**1. Introduction**

204 Advances in Hematopoietic Stem Cell Research

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5472.

Stem cells are characterized by their capacity to self renew and differentiate into progressively restricted cells that ultimately become limited to a specific cell fate. The two broad types of mammalian stem cells are: embryonic stem cells and adult stem cells.

Embryonic stem cells (ESC) are mostly derived from the undifferentiated inner mass cells of a blastocyst. These cells give rise during development of the embryo to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. They do not contribute to the extra-embryonic membranes or the placenta. Ex-vivo, they can be cultured for extended periods of time and under the appropriate conditions, they can be also directed to differentiate into many specialized types of cells. These particular features are being exploited to use ESC as starting material for treatment of degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, the quite small production of ESCderived cells obtained or the active immune rejection of the ESC-derived graft.

Unlike embryonic stem cells, the adult stem cells are already partially specialized. They have been found in most self-renewing tissues, including the skin, the brain, the intestinal epithelium and the hematopoietic system and have the primary role of maintaining and repairing the tissue in which they are found. They are located deep within organs in specialized areas known as the "stem cell niche" (Scadden, 2006). This microenvironment allows for their survival, self renewal, regulated proliferation and maintenance of their quiescence for long periods of time until the moment in which they are activated. *Ex vivo*, however, the capacity of stem cells to self-renew is limited, they exhibit poor survival and consequently their numbers sharply declines during experimental manipulation.

One of the more intriguing but highly debated areas of stem cell biology was the phenomenon described as plasticity or transdifferentiation. Numerous reports expressed opposing views

Searching for the Key to Expand Hematopoietic Stem Cells 207

To maintain the steady-state of the stem cell compartment and to allow the regeneration of hematopoietic cells after transplantation or after hematopoietic injury, HSC divide asymmetrically or symmetrically. In an asymmetric self-renewing division, the two daughter cells adopt different fates, resulting in only one cell maintaining stem-cell properties. The symmetric self-renewing division refers to the process whereby both daughter cells retain stem cell properties. This type of cell division expands the stem-cell pool and is therefore critical for

The HSC fate decisions are dependent on concomitantly intrinsic HSC fate determinants and extrinsic signals delivered by the bone marrow (BM) niches were HSC resides. These niches are small cavities formed by heterogeneous types of cells, named stroma, that are positioned close to the BM longitudinal axis of the femur with more differentiated cells disposed in a graduated manner as the central longitudinal axis of the bone is approached. The attachment of HSC to the stroma via a network of adhesion molecules provide an environment that optimally balances signals that control self-renewal, proliferation and differentiation. Under normal physiological conditions, HSC are kept in a relatively low proliferative, quiescent state, protecting them from stress and preventing their depletion due to excessive proliferation (Jang and Sharkis, 2007). Recent data imply that these areas

To take advantage of the HSC plasticity capacities for therapeutic use, HSC may be withdrawn from their original niches, and placed on a novel non-hematopoietic environment. Once located in this novel medium, the reprogramming of the cell genome occurs and directs and/or contributes to their conversion into unrelated cell types (Fig. 2). The unexpected flexibility of HSC to produce non-hematopoietic cells was described for several cells/tissues (Quesenberry et al., 2010) including liver cells (Almeida-Porada et al., 2010; Jang et al., 2004), neurones (Mezey et al., 2000), lung epithelial (Abe et al., 2003) or

Fig. 2. A schematic representation of HSC plasticity. Hematopoietic cell are removed from the femoral bone (1) and the HSC-enriched population is cultured *in vitro*. Following an optional genetic modification, cells may be used to generate *in vivo* non-hematopoietic cell types.

sustaining the HSC compartment and thus is a requirement for lifelong hematopoiesis.

where HSC reside are hypoxic (Parmar et al., 2007).

connective tissues (Ogawa et al., 2010).

about this ability of stem cells to cross organ/tissue boundaries. These discrepancies have now been mostly passed over by current research showing that cell populations of one lineage might produce cells from other lineages by changing gene expression in response to microenvironmental cues (Jang and Sharkis, 2005; Theise, 2010).

Owing to their unique characteristic of plasticity, self-renewal capacity and potential to generate functional cell types, stem cells are particularly attractive for developing therapeutic settings that range from drug discovery protocols to cell transplantation and regenerative therapies. Nevertheless, several challenges including the need to identify the signals that influence the stem cell fate decisions and the application of this information towards the design of stem cell bioprocesses have to be overcome to accomplish the transition from fundamental science to functional technologies.

#### **1.1 Hematopoietic stem cells**

Hematopoietic stem cells (HSC) are probably the best characterized adult stem cell and often serve as a paradigm for other stem cells. Even though no morphological criteria to unequivocally identify such cells exist, HSC have been proven to be invaluable in the clinic. They are the only stem cells used routinely in cell based therapies, to treat numerous hematologic and non-hematologic malignancies as well as a range of both inherited and acquired diseases. This is typically due i) to the availability of a straight forward purification protocols using cell surface antigen selection and ii) to the possibility to perform reconstitution assays that rely on their clonal ability to reconstitute the entire hematopoietic system following transplantation into myeloablated recipients (Fig. 1). The same cell surface antigens, however, do not always conform to the same stem cell functional phenotype (Simonnet et al., 2009) and therefore the transplantation procedure constitutes undoubtedly the "gold standard" method for proving that a cell is indeed an HSC.

Fig. 1. A diagrammatic representation of a stem cell in its micro-environment and one stem cell induced to move out of the niche where it will undergo development. Following BM removal and cell surface antigen selection (1), cells are cultured *in vitro* and infused in a myeloablated mouse. Several weeks after (2), blood cells are regenerated in the transplanted mouse.

about this ability of stem cells to cross organ/tissue boundaries. These discrepancies have now been mostly passed over by current research showing that cell populations of one lineage might produce cells from other lineages by changing gene expression in response to micro-

Owing to their unique characteristic of plasticity, self-renewal capacity and potential to generate functional cell types, stem cells are particularly attractive for developing therapeutic settings that range from drug discovery protocols to cell transplantation and regenerative therapies. Nevertheless, several challenges including the need to identify the signals that influence the stem cell fate decisions and the application of this information towards the design of stem cell bioprocesses have to be overcome to accomplish the

Hematopoietic stem cells (HSC) are probably the best characterized adult stem cell and often serve as a paradigm for other stem cells. Even though no morphological criteria to unequivocally identify such cells exist, HSC have been proven to be invaluable in the clinic. They are the only stem cells used routinely in cell based therapies, to treat numerous hematologic and non-hematologic malignancies as well as a range of both inherited and acquired diseases. This is typically due i) to the availability of a straight forward purification protocols using cell surface antigen selection and ii) to the possibility to perform reconstitution assays that rely on their clonal ability to reconstitute the entire hematopoietic system following transplantation into myeloablated recipients (Fig. 1). The same cell surface antigens, however, do not always conform to the same stem cell functional phenotype (Simonnet et al., 2009) and therefore the transplantation procedure constitutes undoubtedly

Fig. 1. A diagrammatic representation of a stem cell in its micro-environment and one stem cell induced to move out of the niche where it will undergo development. Following BM removal and cell surface antigen selection (1), cells are cultured *in vitro* and infused in a myeloablated mouse. Several weeks after (2), blood cells are regenerated in the transplanted mouse.

environmental cues (Jang and Sharkis, 2005; Theise, 2010).

transition from fundamental science to functional technologies.

the "gold standard" method for proving that a cell is indeed an HSC.

**1.1 Hematopoietic stem cells** 

To maintain the steady-state of the stem cell compartment and to allow the regeneration of hematopoietic cells after transplantation or after hematopoietic injury, HSC divide asymmetrically or symmetrically. In an asymmetric self-renewing division, the two daughter cells adopt different fates, resulting in only one cell maintaining stem-cell properties. The symmetric self-renewing division refers to the process whereby both daughter cells retain stem cell properties. This type of cell division expands the stem-cell pool and is therefore critical for sustaining the HSC compartment and thus is a requirement for lifelong hematopoiesis.

The HSC fate decisions are dependent on concomitantly intrinsic HSC fate determinants and extrinsic signals delivered by the bone marrow (BM) niches were HSC resides. These niches are small cavities formed by heterogeneous types of cells, named stroma, that are positioned close to the BM longitudinal axis of the femur with more differentiated cells disposed in a graduated manner as the central longitudinal axis of the bone is approached. The attachment of HSC to the stroma via a network of adhesion molecules provide an environment that optimally balances signals that control self-renewal, proliferation and differentiation. Under normal physiological conditions, HSC are kept in a relatively low proliferative, quiescent state, protecting them from stress and preventing their depletion due to excessive proliferation (Jang and Sharkis, 2007). Recent data imply that these areas where HSC reside are hypoxic (Parmar et al., 2007).

To take advantage of the HSC plasticity capacities for therapeutic use, HSC may be withdrawn from their original niches, and placed on a novel non-hematopoietic environment. Once located in this novel medium, the reprogramming of the cell genome occurs and directs and/or contributes to their conversion into unrelated cell types (Fig. 2). The unexpected flexibility of HSC to produce non-hematopoietic cells was described for several cells/tissues (Quesenberry et al., 2010) including liver cells (Almeida-Porada et al., 2010; Jang et al., 2004), neurones (Mezey et al., 2000), lung epithelial (Abe et al., 2003) or connective tissues (Ogawa et al., 2010).

Fig. 2. A schematic representation of HSC plasticity. Hematopoietic cell are removed from the femoral bone (1) and the HSC-enriched population is cultured *in vitro*. Following an optional genetic modification, cells may be used to generate *in vivo* non-hematopoietic cell types.

Searching for the Key to Expand Hematopoietic Stem Cells 209

The role of Hh signaling in HSC is controversial. Bhardwaj et al provided evidence for a role of Hh signaling in HSC (Bhardwaj et al., 2001). In this study, suppression of Hh signaling inhibited proliferation of HSC and addition of soluble SHh induced expansion of hematopoietic repopulating cells (Bhardwaj et al., 2001). More recent reports confirmed that suppression of the Hh pathway leads to a severe defect in HSC functions (Merchant et al., 2010; Trowbridge et al., 2006) whereas others reported that this pathway can be dispensable for HSC biology (Gao et al., 2009; Hofmann et al., 2009). In *Ptc1*+/−mice, which have increased Hh activity, activation of the Hh signaling pathway induces expansion of primitive blood cells under homeostatic conditions. However, when HSC are challenged to regenerate the blood system, persistent Hh activation leads to HSC exhaustion (Trowbridge et al., 2006). Furthermore, Indian Hh gene transfer can confer enhanced hematopoietic support ability to BM stromal cells, suggesting that it is involved in the interaction between HSC and the stromal cells. This leads to an increase in proliferation and repopulating capacity of primitive hematopoietic cells (Kobune et al., 2004). These results suggest a role for Hh signaling in balancing homeostasis and regeneration *in vivo*. In contrast, other reports show that Hh signaling is dispensable for adult HSC functions (Gao et al., 2009; Hofmann et al., 2009). In these studies conditional deletion of SMO, the only non redundant component of the Hh cascade, or pharmacologic inhibition of Hh signaling have no apparent effect on adult hematopoietic, including peripheral blood count, number or cell cycle status of stem or progenitor cells, hematopoietic colony-forming potential or long-term repopulating activity in *in vivo* assays. In agreement with this notion, genome-wide transcriptome analysis revealed that silencing the Hh signaling does not significantly alter the HSC-specific gene expression ''signature.'' Taken together, these conflicting data suggest that Hh signaling may influence

HSC through more complex networks such as cell-niche interactions.

soluble growth factors as well as adhesion proteins (Walenda et al., 2011).

FGF belongs to a family of heparin-binding polypeptides that shows multiple functions, including effects on cell proliferation, differentiation and survival (Baird, 1994). Twenty-four members of the FGF family have been identified in human and mice. FGFs bind and activate their cognate FGFRs that are encoded by four genes (FGFR1– 4). This results in receptor dimerization, tyrosine kinase autophosphorylation, and recruitment of signaling complexes. The FGF signal transduction proceeds by one, or a combination, of three main pathways: Ras/mitogen-activated protein kinase (MAPK) signaling; planar cell polarity/calcium; phosphoinotitide-3-kinase (PI3K)/Akt (extensively reviewed by Bottcher and Niehrs, 2005). Both FGF-1 and FGF-2 support HSC expansion when unfractionated mouse BM cells are cultured in serum-free medium (de Haan et al., 2003; Yeoh et al., 2006). Crcareva et al*.* confirmed that FGF-1 stimulates ex-vivo expansion of HSC (Crcareva et al., 2005). Conditional derivatives of FGFR-1 have also been used to support short-term HSC expansion and long-term HSC survival (Weinreich et al., 2006). This factor seems to also support *ex vivo* expansion of murine and human HSC in combination with other cytokines, i.e stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1] (Zhang and Lodish, 2005). Moreover, a recent study showed that addition of SCF, TPO, and FGF-1 to a mesenchymal stem cells (MSC) culture stimulates proliferation, maintenance of primitive immunophenotype, and expansion of CFU-initiating cells. This supports the notion that expansion of HSC requires complex stimulation of different signal cascades activated by

**2.2 Fibroblast growth factor (FGF) signaling** 

The development of HSC-based therapies however, is to some extent prevented by the scarce representation of HSC in the BM and their finite lifespan *ex vivo*. Increasing their utilisation needs enhancement of hematopoietic stem cells availability or *de novo* generation of HSC. This presumes i) the development of robust methods to efficiently control HSC regulatory processes; ii) the therapeutic *in vivo* or *in vitro* expansion of HSC number and iii) the utilisation of optimized protocols to generate available HSC from ESC or IPSC.
