**7. Role of telomerase in hematopoietic stem cell**

Stem cells self renewal capacity is believed to be closely associated with tissue degeneration during aging. Studies of human genetic diseases and gene-targeted animal models have provided evidence that functional decline of telomeres and deregulation of cell cycle checkpoints contribute to the aging process of tissue stem cells. Telomere dysfunction can induce DNA damage response via key cell cycle checkpoints, leading to cellular senescence or apoptosis depending on the tissue type and developmental stage of a specific stem cell compartment (Ju Z et al., 2011).

Studies in hematopoietic stem cell (HSC) biology are often focused on "self-renewal" and differentiation. Implicit in the word self-renewal is that the two daughter cells generated by

Recently we demonstrated that another polycomb group member, Suz12 gene, is activated by the non canonical Wnt pathway and may epigenetically inhibit genes involved in hematopoietic differentiation. These data pointed to cell cycle changes, deregulation of early differentiation genes and regulation of PRC2 polycomb complex genes, due to Suz12 role in CML blast crisis. This observation indicates that the cross talk between Wnt and Polycomb pathways may promotes hematopoietic differentiation. (Pizzatti et al., 2010).

Taken together all these data fits in a model were HSCs fated to self renew are in contact in the endosteal niches with osteoblasts expressing *Notch* legands (*Jagged*) so the pathway that

The *Hedgehog* (*Hh*) is a ligand that binds and represses the *Patched* receptor and thereby releases the latent activity of the multipass membrane protein Smoothened, which is essential for transducing the *Hh* signal. Using *Patched*+/− mouse with increased *Hh* signaling activity, it was demonstrated that constitutive activation of the *Hh* signaling pathway results in the steady-state accumulation of phenotypically defined HSCs and an increase in the proportion of cycling cells within this population (Kuhn et al., 1995). However, HSC activity on secondary transplantation is reduced 3-fold, indicating the functional exhaustion of the HSC pool in this mutant. In vivo treatment with an inhibitor of the *Hh* pathway rescues these transcriptional and functional defects in HSCs. This study establishes *Hh* signaling as a negative regulator of the HSC quiescence. In contrast to the germline *Patched*+/− mode, the mode of conditional deletion of Smoothened in the adult hematopoietic compartment was used in other two studies. However, the negative effects of the Hh pathway on HSC quiescence were shown in one study (Walkley & Orkin, 2006) and not in another (Stead et al., 2002). The discrepancy is possibly due to a distinct mode of deletion. How this pathway collaborate with the two others is not clear although interaction through GSK3 have been

The outcome of *Hh* signaling varies according to the receiving cell type. GLI, the cytoplasmic effector of *Hh* signaling activates the transcription of several target genes as *CyclinD1 and D2,N-Myc,Wnts, FoxM1,Hes 1,Bcl2,Osteopontin* and others. If these genes are activated in HSCs has not yet been defined but if they are, a clear interconnection between

One important point when talking about self-renewal is how to prevent exhaustion of the

Stem cells self renewal capacity is believed to be closely associated with tissue degeneration during aging. Studies of human genetic diseases and gene-targeted animal models have provided evidence that functional decline of telomeres and deregulation of cell cycle checkpoints contribute to the aging process of tissue stem cells. Telomere dysfunction can induce DNA damage response via key cell cycle checkpoints, leading to cellular senescence or apoptosis depending on the tissue type and developmental stage of a specific stem cell

Studies in hematopoietic stem cell (HSC) biology are often focused on "self-renewal" and differentiation. Implicit in the word self-renewal is that the two daughter cells generated by

will be induced is *Notch* pathway.

already proposed.

HSC pool.

compartment (Ju Z et al., 2011).

several important signaling pathways is visualized.

**7. Role of telomerase in hematopoietic stem cell** 

a self-renewal division are identical to the parental cell. Strictly speaking, this is not possible because DNA is continuously damaged and repaired by DNA-repair mechanisms that are not 100% efficient (Lansdorp et al., 2005).

It is important to note that the efficiency of DNA repair varies greatly among different stem cell types. For example, embryonic stem cells are quite resistant to DNA damage and maintain the length of telomere repeats on serial passage, whereas HSCs are quite sensitive to DNA damage and less able to maintain telomere length. This idea has given rise to the notion that many aspects of normal aging could primarily reflect limitations in DNA repair and telomere-maintenance pathways in the (stem) cells of the soma (Lansdorp et al., 2005).

The loss of telomere repeats in adult hematopoietic cells (including purified "candidate" HSCs) relative to fetal hematopoietic cells also fits a model that postulates a finite and limited replicative potential of HSCs (Vaziri et al., 1994; Lansdorp et al., 1995; Lansdorp et al., 1997). How this collaborates with the model of LT-HSCs given rise to ST-HSCs has not still been addressed.

Eukaryotic chromosomes are capped by special structures called telomeres, which are guanine-rich, simple repeat sequences. Telomeres act to guarantee chromosome integrity by preventing illegitimate recombination, degradation, and end fusions (Blackburn et al., 1991; Stain et al ., 2004).

Synthesis and maintenance of telomeric repeats are accomplished by a specialized ribonucleoprotein complex known as telomerase. Telomerase consists of an essential RNA template and protein components, one of which appears to resemble reverse transcriptase. In the absence of telomerase, the failure of DNA polymerase to fully synthesize DNA termini leads to chromosome shortening (Stain et al., 2004; Lee et al., 1998).

In contrast to mice were short telomeres maintain cell survival for some generations, a modest two fold reduction in telomerase levels in humans (resulting from haploinsufficiency for the telomerase RNA template gene) typically results in premature death from complications of aplastic anemia or immune deficiency. Recent studies indicate that short telomeres and eventual marrow failure may also result from haploinsufficiency for the telomerase reverse transcriptase(hTERT) gene (Yamaguchi et al., 2005).

Moreover the large number of HSCs typically used in clinical transplant settings may effectively prevent their replicative exhaustion. Variations in telomere length between cells and individuals have even made it difficult to reproducibly document a decline in telomere length following transplantation. Nevertheless, a significant shortening of telomeres was observed in the first year after allogeneic bone marrow transplantation (Landsdorp et al., 2005). Furthermore, marrow failure with pronounced telomere shortening has been described in a few long-term survivors of HSC transplants. Although there is little evidence to suggest that telomere shortening will result in an epidemic of marrow failure in HSC transplant recipients, caution remains warranted when the cell number available for transplantation is limited or when the telomere length in HSCs for transplantation is short, as in cells from old donors or patients with telomerase deficiencies. It is tempting to speculate that some of the advantages of cord blood HSC transplants are related to the longer telomeres in individual cord blood HSCs (Awaya et al ., 2002).

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 25

Asymmetric stem cell divisions can be controlled by intrinsic mechanisms or the asymmetric exposure to extrinsic cues. Intrinsic mechanisms use apical-basal or planar polarity along the mitotic spindle to asymmetrically segregate cell fate determinants into only one daughter cell. Extrinsic mechanisms rely on contact with the so called stem cell niche, a cellular microenvironment that provides external cues (Doe, 2008; Li & Xie, 2005; Morrison & Spradling, 2008). Orientation of its mitotic spindle perpendicular to the niche surface allows the asymmetric segregation of cell fate determinants relative to the external stimuli to

Much progress has been made in understanding intercellular mechanisms, especially the identification of niches for various types of tissue stem cells and elucidation of the role of the

Although the role of niche in the asymmetric division of mammalian stem cells has not been clearly illustrated, Fuchs (2008) have shown that embryonic basal epidermal cells use their polarity to divide asymmetrically with respect to the underlying basal lamina, generating a committed suprabasal cell and a proliferative basal cell. Because skin stem cells are a subpopulation of mitotically active basal epidermal cells, it is conceivable that these stem cells divide in an asymmetric fashion to self-renew and to produce differentiated keratinocytes. Moreover, integrins and cadherins in the basal lamina are essential for the proper localization of apical complexes containing atypical PKC (aPKC), the Par3 – LGN – Inscuteable protein, and NuMA (nuclear mitotic apparatus protein) – dynactin. This asymmetric localization may be functionally important because similar complexes in

In addition to basal epidermal cells, mouse neuroepithelial stem cells and hematopoietic precursor cells undergo both asymmetric and symmetric divisions. In the mammalian central nervous system, embryonic neuroepithelial cells first undergo symmetric division to expand their population and then switch to asymmetric divisions for neurogenesis. This switch involves a change in cleavage plane orientation from perpendicular to parallel to the plane of the apical lamina, leading to an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1 – 2%) of the entire neuroepithelial cell plasma membrane (Kosodo et al., 2004). Somewhat similarly, mouse hematopoietic progenitor cells are capable of both symmetric and asymmetric divisions in cultures supported by stromal cells (Wu et al., 2007). A pro-differentiation stromal cell line increased the frequency of asymmetric division, whereas a pro-proliferation stromal cell line promoted symmetric division. These observations indicate that niche signaling can also

Although niche induction accounts for asymmetric division in some types of stem cells, it may not play a role in all types of stem cells. In some stem cells there is an intrinsic polarity where molecules are segregated along an axis and serves as determinant of cell fate after cell division. In these cases the orientation of mitotic spindle has to be coordinated with the asymmetric localization of the cell fate regulators. This is the case, for example of *Numb* homologue during hematopoietic precursor cell division. So looking for hematopoietic niche and signaling between microenviroment cells and the stem cell we could " envisage"(imagine; idealize, construct) a model for stem cell to decide between symmetric

*Drosophila* neuroblasts are essential for asymmetric division (Chia et al., 2008).

control the asymmetry of stem cell division at a populational level.

maintain self-renewal potential.

or asymmetric division.

niche in regulating asymmetric stem cell division.

It has been proposed that telomeres can switch between an open state (in principle allowing elongation by telomerase) and a closed state (inaccessible to telomerase) with the likelihood of the open state inversely related to the length of the repeat track (Blackburn et al., 2001). In most human cells, telomerase appears to be present at limiting levels, allowing elongation of only a limited number of critically short telomeres. Accumulation of short telomeres before replicative senescence has been observed and replicative senescence or apoptosis could result when the number of critically short telomeres exceeds the telomere repair capacity in a cell (Ju Z et al., 2011). In this context the ability of HSCs to modulate telomerase activity may be crucial in maintaining the self-renewal process. In human BM cells, low telomerase activity levels were demonstrated in multipotent HSCs, whereas significant upregulation of enzyme activity was apparent in the presence of proliferation-inducing cytokines (Samper et al., 2002; Stein et al., 2004). So cytokines and JAK-STAT signaling pathway may contribute to self renewal by maintaining telomerase activity.

The role of DNA repair pathways and telomeres/telomerase in the biology of normal and malignant human HSCs cells as well as the biology of aging clearly needs further study. New insight in the role of telomerase in HSCs has been provided by recent studies of patients with inborn errors in telomerase activity. Therefore, a further understanding of the molecular mechanisms underlying HSC aging may help identity new therapeutic targets for stem cell-based regenerative medicine.

#### **8. Asymmetric cell division – A mechanism to generate progenitors while maintaining HSC**

In both invertebrates like the insect *Drosophila*, and mammals, the major characteristic of stem cells is their ability to self-renew. Using various modes of proliferation, stem cells maintain or expand the available stem cell pool, but they can also generate more specialized progeny that constitute the majority of cells in an adult individual. In multi-cellular organisms, totipotent zygotes generate pluripotent stem cells, which become increasingly restricted in their lineage potential during development, and subsequently give rise to mature tissue-specific, multipotent stem cells. Stem cells show either 'proliferative' symmetric divisions or 'differentiative' asymmetric divisions to regulate a balance between the maintenance of stem cell pool and the supply of mature cells. It is critical for stem cells to tightly control this balance between the two different modes of division, both during development and adulthood, because, failure in maintaining cellular homeostasis may lead to incomplete tissue or organ development, whereas uncontrolled proliferation can lead to tumorigenesis.

Symmetric cell divisions commonly occur during development of invertebrates and vertebrates, phenomena that can also be observed during wound healing and regeneration of tissues. This mode of division is defined by the generation of two daughter cells that acquire the same fate, thereby expanding the pool of stem cells required or generating two differentiating daughter cells. Asymmetric cell divisions play a key role in generating cellular diversity during development by generating two daughter cells that are committed to different fates in a single division, simultaneously self-renewing to generate a daughter cell with stem cell properties, as well as to give rise to a more differentiated progeny.

It has been proposed that telomeres can switch between an open state (in principle allowing elongation by telomerase) and a closed state (inaccessible to telomerase) with the likelihood of the open state inversely related to the length of the repeat track (Blackburn et al., 2001). In most human cells, telomerase appears to be present at limiting levels, allowing elongation of only a limited number of critically short telomeres. Accumulation of short telomeres before replicative senescence has been observed and replicative senescence or apoptosis could result when the number of critically short telomeres exceeds the telomere repair capacity in a cell (Ju Z et al., 2011). In this context the ability of HSCs to modulate telomerase activity may be crucial in maintaining the self-renewal process. In human BM cells, low telomerase activity levels were demonstrated in multipotent HSCs, whereas significant upregulation of enzyme activity was apparent in the presence of proliferation-inducing cytokines (Samper et al., 2002; Stein et al., 2004). So cytokines and JAK-STAT signaling pathway may contribute to

The role of DNA repair pathways and telomeres/telomerase in the biology of normal and malignant human HSCs cells as well as the biology of aging clearly needs further study. New insight in the role of telomerase in HSCs has been provided by recent studies of patients with inborn errors in telomerase activity. Therefore, a further understanding of the molecular mechanisms underlying HSC aging may help identity new therapeutic targets for

**8. Asymmetric cell division – A mechanism to generate progenitors while** 

In both invertebrates like the insect *Drosophila*, and mammals, the major characteristic of stem cells is their ability to self-renew. Using various modes of proliferation, stem cells maintain or expand the available stem cell pool, but they can also generate more specialized progeny that constitute the majority of cells in an adult individual. In multi-cellular organisms, totipotent zygotes generate pluripotent stem cells, which become increasingly restricted in their lineage potential during development, and subsequently give rise to mature tissue-specific, multipotent stem cells. Stem cells show either 'proliferative' symmetric divisions or 'differentiative' asymmetric divisions to regulate a balance between the maintenance of stem cell pool and the supply of mature cells. It is critical for stem cells to tightly control this balance between the two different modes of division, both during development and adulthood, because, failure in maintaining cellular homeostasis may lead to incomplete tissue or organ development, whereas uncontrolled proliferation can lead to

Symmetric cell divisions commonly occur during development of invertebrates and vertebrates, phenomena that can also be observed during wound healing and regeneration of tissues. This mode of division is defined by the generation of two daughter cells that acquire the same fate, thereby expanding the pool of stem cells required or generating two differentiating daughter cells. Asymmetric cell divisions play a key role in generating cellular diversity during development by generating two daughter cells that are committed to different fates in a single division, simultaneously self-renewing to generate a daughter cell with stem cell properties, as well as to give rise

self renewal by maintaining telomerase activity.

stem cell-based regenerative medicine.

**maintaining HSC** 

tumorigenesis.

to a more differentiated progeny.

Asymmetric stem cell divisions can be controlled by intrinsic mechanisms or the asymmetric exposure to extrinsic cues. Intrinsic mechanisms use apical-basal or planar polarity along the mitotic spindle to asymmetrically segregate cell fate determinants into only one daughter cell. Extrinsic mechanisms rely on contact with the so called stem cell niche, a cellular microenvironment that provides external cues (Doe, 2008; Li & Xie, 2005; Morrison & Spradling, 2008). Orientation of its mitotic spindle perpendicular to the niche surface allows the asymmetric segregation of cell fate determinants relative to the external stimuli to maintain self-renewal potential.

Much progress has been made in understanding intercellular mechanisms, especially the identification of niches for various types of tissue stem cells and elucidation of the role of the niche in regulating asymmetric stem cell division.

Although the role of niche in the asymmetric division of mammalian stem cells has not been clearly illustrated, Fuchs (2008) have shown that embryonic basal epidermal cells use their polarity to divide asymmetrically with respect to the underlying basal lamina, generating a committed suprabasal cell and a proliferative basal cell. Because skin stem cells are a subpopulation of mitotically active basal epidermal cells, it is conceivable that these stem cells divide in an asymmetric fashion to self-renew and to produce differentiated keratinocytes. Moreover, integrins and cadherins in the basal lamina are essential for the proper localization of apical complexes containing atypical PKC (aPKC), the Par3 – LGN – Inscuteable protein, and NuMA (nuclear mitotic apparatus protein) – dynactin. This asymmetric localization may be functionally important because similar complexes in *Drosophila* neuroblasts are essential for asymmetric division (Chia et al., 2008).

In addition to basal epidermal cells, mouse neuroepithelial stem cells and hematopoietic precursor cells undergo both asymmetric and symmetric divisions. In the mammalian central nervous system, embryonic neuroepithelial cells first undergo symmetric division to expand their population and then switch to asymmetric divisions for neurogenesis. This switch involves a change in cleavage plane orientation from perpendicular to parallel to the plane of the apical lamina, leading to an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1 – 2%) of the entire neuroepithelial cell plasma membrane (Kosodo et al., 2004). Somewhat similarly, mouse hematopoietic progenitor cells are capable of both symmetric and asymmetric divisions in cultures supported by stromal cells (Wu et al., 2007). A pro-differentiation stromal cell line increased the frequency of asymmetric division, whereas a pro-proliferation stromal cell line promoted symmetric division. These observations indicate that niche signaling can also control the asymmetry of stem cell division at a populational level.

Although niche induction accounts for asymmetric division in some types of stem cells, it may not play a role in all types of stem cells. In some stem cells there is an intrinsic polarity where molecules are segregated along an axis and serves as determinant of cell fate after cell division. In these cases the orientation of mitotic spindle has to be coordinated with the asymmetric localization of the cell fate regulators. This is the case, for example of *Numb* homologue during hematopoietic precursor cell division. So looking for hematopoietic niche and signaling between microenviroment cells and the stem cell we could " envisage"(imagine; idealize, construct) a model for stem cell to decide between symmetric or asymmetric division.

Networks Establishing Hematopoietic Stem Cell Multipotency and Self-Renewal 27

Arai, F.; Hirao, A.; Ohmura, M.; Sato, H.; Matsuoka, S.; Takubo, K.; Ito, K.; Koh, G.Y. &

Arai, F.& Suda T.(2007). Maintenance of quiescent hematopoietic stem cells in the

Avecilla, S.T.; Hattori, K.; Heissig, B.; Tejada, R.; Liao, F.; Shido, K.; Jin, D.K.; Dias, S.; Zhang,

Awaya, N.; Baerlocher, G.M.; Manley, T.J.; Sanders, J.E.; Mielcarek ,M.; Torok-Storb, B. &

Baron, M.(2001). Induction of embryonic hematopoietic and endothelial stem/progenitor cells by hedgehog-mediated signals. *Differentiation*. Oct;68(4-5):175-85. Bendall, S.C.; Stewart, M.H.; Menendez, P. George,D.; Vijayaragavan, K.; Werbowetski-

Bijl, J.; Thompson, A.; Ramirez-Solis, R.; Krosl, J.; Grier, D.G.; Lawrence, H.J. &

Blank, U.; Karlsson, G. & Karlsson, S.(2008). Signaling pathways governing stem-cell fate.

Bowie, M.B.; McKnight, K.D.; Kent, D.G.; McCaffrey, L.; Hoodless, P.A. & Eaves,

regulators in murine embryonic stem cells. *Nature*. May 18;441(7091):349-53. Bradford, G.B.; Williams, B.; Rossi, R. & Bertoncello, I.(1997). Quiescence, cycling, and

Brun, A.C.; Björnsson, J.M.; Magnusson, M.; Larsson, N.; Leveén, P.; Ehinger, M.; Nilsson, E.

Blackburn, E.H.(1991). Structure and function of telomeres. *Nature*. 350:569–573. Blackburn, E.H. (2001). Switching and signaling at the telomere. *Cell* 106: 661–673.

pluripotent human cells in vitro. *Nature*. Aug 30; 448 (7157):1015-21. Bhardwaj, G.; Murdoch, B.; Wu, D.; Baker, D.P.; Williams, K.P.; Chadwick, K.; Ling, L.E.;

quiescence in the bone marrow niche. *Cell*. Jul 23;118(2):149-61.

osteoblastic niche. *Ann N Y Acad Sci*. Jun;1106:41-53.

required for thrombopoiesis. *Nat Med*. Jan;10(1):64-71.

*Transplant*. 8(11):597-600.

Feb;2(2):172-80.

May;25(5):445-53.

*Blood*. Jun1;103(11):4126-33.

*Blood*. Jan 15;111(2):492-503.

Suda,T.(2004). Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell

F.; Hartman. T.E.; Hackett, N.R.; Crystal, R.G.; Witte, L.; Hicklin, D.J.; Bohlen, P.; Eaton, D.; Lyden, D.; de Sauvage, F.& Rafii, S.(2004). Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is

Lansdorp, P.M. (2002). Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure*? Biol Blood Marrow* 

Ogilvie, T.; Ramos-Mejia, V.; Rouleau, A.; Yang, J.; Bossé, M.; Lajoie, G.& Bhatia, M.(2007). IGF and FGF cooperatively establish the regulatory stem cell niche of

Karanu, F.N. & Bhatia, M.(2001). Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. *Nat Immunol*.

Sauvageau,G.(2006). Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells. *Blood*. Jul 1;108(1):116-22.

C.J.(2006).Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect. *J Clin Invest.* Oct;116(10):2808-16. Boyer, L.A.; Plath, K.; Zeitlinger, J.; Brambrink, T.; Medeiros, L.A.; Lee, T.I.; Levine, S.S.;

Wernig, M.; Tajonar, A.; Ray, M.K.; Bell, G.W.; Otte, A.P.; Vidal, M.; Gifford, D.K.; Young, R.A.& Jaenisch, R.(2006). Polycomb complexes repress developmental

turnover in the primitive hematopoietic stem cell compartment. *Exp Hematol*.

& Karlsson, S.(2004). Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells.

A particularly exciting development in basic stem cell research in the past few years is the discovery of novel functions of cell cycle regulators in controlling the asymmetry of stem cell division, as timely reviewed by Chia et al (2008). For example, the *cdc2/cdk1* level controls whether a neural or muscle progenitor undergoes symmetric or asymmetric division. In neuroblasts, high levels of CDK1 during mitosis are required for the asymmetric localization of apical and basal protein complexes. In addition, Aurora and Polo kinases act as tumor suppressors in neuroblasts by preventing excess self-renewal, implicating the function of asymmetric division in restricting over-proliferation. The mutations of these two kinase genes affect the asymmetric localization of aPKC, Numb, Partner of Numb, and Notch, causing symmetric division to generate two daughter neuroblasts. In addition, anaphase-promoting complex/cyclosome is also required for the localization of Miranda and its cargo proteins (Prospero, Brain Tumor, and Staufen). More surprisingly, even cyclin E, a G1 cyclin, is involved in asymmetric neuroblast division.

Interestingly in epidermal progenitors the decision of choosing between symmetric cell division or asymmetric cell decision is tightly regulated. Two control points have been indentified: expression of Inscutable and recruitment of NuMA to the apical cell cortex. Moreover in embryonic lung distal epithelium Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling with the participation of NuMa protein (El-Hashad et al , 2011).
