intensely proliferating and renewing tissue should contain a stem cell pool. Since the stratum basalis of the endometrium is necessary for monthly renewal of the functional layer [48], characterized by intense prolif‐ eration under stimulation of rising estradiol levels, it is a prime candidate for harboring stem cells. Still, there is no common agreement yet where adult endometrial stem cells reside in vivo. LRCs were first identified by BrdU labeling in the epithelial layer and underlying stroma in postnatal mice [2]. However, by 12 weeks post labeling, no BrdU positive cells were left. Suboptimal timing of the pulse could mean that division of slow-cycling stem cells was missed such that only transitory amplifying progeny was labeled. Nevertheless, the study provided

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147

the first in vivo evidence for the existence of long-lived cells in the endometrium.

A significant advance over BrdU labeling for localizing stem cells in the mouse was recently achieved with the development of a histone2B-GFP (H2B-GFP) reporter in a Tet-inducible system, in which a doxyclicline pulse leads to fluorescent labeling of chromosomes, without mutagenic stress. This method allows induction of the construct during embryonic develop‐ ment, which increases the likelihood that stem cells will be labeled. With each cell division the GFP signal is reduced until after ~ 12 weeks it can only be detected in LRCs. In the first study using this system [49], the doxycyclin pulse was administred in adult animals and 12 weeks after pulse withdrawal LRCs were identified only in the distal oviduct segment of fimbrium and ampulla. Their presence was stable even after 47 weeks. In a follow-up study, labeling was extended to the embryonic period, confirming localization of LRCs to the distal oviduct, and identifying an additional population at the endocervical transition region but not within the endometrium. Only a pulse during the pre-pubertal period (post natal day 21-42) resulted in labeling rare cells in the glandular endometrial epithelium [4]. It is perhaps due to these experimental difficulties in identifying the adult stem cells in the uterus of a living organism, despite the undisputedly enormous capacity for endometrial regeneration and plasticity, that alternative models have been proposed to explain regenerative capacity of the endometrium.

An interesting hypothesis that has gained some traction within the scientific community is the possibility that bone marrow could be a source of endometrial stem cells. Previously, bone marrow stem cells were reported to be able to differentiate into a wide variety of cell types [50-52]. Analysis of bone marrow transplant recipients revealed the presence of donor cells in the opening of endometrial glands, raising the possibility that a stem cell pool outside of the reproductive system may contribute to the regeneration processes [53,54], mirroring the findings with female GSCs. Ikoma and colleagues used in situ hybridization to confirm the localization of Y chromosome-positive donor cells in the endometrium of a female patient who had undergone bone marrow transplant from a male donor [55]. However newer studies have questioned the functional importance of such findings, as bone marrow cells residing within

While a lineage tree analysis [39] based on somatic mutations accumulating in microsatellites found that oocytes form a cluster which is entirely distinct from other cell populations – suggesting that there is no intermixing of the germ cell precursor pool with that of any other cell type – it is conceivable that a very rare subpopulation of bone marrow stem cells would be missed in such an analysis. In fact their results also show that the number of mitotic divisions oocytes have undergone increases with age and following unilateral ovarectomy. This may be explained by recruitment of oocytes in the order in which they first differentiated during development – but is also consistent with the notion of continuous oocyte production from cycling stem cells. Many observers have suggested that if GSCs do exist, they are most likely to be derived from the normal developmental precursors of oocytes, i.e. PGCs or oogonia – which have not yet differentiated into oocytes and are still able to undergo mitosis [40-42]. The close relationship of PGCs to pluripotent cells is demonstrated by the fact that following isolation from the embryo, they can be converted back to a pluripotent phenotype termed embryonic germ cells in vitro without genetic manipulation [43-45].

Even if one accepts the existence of GSCs, there are a number of unanswered questions apart from their exact location. Firstly, it is not clear whether they contribute to oocyte production under normal physiological conditions, or only after injury. Secondly, if the follicle pool is replenished by GSCs, why does this replenishment eventually cease, leading to menopause? Niikura et al [27] have suggested that the aging niche environment itself may be responsible – however, the life-long production of spermatozoa in the testis indicates that this is not in itself a sufficient explanation. A large number of germ cells in neonatal mouse ovaries have not yet entered meiosis and can be induced to proliferate, increasing the follicular pool – but by the time animals enter reproductive age the numbers have returned to "normal" levels [46]. This suggests that mechanisms exist within the ovary to actively regulate the number of follicles. Together with the fact that a large proportion of oocytes are eliminated shortly after birth [47] this indicates a highly selective process to ensure the removal of oocytes with reduced meiotic fitness, which runs counter to the idea of continued oocyte replenishment from cycling precursors.

Whether or not GSCs exist in vivo, the presence of cells that can be expanded and differentiated to functioning oocytes in vitro – as suggested by Zou et al [7] and others [28] – would in itself be of huge potential benefit for the treatment of infertility. Nonetheless, the long time required to induce proliferation of these cells in vitro (around 10 weeks), compared to other adult stem cells, suggests that transformation of the cells in vitro may be responsible for the observed phenotype – similar to findings which describe the production of oocytes from other somatic stem cells in vitro – or that indeed the results may be explained by rare primordial oocytes that are carried over during the in vitro period.

#### **4. Uterine endometrial stem cells**

but appear to contribute also to other tissues [38]. These observations, as well as the fact that hematopoietic stem cells also migrate from the proximal epiblast to the embryonic aortagonad-mesonephric region during the same period of development, imply that it is theoreti‐ cally possible that there may be "intermixing" or indeed that there is a common precursor pool

While a lineage tree analysis [39] based on somatic mutations accumulating in microsatellites found that oocytes form a cluster which is entirely distinct from other cell populations – suggesting that there is no intermixing of the germ cell precursor pool with that of any other cell type – it is conceivable that a very rare subpopulation of bone marrow stem cells would be missed in such an analysis. In fact their results also show that the number of mitotic divisions oocytes have undergone increases with age and following unilateral ovarectomy. This may be explained by recruitment of oocytes in the order in which they first differentiated during development – but is also consistent with the notion of continuous oocyte production from cycling stem cells. Many observers have suggested that if GSCs do exist, they are most likely to be derived from the normal developmental precursors of oocytes, i.e. PGCs or oogonia – which have not yet differentiated into oocytes and are still able to undergo mitosis [40-42]. The close relationship of PGCs to pluripotent cells is demonstrated by the fact that following isolation from the embryo, they can be converted back to a pluripotent phenotype termed

Even if one accepts the existence of GSCs, there are a number of unanswered questions apart from their exact location. Firstly, it is not clear whether they contribute to oocyte production under normal physiological conditions, or only after injury. Secondly, if the follicle pool is replenished by GSCs, why does this replenishment eventually cease, leading to menopause? Niikura et al [27] have suggested that the aging niche environment itself may be responsible – however, the life-long production of spermatozoa in the testis indicates that this is not in itself a sufficient explanation. A large number of germ cells in neonatal mouse ovaries have not yet entered meiosis and can be induced to proliferate, increasing the follicular pool – but by the time animals enter reproductive age the numbers have returned to "normal" levels [46]. This suggests that mechanisms exist within the ovary to actively regulate the number of follicles. Together with the fact that a large proportion of oocytes are eliminated shortly after birth [47] this indicates a highly selective process to ensure the removal of oocytes with reduced meiotic fitness, which runs counter to the idea of continued oocyte replenishment from cycling

Whether or not GSCs exist in vivo, the presence of cells that can be expanded and differentiated to functioning oocytes in vitro – as suggested by Zou et al [7] and others [28] – would in itself be of huge potential benefit for the treatment of infertility. Nonetheless, the long time required to induce proliferation of these cells in vitro (around 10 weeks), compared to other adult stem cells, suggests that transformation of the cells in vitro may be responsible for the observed phenotype – similar to findings which describe the production of oocytes from other somatic stem cells in vitro – or that indeed the results may be explained by rare primordial oocytes that

for PGCs and a subpopulation of bone marrow stem cells.

embryonic germ cells in vitro without genetic manipulation [43-45].

precursors.

146 Adult Stem Cell Niches

are carried over during the in vitro period.

From the volume of work, it is fair to say that human endometrium has been the most intensively studied portion of the female genital tract in the field of adult stem cell research. This is partly due to the fact that the uterus is by far the most accessible portion of the tract, where sample collection and analysis is much less invasive compared to investigating ovary or fallopian tubes, and partly to the logic assumption that such intensely proliferating and renewing tissue should contain a stem cell pool. Since the stratum basalis of the endometrium is necessary for monthly renewal of the functional layer [48], characterized by intense prolif‐ eration under stimulation of rising estradiol levels, it is a prime candidate for harboring stem cells. Still, there is no common agreement yet where adult endometrial stem cells reside in vivo. LRCs were first identified by BrdU labeling in the epithelial layer and underlying stroma in postnatal mice [2]. However, by 12 weeks post labeling, no BrdU positive cells were left. Suboptimal timing of the pulse could mean that division of slow-cycling stem cells was missed such that only transitory amplifying progeny was labeled. Nevertheless, the study provided the first in vivo evidence for the existence of long-lived cells in the endometrium.

A significant advance over BrdU labeling for localizing stem cells in the mouse was recently achieved with the development of a histone2B-GFP (H2B-GFP) reporter in a Tet-inducible system, in which a doxyclicline pulse leads to fluorescent labeling of chromosomes, without mutagenic stress. This method allows induction of the construct during embryonic develop‐ ment, which increases the likelihood that stem cells will be labeled. With each cell division the GFP signal is reduced until after ~ 12 weeks it can only be detected in LRCs. In the first study using this system [49], the doxycyclin pulse was administred in adult animals and 12 weeks after pulse withdrawal LRCs were identified only in the distal oviduct segment of fimbrium and ampulla. Their presence was stable even after 47 weeks. In a follow-up study, labeling was extended to the embryonic period, confirming localization of LRCs to the distal oviduct, and identifying an additional population at the endocervical transition region but not within the endometrium. Only a pulse during the pre-pubertal period (post natal day 21-42) resulted in labeling rare cells in the glandular endometrial epithelium [4]. It is perhaps due to these experimental difficulties in identifying the adult stem cells in the uterus of a living organism, despite the undisputedly enormous capacity for endometrial regeneration and plasticity, that alternative models have been proposed to explain regenerative capacity of the endometrium.

An interesting hypothesis that has gained some traction within the scientific community is the possibility that bone marrow could be a source of endometrial stem cells. Previously, bone marrow stem cells were reported to be able to differentiate into a wide variety of cell types [50-52]. Analysis of bone marrow transplant recipients revealed the presence of donor cells in the opening of endometrial glands, raising the possibility that a stem cell pool outside of the reproductive system may contribute to the regeneration processes [53,54], mirroring the findings with female GSCs. Ikoma and colleagues used in situ hybridization to confirm the localization of Y chromosome-positive donor cells in the endometrium of a female patient who had undergone bone marrow transplant from a male donor [55]. However newer studies have questioned the functional importance of such findings, as bone marrow cells residing within the endometrium do not appear to contribute to the stem cell pool, which exhibits high clonogenic capacity in vitro, but are instead reminiscent of terminally differentiated cells [56], which could be explained by the capacity of bone marrow-derived cells to fuse with differen‐ tiated cells from a variety of organs [57].

signaling activity and increased expression of the Wnt target genes Axin2, Cyclin D1, ID2,

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As well as studying the role of endometrial stem cells in regeneration of the functional endometrium, a number of methods have been developed to use endometrial progenitors in translational approaches for therapeutic purposes. In vitro differentiation assays suggest that mesenchymal stromal stem cells may be able to differentiate into diverse cell types [58,65,66], and can be efficiently isolated from menstrual blood. A clinical trial of endometrial stem cells as a source of autologous regenerative cardiomyocytes to treat ischemic cardiac injury is underway [67] and promising results were also reported for pancreatic island replacement in

In parallel to the translational approaches, we need to get a better understanding of the biology of endometrial stem cells in vivo to understand how these cells influence the development of pathologies in the uterus. There are strong indications that deregulation of the stem cell niche plays a role in endometriosis, a disease which affects up to 10% of all women and is present in nearly half of those experiencing fertility problems or pelvic pain after the age of 35 [69]. The hallmark of endometriosis is the presence of ectopic explants of endometrial tissue outside of the uterus, which can affect the fallopian tubes, the ovary but also more distant regions of the pelvic cavity, causing pain and discomfort. Endometri‐ otic lesions have identical responses to hormonal stimuli as the endometrium of the uterus. The model of "retrograde menstruation" is accepted as the most plausible explanation for the spread of endometriosis tissue through the genital tract, but the molecular mecha‐ nisms which trigger and control the disease remain obscure. Analysis of menstrual tissue samples from affected women showed that endometriosis patients have significantly more fragments of the basalis layer in the flow than healthy controls [70]. Cells from menstrual blood of patients also have longer than average telomeres, which is in agreement with an enhanced stem cell presence [71]. According to this model, long-lived stem cells from the basalis are disseminated and give rise to endometriotic lesions. In healthy women, on the other hand, only the upper layer of the endometrium is ablated, leaving the basalis layer intact. These data offer hope that characterization of the endometrial stem cell niche and its regulatory mechanisms will lead to a breakthrough in prevention and treatment of endometriosis – a disease that poses an enormous burden for patients' quality of life as

Our understanding of the biology of fallopian tube epithelium is still rudimentary. From the perspective of medical diagnostics, it is the least accessible portion of the female reproductive tract. The fallopian tube is barely visible by ultrasound and even exploratory laparoscopy offers only information about tubal patency, and no insight into the condition of the mucosa. Current knowledge about tubal histology and pathology comes solely from patients of salpingectomy procedures, where tubes are surgically removed, usually as part of a total

CD44.

a xenograft diabetic mouse model [68].

well as high costs for the healthcare system.

**5. Fallopian tube stem cells**

Regardless of the difficulties in identifying and characterizing the adult stem cell niche in the intact epithelium in vivo, numerous studies in recent years have isolated distinct populations of putative adult stem or progenitor cells from uterine tissue and demonstrat‐ ed their proliferative capacity and broad differentiation potential in vitro. Two different classes of adult stem cells are found in the endometrium: mesenchymal and epithelial. Mesenchymal stem cells have also been named Endometrial Regenerative Cells (ERC) due to their high proliferative potential. Although the ability to produce a clonal population of cells when seeded at very low dilution is much greater for the stromal mesenchymal subpopulation, the epithelium also contains cells capable of clonal expansion in vitro. Self renewal of endometrial stem cells in vitro was first demonstrated in pioneering work by the group of Caroline Gargett [2,58,59]. In the absence of a widely accepted surface marker for endometrial stem cells, they demonstrated that multipotent cells can be efficiently isolated based on the uptake of Hoechst 33342 [60]. The method of SP cells was original‐ ly established in procedures for adult stem cells isolation from the hematoopetic tissue [61]. It made use of the discovery that the small population of adult stem cells in the tissue differs from the differentiated cells by their capacity to efficiently eject Hoechst dye from the cytoplasm, presumably due to the high expression of the ABCG2 transporter [62]. Designated endometrial side population cells (ESP), consisting of both epithelial and mesenchymal SP cells, are able to generate endometrial glandular structures in vivo if transplanted under the kidney capsule of NOD/SCID immunocompromised mice [63]. Interestingly, a fraction of the ESPs migrated and generated blood vessels to supply the endometrium, showing potency to generate different types of tissue in the functional organ. Still, the overall efficiency of the procedure was rather low as endometrium formation occurred in only 2 out of 24 injected animals. Dramatic improvements in the outcome of this type of xenograft was achieved by expanding and cloning lines of SP cells from both epithelial and stromal compartments. Transplantation of individual lines supported by the administration of estrogen (E2) and progesterone (P) to mimic the menstrual cycle gave rise to endometrial tissue in all animals [56]. This is a particularly significant finding since endometrial stem cells do not express E2 and P receptors. Nevertheless, the hormonal environment greatly affects the outcome of the tissue regeneration process. This phenomen‐ on was closely analyzed by Janzen and colleagues [64], who showed that hormonal withdrawal in the xenograft mouse model leads to a pronounced decrease in the size of endometrial tissue while the remaining cells are highly enriched in stem cells. This suggests that the stem cell niche is activated at the beginning of each cycle, when both estradiol and progesterone levels are low, while the subsequent hormonal stimulation drives regenera‐ tion and proliferation of transitory amplifying cells up to their final differentiation in response to progesterone stimulation from the corpus luteum in the second phase of menstrual cycle. On the molecular level, endometrial stem cells showed elevated Wnt signaling activity and increased expression of the Wnt target genes Axin2, Cyclin D1, ID2, CD44.

As well as studying the role of endometrial stem cells in regeneration of the functional endometrium, a number of methods have been developed to use endometrial progenitors in translational approaches for therapeutic purposes. In vitro differentiation assays suggest that mesenchymal stromal stem cells may be able to differentiate into diverse cell types [58,65,66], and can be efficiently isolated from menstrual blood. A clinical trial of endometrial stem cells as a source of autologous regenerative cardiomyocytes to treat ischemic cardiac injury is underway [67] and promising results were also reported for pancreatic island replacement in a xenograft diabetic mouse model [68].

In parallel to the translational approaches, we need to get a better understanding of the biology of endometrial stem cells in vivo to understand how these cells influence the development of pathologies in the uterus. There are strong indications that deregulation of the stem cell niche plays a role in endometriosis, a disease which affects up to 10% of all women and is present in nearly half of those experiencing fertility problems or pelvic pain after the age of 35 [69]. The hallmark of endometriosis is the presence of ectopic explants of endometrial tissue outside of the uterus, which can affect the fallopian tubes, the ovary but also more distant regions of the pelvic cavity, causing pain and discomfort. Endometri‐ otic lesions have identical responses to hormonal stimuli as the endometrium of the uterus. The model of "retrograde menstruation" is accepted as the most plausible explanation for the spread of endometriosis tissue through the genital tract, but the molecular mecha‐ nisms which trigger and control the disease remain obscure. Analysis of menstrual tissue samples from affected women showed that endometriosis patients have significantly more fragments of the basalis layer in the flow than healthy controls [70]. Cells from menstrual blood of patients also have longer than average telomeres, which is in agreement with an enhanced stem cell presence [71]. According to this model, long-lived stem cells from the basalis are disseminated and give rise to endometriotic lesions. In healthy women, on the other hand, only the upper layer of the endometrium is ablated, leaving the basalis layer intact. These data offer hope that characterization of the endometrial stem cell niche and its regulatory mechanisms will lead to a breakthrough in prevention and treatment of endometriosis – a disease that poses an enormous burden for patients' quality of life as well as high costs for the healthcare system.

#### **5. Fallopian tube stem cells**

the endometrium do not appear to contribute to the stem cell pool, which exhibits high clonogenic capacity in vitro, but are instead reminiscent of terminally differentiated cells [56], which could be explained by the capacity of bone marrow-derived cells to fuse with differen‐

Regardless of the difficulties in identifying and characterizing the adult stem cell niche in the intact epithelium in vivo, numerous studies in recent years have isolated distinct populations of putative adult stem or progenitor cells from uterine tissue and demonstrat‐ ed their proliferative capacity and broad differentiation potential in vitro. Two different classes of adult stem cells are found in the endometrium: mesenchymal and epithelial. Mesenchymal stem cells have also been named Endometrial Regenerative Cells (ERC) due to their high proliferative potential. Although the ability to produce a clonal population of cells when seeded at very low dilution is much greater for the stromal mesenchymal subpopulation, the epithelium also contains cells capable of clonal expansion in vitro. Self renewal of endometrial stem cells in vitro was first demonstrated in pioneering work by the group of Caroline Gargett [2,58,59]. In the absence of a widely accepted surface marker for endometrial stem cells, they demonstrated that multipotent cells can be efficiently isolated based on the uptake of Hoechst 33342 [60]. The method of SP cells was original‐ ly established in procedures for adult stem cells isolation from the hematoopetic tissue [61]. It made use of the discovery that the small population of adult stem cells in the tissue differs from the differentiated cells by their capacity to efficiently eject Hoechst dye from the cytoplasm, presumably due to the high expression of the ABCG2 transporter [62]. Designated endometrial side population cells (ESP), consisting of both epithelial and mesenchymal SP cells, are able to generate endometrial glandular structures in vivo if transplanted under the kidney capsule of NOD/SCID immunocompromised mice [63]. Interestingly, a fraction of the ESPs migrated and generated blood vessels to supply the endometrium, showing potency to generate different types of tissue in the functional organ. Still, the overall efficiency of the procedure was rather low as endometrium formation occurred in only 2 out of 24 injected animals. Dramatic improvements in the outcome of this type of xenograft was achieved by expanding and cloning lines of SP cells from both epithelial and stromal compartments. Transplantation of individual lines supported by the administration of estrogen (E2) and progesterone (P) to mimic the menstrual cycle gave rise to endometrial tissue in all animals [56]. This is a particularly significant finding since endometrial stem cells do not express E2 and P receptors. Nevertheless, the hormonal environment greatly affects the outcome of the tissue regeneration process. This phenomen‐ on was closely analyzed by Janzen and colleagues [64], who showed that hormonal withdrawal in the xenograft mouse model leads to a pronounced decrease in the size of endometrial tissue while the remaining cells are highly enriched in stem cells. This suggests that the stem cell niche is activated at the beginning of each cycle, when both estradiol and progesterone levels are low, while the subsequent hormonal stimulation drives regenera‐ tion and proliferation of transitory amplifying cells up to their final differentiation in response to progesterone stimulation from the corpus luteum in the second phase of menstrual cycle. On the molecular level, endometrial stem cells showed elevated Wnt

tiated cells from a variety of organs [57].

148 Adult Stem Cell Niches

Our understanding of the biology of fallopian tube epithelium is still rudimentary. From the perspective of medical diagnostics, it is the least accessible portion of the female reproductive tract. The fallopian tube is barely visible by ultrasound and even exploratory laparoscopy offers only information about tubal patency, and no insight into the condition of the mucosa. Current knowledge about tubal histology and pathology comes solely from patients of salpingectomy procedures, where tubes are surgically removed, usually as part of a total hysterectomy. The importance of making progress in this area has been highlighted in recent years, as the potential significance of this tissue in disease initiation has become clear. The distal portion of the tubal fimbrium, and the ampulla with its abundant mucosal folds, are of critical importance for oocyte capture and fertilization. Intimate contact of the fimbrium with the surface of the ovary exposes the epithelial layer of the tube to the inflammatory signals associated with ovulation which are present in follicular fluid. Thus, there is an increased requirement for robust renewal mechanisms. Indeed, in vitro experiments with cell isolates from the fallopian tube have defined a population that is positive for CD44 and integrin α6 and has the capacity for clonal growth, self renewal and differentiation into the secretory and ciliated cells found in the tubal epithelial monolayer-specifically in the distal region of the tube [72]. Strong clonogenic potential of a H2B-GFP-retaining subset of epithelial cells in the distal oviduct and their capacity to form and maintain spheroids in vitro long-term has been demonstrated in a mouse model [49]. These spheroids are able to differentiate into more complex structures of the mucosa, strongly suggesting that they do indeed contain progenitor cells of the tube with the potential to proliferate and differentiate in vitro. However, the exact organization of the fallopian tube stem cell niche in vivo remains unknown apart from the positive identification of candidate cells in label-retaining experiments in vivo mentioned above. Moreover, the exact turnover rate of the epithelium and how it responds to different physiological and environmental stimuli or stresses is yet to be established.

Label-retaining cells on the ovarian surface were identified in vivo by comparing a histone-GFP inducible transgenic model and BrdU labeling of animals [78]. It is important to note that both methods resulted in identification of positive cells in the OSE epithelium of the ovary but the two populations overlapped only in a minority of cases. This further underscores the complexity and difficulty of identifying adult stem cells solely on the basis of cell division rate. Recently, the ovarian surface was subjected to a more detailed analysis, and the highest proportion of LRCs was detected in the hilum region close to the fallopian tube. These cells expressed high levels of aldehyde dehydrogenase 1 (ALDH1), and were able to proliferate long-term in vitro [3], forming spheres. Long-term pulse experiments and staining with the proliferation marker Ki67 also suggested that slow-cycling cells are activated in a cyclical fashion during the estrus phase, which supports a repair mechanism for the damaged OSE monolayer. Finally, genetic lineage tracing of Lgr5 expression in the ovary was performed [3], by using the Lgr5tm1(cre/ERT2)Cle/J mouse which harbors a genetic construct that enables GFP labeling of Lgr5 expressing cells and tamoxifen-inducible expression of Cre recombinase. When crossed with a strain harboring a stop codon in a dTomato sequence that can be excised by Cre recombinase, offspring mice are produced in which easy tracing of the progeny of Lgr5 expressing cells is possible by a simple tamoxifen pulse at the desired time point [79]. This

monolayer during renewal of the epithelium in the course of 1 month [3]. Notably, this study was focused on the ovary and no lineage tracing was analyzed in other portions of the genital tract. A similar approach, with the SOX2 gene promoter controlling expression of CRE recombinase in a tamoxifen-inducible fashion, revealed the existence of long-term lineage labeling in the cervical epithelium of mice (Arnold et al 2012). It remains unclear, however, if other parts of the female genital system contain epithelial cells originating from SOX2+ progenitors and it will be interesting to find out how these populations of putative stem cell candidates identified by independent methods correlate with each other. It is of course feasible that different tissue compartments harbor stem cells which are defined by different molecular markers. Although these two studies represent a methodological breakthrough in the detection of adult stem cells, by demonstrating the in vivo capacity of these cells to give rise to differ‐ entiated progeny in the tissue through tracing cellular markers in physiological conditions, they are not entirely comprehensive. This approach is hypothesis-driven by selecting candi‐ date genes; however, the list of potential stemness regulators that could be tested is much longer. For example, Lgr5 is only one member of the Lgr receptor family, and other members are also involved in regulating tissue regeneration, e.g. Lgr6 in the hair follicle [80] and Lgr4 in the prostate [81]. More detailed studies are needed to confirm whether the LGR5+ cells detected in the hilum represent the pool of true stem cells or a more dynamic population of amplifying cells that have already undergone a degree of lineage commitment. The SOX2+ cells identified in the cervix are a good starting point to define the molecular characteristics of the long-lived basal cells which have so far proven to be elusive using other tracing methods, and this genetic system will provide a valuable tool for a more detailed analysis of the adult stem

population at the hilum, which contributes to the whole OSE

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151

experiment revealed an Lgr5+

cell niche in the future.

Beyond the requirement for Wnt signaling for normal development of the genital tract, several studies have reported changes in Wnt signaling associated with pathology of the fallopian tube. For example, activation of β-catenin signaling is implicated as a contributing factor in ectopic pregnancy [73,74] and endometriosis [75]. Increased Wnt signaling is a mucosal response to infection [76], confirming that this paracrine pathway plays a role in homeostasis inside the fallopian tube. More studies are needed, however, to illuminate all aspects of paracrine signaling in the fallopian tube epithelium in health and disease. As mentioned above, Wnt signaling alterations are one of the hallmarks of ovarian cancer. Since there is now consensus in the medical community, based on cumulative molecular and clinical evidence, that a significant portion of high grade serous ovarian carcinoma originates from the fallopian tube fimbrium rather than the ovarian surface epithelium, it has become imperative to illuminate the regulatory mechanisms involved in fallopian tube epithelium homeostasis. Different models of ovarian carcinogenesis and the potential role of adult stem cells in this process will be reviewed fully later in this chapter.

#### **6. Ovarian and cervical stem cells**

In contrast to the uterus, direct evidence for adult stem cells in the ovary is sparse. However, the tissue remodeling processes involved in ovulation require a considerable regeneration potential within this organ that has to be maintained for several decades. Beyond the healing of the ovulatory wound created in the epithelial surface during follicle rupture, ovarian epithelial cells also undergo proliferation at the beginning of the menstrual cycle [77].

Label-retaining cells on the ovarian surface were identified in vivo by comparing a histone-GFP inducible transgenic model and BrdU labeling of animals [78]. It is important to note that both methods resulted in identification of positive cells in the OSE epithelium of the ovary but the two populations overlapped only in a minority of cases. This further underscores the complexity and difficulty of identifying adult stem cells solely on the basis of cell division rate. Recently, the ovarian surface was subjected to a more detailed analysis, and the highest proportion of LRCs was detected in the hilum region close to the fallopian tube. These cells expressed high levels of aldehyde dehydrogenase 1 (ALDH1), and were able to proliferate long-term in vitro [3], forming spheres. Long-term pulse experiments and staining with the proliferation marker Ki67 also suggested that slow-cycling cells are activated in a cyclical fashion during the estrus phase, which supports a repair mechanism for the damaged OSE monolayer. Finally, genetic lineage tracing of Lgr5 expression in the ovary was performed [3], by using the Lgr5tm1(cre/ERT2)Cle/J mouse which harbors a genetic construct that enables GFP labeling of Lgr5 expressing cells and tamoxifen-inducible expression of Cre recombinase. When crossed with a strain harboring a stop codon in a dTomato sequence that can be excised by Cre recombinase, offspring mice are produced in which easy tracing of the progeny of Lgr5 expressing cells is possible by a simple tamoxifen pulse at the desired time point [79]. This experiment revealed an Lgr5+ population at the hilum, which contributes to the whole OSE monolayer during renewal of the epithelium in the course of 1 month [3]. Notably, this study was focused on the ovary and no lineage tracing was analyzed in other portions of the genital tract. A similar approach, with the SOX2 gene promoter controlling expression of CRE recombinase in a tamoxifen-inducible fashion, revealed the existence of long-term lineage labeling in the cervical epithelium of mice (Arnold et al 2012). It remains unclear, however, if other parts of the female genital system contain epithelial cells originating from SOX2+ progenitors and it will be interesting to find out how these populations of putative stem cell candidates identified by independent methods correlate with each other. It is of course feasible that different tissue compartments harbor stem cells which are defined by different molecular markers. Although these two studies represent a methodological breakthrough in the detection of adult stem cells, by demonstrating the in vivo capacity of these cells to give rise to differ‐ entiated progeny in the tissue through tracing cellular markers in physiological conditions, they are not entirely comprehensive. This approach is hypothesis-driven by selecting candi‐ date genes; however, the list of potential stemness regulators that could be tested is much longer. For example, Lgr5 is only one member of the Lgr receptor family, and other members are also involved in regulating tissue regeneration, e.g. Lgr6 in the hair follicle [80] and Lgr4 in the prostate [81]. More detailed studies are needed to confirm whether the LGR5+ cells detected in the hilum represent the pool of true stem cells or a more dynamic population of amplifying cells that have already undergone a degree of lineage commitment. The SOX2+ cells identified in the cervix are a good starting point to define the molecular characteristics of the long-lived basal cells which have so far proven to be elusive using other tracing methods, and this genetic system will provide a valuable tool for a more detailed analysis of the adult stem cell niche in the future.

hysterectomy. The importance of making progress in this area has been highlighted in recent years, as the potential significance of this tissue in disease initiation has become clear. The distal portion of the tubal fimbrium, and the ampulla with its abundant mucosal folds, are of critical importance for oocyte capture and fertilization. Intimate contact of the fimbrium with the surface of the ovary exposes the epithelial layer of the tube to the inflammatory signals associated with ovulation which are present in follicular fluid. Thus, there is an increased requirement for robust renewal mechanisms. Indeed, in vitro experiments with cell isolates from the fallopian tube have defined a population that is positive for CD44 and integrin α6 and has the capacity for clonal growth, self renewal and differentiation into the secretory and ciliated cells found in the tubal epithelial monolayer-specifically in the distal region of the tube [72]. Strong clonogenic potential of a H2B-GFP-retaining subset of epithelial cells in the distal oviduct and their capacity to form and maintain spheroids in vitro long-term has been demonstrated in a mouse model [49]. These spheroids are able to differentiate into more complex structures of the mucosa, strongly suggesting that they do indeed contain progenitor cells of the tube with the potential to proliferate and differentiate in vitro. However, the exact organization of the fallopian tube stem cell niche in vivo remains unknown apart from the positive identification of candidate cells in label-retaining experiments in vivo mentioned above. Moreover, the exact turnover rate of the epithelium and how it responds to different

physiological and environmental stimuli or stresses is yet to be established.

process will be reviewed fully later in this chapter.

**6. Ovarian and cervical stem cells**

150 Adult Stem Cell Niches

Beyond the requirement for Wnt signaling for normal development of the genital tract, several studies have reported changes in Wnt signaling associated with pathology of the fallopian tube. For example, activation of β-catenin signaling is implicated as a contributing factor in ectopic pregnancy [73,74] and endometriosis [75]. Increased Wnt signaling is a mucosal response to infection [76], confirming that this paracrine pathway plays a role in homeostasis inside the fallopian tube. More studies are needed, however, to illuminate all aspects of paracrine signaling in the fallopian tube epithelium in health and disease. As mentioned above, Wnt signaling alterations are one of the hallmarks of ovarian cancer. Since there is now consensus in the medical community, based on cumulative molecular and clinical evidence, that a significant portion of high grade serous ovarian carcinoma originates from the fallopian tube fimbrium rather than the ovarian surface epithelium, it has become imperative to illuminate the regulatory mechanisms involved in fallopian tube epithelium homeostasis. Different models of ovarian carcinogenesis and the potential role of adult stem cells in this

In contrast to the uterus, direct evidence for adult stem cells in the ovary is sparse. However, the tissue remodeling processes involved in ovulation require a considerable regeneration potential within this organ that has to be maintained for several decades. Beyond the healing of the ovulatory wound created in the epithelial surface during follicle rupture, ovarian

epithelial cells also undergo proliferation at the beginning of the menstrual cycle [77].

## **7. Adult stem cells between development and disease — Role of the Wnt pathway**

The precise signaling pathways controlling the renewal processes of the fallopian tube epithelium, endometrium and ovary remain unknown, but there are several reasons to assume that they depend on paracrine Wnt, Notch and BMP signaling. Wnt signaling controls crucial developmental processes during all phases of embryogenesis and during adult life. Wnt ligands interact with a family of receptors, inducing a variety of responses depending on the cellular context. The main transducer of Wnt signalling within the cell is β-catenin, which translocates to the nucleus and induces expression of target genes via several transcription factors (Figure 2).

Although a detailed overview of Wnt signaling is beyond the scope of this chapter, we will briefly outline its involvement in the control of the cell behavior within a tissue, affecting, among other processes, proliferation, establishment of polarity, differentiation, morphoge‐ netic movements and apoptosis. With respect to tissue homeostasis, Wnt signaling acts as a key cell-cell communication network during tissue formation in development, as well as for maintaining tissue function. The embryological development of the female genital tract relies on active Wnt signaling, as evidenced by Müllerian aplasia. This autosomal mutation in the Wnt4 gene leads to a severely underdeveloped or absent uterus. Mouse models have further revealed a strong dependence of developmental processes on Wnt 5a, 7a, and 9b, since mutant animals showed severe malformations of different parts of the genital tract raging from defective coiling of oviducts to absence of the upper vagina or uterine glands [82-84]. The importance of the Wnt pathway for maintenance of homeostasis is well-documented by molecular and genetic analysis of numerous human malignancies. Perhaps the most startling example of the tight relationship between Wnt signaling and control of proliferation in mucosal epithelium is provided by the relationship between Adenomatous polyposis coli (APC) mutations and colon cancer. APC protein in complex with Axin1 promotes degradation of βcatenin and thereby inhibits Wnt signaling transduction (see Fig 2). In familial adenomatous polyposis patients, who carry an APC functional deletion mutation, the risk of developing colon cancer is almost 100% and 5% of sporadic colon cancer patients harbor either APC lossof-function or β-catenin activating mutations [85]. Mutations in components of the Wnt pathway are frequently found in numerous other malignancies as well, e.g. pancreas, liver, kidney, pituitary, and notably also in the ovary and endometrium [86].

Although the significance of Wnt signaling for tissue maintenance has been known for the last couple of decades, it is the discovery of adult tissue stem cells which led to a breakthrough in our understanding of the regulatory mechanisms at the molecular level. Lgr5-expressing stem cells of the intestine ensure renewal of the mucosa every 3-5 days. Although Lgr5<sup>+</sup> cells at the base of intestinal crypts are sufficient to recreate the complete epithelial layer in vitro, an alternative model attributes true "stemness" to rare, more quiescent cells in the wall of the crypt, which express Bmi1, HOPX and mTERT [87-89]. Ablation of Lgr5+ cells does not disrupt homeostasis, as Bmi1+ cells compensate for the loss [90]. This illustrates the complexity of the regulatory stem cell niche, where different cells can be recruited and even reprogrammed,

depending on the conditions during epithelial renewal. Although Lgr5 is a Wnt signaling target, crypt organization, cell fate determination and differentiation are also dependent on integration of signals from the Notch [91] and TGF-β pathways. Such paracrine signaling pathways have emerged as a common principle of functioning stem cell niches in other organs [92-95]. The inherent longevity of adult stem is a potent mechanism for dissemination of

**Figure 2.** Paracrine regulation of the adult stem cell niche. A) Schematic representation of the cellular mechanism of β-catenin turnover in the cytoplasm. After the Wnt signal triggers dimerization of receptors, β-catenin is released from the degradation complex and translocates to the nucleus, where it activates Wnt target genes; B) Hierarchical organi‐ zation of the intestinal crypt, with Lgr5+stem cells localizing to the bottom off the crypt between nurturing Paneth cells. Above the stem cells, there is a zone of intense proliferation (transitory amplifying cells), followed by terminally

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accumulated mutations within the tissue.

differentiated epithelium, which has short life span (1-2 days)

Adult Stem Cell Niches — Stem Cells in the Female Reproductive System http://dx.doi.org/10.5772/58842 153

**7. Adult stem cells between development and disease — Role of the Wnt**

The precise signaling pathways controlling the renewal processes of the fallopian tube epithelium, endometrium and ovary remain unknown, but there are several reasons to assume that they depend on paracrine Wnt, Notch and BMP signaling. Wnt signaling controls crucial developmental processes during all phases of embryogenesis and during adult life. Wnt ligands interact with a family of receptors, inducing a variety of responses depending on the cellular context. The main transducer of Wnt signalling within the cell is β-catenin, which translocates to the nucleus and induces expression of target genes via

Although a detailed overview of Wnt signaling is beyond the scope of this chapter, we will briefly outline its involvement in the control of the cell behavior within a tissue, affecting, among other processes, proliferation, establishment of polarity, differentiation, morphoge‐ netic movements and apoptosis. With respect to tissue homeostasis, Wnt signaling acts as a key cell-cell communication network during tissue formation in development, as well as for maintaining tissue function. The embryological development of the female genital tract relies on active Wnt signaling, as evidenced by Müllerian aplasia. This autosomal mutation in the Wnt4 gene leads to a severely underdeveloped or absent uterus. Mouse models have further revealed a strong dependence of developmental processes on Wnt 5a, 7a, and 9b, since mutant animals showed severe malformations of different parts of the genital tract raging from defective coiling of oviducts to absence of the upper vagina or uterine glands [82-84]. The importance of the Wnt pathway for maintenance of homeostasis is well-documented by molecular and genetic analysis of numerous human malignancies. Perhaps the most startling example of the tight relationship between Wnt signaling and control of proliferation in mucosal epithelium is provided by the relationship between Adenomatous polyposis coli (APC) mutations and colon cancer. APC protein in complex with Axin1 promotes degradation of βcatenin and thereby inhibits Wnt signaling transduction (see Fig 2). In familial adenomatous polyposis patients, who carry an APC functional deletion mutation, the risk of developing colon cancer is almost 100% and 5% of sporadic colon cancer patients harbor either APC lossof-function or β-catenin activating mutations [85]. Mutations in components of the Wnt pathway are frequently found in numerous other malignancies as well, e.g. pancreas, liver,

kidney, pituitary, and notably also in the ovary and endometrium [86].

Although the significance of Wnt signaling for tissue maintenance has been known for the last couple of decades, it is the discovery of adult tissue stem cells which led to a breakthrough in our understanding of the regulatory mechanisms at the molecular level. Lgr5-expressing stem

base of intestinal crypts are sufficient to recreate the complete epithelial layer in vitro, an alternative model attributes true "stemness" to rare, more quiescent cells in the wall of the crypt, which express Bmi1, HOPX and mTERT [87-89]. Ablation of Lgr5+ cells does not disrupt

regulatory stem cell niche, where different cells can be recruited and even reprogrammed,

cells compensate for the loss [90]. This illustrates the complexity of the

cells at the

cells of the intestine ensure renewal of the mucosa every 3-5 days. Although Lgr5<sup>+</sup>

**pathway**

152 Adult Stem Cell Niches

several transcription factors (Figure 2).

homeostasis, as Bmi1+

**Figure 2.** Paracrine regulation of the adult stem cell niche. A) Schematic representation of the cellular mechanism of β-catenin turnover in the cytoplasm. After the Wnt signal triggers dimerization of receptors, β-catenin is released from the degradation complex and translocates to the nucleus, where it activates Wnt target genes; B) Hierarchical organi‐ zation of the intestinal crypt, with Lgr5+stem cells localizing to the bottom off the crypt between nurturing Paneth cells. Above the stem cells, there is a zone of intense proliferation (transitory amplifying cells), followed by terminally differentiated epithelium, which has short life span (1-2 days)

depending on the conditions during epithelial renewal. Although Lgr5 is a Wnt signaling target, crypt organization, cell fate determination and differentiation are also dependent on integration of signals from the Notch [91] and TGF-β pathways. Such paracrine signaling pathways have emerged as a common principle of functioning stem cell niches in other organs [92-95]. The inherent longevity of adult stem is a potent mechanism for dissemination of accumulated mutations within the tissue.

#### **8. Cancer stem cells and the putative link to adult stem cells**

could prove to be of pivotal importance for resolving remaining questions about tumor spread and the role of cancer stem cells. In this light, we will address changes in tissue architecture and physiology of the female reproductive tract that could favor malignant transformation and bring it into context of therapeutic implications, particularly in light of recent evidence that dysregulation of tissue homeostasis could be the result of infection with certain sexually

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Malignant tumors of the genital tract are the third leading cause of cancer related deaths after breast cancer and lung cancer [107]. Based on the primary organ, affected cancers are classified as ovarian, uterine, cervical, vulval and vaginal. The high prevalence of these cancers is likely to be related to the great plasticity of these tissues, their regenerative potential and thus probably their high cell turnover as well. On top of this, openness to the external environment and exposure to pathogens makes them potentially vulnerable to the transformation. The latter has been demonstrated by the dramatic correlation between HPV infection cervical cancers,

From the perspective of patient care and long-term prognosis, one of the biggest challenges for medicine represents high grade serous ovarian cancer, an aggressive form of epithelial ovarian cancer which has a survival rate of under 40 % after 10 years [109]. This deadly malignancy takes more than 14,000 lives per year in the US alone and no improvement can be expected in the foreseeable future, due to the absence of early screening methods and the aggressive nature of the cancer. The origin of high-grade serous ovarian cancer (HG-SOC) has puzzled medical doctors and pathologists for decades. Naturally, the site of carcinogenesis was initially attributed to OSE cells [110]. Numerous examples of developmental genetics show that relatively complex changes in phenotype are frequently induced by expression of only one master regulator gene. In the case of OSE cells, ectopic expression of homeobox transcrip‐ tion factor HOX9 in the xenograft mouse model causes Müllerean metaplasia-transformation of cuboidal to papillary columnar epithelium resembling fallopian tube mucosa and serous ovarian cancer [111]. However, there is as yet no clinical evidence that this conversion also occurs in vivo. The scarcity of precancerous lesions or early stage carcinoma in situ detected by pathologies [112], raises questions whether this hypothesis is supported by clinical data. In parallel, the hypothesis that the etiology of HG-SOC could be explained by malignant transformation in neighboring tubal epithelium has gained increasing support. The fallopian tube develops from the Müllerian duct tissue, encompassing a columnar epithelial monolayer, which continues to express PAX8, the main cellular marker of "Müllerian differentiation" – a phenotype which is reminiscent of cancer tissue from HG-SOC patients. A breakthrough came from a cohort of clinical studies in BRCA mutation carriers, who have a very high hereditary risk of developing ovarian and breast cancer later in life. Small malignancies within the tubal epithelium (so-called serous tubal intraepithelial carcinoma – STIC) were discovered in up to 10% of patients who underwent prophylactic surgery to remove both fallopian tubes and ovaries [113]. These apparently healthy patients thus already had cancer in their fallopian

transmitted pathogens.

since nearly 100% of patients are HPV positive [108].

**9. Serous ovarian cancer and stem cells of the fallopian tube**

Parallel to the discovery of adult stem cells in healthy tissues, cancer research has produced a bulk of evidence showing that most malignancies are not homogenous cell masses but rather heterogeneous tissues whose progression depends on the fitness of a distinct cellular fraction: cancer stem cells (CSCs). CSCs can confer resistance to chemotherapeutic agents or exhibit other characteristics that provide a competitive advantage to the cancer and ensure its progression [96-99]. In many cases, long-term prognosis and patient survival can be correlated with the frequency of cancer stem cells in the tumor at the time of diagnosis [100]. CSCs exhibit the characteristics of adult stem cells: self renewal and differentiation capacity. They can be distinguished from the bulk of the tumor tissue as the only fraction that is able to generate new tumors when transplanted into immunocompromised mice [101,102]. For this reason, they are sometimes referred to as tumor initiating cells. This property of cancer stem cells has been demonstrated for numerous malignancies, including ovarian and endometrial cancer. As with markers of adult stem cells, markers specifically associated with cancer stem cells are also proving difficult to identify. It is of course possible that there is no unique cancer stem cell for ovarian, endometrial or other genital malignancies and that stemness can be achieved by alternative routes in individual cases, thereby resulting in different combinations of surface markers such as CD44, EpCAM, ALDH1, CD117, CD133 etc, which have been identified by different studies [81,103,104]. Cancer stem cells are intermixed with the bulk of the tumor tissue, but are found enriched in advanced stages of metastasis, in spheres present in effusions recovered from ascites [105]. Spheres exhibit anchorage-independent growth but can efficient‐ ly adhere to mesothelial cells in the peritoneum via integrin 1, which may play a role in metastatic spread. It is not yet known what triggers formation of spheres, although the physiological implications for disease progression and response to treatment are immense. The compact organization of the cells in a cluster makes drug delivery ineffective, either due to the difficulty of drugs to penetrate, or induction of a quiescent state in cells at the core through tight cell-cell contacts, making them insensitive to agents targeting actively replicating cells. Either way, it is clear that 3D organization represents another layer of protection for cancer cells from chemotherapy. This effect has been demonstrated by comparing the response to the standard therapeutic drugs cisplatin and paclitaxel for 11 different ovarian cancer cell lines in 2D in 3D [106].

Importantly, however, it remains unknown if and how cancer stem cells are related to normal adult stem cells. Cancer stem cells may develop from adult stem cells by losing dependence on niche regulatory factors, or they may simply be the progeny of differentiated cells that have acquired "stemness" characteristics during the accumulation of mutations and cellular transformation. It is conceivable that both mechanisms could occur in all or some types of cancer. If there is indeed a causal relationship between dysregulation of the adult stem cell niche and carcinogenesis, this may open up new possibilities for early diagnosis and timely intervention. Since the research field of cancer stem cells is currently offering a variety of sometimes competing models of what defines this population in endometrial, cervical and serous ovarian cancer, we focus our attention on the current understanding of carcinogenesis in the genital tract. Understanding of the cellular events that lead to initial transformation could prove to be of pivotal importance for resolving remaining questions about tumor spread and the role of cancer stem cells. In this light, we will address changes in tissue architecture and physiology of the female reproductive tract that could favor malignant transformation and bring it into context of therapeutic implications, particularly in light of recent evidence that dysregulation of tissue homeostasis could be the result of infection with certain sexually transmitted pathogens.

Malignant tumors of the genital tract are the third leading cause of cancer related deaths after breast cancer and lung cancer [107]. Based on the primary organ, affected cancers are classified as ovarian, uterine, cervical, vulval and vaginal. The high prevalence of these cancers is likely to be related to the great plasticity of these tissues, their regenerative potential and thus probably their high cell turnover as well. On top of this, openness to the external environment and exposure to pathogens makes them potentially vulnerable to the transformation. The latter has been demonstrated by the dramatic correlation between HPV infection cervical cancers, since nearly 100% of patients are HPV positive [108].

#### **9. Serous ovarian cancer and stem cells of the fallopian tube**

**8. Cancer stem cells and the putative link to adult stem cells**

lines in 2D in 3D [106].

154 Adult Stem Cell Niches

Parallel to the discovery of adult stem cells in healthy tissues, cancer research has produced a bulk of evidence showing that most malignancies are not homogenous cell masses but rather heterogeneous tissues whose progression depends on the fitness of a distinct cellular fraction: cancer stem cells (CSCs). CSCs can confer resistance to chemotherapeutic agents or exhibit other characteristics that provide a competitive advantage to the cancer and ensure its progression [96-99]. In many cases, long-term prognosis and patient survival can be correlated with the frequency of cancer stem cells in the tumor at the time of diagnosis [100]. CSCs exhibit the characteristics of adult stem cells: self renewal and differentiation capacity. They can be distinguished from the bulk of the tumor tissue as the only fraction that is able to generate new tumors when transplanted into immunocompromised mice [101,102]. For this reason, they are sometimes referred to as tumor initiating cells. This property of cancer stem cells has been demonstrated for numerous malignancies, including ovarian and endometrial cancer. As with markers of adult stem cells, markers specifically associated with cancer stem cells are also proving difficult to identify. It is of course possible that there is no unique cancer stem cell for ovarian, endometrial or other genital malignancies and that stemness can be achieved by alternative routes in individual cases, thereby resulting in different combinations of surface markers such as CD44, EpCAM, ALDH1, CD117, CD133 etc, which have been identified by different studies [81,103,104]. Cancer stem cells are intermixed with the bulk of the tumor tissue, but are found enriched in advanced stages of metastasis, in spheres present in effusions recovered from ascites [105]. Spheres exhibit anchorage-independent growth but can efficient‐ ly adhere to mesothelial cells in the peritoneum via integrin 1, which may play a role in metastatic spread. It is not yet known what triggers formation of spheres, although the physiological implications for disease progression and response to treatment are immense. The compact organization of the cells in a cluster makes drug delivery ineffective, either due to the difficulty of drugs to penetrate, or induction of a quiescent state in cells at the core through tight cell-cell contacts, making them insensitive to agents targeting actively replicating cells. Either way, it is clear that 3D organization represents another layer of protection for cancer cells from chemotherapy. This effect has been demonstrated by comparing the response to the standard therapeutic drugs cisplatin and paclitaxel for 11 different ovarian cancer cell

Importantly, however, it remains unknown if and how cancer stem cells are related to normal adult stem cells. Cancer stem cells may develop from adult stem cells by losing dependence on niche regulatory factors, or they may simply be the progeny of differentiated cells that have acquired "stemness" characteristics during the accumulation of mutations and cellular transformation. It is conceivable that both mechanisms could occur in all or some types of cancer. If there is indeed a causal relationship between dysregulation of the adult stem cell niche and carcinogenesis, this may open up new possibilities for early diagnosis and timely intervention. Since the research field of cancer stem cells is currently offering a variety of sometimes competing models of what defines this population in endometrial, cervical and serous ovarian cancer, we focus our attention on the current understanding of carcinogenesis in the genital tract. Understanding of the cellular events that lead to initial transformation From the perspective of patient care and long-term prognosis, one of the biggest challenges for medicine represents high grade serous ovarian cancer, an aggressive form of epithelial ovarian cancer which has a survival rate of under 40 % after 10 years [109]. This deadly malignancy takes more than 14,000 lives per year in the US alone and no improvement can be expected in the foreseeable future, due to the absence of early screening methods and the aggressive nature of the cancer. The origin of high-grade serous ovarian cancer (HG-SOC) has puzzled medical doctors and pathologists for decades. Naturally, the site of carcinogenesis was initially attributed to OSE cells [110]. Numerous examples of developmental genetics show that relatively complex changes in phenotype are frequently induced by expression of only one master regulator gene. In the case of OSE cells, ectopic expression of homeobox transcrip‐ tion factor HOX9 in the xenograft mouse model causes Müllerean metaplasia-transformation of cuboidal to papillary columnar epithelium resembling fallopian tube mucosa and serous ovarian cancer [111]. However, there is as yet no clinical evidence that this conversion also occurs in vivo. The scarcity of precancerous lesions or early stage carcinoma in situ detected by pathologies [112], raises questions whether this hypothesis is supported by clinical data. In parallel, the hypothesis that the etiology of HG-SOC could be explained by malignant transformation in neighboring tubal epithelium has gained increasing support. The fallopian tube develops from the Müllerian duct tissue, encompassing a columnar epithelial monolayer, which continues to express PAX8, the main cellular marker of "Müllerian differentiation" – a phenotype which is reminiscent of cancer tissue from HG-SOC patients. A breakthrough came from a cohort of clinical studies in BRCA mutation carriers, who have a very high hereditary risk of developing ovarian and breast cancer later in life. Small malignancies within the tubal epithelium (so-called serous tubal intraepithelial carcinoma – STIC) were discovered in up to 10% of patients who underwent prophylactic surgery to remove both fallopian tubes and ovaries [113]. These apparently healthy patients thus already had cancer in their fallopian tubes, which had not yet metastasized to the ovary. Samples from HG-SOC patients subse‐ quently confirmed that STICS were present in up to 60% of cases [114]. Although not definitive proof, these findings strongly support the hypothesis that the fallopian tube epithelium is the tissue of origin of serous ovarian cancer. Recently, malignant transformation of the fallopian epithelium into full-blown ovarian cancer has been successfully triggered in a transgenic mouse model [115]. Here, the Cre recombinase system was used under control of the PAX8 promoter to introduce mutations into Brca1/2, Tp53 and Pten, which are frequently found mutated in HG-SOC. The animals developed malignant disease with a HG-SOC phenotype, which metastasized to the ovary, peritoneum or liver – modes of spread that are also found in patients, and were for a long time considered as evidence for separate origins of the disease. Regardless of the important function that BRCA1/2-mediated DNA repair mechanisms have in fallopian tube cells, in order to solve the complex problem of HGSC etiology it is necessary to understand the role of the p53 tumor suppressor. p53 is mutated in nearly all HG-SOC patients, but somatic mutations of p53 on their own do not increase the likelihood of HGSC development [116]. Nevertheless, nuclear accumulation of p53 is much more frequent in tubes where STICs are also found, arguing that this phenotype is perhaps characteristic of a cellular state which is prone to transformation upon further "hits". However, a valid causal relation‐ ship between these cellular atypias and the appearance of fully transformed malignant serous tubal intraepithelial carcinoma (STICs) is not yet conclusively proven. Therefore, true prema‐ lignant lesions in the fallopian tube as precursors of HG-SOC are yet to be defined.

tissue, or indeed the role played by adult stem cells. Approximately 75-80% of endometrial cancers are classified as type 1-endometrioid endometrial cancer (EEC) – with the remaining cases belonging to papillary, mucinous or clear cell types. Histologically, they consist of malignant endometrial cells in the columnar monolayer, although squamous metaplasia is sometimes observed [117]. They frequently harbor alterations in PTEN and Wnt signaling pathways, and a prevalent staining pattern of nuclear catenin localization is found in up to 60% of endometrial hyperplasias and 30% of endometrial cancers [118]. In a mouse model with constitutively active Wnt signaling due to APC deletion in the genital tract, loss of PTEN function was found to be the rate-limiting step of carcinogenesis inside the uterus, while activation of β-catenin signaling increased the severity of the disease [119]. Strikingly, a followup study of the mice that were initially declared tumor free by inspection of the uterus revealed that 62% did get sendometrioid cancer, but not in the uterus, as would be expected, but rather in the neighboring distal oviduct [120]. Thus, this recent study argues that the fallopian tube may participate not only in the carcinogenesis HG-SOC but also in a subset of endometrial cancers. Notably, in human patients loss of PTEN associates with better prognosis and reduced risk of metastasis, since the tumor appears more differentiated [121]. These at first sight contradictory facts might be explained by the different requirements of cancer cells in the early and late stages of progression. While PTEN is a tumor suppressor, its major downstream effector, p-Akt, has dual functions in tumor cells. In mammary carcinoma, activated Akt enhances carcinogenesis via the ErbB-2 pathway, but inhibits invasion of the tumor and its metastasis [122]. Of course, mutations in the PTEN-PI3 kinase pathway components represent only one of the important signaling routes that have been found to be altered in endometrial cancer. Similarly, as in ovarian cancer, it remains to be seen how discrete cytological changes progress into full-blown cancer and which cells give rise to the cancer stem cells that are detected in the later stages of malignant disease. A recent study, based on a detailed analysis of 113 cases of endometrial cancer, postulated an important role for SALL4 protein which is known as a strong determinator of pluripotency in human embryonic cells [123]. The authors found SALL4 expressed in 47% of cancer samples while no expression was detected either in healthy controls or in the hyperplastic tissue, and increased levels of expression negatively influenced prognosis and patient survival. SALL4 increases c-Myc transcriptional activity and reduces the response of affected cells to carboplatin treatment. While it cannot be excluded that SALL4 expression in precancerous stages is limited to very low levels in sparse stem cells below detection limit of whole tissue sample, these results strongly indicate that pluripotent capacity of the tumor is acquired at later stages of disease development. The existence of cells with stem cell characteristics has recently also been demonstrated for cervical cancer. Sox2 expressing cells constitute a small percentage of cells in cervical cancer cell lines, but they show much greater tumor forming capacity in the xenograft mouse model than the remaining cells [124]. This correlates well with findings of tracing experiments from the Sox2 mouse model mentioned above, which identified stem cell lineages in the cervix [125]. It is becoming increasingly clear that the basal layer of cervical epithelium plays a decisive role in the initiation of cervical cancer, as these are the only dividing cells in squamous stratified epithelium. Active progression of the cell cycle is a prerequisite for HPV-driven cellular transformation, as host cells have to pass through the prophase of mitosis for transcription of viral genes to be initiated

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An understanding of the molecular mechanisms of epithelial homeostasis and renewal in healthy fallopian tube tissue is thus likely to be essential for successful resolution of the molecular events that lead to serous ovarian cancer. Of particular interest in this context is how slow-cycling adult stem cells in the distal fallopian tube respond to long-term changes in the tissue microenvironment from numerous different stimuli such as inflammation, genotoxic stress changes in extracellular matrix, or the presence of pathogens. The role of the microen‐ viroment via cooperation of different cellular compartments and extracellular matrix not only provides conditions for physiologically regulated responses, such are repair and healing, but can also decisively influence the progression of disease. As discussed in the section on cancer stem cells, cancer tissue is heterogeneous and keeping in mind the monoclonal origin of cancers, it is unclear whether differentiated cells are subject to transformation followed by expansion, whether some of them are reprogrammed into cancer stem cells, or whether deregulated stem cells are the source of the malignancy after additional somatic mutations. The latter hypothesis is somewhat more likely, given the presence of cells with stem cell characteristics in tumors, and that the alternative would mean acquisition of pluripotency. However, at this stage reverse reprogramming of differentiated cells cannot be excluded as the underlying mechanism.

The same is true for endometrial cancer. In contrast to the complete absence of diagnostic tools for early detection of ovarian cancer, there is a detailed classification of neoplastic changes in the endometrium – known as endometrial intraepithelial neoplasia (EIN), although there is a lot of controversy among pathologists how reliable the existing methodology is as a prognostic factor. Moreover, there is still no consensus regarding the cellular origin of the malignant tissue, or indeed the role played by adult stem cells. Approximately 75-80% of endometrial cancers are classified as type 1-endometrioid endometrial cancer (EEC) – with the remaining cases belonging to papillary, mucinous or clear cell types. Histologically, they consist of malignant endometrial cells in the columnar monolayer, although squamous metaplasia is sometimes observed [117]. They frequently harbor alterations in PTEN and Wnt signaling pathways, and a prevalent staining pattern of nuclear catenin localization is found in up to 60% of endometrial hyperplasias and 30% of endometrial cancers [118]. In a mouse model with constitutively active Wnt signaling due to APC deletion in the genital tract, loss of PTEN function was found to be the rate-limiting step of carcinogenesis inside the uterus, while activation of β-catenin signaling increased the severity of the disease [119]. Strikingly, a followup study of the mice that were initially declared tumor free by inspection of the uterus revealed that 62% did get sendometrioid cancer, but not in the uterus, as would be expected, but rather in the neighboring distal oviduct [120]. Thus, this recent study argues that the fallopian tube may participate not only in the carcinogenesis HG-SOC but also in a subset of endometrial cancers. Notably, in human patients loss of PTEN associates with better prognosis and reduced risk of metastasis, since the tumor appears more differentiated [121]. These at first sight contradictory facts might be explained by the different requirements of cancer cells in the early and late stages of progression. While PTEN is a tumor suppressor, its major downstream effector, p-Akt, has dual functions in tumor cells. In mammary carcinoma, activated Akt enhances carcinogenesis via the ErbB-2 pathway, but inhibits invasion of the tumor and its metastasis [122]. Of course, mutations in the PTEN-PI3 kinase pathway components represent only one of the important signaling routes that have been found to be altered in endometrial cancer. Similarly, as in ovarian cancer, it remains to be seen how discrete cytological changes progress into full-blown cancer and which cells give rise to the cancer stem cells that are detected in the later stages of malignant disease. A recent study, based on a detailed analysis of 113 cases of endometrial cancer, postulated an important role for SALL4 protein which is known as a strong determinator of pluripotency in human embryonic cells [123]. The authors found SALL4 expressed in 47% of cancer samples while no expression was detected either in healthy controls or in the hyperplastic tissue, and increased levels of expression negatively influenced prognosis and patient survival. SALL4 increases c-Myc transcriptional activity and reduces the response of affected cells to carboplatin treatment. While it cannot be excluded that SALL4 expression in precancerous stages is limited to very low levels in sparse stem cells below detection limit of whole tissue sample, these results strongly indicate that pluripotent capacity of the tumor is acquired at later stages of disease development. The existence of cells with stem cell characteristics has recently also been demonstrated for cervical cancer. Sox2 expressing cells constitute a small percentage of cells in cervical cancer cell lines, but they show much greater tumor forming capacity in the xenograft mouse model than the remaining cells [124]. This correlates well with findings of tracing experiments from the Sox2 mouse model mentioned above, which identified stem cell lineages in the cervix [125]. It is becoming increasingly clear that the basal layer of cervical epithelium plays a decisive role in the initiation of cervical cancer, as these are the only dividing cells in squamous stratified epithelium. Active progression of the cell cycle is a prerequisite for HPV-driven cellular transformation, as host cells have to pass through the prophase of mitosis for transcription of viral genes to be initiated

tubes, which had not yet metastasized to the ovary. Samples from HG-SOC patients subse‐ quently confirmed that STICS were present in up to 60% of cases [114]. Although not definitive proof, these findings strongly support the hypothesis that the fallopian tube epithelium is the tissue of origin of serous ovarian cancer. Recently, malignant transformation of the fallopian epithelium into full-blown ovarian cancer has been successfully triggered in a transgenic mouse model [115]. Here, the Cre recombinase system was used under control of the PAX8 promoter to introduce mutations into Brca1/2, Tp53 and Pten, which are frequently found mutated in HG-SOC. The animals developed malignant disease with a HG-SOC phenotype, which metastasized to the ovary, peritoneum or liver – modes of spread that are also found in patients, and were for a long time considered as evidence for separate origins of the disease. Regardless of the important function that BRCA1/2-mediated DNA repair mechanisms have in fallopian tube cells, in order to solve the complex problem of HGSC etiology it is necessary to understand the role of the p53 tumor suppressor. p53 is mutated in nearly all HG-SOC patients, but somatic mutations of p53 on their own do not increase the likelihood of HGSC development [116]. Nevertheless, nuclear accumulation of p53 is much more frequent in tubes where STICs are also found, arguing that this phenotype is perhaps characteristic of a cellular state which is prone to transformation upon further "hits". However, a valid causal relation‐ ship between these cellular atypias and the appearance of fully transformed malignant serous tubal intraepithelial carcinoma (STICs) is not yet conclusively proven. Therefore, true prema‐

lignant lesions in the fallopian tube as precursors of HG-SOC are yet to be defined.

the underlying mechanism.

156 Adult Stem Cell Niches

An understanding of the molecular mechanisms of epithelial homeostasis and renewal in healthy fallopian tube tissue is thus likely to be essential for successful resolution of the molecular events that lead to serous ovarian cancer. Of particular interest in this context is how slow-cycling adult stem cells in the distal fallopian tube respond to long-term changes in the tissue microenvironment from numerous different stimuli such as inflammation, genotoxic stress changes in extracellular matrix, or the presence of pathogens. The role of the microen‐ viroment via cooperation of different cellular compartments and extracellular matrix not only provides conditions for physiologically regulated responses, such are repair and healing, but can also decisively influence the progression of disease. As discussed in the section on cancer stem cells, cancer tissue is heterogeneous and keeping in mind the monoclonal origin of cancers, it is unclear whether differentiated cells are subject to transformation followed by expansion, whether some of them are reprogrammed into cancer stem cells, or whether deregulated stem cells are the source of the malignancy after additional somatic mutations. The latter hypothesis is somewhat more likely, given the presence of cells with stem cell characteristics in tumors, and that the alternative would mean acquisition of pluripotency. However, at this stage reverse reprogramming of differentiated cells cannot be excluded as

The same is true for endometrial cancer. In contrast to the complete absence of diagnostic tools for early detection of ovarian cancer, there is a detailed classification of neoplastic changes in the endometrium – known as endometrial intraepithelial neoplasia (EIN), although there is a lot of controversy among pathologists how reliable the existing methodology is as a prognostic factor. Moreover, there is still no consensus regarding the cellular origin of the malignant and infection established [126]. Cervical cancer develops from premalignant lesions called cervical intraepithelial neoplasia (CIN), which are routinely detected by regular Pap smears. Importantly, CINs occur almost exclusively within the transformation zone of the cervix, a region of metaplastic conversion of columnar to squamous epithelium. Regular screening has greatly improved the long-term prognosis and survival rates of cervical cancer [127].

treatment and diagnosis of female reproductive cancers and endometriosis can be achieved. As we have outlined, the high activity of adult stem cells in the female reproductive tract that is required to maintain cyclical changes in tissue architecture put these tissues at a particularly high risk for accumulating mutations with the potential for transformation. This is likely to be exacerbated by the fact that the genital system is exposed to a variety of sexually transmitted pathogens. There is mounting evidence that these infections may play a role in initiation of malignancies in the ovary, uterus or as co-factors to HPV in the cervix [18-20]. Although deciphering the behavior of adult stem cells in disease remains a very challenging research area, a dynamic field of translational approaches has emerged, using stem cells as a source of healthy tissue in different models of in vitro differentiation and transplantation. There is justified optimism that such models will help to elucidate not only the events involved in deregulation of the adult stem cell niche, but also the interaction of human pathogens with this niche, as well as the somatic tissue derived from it. A more complete understanding of these events will provide a basis for the development of more effective preventative and therapeutic

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measures for diseases of the female reproductive tract.

, Rike Zietlow and Thomas F. Meyer

Max Planck Institute for Infection Biology, Department of Molecular Biology, Berlin, Germany

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within the female reproductive epithelium. Cell Cycle 12: 2888-2898.

\*Address all correspondence to: kessler@mpiib-berlin.mpg.de

**Author details**

Mirjana Kessler\*

**References**

238-242.

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The mounting evidence for an involvement of the fallopian tube in development of ovarian and potentially other cancers thus highlights the importance of developing methodologies to improve diagnostic sampling and visualization of this organ. In particular, there is an imper‐ ative to improve diagnostics of the tube in patients. Ideally, some kind of endoscopy would enable detailed exploration of the mucosa in vivo, including the taking of biopsies. This would improve our understanding of phenotypical changes that take place in the epithelium, better define and categorize alterations and discover real premalignant lesions or early malignancies. The result would hopefully be comparable to the advancement that routine colonoscopy brought to early diagnosis and proper management of colon cancer.

#### **10. Conclusion**

We have presented in detail the current "state of the art" in the field of adult stem cells of the female reproductive system, their role in maintaining healthy mucosal tissue and changes that occur during disease. Moreover, we have focused on the phenomenon of cancer stem cells, which appears to be very important for the progression of malignancies by giving tumor tissue a competitive advantage. Still, the question remains how adult stem cells relate to cancer stem cells, and the models which have been postulated so far, as in the case of cervical cancer, have yet to be experimentally proven.

Regardless of the final outcome, it is almost certain that adult stem cells play an important role in the process of cancer initiation. Carcinogenesis is considered to be a long, stepwise process of accumulation of mutations. Therefore, differentiated cells with short life spans are unlikely to pass on mutations unless the initial acquired change leads to immortalization. Adult stem cells on the other hand, are long-lived and thereby inevitably accumulate mutations over decades. With each asymmetric division, mutations are passed to the differentiating progeny. The final "transformation step" may occur afterwards in the differentiated cell, or the stem cell itself may reach the stage where it becomes cancerous. The latter model presumes that as tumor tissue grows, the stem cell continues to give rise to differentiated progeny, while becoming itself a cancer stem cell. In both cases, a definitive premalignant molecular fingerprint should be found in the original adult stem cell. Therefore, it will be of great importance to further characterize adult stem cells in the ovary, fallopian tube and uterus and elucidate the molecular mechanisms of epithelial renewal in healthy tissue, but also to determine the genomic changes which occur over time in response to altered hormonal stimulation, tissue injury or infection.

Recent studies with novel genetic lineage tracing tools in the mouse give a promising outlook that concrete molecular pathways controlling stemness will be defined in the near future. Only when this goal has been achieved is it conceivable that a major breakthrough in the early treatment and diagnosis of female reproductive cancers and endometriosis can be achieved. As we have outlined, the high activity of adult stem cells in the female reproductive tract that is required to maintain cyclical changes in tissue architecture put these tissues at a particularly high risk for accumulating mutations with the potential for transformation. This is likely to be exacerbated by the fact that the genital system is exposed to a variety of sexually transmitted pathogens. There is mounting evidence that these infections may play a role in initiation of malignancies in the ovary, uterus or as co-factors to HPV in the cervix [18-20]. Although deciphering the behavior of adult stem cells in disease remains a very challenging research area, a dynamic field of translational approaches has emerged, using stem cells as a source of healthy tissue in different models of in vitro differentiation and transplantation. There is justified optimism that such models will help to elucidate not only the events involved in deregulation of the adult stem cell niche, but also the interaction of human pathogens with this niche, as well as the somatic tissue derived from it. A more complete understanding of these events will provide a basis for the development of more effective preventative and therapeutic measures for diseases of the female reproductive tract.

#### **Author details**

and infection established [126]. Cervical cancer develops from premalignant lesions called cervical intraepithelial neoplasia (CIN), which are routinely detected by regular Pap smears. Importantly, CINs occur almost exclusively within the transformation zone of the cervix, a region of metaplastic conversion of columnar to squamous epithelium. Regular screening has

The mounting evidence for an involvement of the fallopian tube in development of ovarian and potentially other cancers thus highlights the importance of developing methodologies to improve diagnostic sampling and visualization of this organ. In particular, there is an imper‐ ative to improve diagnostics of the tube in patients. Ideally, some kind of endoscopy would enable detailed exploration of the mucosa in vivo, including the taking of biopsies. This would improve our understanding of phenotypical changes that take place in the epithelium, better define and categorize alterations and discover real premalignant lesions or early malignancies. The result would hopefully be comparable to the advancement that routine colonoscopy

We have presented in detail the current "state of the art" in the field of adult stem cells of the female reproductive system, their role in maintaining healthy mucosal tissue and changes that occur during disease. Moreover, we have focused on the phenomenon of cancer stem cells, which appears to be very important for the progression of malignancies by giving tumor tissue a competitive advantage. Still, the question remains how adult stem cells relate to cancer stem cells, and the models which have been postulated so far, as in the case of cervical cancer, have

Regardless of the final outcome, it is almost certain that adult stem cells play an important role in the process of cancer initiation. Carcinogenesis is considered to be a long, stepwise process of accumulation of mutations. Therefore, differentiated cells with short life spans are unlikely to pass on mutations unless the initial acquired change leads to immortalization. Adult stem cells on the other hand, are long-lived and thereby inevitably accumulate mutations over decades. With each asymmetric division, mutations are passed to the differentiating progeny. The final "transformation step" may occur afterwards in the differentiated cell, or the stem cell itself may reach the stage where it becomes cancerous. The latter model presumes that as tumor tissue grows, the stem cell continues to give rise to differentiated progeny, while becoming itself a cancer stem cell. In both cases, a definitive premalignant molecular fingerprint should be found in the original adult stem cell. Therefore, it will be of great importance to further characterize adult stem cells in the ovary, fallopian tube and uterus and elucidate the molecular mechanisms of epithelial renewal in healthy tissue, but also to determine the genomic changes which occur over time in response to altered hormonal stimulation, tissue injury or infection. Recent studies with novel genetic lineage tracing tools in the mouse give a promising outlook that concrete molecular pathways controlling stemness will be defined in the near future. Only when this goal has been achieved is it conceivable that a major breakthrough in the early

greatly improved the long-term prognosis and survival rates of cervical cancer [127].

brought to early diagnosis and proper management of colon cancer.

**10. Conclusion**

158 Adult Stem Cell Niches

yet to be experimentally proven.

Mirjana Kessler\* , Rike Zietlow and Thomas F. Meyer

\*Address all correspondence to: kessler@mpiib-berlin.mpg.de

Max Planck Institute for Infection Biology, Department of Molecular Biology, Berlin, Germany

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**Section 3**

**Hepatic Stem Cell Niches**

## **Hepatic Stem Cell Niches**

**Chapter 7**

**Adult Hepatic Progenitor Cells**

Additional information is available at the end of the chapter

The liver is the largest internal organ of the body only second in size compared to the skin. The liver not only functions as an endocrine and exocrine organ, but it also performs a multitude of vital functions including glycogen storage, detoxification and plasma protein synthesis [1–4]. The liver receives nutrients and environmental toxins from the digestive tract through the portal vein. This direct transport of potentially harmful agents is hypothesized to have exerted an evolutionary pressure on the liver to possess multiple pathways for regener‐ ation [4,5]. In fact, the regenerative capacity of the liver is so enormous that this was renowned in ancient times and described in Mediterranean folklore. According to Greek mythology the Titan Prometheus stole fire from the Gods of Olympia and gave it to the mortals. As a consequence, Zeus, the king of Gods, chained Prometheus to a rock. An eagle would then appear each day and pecked out part of Prometheus' liver only to let it regenerate overnight [1]. This punishment was to be repeated for eternity, but according to one version of the story,

Despite of the famed renewal capacity of the liver, hepatic diseases constitute a worldwide problem. Hepatic diseases can broadly be divided into two major groups: acute and chronic liver diseases. Acute liver failure is characterized by the manifestation of sudden severe hepatic injury that can have several etiologies [6,7]. Frequent causes included viral hepatitis or drug intoxication, commonly paracetamol, leading to hepatic encephalopathy, coagulopathy and often progressive multiorgan failure [6,8–11]. In developed countries, acute liver failure is relatively rare with an incidence estimated between 1-6 cases per million people each year [6, 12]. In contrast, chronic liver diseases are caused by prolonged insults. Common causes include sustained alcohol consumption, non-alcoholic fatty liver disease and hepatitis B or C virus infection [2]. These insults can lead to hepatic fibrosis, a form of wound healing characterized by the presence of collagen-rich septae connecting the so-called portal areas. If untreated, this potentially reversible manifestation, can progress to end stage cirrhosis, where hepatic

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Heracles (Hercules) eventually killed the eagle and freed Prometheus.

Peter Siig Vestentoft

http://dx.doi.org/10.5772/58814

**1. Introduction**

**Chapter 7**

## **Adult Hepatic Progenitor Cells**

Peter Siig Vestentoft

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58814

#### **1. Introduction**

The liver is the largest internal organ of the body only second in size compared to the skin. The liver not only functions as an endocrine and exocrine organ, but it also performs a multitude of vital functions including glycogen storage, detoxification and plasma protein synthesis [1–4]. The liver receives nutrients and environmental toxins from the digestive tract through the portal vein. This direct transport of potentially harmful agents is hypothesized to have exerted an evolutionary pressure on the liver to possess multiple pathways for regener‐ ation [4,5]. In fact, the regenerative capacity of the liver is so enormous that this was renowned in ancient times and described in Mediterranean folklore. According to Greek mythology the Titan Prometheus stole fire from the Gods of Olympia and gave it to the mortals. As a consequence, Zeus, the king of Gods, chained Prometheus to a rock. An eagle would then appear each day and pecked out part of Prometheus' liver only to let it regenerate overnight [1]. This punishment was to be repeated for eternity, but according to one version of the story, Heracles (Hercules) eventually killed the eagle and freed Prometheus.

Despite of the famed renewal capacity of the liver, hepatic diseases constitute a worldwide problem. Hepatic diseases can broadly be divided into two major groups: acute and chronic liver diseases. Acute liver failure is characterized by the manifestation of sudden severe hepatic injury that can have several etiologies [6,7]. Frequent causes included viral hepatitis or drug intoxication, commonly paracetamol, leading to hepatic encephalopathy, coagulopathy and often progressive multiorgan failure [6,8–11]. In developed countries, acute liver failure is relatively rare with an incidence estimated between 1-6 cases per million people each year [6, 12]. In contrast, chronic liver diseases are caused by prolonged insults. Common causes include sustained alcohol consumption, non-alcoholic fatty liver disease and hepatitis B or C virus infection [2]. These insults can lead to hepatic fibrosis, a form of wound healing characterized by the presence of collagen-rich septae connecting the so-called portal areas. If untreated, this potentially reversible manifestation, can progress to end stage cirrhosis, where hepatic

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

architecture is greatly disturbed and scar tissue encircles nodules of remaining hepatocytes [7,13–17]. Chronic liver diseases are estimated to affect 170 million patients worldwide. Those cases eventually progress to fibrosis and possibly cirrhosis in 25-30 % of these patients [2]. Where acute hepatic failure involves sudden massive cell death, chronic liver diseases are conversely characterized by continuous cell death [18–20].

perform either symmetric or asymmetric cell division [30]. Symmetric stem cell division give rise to two daughter cells that themselves are stem cells, thereby maintaining the stem cell pool. Alternatively, this form for division may result in two daughter cells committed for differentiation. Asymmetric stem cell division, on the other hand, produces one stem cell and one differentiated daughter cell. Differentiated daughter cells are also known as progenitor cells or transit amplifying cells. They divide rapidly in order to generate a pool of continually more differentiated cells en route to replace senescent or damaged tissue cells [34]. Early progenitor cells hold multi-lineage potential and have characteristics similar to the parent stem cell whereas late progenitor cells are more differentiated and produce single-lineage progeny [32]. Therefore, even though stem cells have a high self-renewal capacity, they may divide relatively infrequently, whereas the transit amplifying cells greatly increase in number and

The potency of stem cells requires tight regulation of their behavior. Stem cell quiescence and activation must be regulated according to the needs of the organism. A critical actor in mediating the balanced response of stem cells to the needs of the organism is the stem cell

The stem cell niche concept was first proposed by Schofield who conducted bone marrow studies [35]. It was suggested that stem cells reside in compartments that promote and maintain their characteristics [35]. It is believed that once postnatal tissues are formed, intra-organ stem cells reside in these special tissue microenvironments or niches. However, upon activation, the niche must change the composition of its microenvironment from favoring stem cell quiescence to induce stem cell activation and proliferation. Studies on *Drosophila spp*. gonads have helped understanding the factors constituting the stem cell niche and greatly expanded knowledge of stem cell activation and the generation of transit amplifying cells [36]. These studies have revealed a basal theme to reoccur. Structurally, the typical stem cell niche consists of stem cells resting on a scaffold of extracellular matrix components, having cell-cell interactions with differentiated neighboring cells [36–38]. In *Drosophila spp.* gonads, the extracellular matrix forms a repressing environment to stem cell differentiation, while promoting cellular adhesion [36]. Immediately outside this repressive zone, stem cell adherence is reduced while cellular differentiation is stimulated [36]. More specifically, integrins have been identified as key elements in this adhesion process. These transmembrane proteins that mediate adhesion to the extracellular matrix, are often highly expressed in stem cells and can suppress terminal differentiation in epidermal stem cells, for instance [39,40]. Conversely, the loss of integrins is associated with the epidermal stem cell niche disappearance, characterized by cellular

The key factor to identify stem cell niches is the stem cell localization itself. For this pur‐

thymidine analog BrdU. Upon asymmetric cellular division, stem cells may incorporate either

H-thymidine and the

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 175

pose,label-retention assays may be applied, two common labels being 3

differentiate into given tissue cells.

**3. Stem cell niche**

differentiation [30].

niche.

When hepatic regeneration is hindered orthotopic liver transplantation is the only treatment that radically improves the outcome of hepatic failure [2,21]. However, the worldwide shortage of liver donors result in death of many patients waiting for transplantation [22]. Research into alternative methods of therapeutic treatment is therefore highly needed. The possibility of culturing hepatic stem cells holds the promise to treat certain liver diseases, even with autologous stem cells. This include correcting metabolic diseases characterized by inherited defects of hepatic enzymes or treating fulminant hepatic failure characterized by rapid onset of liver failure and death, when donor organs are unavailable [23]. The use of autologous stem cells would additionally prevent the lifespan administration of immunosuppressive agents currently employed to prevent allograft rejection. Therefore, there has been an increasing interest into using hepatic stem cell-based therapies as novel alternatives to traditional liver treatments. However, the stem cell biology of the liver is not well understood. In particular, the lack of specific markers for hepatic stem cell identification has hindered their characteri‐ zation and isolation [24–28].

The present chapter will provide an overview of current knowledge of the rodent and human hepatic stem cell niche.

In particular, the chapter will go through the development of the hepatic stem cell niche, the associated extracellular matrix molecules and support cells. Attention will also be given to the various modes of hepatic regeneration and the involvement of hepatic stem cells in cancerous disease states.

#### **2. Stem cells**

Even though stem cells have been identified and characterized in several organs, no universally accepted definition of what constitutes a stem cell has been defined [29]. However, a broadly accepted view is that stem cells are cells that hold a capacity for unlimited or prolonged selfrenewal and can also give rise to at least one type of highly differentiated progeny [30]. However, many classes of stem cell exist with different potentials. These range from the totipotent fertilized egg from which entire organisms develop over pluripotent embryonic stem cells that can give rise to the three germ layers to the unipotent tissue stem cells.

Typically, tissue or intra-organ stem cells are less differentiated cells that exist in a mitotically quiescent form [31]. This class of stem cells are so-called "determined", meaning that they lack markers of final differentiation, but are able to divide and differentiate into highly specialized effector cells [32,33]. When needed tissue stem cells are activated to divide and clonally regenerate the tissue in which they are located [32,34]. Upon activation, tissue stem cells would perform either symmetric or asymmetric cell division [30]. Symmetric stem cell division give rise to two daughter cells that themselves are stem cells, thereby maintaining the stem cell pool. Alternatively, this form for division may result in two daughter cells committed for differentiation. Asymmetric stem cell division, on the other hand, produces one stem cell and one differentiated daughter cell. Differentiated daughter cells are also known as progenitor cells or transit amplifying cells. They divide rapidly in order to generate a pool of continually more differentiated cells en route to replace senescent or damaged tissue cells [34]. Early progenitor cells hold multi-lineage potential and have characteristics similar to the parent stem cell whereas late progenitor cells are more differentiated and produce single-lineage progeny [32]. Therefore, even though stem cells have a high self-renewal capacity, they may divide relatively infrequently, whereas the transit amplifying cells greatly increase in number and differentiate into given tissue cells.

#### **3. Stem cell niche**

architecture is greatly disturbed and scar tissue encircles nodules of remaining hepatocytes [7,13–17]. Chronic liver diseases are estimated to affect 170 million patients worldwide. Those cases eventually progress to fibrosis and possibly cirrhosis in 25-30 % of these patients [2]. Where acute hepatic failure involves sudden massive cell death, chronic liver diseases are

When hepatic regeneration is hindered orthotopic liver transplantation is the only treatment that radically improves the outcome of hepatic failure [2,21]. However, the worldwide shortage of liver donors result in death of many patients waiting for transplantation [22]. Research into alternative methods of therapeutic treatment is therefore highly needed. The possibility of culturing hepatic stem cells holds the promise to treat certain liver diseases, even with autologous stem cells. This include correcting metabolic diseases characterized by inherited defects of hepatic enzymes or treating fulminant hepatic failure characterized by rapid onset of liver failure and death, when donor organs are unavailable [23]. The use of autologous stem cells would additionally prevent the lifespan administration of immunosuppressive agents currently employed to prevent allograft rejection. Therefore, there has been an increasing interest into using hepatic stem cell-based therapies as novel alternatives to traditional liver treatments. However, the stem cell biology of the liver is not well understood. In particular, the lack of specific markers for hepatic stem cell identification has hindered their characteri‐

The present chapter will provide an overview of current knowledge of the rodent and human

In particular, the chapter will go through the development of the hepatic stem cell niche, the associated extracellular matrix molecules and support cells. Attention will also be given to the various modes of hepatic regeneration and the involvement of hepatic stem cells in cancerous

Even though stem cells have been identified and characterized in several organs, no universally accepted definition of what constitutes a stem cell has been defined [29]. However, a broadly accepted view is that stem cells are cells that hold a capacity for unlimited or prolonged selfrenewal and can also give rise to at least one type of highly differentiated progeny [30]. However, many classes of stem cell exist with different potentials. These range from the totipotent fertilized egg from which entire organisms develop over pluripotent embryonic

Typically, tissue or intra-organ stem cells are less differentiated cells that exist in a mitotically quiescent form [31]. This class of stem cells are so-called "determined", meaning that they lack markers of final differentiation, but are able to divide and differentiate into highly specialized effector cells [32,33]. When needed tissue stem cells are activated to divide and clonally regenerate the tissue in which they are located [32,34]. Upon activation, tissue stem cells would

stem cells that can give rise to the three germ layers to the unipotent tissue stem cells.

conversely characterized by continuous cell death [18–20].

zation and isolation [24–28].

hepatic stem cell niche.

disease states.

174 Adult Stem Cell Niches

**2. Stem cells**

The potency of stem cells requires tight regulation of their behavior. Stem cell quiescence and activation must be regulated according to the needs of the organism. A critical actor in mediating the balanced response of stem cells to the needs of the organism is the stem cell niche.

The stem cell niche concept was first proposed by Schofield who conducted bone marrow studies [35]. It was suggested that stem cells reside in compartments that promote and maintain their characteristics [35]. It is believed that once postnatal tissues are formed, intra-organ stem cells reside in these special tissue microenvironments or niches. However, upon activation, the niche must change the composition of its microenvironment from favoring stem cell quiescence to induce stem cell activation and proliferation. Studies on *Drosophila spp*. gonads have helped understanding the factors constituting the stem cell niche and greatly expanded knowledge of stem cell activation and the generation of transit amplifying cells [36]. These studies have revealed a basal theme to reoccur. Structurally, the typical stem cell niche consists of stem cells resting on a scaffold of extracellular matrix components, having cell-cell interactions with differentiated neighboring cells [36–38]. In *Drosophila spp.* gonads, the extracellular matrix forms a repressing environment to stem cell differentiation, while promoting cellular adhesion [36]. Immediately outside this repressive zone, stem cell adherence is reduced while cellular differentiation is stimulated [36]. More specifically, integrins have been identified as key elements in this adhesion process. These transmembrane proteins that mediate adhesion to the extracellular matrix, are often highly expressed in stem cells and can suppress terminal differentiation in epidermal stem cells, for instance [39,40]. Conversely, the loss of integrins is associated with the epidermal stem cell niche disappearance, characterized by cellular differentiation [30].

The key factor to identify stem cell niches is the stem cell localization itself. For this pur‐ pose,label-retention assays may be applied, two common labels being 3 H-thymidine and the thymidine analog BrdU. Upon asymmetric cellular division, stem cells may incorporate either of these labels into their DNA thereby retaining 50% of the label with the resulting daughter stem cell and 50% with the transit amplifying cell. As transit amplifying cells are fast cycling the label is gradually diluted in the following chase period while the slow cycling daughter stem cell retain the marker. Use of these and similar label-retaining assays have been employed to identify the stem cell niche of the skin, hair follicle and peripheral cornea [41–45]. Though being an intriguing method for locating stem cells, label-retention techniques has certain disadvantages. Stem cells that did not enter the cell cycle during the labelling period will for instance remain unmarked, while progenitor cells that terminally differentiate and stop cell division can retain markers for longer periods of time [46]. Stem cells and their niches have, never the less, been identified in several organs. In vertebrates these include the bulge region of the hair follicle, the bone marrow and the lover region of the crypts in the small intestine [41,47–49]. The common denominator of these organs, however, is that they are characterized by a continuous supply of cells descending from the stem cells. Stem cells in tissues charac‐ terized by a lower cellular turnover are, on the contrary, more difficult to identify. One such organ is the liver, where mitotically quiescent hepatocytes have relatively long life spans and high proliferative capacities [50,51].

#### **4. Hepatic anatomy**

Although many cell types are present, the liver is characterized by two epithelial tissue components; cholangiocytes and hepatic cords containing hepatocytes, respectively. The hepatocytes secrete serum proteins, including albumin, and express monooxygenases from the cytochrome P450 family, the major enzymes involved in oxidative metabolism of xenobi‐ otics [52]. Cholangiocytes, on the other hand, form biliary channels transporting bile from the liver towards the bile bladder.

and water between sinusoidal blood and hepatocytes [57]. The sinusoidal wall is additionally separated from the hepatocytes by a lumen termed the space of Disse. The predominant view is that blood drain from the portal vein and portal artery branches and blends in the sinusoids from where it drains into the central vein [54,55]. Lymph, on the other hand, is thought to be generated by filtration of sinusoidal blood into the space of Disse from where it flows towards lymphatic vessels located in the portal tracts [54]. Bile canaliculi are narrow spaces formed from the apical membranes of adjacent hepatocytes in the hepatic cords [54]. Bile originating from the bile canaliculi is transported towards terminal bile ducts in the portal tracts through

**Figure 1.** A. Cartoon of a stylized hepatic lobule. Each hexagonal corner of the hepatic lobule is marked by a portal area containing a portal vein, a portal artery and a bile duct. A central vein mark the center of the lobule. B. Hematoxy‐ lin and eosin staining of a tissue section from adult normal human liver. A portal area containing a portal vein, portal

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 177

Non-parenchymal cell types also present in the liver include stellate cells and Kupffer cells. Hepatic stellate cells, also known as Ito cells, are starshaped and contain lipid droplets with vast amounts of vitamin A [58]. In normal liver they are located to the space of Disse which is suggested to constitute the hepatic stellate cell niche [59]. Kupffer cells, on the other hand, are hepatic macrophages involved in the phagocytosis of cellular debris, extracellular matrix

The liver in an endodermal derived organ with hepatocytes and cholangiocytes originating from a common progenitor termed "hepatoblast" or "primitive hepatocyte" [61]. Development of the liver goes through sequential stages including induction, specification, proliferation and maturation steps. The endoderm is important for inducing development of the neighboring cardiogenic mesoderm followed by maturation of the heart. Embryonic development of the liver is initiated in the ventral part of the anterior endoderm, whereas pancreas coordinately

the canal of Hering [54,56].

artery and bile duct is discernible. Magnification x100.

**5. Liver development**

components and release of inflammatory factors [60].

Examination of hepatic tissue sections reveal an unvarying landscape of cords of hepatocytes with scattered central veins and so-called portal triads or portal tracts. The latter contain bile ducts and branches from the portal vein and portal artery, thereby forming a triad [53]. However, from a three-dimensional perspective, this dull landscape masks a highly complex organ [53,54]. Accurately defining the livers functional entities have historically been difficult, as multiple functions could be applied based on either enzymatic expression patterns or histological observations. A frequently used definition is the simple histological unit "lobule" (figure 1). The classic lobule is envisioned as a two-dimensional hexagonal structure centered around a central vein [55]. Each hexagonal corner contain a portal tract and cords of hepato‐ cytes extend from the hepatocytic limiting plate at the periportal space, towards the central vein [55]. The terminal segments of the biliary system in the portal tracts connect directly with the hepatic cords through a specialized structure known as the Canal of Hering – thought to constitute the hepatic progenitor cell niche [56]. Canals of Hering are formed partly by biliary cells and partly by hepatocytes near the limiting plate [56]. Hepatic cords are separated from each other by a special form of blood vessels called sinusoids [55]. The sinusoids are lined by endothelial cells with open pores, or fenestrae, lacking a diaphragm and a basal lamina [57]. The resulting high endothelial permeability facilitate the exchange of macromolecules, solutes

**Figure 1.** A. Cartoon of a stylized hepatic lobule. Each hexagonal corner of the hepatic lobule is marked by a portal area containing a portal vein, a portal artery and a bile duct. A central vein mark the center of the lobule. B. Hematoxy‐ lin and eosin staining of a tissue section from adult normal human liver. A portal area containing a portal vein, portal artery and bile duct is discernible. Magnification x100.

and water between sinusoidal blood and hepatocytes [57]. The sinusoidal wall is additionally separated from the hepatocytes by a lumen termed the space of Disse. The predominant view is that blood drain from the portal vein and portal artery branches and blends in the sinusoids from where it drains into the central vein [54,55]. Lymph, on the other hand, is thought to be generated by filtration of sinusoidal blood into the space of Disse from where it flows towards lymphatic vessels located in the portal tracts [54]. Bile canaliculi are narrow spaces formed from the apical membranes of adjacent hepatocytes in the hepatic cords [54]. Bile originating from the bile canaliculi is transported towards terminal bile ducts in the portal tracts through the canal of Hering [54,56].

Non-parenchymal cell types also present in the liver include stellate cells and Kupffer cells. Hepatic stellate cells, also known as Ito cells, are starshaped and contain lipid droplets with vast amounts of vitamin A [58]. In normal liver they are located to the space of Disse which is suggested to constitute the hepatic stellate cell niche [59]. Kupffer cells, on the other hand, are hepatic macrophages involved in the phagocytosis of cellular debris, extracellular matrix components and release of inflammatory factors [60].

#### **5. Liver development**

of these labels into their DNA thereby retaining 50% of the label with the resulting daughter stem cell and 50% with the transit amplifying cell. As transit amplifying cells are fast cycling the label is gradually diluted in the following chase period while the slow cycling daughter stem cell retain the marker. Use of these and similar label-retaining assays have been employed to identify the stem cell niche of the skin, hair follicle and peripheral cornea [41–45]. Though being an intriguing method for locating stem cells, label-retention techniques has certain disadvantages. Stem cells that did not enter the cell cycle during the labelling period will for instance remain unmarked, while progenitor cells that terminally differentiate and stop cell division can retain markers for longer periods of time [46]. Stem cells and their niches have, never the less, been identified in several organs. In vertebrates these include the bulge region of the hair follicle, the bone marrow and the lover region of the crypts in the small intestine [41,47–49]. The common denominator of these organs, however, is that they are characterized by a continuous supply of cells descending from the stem cells. Stem cells in tissues charac‐ terized by a lower cellular turnover are, on the contrary, more difficult to identify. One such organ is the liver, where mitotically quiescent hepatocytes have relatively long life spans and

Although many cell types are present, the liver is characterized by two epithelial tissue components; cholangiocytes and hepatic cords containing hepatocytes, respectively. The hepatocytes secrete serum proteins, including albumin, and express monooxygenases from the cytochrome P450 family, the major enzymes involved in oxidative metabolism of xenobi‐ otics [52]. Cholangiocytes, on the other hand, form biliary channels transporting bile from the

Examination of hepatic tissue sections reveal an unvarying landscape of cords of hepatocytes with scattered central veins and so-called portal triads or portal tracts. The latter contain bile ducts and branches from the portal vein and portal artery, thereby forming a triad [53]. However, from a three-dimensional perspective, this dull landscape masks a highly complex organ [53,54]. Accurately defining the livers functional entities have historically been difficult, as multiple functions could be applied based on either enzymatic expression patterns or histological observations. A frequently used definition is the simple histological unit "lobule" (figure 1). The classic lobule is envisioned as a two-dimensional hexagonal structure centered around a central vein [55]. Each hexagonal corner contain a portal tract and cords of hepato‐ cytes extend from the hepatocytic limiting plate at the periportal space, towards the central vein [55]. The terminal segments of the biliary system in the portal tracts connect directly with the hepatic cords through a specialized structure known as the Canal of Hering – thought to constitute the hepatic progenitor cell niche [56]. Canals of Hering are formed partly by biliary cells and partly by hepatocytes near the limiting plate [56]. Hepatic cords are separated from each other by a special form of blood vessels called sinusoids [55]. The sinusoids are lined by endothelial cells with open pores, or fenestrae, lacking a diaphragm and a basal lamina [57]. The resulting high endothelial permeability facilitate the exchange of macromolecules, solutes

high proliferative capacities [50,51].

**4. Hepatic anatomy**

176 Adult Stem Cell Niches

liver towards the bile bladder.

The liver in an endodermal derived organ with hepatocytes and cholangiocytes originating from a common progenitor termed "hepatoblast" or "primitive hepatocyte" [61]. Development of the liver goes through sequential stages including induction, specification, proliferation and maturation steps. The endoderm is important for inducing development of the neighboring cardiogenic mesoderm followed by maturation of the heart. Embryonic development of the liver is initiated in the ventral part of the anterior endoderm, whereas pancreas coordinately develops from the dorsal part. Within a short period of time, in a so-called "window of opportunity" around embryonic day (E) 8.5-11.5 in mouse endoderm, the anterior endoderm is competent for activation of a hepatic development gene program [62]. At the time of hepatic induction, the adjacent mesenchymal tissue, comprising the cardiogenic mesoderm and septum transversum, produces subtypes of growth factors: fibroblast growth factor (FGF) and bone morphogenetic protein (BMP), respectively [63]. Growth factors acid FGF, basic FGF, FGF4, BMP 2, and BMP4 initiate a hepatic gene expression program, while FGF or cardiogenic mesoderm suppresses the pancreatic gene expression program [62]. In the absence of BMPs or FGFs, the pancreatic gene expression program is initiated while the hepatic gene program is suppressed [62].

stem cell compartment [81]. SOX9 is expressed in the hepatic diverticulum but disappears during the endodermal invasion of the septum transversum. At E11.5, however, SOX9 is reexpressed in hepatoblasts located near the developing portal veins [82]. These prospective cholangiocytes lining the mesenchyme surrounding developing portal veins form a singlelayered ring at E14.5 termed the "ductal plate". Studies on cells isolated from the ductal plate and from adult livers have shed important information on this structure. Cells from adult livers and the ductal plate, positive for epithelial cell adhesion molecule (EpCAM) and CK19 and negative for AFP can give rise to both the hepatic and biliary lineages, when injected into immunodeficient NOD/SCID mice [83,84]. The ductal plate is therefore not only suggested to constitute the pre-and perinatal hepatic progenitor cell niche, but also to be directly antecedent

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 179

The ductal plate, which can be envisioned as a biliary sleeve, increase expression of CK8, 18 and 19 relative to the remaining parenchymal cells [86,87]. Through a unique mode of tubulogenesis the cholangiocytes induce neighboring hepatoblast to differentiate into cholan‐ giocytes themselves thereby developing a two-layered transiently asymmetric ductal plate around E16.5 [79,82]. Focal lumina appear between the mesenchymal and parenchymal ductal plate facing layers, thereby giving rise to early bile ducts at E16.5 [79]. In the following remodeling phase, these primitive bile ducts migrate into the portal mesenchyme in a complex process timely coordinated with the formation of hepatic portal arteries [65]. The parts of the ductal plate, which are not involved in bile duct formation, possibly regress as a result of apoptosis [88]. As of a result, the intrahepatic bile ducts loose contact with the ductal plate and become fully embedded in the portal mesenchyme in the remodeled stage. However, the intrahepatic bile duct system is still immature until several weeks after birth and remnants of the ductal plate can be identified, in particular, at the smaller vein branches [86,89]. As a final step in the maturation process, developing cholangiocytes initiate expression of CK7, a marker of adult bile duct cells [53,86]. The outlined development of the intrahepatic bile duct system is initiated at the hepatic hilum from where it gradually progresses towards the periphery of

The wide range of important metabolic functions performed by the liver and its proximity to ingested environmental toxins are hypothesized to have imparted the livers tremendous

Hepatocytes are the main component of liver and therefore, the most vulnerable to damage. The generation of adult hepatocytes, under non-pathogenic conditions, has been widely disputed. In normal liver, parenchymal turnover is slow with hepatocyte lifespans estimated 150 to 450 days in rat [50,51,91,92]. With a turnover rate of normal liver cells of approximately 1 in 20,000-40,000 at any given time the entire liver is estimated to be replaced by normal tissue at least once a year [93]. As hepatocytes supposedly are terminally differentiated cells, they were once hypothesized only to possess the capacity for one or two cell divisions. A number

to the canal of Hering, the presumed adult hepatic progenitor cell niche [83–85].

the liver, where the smaller portal branches reside [89].

**6. Hepatic tissue homeostasis**

capacity for adaptation and regeneration [3,90].

Following induction of hepatic gene expression, the endodermal cells adopt a columnar appearance at E8.5 and express albumin. At E9.5 a thickening of the endoderm is observed, interceded by primitive endothelial cells from the septum transversum [62,64]. This prospec‐ tive liver, termed the hepatic diverticulum or "liver bud", is visible in the human embryo at the 17 somite stage, corresponding to 3 weeks and 5 days post conception [53,64]. Signaling molecules, including BMPs, hepatocyte growth factor (Hgf) and vascular endothelial growth factor receptor 2 (Vegfr-2) from the septum transversum and endothelial cells induce prolif‐ eration and migration of hepatoblast positive for cytokeratin (CK19), Hepatocyte Paraffin 1 (HepPar1), α-fetoprotein (AFP) and albumin, into the adjacent septum transversum [62,65– 69]. At E11 the hepatoblasts addionationally stain for the intermediate filament proteins CK8, CK14 and CK18 [70–72]. Concurrent with the hepatoblast invasion the endothelial cells coalesce around spaces in the septum transversum thereby forming anastomosing primitive blood vessels around which hepatoblast are situated. The endodermal invasion displaces the septum transversum that eventually form the liver capsule, mesenchyme and possibly the hepatic stellate cells [62,68,69,73,74].

At E14 (in mouse) hematopoietic cells colonize the liver, making it a prenatal site for hemato‐ poiesis. Concomitantly, hepatoblasts express markers of both the hepatocytic and cholangio‐ cytic lineages and are capable of differentiating into either of the two epithelial cell types. The hepatoblasts, however, gradually commit to either the hepatocytic or cholangiocytic lineages. Three transcriptions factors, Hepatocyte Nuclear Factor (HNF)-4α, HNF-6 and HNF-1β, are found to be particularly important in this process. Microarray data have demonstrated that HNF-4α bind approximately half of the active genes in liver and is essential for determination toward a hepatocytic fate [75,76]. On the other hand, HNF-6 and HNF-1β are essential for development of the biliary lineage. Knockout mice for HNF6 and its downstream target HNF1β, develop no gallbladder and display abnormal development of the intrahepatic and extrahepatic bile ducts [77,78]. Around E16 (mouse) the hepatoblast are commited to either the hepatocytic or cholangiocytic lineages and are thereby no longer bipotential [62,79,80].

During development of the liver, morphogenesis of the biliary tree is also said to proceed through a series of developmental stages. These are categorized as the ductal plate, remodeling bile duct and remodeled bile duct stages [79]. The earliest indicator of biliary development comes from studies of the transcription factor SRY-related HMG box transcription factor 9 (SOX9). SOX9 is essential for the formation of certain stem cell niches, such as the hair follicle stem cell compartment [81]. SOX9 is expressed in the hepatic diverticulum but disappears during the endodermal invasion of the septum transversum. At E11.5, however, SOX9 is reexpressed in hepatoblasts located near the developing portal veins [82]. These prospective cholangiocytes lining the mesenchyme surrounding developing portal veins form a singlelayered ring at E14.5 termed the "ductal plate". Studies on cells isolated from the ductal plate and from adult livers have shed important information on this structure. Cells from adult livers and the ductal plate, positive for epithelial cell adhesion molecule (EpCAM) and CK19 and negative for AFP can give rise to both the hepatic and biliary lineages, when injected into immunodeficient NOD/SCID mice [83,84]. The ductal plate is therefore not only suggested to constitute the pre-and perinatal hepatic progenitor cell niche, but also to be directly antecedent to the canal of Hering, the presumed adult hepatic progenitor cell niche [83–85].

The ductal plate, which can be envisioned as a biliary sleeve, increase expression of CK8, 18 and 19 relative to the remaining parenchymal cells [86,87]. Through a unique mode of tubulogenesis the cholangiocytes induce neighboring hepatoblast to differentiate into cholan‐ giocytes themselves thereby developing a two-layered transiently asymmetric ductal plate around E16.5 [79,82]. Focal lumina appear between the mesenchymal and parenchymal ductal plate facing layers, thereby giving rise to early bile ducts at E16.5 [79]. In the following remodeling phase, these primitive bile ducts migrate into the portal mesenchyme in a complex process timely coordinated with the formation of hepatic portal arteries [65]. The parts of the ductal plate, which are not involved in bile duct formation, possibly regress as a result of apoptosis [88]. As of a result, the intrahepatic bile ducts loose contact with the ductal plate and become fully embedded in the portal mesenchyme in the remodeled stage. However, the intrahepatic bile duct system is still immature until several weeks after birth and remnants of the ductal plate can be identified, in particular, at the smaller vein branches [86,89]. As a final step in the maturation process, developing cholangiocytes initiate expression of CK7, a marker of adult bile duct cells [53,86]. The outlined development of the intrahepatic bile duct system is initiated at the hepatic hilum from where it gradually progresses towards the periphery of the liver, where the smaller portal branches reside [89].

#### **6. Hepatic tissue homeostasis**

develops from the dorsal part. Within a short period of time, in a so-called "window of opportunity" around embryonic day (E) 8.5-11.5 in mouse endoderm, the anterior endoderm is competent for activation of a hepatic development gene program [62]. At the time of hepatic induction, the adjacent mesenchymal tissue, comprising the cardiogenic mesoderm and septum transversum, produces subtypes of growth factors: fibroblast growth factor (FGF) and bone morphogenetic protein (BMP), respectively [63]. Growth factors acid FGF, basic FGF, FGF4, BMP 2, and BMP4 initiate a hepatic gene expression program, while FGF or cardiogenic mesoderm suppresses the pancreatic gene expression program [62]. In the absence of BMPs or FGFs, the pancreatic gene expression program is initiated while the hepatic gene program

Following induction of hepatic gene expression, the endodermal cells adopt a columnar appearance at E8.5 and express albumin. At E9.5 a thickening of the endoderm is observed, interceded by primitive endothelial cells from the septum transversum [62,64]. This prospec‐ tive liver, termed the hepatic diverticulum or "liver bud", is visible in the human embryo at the 17 somite stage, corresponding to 3 weeks and 5 days post conception [53,64]. Signaling molecules, including BMPs, hepatocyte growth factor (Hgf) and vascular endothelial growth factor receptor 2 (Vegfr-2) from the septum transversum and endothelial cells induce prolif‐ eration and migration of hepatoblast positive for cytokeratin (CK19), Hepatocyte Paraffin 1 (HepPar1), α-fetoprotein (AFP) and albumin, into the adjacent septum transversum [62,65– 69]. At E11 the hepatoblasts addionationally stain for the intermediate filament proteins CK8, CK14 and CK18 [70–72]. Concurrent with the hepatoblast invasion the endothelial cells coalesce around spaces in the septum transversum thereby forming anastomosing primitive blood vessels around which hepatoblast are situated. The endodermal invasion displaces the septum transversum that eventually form the liver capsule, mesenchyme and possibly the

At E14 (in mouse) hematopoietic cells colonize the liver, making it a prenatal site for hemato‐ poiesis. Concomitantly, hepatoblasts express markers of both the hepatocytic and cholangio‐ cytic lineages and are capable of differentiating into either of the two epithelial cell types. The hepatoblasts, however, gradually commit to either the hepatocytic or cholangiocytic lineages. Three transcriptions factors, Hepatocyte Nuclear Factor (HNF)-4α, HNF-6 and HNF-1β, are found to be particularly important in this process. Microarray data have demonstrated that HNF-4α bind approximately half of the active genes in liver and is essential for determination toward a hepatocytic fate [75,76]. On the other hand, HNF-6 and HNF-1β are essential for development of the biliary lineage. Knockout mice for HNF6 and its downstream target HNF1β, develop no gallbladder and display abnormal development of the intrahepatic and extrahepatic bile ducts [77,78]. Around E16 (mouse) the hepatoblast are commited to either the hepatocytic or cholangiocytic lineages and are thereby no longer bipotential [62,79,80].

During development of the liver, morphogenesis of the biliary tree is also said to proceed through a series of developmental stages. These are categorized as the ductal plate, remodeling bile duct and remodeled bile duct stages [79]. The earliest indicator of biliary development comes from studies of the transcription factor SRY-related HMG box transcription factor 9 (SOX9). SOX9 is essential for the formation of certain stem cell niches, such as the hair follicle

is suppressed [62].

178 Adult Stem Cell Niches

hepatic stellate cells [62,68,69,73,74].

The wide range of important metabolic functions performed by the liver and its proximity to ingested environmental toxins are hypothesized to have imparted the livers tremendous capacity for adaptation and regeneration [3,90].

Hepatocytes are the main component of liver and therefore, the most vulnerable to damage. The generation of adult hepatocytes, under non-pathogenic conditions, has been widely disputed. In normal liver, parenchymal turnover is slow with hepatocyte lifespans estimated 150 to 450 days in rat [50,51,91,92]. With a turnover rate of normal liver cells of approximately 1 in 20,000-40,000 at any given time the entire liver is estimated to be replaced by normal tissue at least once a year [93]. As hepatocytes supposedly are terminally differentiated cells, they were once hypothesized only to possess the capacity for one or two cell divisions. A number of studies of label-retaining markers of cells based on the incorporation of markers such as tritiated thymidine into DNA in rats or lack of markers such as cytochrome c in humans have located proliferative hepatocytes in the periportal region [94,95]. Cell tracking has illustrated a gradual invasion of these recognizable cells from the portal tract towards the terminal central vein. Based on these and similar experiments the "streaming liver" hypothesis was suggested in which mitotically active hepatocytes at the limiting plate in the periportal region continu‐ ously provided hepatocytic offspring. In a unidirectional fashion, these hepatocytes are hypothesized to stream along the sinusoids as they gradually change enzymatic expression and eventually replace dead hepatocytes in the perivenous region [94]. However, this model is still quite controversal. Long-term labelling of hepatocytes with beta-galactosidase, an enzyme capable of converting X-gal into an insoluble blue compound, found positive clusters of hepatocytes, ergo cells that had divided, throughout the liver lobule thereby contradicting the streaming liver hypothesis [51].

system and in the intestinal crypts [42,44,49,102–105]. These organs are, unlike the liver, generally under constant renewal and require frequent stem cell division for tissue replenish‐ ment. Stem cells in these organs are therefore fulltime committed to perform stem cell function. However, stem cells in tissues with low turnover have been notoriously difficult to detect. As with arrangements in other stem cell niches the hepatic progenitor cell niche is thought to be structurally composed of a stem or progenitor cell population situated on a basal lamina and in contact with surrounding support cells [36,38]. As cellular turnover in the liver is already low and hepatic homeostasis and regeneration to a large extent is completed by differentiated parenchymal and non-parenchymal cells stem cells in this organ have been difficult to

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 181

While hepatocytes can conceptually be considered as the livers functional stem cells, the contribution of hepatic stem or progenitor cells to liver regeneration has been debated. Clues to a possible existence of stem cells in the liver came from early studies conducted by Farber, Wilson and Leduc [106,107]. Following dietary administration of DL-ethionine or carcinogenic 2-acetylaminofluorene (2-AAF) to rats, Farber observed the presence of pseudoductular structures consisting of small cells near the hepatic portal areas in rat liver (figure 2). These small cells are termed "oval cells" due to their oval shaped nucleus and scant cytoplasm [106]. In a following study Wilson and Leduc examined murine livers following dietary adminis‐ tration of ethionine and bentonite [107]. The presence of small cholangioles apparently giving rise to both bile-duct cells and parenchymal cells suggested the presence or a population of reserve cells or stem cells [107]. In acute liver failure and chronic liver diseases similar so-called "ductular reactions" may be noted at the portal triad interface [7,108–110]. The ductular response is thought to result from proliferating progenitor cells and represent the livers "second tier of defense" or "level 2 response" [4]. These cells termed oval cells in rodents are named progenitor cells in humans as rodent hepatic injury models and human diseases may not be directly comparable. However, we will collectively refer to them as hepatic progenitor cells (HPCs). The resulting arborizing network of ductular structures sprouting from the portal

area is classified as an atypical ductular reaction due to a poorly defined lumen.

A number of rodent hepatic injury models have been developed to investigate various modes of regeneration and to mimic human hepatic diseases. Particularly notable models include partial hepatectomy, which induces proliferation of differentiated hepatocytes and cholan‐ giocytes and thereby represent the first tier of defense [18,111]. Ligation of the common bile duct (Bile Duct Ligation) obstructs bile flow from the liver (figure 2). This surgical technique mimics cholestasis and induces proliferation of hepatocytes and the larger bile ducts without signs of differentiation towards the hepatocytic lineage in the latter [112,113]. Several injury models specifically induce HPC responses and thereby the second tier of defense. For example administration of the choline-deficient ethionine-supplemented (CDE) diet or carcinogenic agents such as 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to rodents induces ductular reactions while carbon tetrachloride (CCl4) administration additionally result in advanced hepatic fibrosis [114–116]. In the 2-AAF/PHx model administration of 2-acetylaminofluorene to rats is followed by two-thirds partial hepatectomy. This procedure block hepatocyte differentiation, ergo the first tier of defense, while at the same time providing a strong stimulus

characterize.

The relative mitotic quiescence of hepatocytes and cholangiocytes mask their huge prolifera‐ tive potential. Resecting two-thirds of the liver in accordance with the partial hepatectomy protocol (PHx) leads to complete regrowth in approximately 10 days [1,3]. This regrowth is, however, not a true regeneration, given that it does not recreate original hepatic morphology but is compensatory hyperplasia in the residual liver lobes [55]. Even with this relatively harsh treatment of the liver, only 1-2 proliferative events of hepatic epithelial cells are needed to lead to complete compensatory regrowth with no or very little hepatic stem cell contribution [1,96]. Impressively, this procedure can be repeated at least 12 times in rats without regenerative failure or endangering liver function as hepatocytes maintain a fully differentiated state [97]. In an experimental animal model, mice deficient for the tyrosine catabolic enzyme, fumaryla‐ cetoacetate hydrolase (FAH), suffer from hepatocyte damage due to accumulation of fumar‐ ylacetoacetate and its precursor maleylacetoacetate [98]. However, wild-type hepatocytes are capable of rescuing this phenotype. In an elegant study serial transplantations of wild-type hepatocytes into FAH deficient mice repopulated 6 generations of livers corresponding to 69 cell doublings [98]. Therefore, during normal tissue homeostasis, hepatocytes could be regarded as the functional unipotent hepatic stem cell, capable of giving rise to more than 50 livers [1]. Furthermore, in some chronic biliary diseases such as primary biliary cirrhosis and primary sclerosing cholangitis, hepatocytes have even been observed differentiating into biliary cells [99,100]. Nonetheless, the replicative activity of even hepatocytes can apparently decrease in chronic hepatic injury in mice and advance cirrhosis in humans, possibly due to telomere shortening [101]. Regeneration through replication of hepatocytes and cholangio‐ cytes is also known as the "first tier of defense" or a "level 1 response" [4]. This form of response was responsible was regenerating Prometheus' liver during night as the eagle essentially conducted partial hepatectomy during the day.

#### **7. Localizing the hepatic progenitor cell niche**

Locating stem cells is the first step into characterizing their niche. Stem cells and their niches have been defined in several tissues, including the hair follicle and skin, the hematopoietic system and in the intestinal crypts [42,44,49,102–105]. These organs are, unlike the liver, generally under constant renewal and require frequent stem cell division for tissue replenish‐ ment. Stem cells in these organs are therefore fulltime committed to perform stem cell function. However, stem cells in tissues with low turnover have been notoriously difficult to detect. As with arrangements in other stem cell niches the hepatic progenitor cell niche is thought to be structurally composed of a stem or progenitor cell population situated on a basal lamina and in contact with surrounding support cells [36,38]. As cellular turnover in the liver is already low and hepatic homeostasis and regeneration to a large extent is completed by differentiated parenchymal and non-parenchymal cells stem cells in this organ have been difficult to characterize.

of studies of label-retaining markers of cells based on the incorporation of markers such as tritiated thymidine into DNA in rats or lack of markers such as cytochrome c in humans have located proliferative hepatocytes in the periportal region [94,95]. Cell tracking has illustrated a gradual invasion of these recognizable cells from the portal tract towards the terminal central vein. Based on these and similar experiments the "streaming liver" hypothesis was suggested in which mitotically active hepatocytes at the limiting plate in the periportal region continu‐ ously provided hepatocytic offspring. In a unidirectional fashion, these hepatocytes are hypothesized to stream along the sinusoids as they gradually change enzymatic expression and eventually replace dead hepatocytes in the perivenous region [94]. However, this model is still quite controversal. Long-term labelling of hepatocytes with beta-galactosidase, an enzyme capable of converting X-gal into an insoluble blue compound, found positive clusters of hepatocytes, ergo cells that had divided, throughout the liver lobule thereby contradicting

The relative mitotic quiescence of hepatocytes and cholangiocytes mask their huge prolifera‐ tive potential. Resecting two-thirds of the liver in accordance with the partial hepatectomy protocol (PHx) leads to complete regrowth in approximately 10 days [1,3]. This regrowth is, however, not a true regeneration, given that it does not recreate original hepatic morphology but is compensatory hyperplasia in the residual liver lobes [55]. Even with this relatively harsh treatment of the liver, only 1-2 proliferative events of hepatic epithelial cells are needed to lead to complete compensatory regrowth with no or very little hepatic stem cell contribution [1,96]. Impressively, this procedure can be repeated at least 12 times in rats without regenerative failure or endangering liver function as hepatocytes maintain a fully differentiated state [97]. In an experimental animal model, mice deficient for the tyrosine catabolic enzyme, fumaryla‐ cetoacetate hydrolase (FAH), suffer from hepatocyte damage due to accumulation of fumar‐ ylacetoacetate and its precursor maleylacetoacetate [98]. However, wild-type hepatocytes are capable of rescuing this phenotype. In an elegant study serial transplantations of wild-type hepatocytes into FAH deficient mice repopulated 6 generations of livers corresponding to 69 cell doublings [98]. Therefore, during normal tissue homeostasis, hepatocytes could be regarded as the functional unipotent hepatic stem cell, capable of giving rise to more than 50 livers [1]. Furthermore, in some chronic biliary diseases such as primary biliary cirrhosis and primary sclerosing cholangitis, hepatocytes have even been observed differentiating into biliary cells [99,100]. Nonetheless, the replicative activity of even hepatocytes can apparently decrease in chronic hepatic injury in mice and advance cirrhosis in humans, possibly due to telomere shortening [101]. Regeneration through replication of hepatocytes and cholangio‐ cytes is also known as the "first tier of defense" or a "level 1 response" [4]. This form of response was responsible was regenerating Prometheus' liver during night as the eagle essentially

Locating stem cells is the first step into characterizing their niche. Stem cells and their niches have been defined in several tissues, including the hair follicle and skin, the hematopoietic

the streaming liver hypothesis [51].

180 Adult Stem Cell Niches

conducted partial hepatectomy during the day.

**7. Localizing the hepatic progenitor cell niche**

While hepatocytes can conceptually be considered as the livers functional stem cells, the contribution of hepatic stem or progenitor cells to liver regeneration has been debated. Clues to a possible existence of stem cells in the liver came from early studies conducted by Farber, Wilson and Leduc [106,107]. Following dietary administration of DL-ethionine or carcinogenic 2-acetylaminofluorene (2-AAF) to rats, Farber observed the presence of pseudoductular structures consisting of small cells near the hepatic portal areas in rat liver (figure 2). These small cells are termed "oval cells" due to their oval shaped nucleus and scant cytoplasm [106]. In a following study Wilson and Leduc examined murine livers following dietary adminis‐ tration of ethionine and bentonite [107]. The presence of small cholangioles apparently giving rise to both bile-duct cells and parenchymal cells suggested the presence or a population of reserve cells or stem cells [107]. In acute liver failure and chronic liver diseases similar so-called "ductular reactions" may be noted at the portal triad interface [7,108–110]. The ductular response is thought to result from proliferating progenitor cells and represent the livers "second tier of defense" or "level 2 response" [4]. These cells termed oval cells in rodents are named progenitor cells in humans as rodent hepatic injury models and human diseases may not be directly comparable. However, we will collectively refer to them as hepatic progenitor cells (HPCs). The resulting arborizing network of ductular structures sprouting from the portal area is classified as an atypical ductular reaction due to a poorly defined lumen.

A number of rodent hepatic injury models have been developed to investigate various modes of regeneration and to mimic human hepatic diseases. Particularly notable models include partial hepatectomy, which induces proliferation of differentiated hepatocytes and cholan‐ giocytes and thereby represent the first tier of defense [18,111]. Ligation of the common bile duct (Bile Duct Ligation) obstructs bile flow from the liver (figure 2). This surgical technique mimics cholestasis and induces proliferation of hepatocytes and the larger bile ducts without signs of differentiation towards the hepatocytic lineage in the latter [112,113]. Several injury models specifically induce HPC responses and thereby the second tier of defense. For example administration of the choline-deficient ethionine-supplemented (CDE) diet or carcinogenic agents such as 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to rodents induces ductular reactions while carbon tetrachloride (CCl4) administration additionally result in advanced hepatic fibrosis [114–116]. In the 2-AAF/PHx model administration of 2-acetylaminofluorene to rats is followed by two-thirds partial hepatectomy. This procedure block hepatocyte differentiation, ergo the first tier of defense, while at the same time providing a strong stimulus

tor cell induction, but has no consequence on proliferation of larger bile ducts [113]. The anatomical location of the Canal of Hering, at the portal triad interface, makes this structure a prime candidate for the adult HPC compartment or "niche" [56,120–122]. Based on these experiments, the Canal of Hering represents, therefore, the HPC niche and the ductular reaction represents the activated HPC niche, respectively. However, the assumed stem cells located in the Canal of Hering may in fact not be "true" stem cells, but rather subpopula‐ tions of biliary or hepatocytic cells with increased stemness compared to other cells of their respective lineage [3]. Although progenitor cells morphologically resemble biliary cells, ductular reactions are phenotypically heterogenous [123]. In the ductular end connected to the biliary tree, the cells display cholangiocytic markers such as CK19, whereas the ductular end facing the parenchyma display hepatocytic markers including HepPar1 and the transcription factor HNF4 [119,123]. Between these extremes, hepatobiliary cells express‐ ing cholangiocytic and hepatocytic markers to various degrees are found [123,124]. It is now clear that the ductular response can be divided into several distinct phases that are evident in the 2-AAF/PHx protocol [117]. In the activation phase, on day 1, few proliferat‐ ing HPCs expressing CK19 are detectable in the biliary ductules. In the early prolifera‐ tion and migration phase on day 5 multiple CK19-positive HPCs can be observed whereas progenitor cell expression of delta-like 1 homolog (DLK1/Pref1) and AFP is rare. In the late proliferation and migration phase on day 9, arborizing ductular structures expand from the

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 183

Even though a number of HPC markers have been reported, none are specific for a pure population of hepatic stem cells [24,123,125,126]. What is more, only few of the reported HPC markers are expressed on the cellular surface and are therefore able to be employed for cellular isolation studies. CK19, OV-6 (an antibody recognizing a shared epitope between CK14 and CK19), EpCAM, CD24, hepatocyte growth factor activator inhibitor type 1 (HAI-1) and suppressor of tumorigenicity 14 (ST14) decorate both the intrahepatic bile ducts and the ductular reactions but only EpCAM, CD24, HAI-1 and ST14 are expressed on the surface [126–129]. AFP and DLK1, however, mark a subpopulation of HPCs suggesting the presence of an established hierarchy amongst the HPCs [129–131]. AFP and DLK1 are normally not expressed in the liver. However, both proteins are observed in hepatoblasts, the embryon‐ ic precursors to the cholangiocytic and hepatocytic lineages, suggesting that oval cells recapitulate a fetal phenotype when activated in hepatic injuries [72,132]. DLK1 is a transmembrane protein often described as an inhibitor of cellular differentiation and is expressed in less differentiated cells [133,134]. For example, forced expression of Dlk1 inhibits adipogenesis, whereas suppression promotes this process [133]. It is therefore conceivable that Dlk1 inhibits HPC differentiation thereby allowing transit amplifying cells to increase in numbers similar to the one observed in other stem cell niches [36]. With regard to AFP, elevated serum levels of this protein are associated with a favorable prognosis for patients with fulminant hepatic failure [135,136]. This observation supports the assumption that AFP marks cells capable of, at least, differentiating towards the

portal area with HPCs expressing CK19, Dlk1 and AFP proteins.

hepatocytic lineage.

**Figure 2.** A. Cartoon of typical constituents of the extracellular matrix. B, C, D. Microphotographic images and car‐ toons of livers from rats subjected to B) sham operation, C) bile duct ligation and D) the 2-AAF-PHx model. Cholangio‐ cytes and progenitor cells are stained for HAI-1 (green) and DLK1 (red). Cartoons in B, C and D portray part of portal areas with bile ducts (green) and their extracellular matrix in the portal mesenchyme bordering the limiting plate. B) In sham operated rat liver cholangiocytes are marked by HAI-1. C) In the bile duct ligation model in rats the larger bile ducts proliferate. D) In the 2-AAF-PHx model in rat liver a ductular reaction contain a subpopulation of hepatic pro‐ genitor cells positive for DLK1. Regardless of injury model extracellular matrix components escort the cholangiocytes. Upon exiting the hepatic progenitor cell niche the progenitor cells differentiate into hepatocytes. Microphotograph magnification x100. Adapted from Vestentoft et al. 2013 [129].

for growth. As a result, a ductular response is mounted (figure 2). Although these proliferating epithelial cells are collectively referred to as oval cells, it remains unclear if the oval cells resulting from different hepatic insults across different species have common characteristics as mice and rat respond differently to the same insults [117].

The ductular response is thought to represent proliferating progenitor cells. However, the origin of these progenitor cells is debated. Ductular reactions initiated, for example in the 2-AAF/PHx protocol, display both biliary and hepatocytic markers [26,118,119]. More‐ over, destruction of the biliary tree through administration of 4,4'-methylenedianiline (MDA) inhibits progenitor cell proliferation, suggesting that progenitor cells originate from the biliary lineage [120]. However, administration of dexamethasone diminishes progeni‐ tor cell induction, but has no consequence on proliferation of larger bile ducts [113]. The anatomical location of the Canal of Hering, at the portal triad interface, makes this structure a prime candidate for the adult HPC compartment or "niche" [56,120–122]. Based on these experiments, the Canal of Hering represents, therefore, the HPC niche and the ductular reaction represents the activated HPC niche, respectively. However, the assumed stem cells located in the Canal of Hering may in fact not be "true" stem cells, but rather subpopula‐ tions of biliary or hepatocytic cells with increased stemness compared to other cells of their respective lineage [3]. Although progenitor cells morphologically resemble biliary cells, ductular reactions are phenotypically heterogenous [123]. In the ductular end connected to the biliary tree, the cells display cholangiocytic markers such as CK19, whereas the ductular end facing the parenchyma display hepatocytic markers including HepPar1 and the transcription factor HNF4 [119,123]. Between these extremes, hepatobiliary cells express‐ ing cholangiocytic and hepatocytic markers to various degrees are found [123,124]. It is now clear that the ductular response can be divided into several distinct phases that are evident in the 2-AAF/PHx protocol [117]. In the activation phase, on day 1, few proliferat‐ ing HPCs expressing CK19 are detectable in the biliary ductules. In the early prolifera‐ tion and migration phase on day 5 multiple CK19-positive HPCs can be observed whereas progenitor cell expression of delta-like 1 homolog (DLK1/Pref1) and AFP is rare. In the late proliferation and migration phase on day 9, arborizing ductular structures expand from the portal area with HPCs expressing CK19, Dlk1 and AFP proteins.

Even though a number of HPC markers have been reported, none are specific for a pure population of hepatic stem cells [24,123,125,126]. What is more, only few of the reported HPC markers are expressed on the cellular surface and are therefore able to be employed for cellular isolation studies. CK19, OV-6 (an antibody recognizing a shared epitope between CK14 and CK19), EpCAM, CD24, hepatocyte growth factor activator inhibitor type 1 (HAI-1) and suppressor of tumorigenicity 14 (ST14) decorate both the intrahepatic bile ducts and the ductular reactions but only EpCAM, CD24, HAI-1 and ST14 are expressed on the surface [126–129]. AFP and DLK1, however, mark a subpopulation of HPCs suggesting the presence of an established hierarchy amongst the HPCs [129–131]. AFP and DLK1 are normally not expressed in the liver. However, both proteins are observed in hepatoblasts, the embryon‐ ic precursors to the cholangiocytic and hepatocytic lineages, suggesting that oval cells recapitulate a fetal phenotype when activated in hepatic injuries [72,132]. DLK1 is a transmembrane protein often described as an inhibitor of cellular differentiation and is expressed in less differentiated cells [133,134]. For example, forced expression of Dlk1 inhibits adipogenesis, whereas suppression promotes this process [133]. It is therefore conceivable that Dlk1 inhibits HPC differentiation thereby allowing transit amplifying cells to increase in numbers similar to the one observed in other stem cell niches [36]. With regard to AFP, elevated serum levels of this protein are associated with a favorable prognosis for patients with fulminant hepatic failure [135,136]. This observation supports the assumption that AFP marks cells capable of, at least, differentiating towards the hepatocytic lineage.

for growth. As a result, a ductular response is mounted (figure 2). Although these proliferating epithelial cells are collectively referred to as oval cells, it remains unclear if the oval cells resulting from different hepatic insults across different species have common characteristics

**Figure 2.** A. Cartoon of typical constituents of the extracellular matrix. B, C, D. Microphotographic images and car‐ toons of livers from rats subjected to B) sham operation, C) bile duct ligation and D) the 2-AAF-PHx model. Cholangio‐ cytes and progenitor cells are stained for HAI-1 (green) and DLK1 (red). Cartoons in B, C and D portray part of portal areas with bile ducts (green) and their extracellular matrix in the portal mesenchyme bordering the limiting plate. B) In sham operated rat liver cholangiocytes are marked by HAI-1. C) In the bile duct ligation model in rats the larger bile ducts proliferate. D) In the 2-AAF-PHx model in rat liver a ductular reaction contain a subpopulation of hepatic pro‐ genitor cells positive for DLK1. Regardless of injury model extracellular matrix components escort the cholangiocytes. Upon exiting the hepatic progenitor cell niche the progenitor cells differentiate into hepatocytes. Microphotograph

The ductular response is thought to represent proliferating progenitor cells. However, the origin of these progenitor cells is debated. Ductular reactions initiated, for example in the 2-AAF/PHx protocol, display both biliary and hepatocytic markers [26,118,119]. More‐ over, destruction of the biliary tree through administration of 4,4'-methylenedianiline (MDA) inhibits progenitor cell proliferation, suggesting that progenitor cells originate from the biliary lineage [120]. However, administration of dexamethasone diminishes progeni‐

as mice and rat respond differently to the same insults [117].

magnification x100. Adapted from Vestentoft et al. 2013 [129].

182 Adult Stem Cell Niches

#### **8. Support cells and the hepatic progenitor cell response**

Proliferation and morphogenesis of cholangiocytes and HPCs is a complex interplay between the biliary cells, surrounding support cells and the extracellular matrix. All of these compo‐ nents contribute to the HPC niche. Cell-cell interactions and cell-matrix interplays are likely to be important for regulating stem cell behavior within niches [37].

other hand, are resident hepatic macrophages. Kupffer cells are greatly activated in the CDE model of HPC response in mice. Before onset of HPC proliferation and parenchymal invasion activated Kupffer cells gradually shift from a more periportal location towards a more centrilobular location. Depletion of Kupffer cells through clodronate injections result in greatly reduced invasion of HPCs into the hepatic parenchyma. However, HPC proliferation is unaltered. In conclusion these data suggest that hepatic stellate cells are involved in the initiation, proliferation and termination of the HPC response, whereas Kupffer cells are needed

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 185

**9. Extracellular matrix components and the hepatic progenitor cell response**

Extracellular matrix can be defined as the complex molecular material surrounding cells and encompass both the basement membrane and the interstitial matrix [152]. Major components include the respective protein families of collagens, laminins, elastins, proteoglycans and

The extracellular matrix is a dynamic scaffold known to affect aspects of stem cell behav‐ ior such as morphology, growth and survival. A proportion of these responses are due to interactions between the extracellular matrix components and integrins, a family of dimeric extracellular matrix receptors that are linked to and transmit signals to the cytoskeleton [156,157]. The extracellular matrix may also contain growth factors which provide growth and morphogenic signals to nearby cells. Even physical features of the matrix, such as rigidity and geometry may influence cellular phenotype and behavior and has been shown to direct stem cell lineage specification [152,158–160]. Studies of *Drosophila spp.* stem cell niches have clarified that the microenvironment, as expected, may promote adherence to the niche and repress stem cell differentiation [36]. What is more, with age the molecular composition of the extracellular matrix change in an unfavorable direction for stem cell function and proliferation. This has been illustrated in experiments where stem cells transplanted from older mice, where stem cell self-renewal and differentiation has deterio‐ rated, to extracellular matrix from younger mice rejuvenate stem cell function to levels

Upon induction of the HPC response, the molecular composition of the HPC niche is thought to change in favor of promoting progenitor cell proliferation. Therefore, a key to understanding HPC biology and to characterize the HPC niche lies within unravelling the extracellular matrix composition of the niche. It is of particular interest to clarify which extracellular matrix molecules regulate the hepatic progenitor cell responses. A number of extracellular matrix molecules taking part in development of the intrahepatic bile ducts or in modulating the HPC response have been identified. Particularly, laminin and collagen I and IV are associated with these processes, but also other extracellular matrix components including tenascin, nidogen 1,

The family of collagen fibrils comprises 28 members, all with at least one triple helical domain and arranged in a rope-like fashion [163,164]. Collagens are deposited in the extracellular space

for HPC invasion into the hepatic parenchyma.

glycosaminoglycans (figure 2) [152–155].

comparable to that observed in younger mice [161,162].

agrin and fibronectin contribute.

Hepatic stellate cells possibly originate from the septum transversum-derived mesothelium lining the liver [137]. They are recognizable in their quiescent state by the expression of desmin and glial fibrillary acidic protein (GFAP), whereas they express alpha smooth muscle actin (α-SMA) when activated, often as a result of hepatic injury [138–141]. In the quiescent state they reside in the space of Disse, which constitute a laminin coated hepatic stellate cell niche, but when activated they give rise to contractile myofibroblast [59]. Both cell types are major producers of extracellular matrix components and activated hepatic stellate cells are the main source of matrix metalloproteinases and their inhibitors. However, so-called portal fibroblasts and vascular myofibroblasts can also transform into myofibroblasts thus giving rise to much confusion about the origins of the latter [142,143]. Additional confusion has been caused by misinterpretation of cellular markers. In particular, Thy-1, a cell surface protein initially suggested to mark oval cells, was later reclassified as a marker for hepatic myofibroblasts [144]

Hepatic stellate cells and myofibroblasts are greatly involved in the HPC response. In both the CDE model of HPC induction in mice, and the 2-AAF/PHx model in rat, hepatic stellate cell and myofibroblast response are invoked [121,145,146]. Hepatic stellate cells and myofibro‐ blasts not only intimately escort the HPC invasion into the parenchyma, but cellular processes from the hepatic stellate cells disrupt the HPC basal lamina and form direct cellular contact [121]. Such direct cell-cell interactions between hepatic stellate cells and liver epithelial cells has been shown to induce differentiation of the latter into a hepatocytic fate *in vitro* [147]. The HPC response is a regulated process undergoing several stages. Both initiation and termination is under tight regulation and hepatic stellate cells may be involved in these processes. HGF is a potent mitogen for hepatocytes whereas TGF-β is a strong inhibitor of their proliferation [148]. TGF-β is additionally identified as a partaker in maintaining quiescence of stem cells in other niches, such as the melanocyte stem cells located to the hair follicle bulge region [149]. Not only are hepatic stellate cells activated and induced to transform into myofibroblasts by TGF-β, but hepatic stellate cells themselves are major producers of this cytokine [148]. Conditioned media harvested from hepatic stellate cells in the early HPC response is rich in hepatocyte growth factor (HGF). This media promote HPC proliferation, possibly due to an override of the antiproliferative effect of TGF-β [150]. In the terminal phases of liver regener‐ ation hepatic stellate cells change cytokine expression profile and produce high levels of TGFβ which inhibits proliferation of hepatocytes [150]. Thus, hepatic stellate cells may be involved in both initiation and termination of the HPC response.

Other cells types involved in the HPC response are macrophages and Kupffer cells. Macro‐ phages can remodel the extracellular matrix, partly through the production of matrix metal‐ loproteinases [151]. As for hepatic stellate cells and myofibroblasts also bone marrow derived macrophages intimately associate with ductular reactions in rats [145]. Kupffer cells, on the other hand, are resident hepatic macrophages. Kupffer cells are greatly activated in the CDE model of HPC response in mice. Before onset of HPC proliferation and parenchymal invasion activated Kupffer cells gradually shift from a more periportal location towards a more centrilobular location. Depletion of Kupffer cells through clodronate injections result in greatly reduced invasion of HPCs into the hepatic parenchyma. However, HPC proliferation is unaltered. In conclusion these data suggest that hepatic stellate cells are involved in the initiation, proliferation and termination of the HPC response, whereas Kupffer cells are needed for HPC invasion into the hepatic parenchyma.

**8. Support cells and the hepatic progenitor cell response**

184 Adult Stem Cell Niches

to be important for regulating stem cell behavior within niches [37].

in both initiation and termination of the HPC response.

Other cells types involved in the HPC response are macrophages and Kupffer cells. Macro‐ phages can remodel the extracellular matrix, partly through the production of matrix metal‐ loproteinases [151]. As for hepatic stellate cells and myofibroblasts also bone marrow derived macrophages intimately associate with ductular reactions in rats [145]. Kupffer cells, on the

Proliferation and morphogenesis of cholangiocytes and HPCs is a complex interplay between the biliary cells, surrounding support cells and the extracellular matrix. All of these compo‐ nents contribute to the HPC niche. Cell-cell interactions and cell-matrix interplays are likely

Hepatic stellate cells possibly originate from the septum transversum-derived mesothelium lining the liver [137]. They are recognizable in their quiescent state by the expression of desmin and glial fibrillary acidic protein (GFAP), whereas they express alpha smooth muscle actin (α-SMA) when activated, often as a result of hepatic injury [138–141]. In the quiescent state they reside in the space of Disse, which constitute a laminin coated hepatic stellate cell niche, but when activated they give rise to contractile myofibroblast [59]. Both cell types are major producers of extracellular matrix components and activated hepatic stellate cells are the main source of matrix metalloproteinases and their inhibitors. However, so-called portal fibroblasts and vascular myofibroblasts can also transform into myofibroblasts thus giving rise to much confusion about the origins of the latter [142,143]. Additional confusion has been caused by misinterpretation of cellular markers. In particular, Thy-1, a cell surface protein initially suggested to mark oval cells, was later reclassified as a marker for hepatic myofibroblasts [144] Hepatic stellate cells and myofibroblasts are greatly involved in the HPC response. In both the CDE model of HPC induction in mice, and the 2-AAF/PHx model in rat, hepatic stellate cell and myofibroblast response are invoked [121,145,146]. Hepatic stellate cells and myofibro‐ blasts not only intimately escort the HPC invasion into the parenchyma, but cellular processes from the hepatic stellate cells disrupt the HPC basal lamina and form direct cellular contact [121]. Such direct cell-cell interactions between hepatic stellate cells and liver epithelial cells has been shown to induce differentiation of the latter into a hepatocytic fate *in vitro* [147]. The HPC response is a regulated process undergoing several stages. Both initiation and termination is under tight regulation and hepatic stellate cells may be involved in these processes. HGF is a potent mitogen for hepatocytes whereas TGF-β is a strong inhibitor of their proliferation [148]. TGF-β is additionally identified as a partaker in maintaining quiescence of stem cells in other niches, such as the melanocyte stem cells located to the hair follicle bulge region [149]. Not only are hepatic stellate cells activated and induced to transform into myofibroblasts by TGF-β, but hepatic stellate cells themselves are major producers of this cytokine [148]. Conditioned media harvested from hepatic stellate cells in the early HPC response is rich in hepatocyte growth factor (HGF). This media promote HPC proliferation, possibly due to an override of the antiproliferative effect of TGF-β [150]. In the terminal phases of liver regener‐ ation hepatic stellate cells change cytokine expression profile and produce high levels of TGFβ which inhibits proliferation of hepatocytes [150]. Thus, hepatic stellate cells may be involved

### **9. Extracellular matrix components and the hepatic progenitor cell response**

Extracellular matrix can be defined as the complex molecular material surrounding cells and encompass both the basement membrane and the interstitial matrix [152]. Major components include the respective protein families of collagens, laminins, elastins, proteoglycans and glycosaminoglycans (figure 2) [152–155].

The extracellular matrix is a dynamic scaffold known to affect aspects of stem cell behav‐ ior such as morphology, growth and survival. A proportion of these responses are due to interactions between the extracellular matrix components and integrins, a family of dimeric extracellular matrix receptors that are linked to and transmit signals to the cytoskeleton [156,157]. The extracellular matrix may also contain growth factors which provide growth and morphogenic signals to nearby cells. Even physical features of the matrix, such as rigidity and geometry may influence cellular phenotype and behavior and has been shown to direct stem cell lineage specification [152,158–160]. Studies of *Drosophila spp.* stem cell niches have clarified that the microenvironment, as expected, may promote adherence to the niche and repress stem cell differentiation [36]. What is more, with age the molecular composition of the extracellular matrix change in an unfavorable direction for stem cell function and proliferation. This has been illustrated in experiments where stem cells transplanted from older mice, where stem cell self-renewal and differentiation has deterio‐ rated, to extracellular matrix from younger mice rejuvenate stem cell function to levels comparable to that observed in younger mice [161,162].

Upon induction of the HPC response, the molecular composition of the HPC niche is thought to change in favor of promoting progenitor cell proliferation. Therefore, a key to understanding HPC biology and to characterize the HPC niche lies within unravelling the extracellular matrix composition of the niche. It is of particular interest to clarify which extracellular matrix molecules regulate the hepatic progenitor cell responses. A number of extracellular matrix molecules taking part in development of the intrahepatic bile ducts or in modulating the HPC response have been identified. Particularly, laminin and collagen I and IV are associated with these processes, but also other extracellular matrix components including tenascin, nidogen 1, agrin and fibronectin contribute.

The family of collagen fibrils comprises 28 members, all with at least one triple helical domain and arranged in a rope-like fashion [163,164]. Collagens are deposited in the extracellular space and particularly collagen I and collagen IV are implicated in hepatic development and regeneration [165]. However, their roles seem quite different. Collagen I is the main component of hepatic fibrosis, where it is laid down by the non-parenchymal hepatic stellate cells and myofibroblasts and contribute to the formation of scaring tissue [165,166]. Collagen IV, on the other hand, is part of the basement membrane of adult biliary cells and contributes to the ductal plate, the prenatal hepatic progenitor cell niche [167,168]. Collagen I and IV delineate expand‐ ing biliary cells not only in the HPC response, but also in the bile duct ligation model [129,146].

**Stem cell niche component Comment**

Laminin Collagen I + IV Nidogen 1 Agrin

**Table 1.** Summary of components associated with the activated hepatic progenitor cell niche.

**10. Activation and aberrant hepatic stem cell activation.**

**Associated extracellular matrix components**

**Progenitor cell niche position** The Canal of Hering Most distal part of the bile duct system. Composed

**Progenitor cell markers** AFP, NCAM, DLK1/Pref1 NCAM and DLK1/Pref1 are cell surface markers. AFP

**Associated cell types** Kupffer cells Necessary for invasion of ductular reactions into the

Animal studies have clarified that when regeneration through hepatocytic division fail HPCs from the canal of Hering contribute to liver regeneration. Despite several protein markers, such as Dlk1, EpCAM, CK19 and AFP, are associated with HPCs a pure population of hepatic stem cells or their niche have not been defined [83,179–182]. However, as elevated levels of AFP are associated with increased survival of patients suffering from acetaminophen-induced liver injury, hepatic stem cells may be activated in acute hepatic diseases [136]. OV6 and CK7 mark ductular reactions and intermediate hepatocytes, i.e. progenitor cells on route to a hepatocytic fate, suggesting stem cell involvement in a variety of human diseases and syndromes. These include hepatitis C virus infection, fatty liver disease and acute processes such as submassive liver cell necrosis, [183]. Generally, HPC activation seems correlated with the severity of inflammation and fibrosis [184]. In addition, the more aggressive the hepatocellular injury is, the larger a proportion of intermediate hepatocytes are observed [184]. Interestingly, the protein deleted in malignant brain tumor 1 (dbmt1) is specifically associated with ductular cell

degrees.

development.

Macrophages Intimately associate with ductular reactions.

phenotype.

hepatic parenchyma. Hepatic stellate cells Intimately associate with ductular reactions. Are

**Progenitor cell origin** Possibly the biliary lineage Administration of dexamethasone selective

**Progenitor cell composition** Phenotypically heterogenous Progenitor cells display biliary (CK19) and

of cholangiocytes and hepatocytes. Link bile ducts

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 187

hepatocytic markers (HepPar1, HNF4α) to various

and DLK1/Pref1 are expressed during hepatic

necessary for initiation, proliferation and termination of ductular reactions.

Laminin is essential for maintaining the biliary

with canaliculi between hepatocytes.

diminishes the progenitor cell response.

Members of the laminin family are trimeric proteins that, as for collagen IV, are part of the basal lamina [169]. In the HPC response laminin expression can be detected in hepatic stellate cells, myofibroblasts, endothelial cells and the progenitor cells themselves [170–172]. As for collagen IV, laminin contribute to the ductal plate during development and form the basal lamina escorting the HPC response in close apposition to stellate cells [121,168]. Several studies have highlighted the importance of remodeling the extracellular matrix in connection with the HPC response. In the CDE-induced murine model of HPC activation α-SMA positive cells and an extracellular matrix rich in collagen I are deposited in the periportal area prior to oval cell proliferation [146]. The ECM is laid down in a portoveinous direction, thereby preforming a niche for the HPCs to invade. However, this invasion process is tightly correlated with ECM remodeling. Hepatic macrophages and stellate cells are sources of a variety of extracellular matrix degrading enzymes, such as matrix metalloproteinases (MMP) 2, 9, 12 and 13, and their inhibitor, tissue inhibitor of metalloproteinase type 1 (TIMP-1) [151,173,174]. Where the CCl4 or CDE-models of HPC activation initiate a florid HPC response in wild-type mice this response is markedly attenuated in mice expressing a degradation resistant form of collagen I [175]. These mice also display a distinct paucity of laminin deposition suggesting that degradation of collagen is a prerequisite for HPC proliferation and parenchymal invasion.

Where ECM remodeling and collagen I degradation is necessary for the HPC response only laminin is important for the biliary phenotype. Primary murine HPCs cultured on laminin upregulate expression of HPC and biliary associated genes, such as DLK1 and aquaporin 1, respectively, while hepatocytic gene expression, exemplified by C/EBPα is inhibited [145]. Collagen I and IV, on the other hand, inhibit or do not influence these biliary genes, whereas fibronectin promote C/EBPα expression. In support of these results, culturing HPCs with laminin support proliferation and expansion *in vitro* whereas culturing HPCs with collagen I result in growth arrest and differentiation [176,177]. The importance of the laminin-rich activated progenitor cell niche for maintaining the biliary/progenitor phenotype *in vivo* is also evident in the HPC response, as disappearance of the basement membrane induces differen‐ tiation [129,178]. Assuming that the canal of Hering truly constitutes the hepatic progenitor cell niche, this niche therefore appear to be sharply limited by the deposition of collagen I, collagen IV, laminin, nidogen 1 and agrin [129]. The niche support maintaining the biliary phenotype and proliferation of HPCs that will differentiate to a hepatocytic phenotype upon exit from the HPC niche, not unlike the scenario of other stem cell niches [36].


**Table 1.** Summary of components associated with the activated hepatic progenitor cell niche.

and particularly collagen I and collagen IV are implicated in hepatic development and regeneration [165]. However, their roles seem quite different. Collagen I is the main component of hepatic fibrosis, where it is laid down by the non-parenchymal hepatic stellate cells and myofibroblasts and contribute to the formation of scaring tissue [165,166]. Collagen IV, on the other hand, is part of the basement membrane of adult biliary cells and contributes to the ductal plate, the prenatal hepatic progenitor cell niche [167,168]. Collagen I and IV delineate expand‐ ing biliary cells not only in the HPC response, but also in the bile duct ligation model [129,146].

186 Adult Stem Cell Niches

Members of the laminin family are trimeric proteins that, as for collagen IV, are part of the basal lamina [169]. In the HPC response laminin expression can be detected in hepatic stellate cells, myofibroblasts, endothelial cells and the progenitor cells themselves [170–172]. As for collagen IV, laminin contribute to the ductal plate during development and form the basal lamina escorting the HPC response in close apposition to stellate cells [121,168]. Several studies have highlighted the importance of remodeling the extracellular matrix in connection with the HPC response. In the CDE-induced murine model of HPC activation α-SMA positive cells and an extracellular matrix rich in collagen I are deposited in the periportal area prior to oval cell proliferation [146]. The ECM is laid down in a portoveinous direction, thereby preforming a niche for the HPCs to invade. However, this invasion process is tightly correlated with ECM remodeling. Hepatic macrophages and stellate cells are sources of a variety of extracellular matrix degrading enzymes, such as matrix metalloproteinases (MMP) 2, 9, 12 and 13, and their inhibitor, tissue inhibitor of metalloproteinase type 1 (TIMP-1) [151,173,174]. Where the CCl4 or CDE-models of HPC activation initiate a florid HPC response in wild-type mice this response is markedly attenuated in mice expressing a degradation resistant form of collagen I [175]. These mice also display a distinct paucity of laminin deposition suggesting that degradation of collagen

Where ECM remodeling and collagen I degradation is necessary for the HPC response only laminin is important for the biliary phenotype. Primary murine HPCs cultured on laminin upregulate expression of HPC and biliary associated genes, such as DLK1 and aquaporin 1, respectively, while hepatocytic gene expression, exemplified by C/EBPα is inhibited [145]. Collagen I and IV, on the other hand, inhibit or do not influence these biliary genes, whereas fibronectin promote C/EBPα expression. In support of these results, culturing HPCs with laminin support proliferation and expansion *in vitro* whereas culturing HPCs with collagen I result in growth arrest and differentiation [176,177]. The importance of the laminin-rich activated progenitor cell niche for maintaining the biliary/progenitor phenotype *in vivo* is also evident in the HPC response, as disappearance of the basement membrane induces differen‐ tiation [129,178]. Assuming that the canal of Hering truly constitutes the hepatic progenitor cell niche, this niche therefore appear to be sharply limited by the deposition of collagen I, collagen IV, laminin, nidogen 1 and agrin [129]. The niche support maintaining the biliary phenotype and proliferation of HPCs that will differentiate to a hepatocytic phenotype upon

is a prerequisite for HPC proliferation and parenchymal invasion.

exit from the HPC niche, not unlike the scenario of other stem cell niches [36].

#### **10. Activation and aberrant hepatic stem cell activation.**

Animal studies have clarified that when regeneration through hepatocytic division fail HPCs from the canal of Hering contribute to liver regeneration. Despite several protein markers, such as Dlk1, EpCAM, CK19 and AFP, are associated with HPCs a pure population of hepatic stem cells or their niche have not been defined [83,179–182]. However, as elevated levels of AFP are associated with increased survival of patients suffering from acetaminophen-induced liver injury, hepatic stem cells may be activated in acute hepatic diseases [136]. OV6 and CK7 mark ductular reactions and intermediate hepatocytes, i.e. progenitor cells on route to a hepatocytic fate, suggesting stem cell involvement in a variety of human diseases and syndromes. These include hepatitis C virus infection, fatty liver disease and acute processes such as submassive liver cell necrosis, [183]. Generally, HPC activation seems correlated with the severity of inflammation and fibrosis [184]. In addition, the more aggressive the hepatocellular injury is, the larger a proportion of intermediate hepatocytes are observed [184]. Interestingly, the protein deleted in malignant brain tumor 1 (dbmt1) is specifically associated with ductular cell populations emerging after acetaminophen intoxication or infection with hepatitis B virus but not in primary biliary cirrhosis or large bile duct obstruction. Dbmt1 therefore may have a role in cellular fate decision [185].

**11. Conclusion and perspectives**

HPCs [123].

reactions.

1 and agrin.

The present chapter has attempted to provide a simplified overview of current knowledge of hepatic stem cells and their niches. Unfortunately, the putative hepatic stem cell has not been identified and therefore not been characterized. Knowledge of the hepatic stem cell therefore mainly originates from analysis of its progeny, the hepatic progenitor cells, in animal models where regeneration through hepatocyte division is impaired. As a result, the location of the HPC niche is unknown. However, evidences point to the canal of Hering as the HPC niche. Assuming that the canal of Hering truly represent the hepatic stem cell niche, and that HPCs are descendants of hepatic stem cell, animal studies and their corresponding human diseases

Adult Hepatic Progenitor Cells http://dx.doi.org/10.5772/58814 189

has provided us with some knowledge of the constituents in the activated HPC niche:

display markers of the biliary and hepatocytic lineages.

**•** Activated hepatic progenitor cells are phenotypically heterogeneous and to various degrees

**•** A cellular hierarchy is present with AFP, DLK1 and NCAM marking subpopulations of

**•** Hepatic stellate cells and macrophages intimately associate with the ductular reactions.

**•** Hepatic stellate cells are necessary for initiation, proliferation and termination of ductular

**•** Kupffer cells are necessary for invasion of ductular reactions into the hepatic parenchyma.

**•** The extracellular matrix in the HPC micromillieu contain laminin, collagen I and IV, nidogen

The establishment of HPC subpopulations suggests the presence of progenitor cells at different stages of differentiation. Identifying additional proteins expressed on the HPC surface could facilitate isolation and characterization of these subpopulations and evaluation of their potential for differentiation. Furthermore, as hepatic stem cells may be implicated in devel‐ opment of primary liver cancers, better characterization of the hepatic progenitor cells could provide new targets for treatment of cancerous diseases. Aberrant progenitor cell activation and proliferation is also dependent on the hepatic microenvironment. As stellate cells and Kupffer cells are involved in the proliferation and invasion of HPCs these cell types could also provide targets for alleviating HPC derived cancers. In addition, targeting stellate cells has the potential to reduce hepatic fibrosis. Therefore, more research is needed into characterizing the hepatic progenitor cell niche and obtaining a better understanding of the activation and

**•** Laminin is necessary for maintaining the biliary phenotype of the HPCs.

differentiation of the hepatic progenitor cells.

**•** HPCs differentiate into hepatocytes upon exit from the activated HPC niche.

Liver cancer is the second most frequent cause of cancer death in men and the sixth leading cause of cancer death in women [186]. Hepatocellular carcinoma (HCC) represent the major histological subtype accounting for 70-85 % of primary livers cancers, followed by an increase in incidents of intrahepatic cholangiocarcinomas (ICC) [186,187]. Given that cancer cells and stem cells share certain characteristics cancer is proposed to represent an abnormal stem cell disease [188,189]. Both categories of cells can self-renew, divide unlimited and give rise to heterogeneous progeny. Indeed, certain gliomas, intestinal adenomas and squamous skin tumours are now attributed to cancer stem cells (CSCs) [190–193]. Liver cancer most frequently arise in chronic liver diseases, such as chronic hepatitis, cirrhosis or both, where hepatocytic regeneration and continuous inflammation occur [194,195]. Hepatocarcinogenesis is consid‐ ered as a slow process in which genomic changes progressively alter the hepatocellular phenotype [194]. In chronic liver diseases, the hepatic microenvironment is substantially altered in a fashion promoting cellular damage. Stellate cells are activated and infiltrating lymphocytes may cause inflammation through the release of free radicals and cytokines, resulting in DNA damage and cell proliferation, factors that may promote aberrant HPC activation [196,197]. However, as the hepatic stem cells are not fully defined, their involve‐ ments in these liver cancers have not been conclusively established. In addition to accumula‐ tion of genomic and epigenetic changes of genes and regulatory pathways the hepatic microenvironment is also involved in promoting liver cancer. For instance, activated hepatic stellate cells locate in the space between endothelial cells and trabeculae of cancer cells in HCC patients [198]. Conditioned media from such activated hepatic stellate cells both increase proliferation and migration of human HCC cells [199]. Thus, activated hepatic stellate cells may both drive fibrosis and proliferation of HCC cells.

A third form of primary liver cancer is the rare HCC-cholangiocarcinoma (HCC-CCA). In addition to the heterogenous cellular morphology also displayed by HCCs and ICCs, HCC-CCA's show signs of both hepatocellular and biliary epithelial differentiation [200]. Indeed, analysis of the expression pattern of hepatocytic marker HepPar1 and cholangiocytic markers CK7, CK19, EpCAM and CD133 in HCC-CCA's reveal subpopulations of cancer cells coex‐ pressing both categories of markers [182,200–203]. These results seemingly confirm the hypothesis that HCC-CCA are of HPC origin and human hepatocarcinogenesis may originate from the transformation of HPCs [200]. The identification of bipotent CSCs possibly originat‐ ing from HPCs is interesting, as stem cell like expression patterns in liver cancers reflect a particularly malignant nature and poor prognostic outcome [182,204–206]. However, the identification of bipotent cancer stem cells also opens for new therapeutic applications. Identification and elimination of CSCs could provide more effective treatment of certain tumors and prevent reoccurrence. Unfortunately, the niche controlling self-renewal, prolifer‐ ation and differentiation of HPCs and CSCs is still not well described and the putative hepatic stem cells remain unidentified.

#### **11. Conclusion and perspectives**

populations emerging after acetaminophen intoxication or infection with hepatitis B virus but not in primary biliary cirrhosis or large bile duct obstruction. Dbmt1 therefore may have a role

Liver cancer is the second most frequent cause of cancer death in men and the sixth leading cause of cancer death in women [186]. Hepatocellular carcinoma (HCC) represent the major histological subtype accounting for 70-85 % of primary livers cancers, followed by an increase in incidents of intrahepatic cholangiocarcinomas (ICC) [186,187]. Given that cancer cells and stem cells share certain characteristics cancer is proposed to represent an abnormal stem cell disease [188,189]. Both categories of cells can self-renew, divide unlimited and give rise to heterogeneous progeny. Indeed, certain gliomas, intestinal adenomas and squamous skin tumours are now attributed to cancer stem cells (CSCs) [190–193]. Liver cancer most frequently arise in chronic liver diseases, such as chronic hepatitis, cirrhosis or both, where hepatocytic regeneration and continuous inflammation occur [194,195]. Hepatocarcinogenesis is consid‐ ered as a slow process in which genomic changes progressively alter the hepatocellular phenotype [194]. In chronic liver diseases, the hepatic microenvironment is substantially altered in a fashion promoting cellular damage. Stellate cells are activated and infiltrating lymphocytes may cause inflammation through the release of free radicals and cytokines, resulting in DNA damage and cell proliferation, factors that may promote aberrant HPC activation [196,197]. However, as the hepatic stem cells are not fully defined, their involve‐ ments in these liver cancers have not been conclusively established. In addition to accumula‐ tion of genomic and epigenetic changes of genes and regulatory pathways the hepatic microenvironment is also involved in promoting liver cancer. For instance, activated hepatic stellate cells locate in the space between endothelial cells and trabeculae of cancer cells in HCC patients [198]. Conditioned media from such activated hepatic stellate cells both increase proliferation and migration of human HCC cells [199]. Thus, activated hepatic stellate cells

A third form of primary liver cancer is the rare HCC-cholangiocarcinoma (HCC-CCA). In addition to the heterogenous cellular morphology also displayed by HCCs and ICCs, HCC-CCA's show signs of both hepatocellular and biliary epithelial differentiation [200]. Indeed, analysis of the expression pattern of hepatocytic marker HepPar1 and cholangiocytic markers CK7, CK19, EpCAM and CD133 in HCC-CCA's reveal subpopulations of cancer cells coex‐ pressing both categories of markers [182,200–203]. These results seemingly confirm the hypothesis that HCC-CCA are of HPC origin and human hepatocarcinogenesis may originate from the transformation of HPCs [200]. The identification of bipotent CSCs possibly originat‐ ing from HPCs is interesting, as stem cell like expression patterns in liver cancers reflect a particularly malignant nature and poor prognostic outcome [182,204–206]. However, the identification of bipotent cancer stem cells also opens for new therapeutic applications. Identification and elimination of CSCs could provide more effective treatment of certain tumors and prevent reoccurrence. Unfortunately, the niche controlling self-renewal, prolifer‐ ation and differentiation of HPCs and CSCs is still not well described and the putative hepatic

in cellular fate decision [185].

188 Adult Stem Cell Niches

may both drive fibrosis and proliferation of HCC cells.

stem cells remain unidentified.

The present chapter has attempted to provide a simplified overview of current knowledge of hepatic stem cells and their niches. Unfortunately, the putative hepatic stem cell has not been identified and therefore not been characterized. Knowledge of the hepatic stem cell therefore mainly originates from analysis of its progeny, the hepatic progenitor cells, in animal models where regeneration through hepatocyte division is impaired. As a result, the location of the HPC niche is unknown. However, evidences point to the canal of Hering as the HPC niche. Assuming that the canal of Hering truly represent the hepatic stem cell niche, and that HPCs are descendants of hepatic stem cell, animal studies and their corresponding human diseases has provided us with some knowledge of the constituents in the activated HPC niche:


The establishment of HPC subpopulations suggests the presence of progenitor cells at different stages of differentiation. Identifying additional proteins expressed on the HPC surface could facilitate isolation and characterization of these subpopulations and evaluation of their potential for differentiation. Furthermore, as hepatic stem cells may be implicated in devel‐ opment of primary liver cancers, better characterization of the hepatic progenitor cells could provide new targets for treatment of cancerous diseases. Aberrant progenitor cell activation and proliferation is also dependent on the hepatic microenvironment. As stellate cells and Kupffer cells are involved in the proliferation and invasion of HPCs these cell types could also provide targets for alleviating HPC derived cancers. In addition, targeting stellate cells has the potential to reduce hepatic fibrosis. Therefore, more research is needed into characterizing the hepatic progenitor cell niche and obtaining a better understanding of the activation and differentiation of the hepatic progenitor cells.

#### **12. Nomenclature**

2-AAF: 2-Acetylaminofluorene.

AFP: α-Fetoprotein.

ASMA: alpha smooth muscle actin.

BMP: Bone morphogenetic protein.

BrdU: 5-Bromo-2´-Deoxyuridine.

CCl4: Carbon tetrachloride.

CDE: Choline-deficient, ethionine supplemented.

SOX9: SRY-related HMG box transcription factor 9.

Vegfr2: Vascular endothelial growth factor receptor-2.

Address all correspondence to: sqp608@alumni.ku.dk

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[10] Ostapowicz G, Fontana RJ, Schiodt FV, Larson A, Davern TJ, Han SH, McCashland TM, Shakil AO, Hay JE, Hynan L, Crippin JS, Blei AT, Samuel G, Reisch J, Lee WM

TIMP-1: tissue inhibitor of metalloproteinase 1.

**Author details**

**References**

Peter Siig Vestentoft\*

Dako, Glostrup, Denmark

21172 [doi].

10.2353/ajpath.2010.090675 [doi].

plant Proc 27: 3519-3520.

CK: Cytokeratin.

CSC: Cancer Stem Cell.

DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

DLK1: Delta-Like 1 Homolog.

Dmbt1: Deleted in malignant brain tumors 1

FGF: Fibroblast growth factor.

GFAP: Glial fibrillary acidic protein.

HAI-1: Hepatocyte Growth Factor Activator Inhibitor Type 1.

HCC: Hepatocellular carcinoma.

HCC-CCA: HCC-cholangiocarcinoma.

HepPar1: Hepatocyte Paraffin 1.

HGF: Hepatocyte growth factor.

HNF: Hepatocyte nuclear factor.

HPC: Hepatic progenitor cell.

ICC: Intrahepatic cholangiocarcinoma.

MDA: 4,4'-Methylenedianiline.

MMP: Matrix metalloproteinase.

NCAM: Neural Cell Adhesion Molecule.

OV6: Oval Cell Marker Antibody 6.

PHx: Partial hepatectomy.

ST14: Suppressor of tumorigenicity 1.

SOX9: SRY-related HMG box transcription factor 9.

TIMP-1: tissue inhibitor of metalloproteinase 1.

Vegfr2: Vascular endothelial growth factor receptor-2.

#### **Author details**

**12. Nomenclature**

190 Adult Stem Cell Niches

AFP: α-Fetoprotein.

CK: Cytokeratin.

CSC: Cancer Stem Cell.

DLK1: Delta-Like 1 Homolog.

FGF: Fibroblast growth factor.

GFAP: Glial fibrillary acidic protein.

HCC: Hepatocellular carcinoma.

HepPar1: Hepatocyte Paraffin 1. HGF: Hepatocyte growth factor. HNF: Hepatocyte nuclear factor.

HPC: Hepatic progenitor cell.

MDA: 4,4'-Methylenedianiline. MMP: Matrix metalloproteinase.

HCC-CCA: HCC-cholangiocarcinoma.

ICC: Intrahepatic cholangiocarcinoma.

NCAM: Neural Cell Adhesion Molecule.

OV6: Oval Cell Marker Antibody 6.

ST14: Suppressor of tumorigenicity 1.

PHx: Partial hepatectomy.

2-AAF: 2-Acetylaminofluorene.

CCl4: Carbon tetrachloride.

ASMA: alpha smooth muscle actin. BMP: Bone morphogenetic protein. BrdU: 5-Bromo-2´-Deoxyuridine.

CDE: Choline-deficient, ethionine supplemented.

DDC: 3,5-diethoxycarbonyl-1,4-dihydrocollidine.

HAI-1: Hepatocyte Growth Factor Activator Inhibitor Type 1.

Dmbt1: Deleted in malignant brain tumors 1

Peter Siig Vestentoft\*

Address all correspondence to: sqp608@alumni.ku.dk

Dako, Glostrup, Denmark

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**Section 4**

**Stem Cell Niches in Adult Nentral Nervous**

**System**


**Stem Cell Niches in Adult Nentral Nervous System**

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208 Adult Stem Cell Niches

**Chapter 8**

**Typical and Atypical Stem Cell Niches of the Adult**

**Spinal Cord Diseases**

http://dx.doi.org/10.5772/58599

**1. Introduction**

Joshua Bernstock, Jeroen Verheyen, Bing Huang,

*may die, nothing may be regenerated."*-Santiago Ramon y Cajal

self-renew, but typically produce only oligodendrocytes [18, 19].

John Hallenbeck and Stefano Pluchino

Additional information is available at the end of the chapter

**Nervous System in Health and Inflammatory Brain and**

*"Once development was ended, the fonts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, and immutable: everything*

The central nervous system (CNS) is inhabited by a heterogeneous population of cells (i.e. neurons and glia) and is marked by a highly complex anatomical structure [1]. In states of host homoeostasis the putative majority of cells in the CNS are long-lived and typically do not require replacement. Nonetheless, neurogenesis in the adult mammalian brain has been shown to occur in a myriad of locations, under a diverse set of physiologic/pathophysiologic condi‐ tions [2-10]. Neurogenesis is driven by stem cells which can be defined by their ability to produce both identical daughter cells (self-renewal) and progeny with more restricted fates (commitment and differentiation) [11]. To be classified as a neural stem cell (NSC), cells should be able to self-renew and give rise to a variety of mature progeny that make up the CNS, including neurons, astrocytes and oligodendrocytes [12-16]. However, fate-restricted precur‐ sor cells capable of self-renewal, but which concurrently display restricted differentiation potential, also reside in the CNS. These cells are often unipotent and are referred to as neural progenitor cells (NPC) [17, 18], for example, oligodendrocyte precursor cells (OPC) are able to

Identification of NSC *in vivo* is clearly complicated and relies on the analysis of cell morphol‐ ogy, mitotic activity, and gene and protein expression. Commonly used NSC markers include nestin, glial fibrillary acidic protein (GFAP), Musashi 1/2, and the Shy-related high mobility

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
