**3.1.3 Pleiotropin (Ptn)**

216 Advances in Hematopoietic Stem Cell Research

The studies described in this section establish that epigenetic alterations can modulate the self-renewal process. Epigenetic state in stem cells can be stably heritable or can be erased (partly or completely) by cell division. These changes might facilitate the transition of a progenitor cell to a self-renewing stem cell, or might prompt a stem cell to differentiate,

As described in the previous section, the strategy for stem-cell expansion involves activation of regulators that encourage HSC self-renewal and/or inhibition of pathways that mediate, differentiation or apoptosis by using primarily genetic modification approaches. An alternative strategy might imply pharmacological intervention by using a variety of small molecules. The term "small molecule" refers to a molecular entity that interacts with one or more molecular targets and effects a change in biological state while having minimal side effects. These small molecules, defined by a known structure, may be chemicals, proteins, small interfering RNAs or antibodies. Some of the most effective compounds for *ex vivo*

Cytokines are secreted proteins that regulate many aspects of hematopoiesis, such as, immune responses and inflammation. Numerous attempts have been made to use classic hematopoietic cytokines for the purpose of expanding HSC *in vitro*. Many interleukins, including interleukin (IL)-3, IL-6, and IL-11, Flt-3 ligand, TPO and SCF have extensively been investigated. In most cases, efforts to expand HSC have failed because of differentiation of HSC and subsequent loss of their reconstitution capacity. The combination of these molecules has however allowed maintaining HSC in culture for several days allowing their use in protocols for gene or cell therapies. Here we describe some examples of

TPO, acting through its receptor c-MPL, is the chief cytokine that regulates megakaryocyte production. However, several studies suggest that TPO can act to increase the *ex-vivo* expansion of HSC (Sitnicka et al., 1996). This effect was far more effective when used in combination with other cytokines including SCF, fms-like tyrosine kinase 3 ligand (FLT3-L), IL-3 or IL-6. Human cord blood cells expanded with this cytokine cocktail were shown to provide good short- and long-term platelet recovery and lymphomyeloid reconstitution in NOD-SCID mice (Ohmizono et al., 1997; Pineault et al., 2010). Further, a non peptidyl molecule agonist of c-MPL, NR-101, was found to be more efficient than TPO in expanding HSC. Indeed, 7 days culture of human cord blood CD34+ or CD34+CD38-, treated with NR-101 induced a 2-fold increase in their number compare to TPO and a 2.9-fold or 2.3-fold increase in SRC numbers compared to freshly isolated CD34+ cells or TPO-expanded cells respectively. As it was not more efficient than TPO in inducing megakaryocyte expansion, its effect seemed to be HSC specific. NR-101 treatment appeared to persistently activate STAT5 and to induce a long-term

divide or lose its ability to self-renew.

**3.1 Regulation by cytokines** 

**3.1.1 Thrombopoietin (TPO)** 

**3. Compounds modifying HSC capacities** 

maintaining or expanding HSC are reviewed below.

cytokines that were used to maintain HSC levels in culture.

accumulation of HIF-1α (Nishino et al., 2009).

Pleiotropin, which have mitogenic and angiogenic activities, has been found to be essential for maintenance of murine HSC. Mice transplanted with LSK CD34- cells treated with Ptn and a standard cocktail of cytokines showed 6-fold increase in HSC frequency compared to cells treated with cytokines alone. *In vivo,* systemic administration of Ptn was found to increase the number of BM LSK cells both in irradiated and nonirradiated mice, suggesting a role for this factor in the *in vivo* regeneration of HSC. Treatment of human cord blood Lin-CD34+CD38- cells with Ptn for 7 days induced a 4-fold increase in CFC content and a 3- or 7 fold improved engraftment at 4 or 7 weeks respectively in NOD-SCID mice compared with controls. This factor may activate the PI3-Kinase/AKT and Notch pathways by alleviating activation of its receptor, RPTP-/ (Himburg et al., 2010).

#### **3.2 Transcription factors: The HOX- family**

#### **3.2.1 HOXB4**

The homeobox gene family member HoxB4 is the most investigated transcription factor for its potential to increase the self-renewal potential of HSC. HOXB4 belongs to a large family of transcription factors that share a highly conserved DNA-binding domain, the homeodomain. In mammals, there are 39 *Hox* genes grouped in four clusters referred to as A, B, C and D. In the hematopoietic system, 16 different *Hox* genes are transcribed during normal hematopoiesis. Primitive subpopulations express primarily genes of the A and B cluster (Giampaolo et al., 1995; Pineault et al., 2002; Sauvageau et al., 1994). Mice transplanted with marrow overexpressing HOXB4 resulted in a 47-fold increase of the competitive repopulating unit (CRU) numbers and did not develop leukemic transformation (Sauvageau et al., 1995). *HOXB4* overexpression in mouse HSC cultured for 14 days induced a primitive cell-specific growth advantage contrary to a progressive depletion of HSC usually observed under these conditions. Total cell growth (mostly mature cells) was enhanced by 2-fold, progenitors by 3-fold and HSC by 1000-fold in cells overexpressing HOXB4 (Antonchuk et al., 2002).

In humans, transient overexpression of HOXB4 in hematopoietic cord blood cells, did not increase proliferation of primitive progenitors, frequency of CFC, and LTC-ICs but induced an iincrease in myeloid differentiation (Brun et al., 2003). Other studies showed that

Searching for the Key to Expand Hematopoietic Stem Cells 219

The capacity of soluble HOXB4 to expand human HSC was verified using several recombinant human HOXB4 proteins. The N-terminal-tat and C-terminal histidine-tagged version of HOXB4 (T-HOXB4-H) had the highest activity in expanding CFC (10-fold) and

Surveys of *Hox* gene expression in HSC enriched populations showed dominancy of the *Hox*A cluster. In d14.5 fetal liver populations enriched for HSC, the expression of HOXA4 is a log higher than that of HOXB4. The fact that during this phase of development HSC undergo their major expansion, combined with the high homology and functional redundancy found within *Hox* paralog groups, suggests a putative role of HOXA4 to expand HSC with negligible or null oncogenic potential. HOXA4 overexpressing HSC expanded 6.6-fold after a week of culture. Although HOXA4 expressing HSC produced mature myeloid and lymphoid progeny in irradiated recipient mice, B-cell progenitors were preferentially expanded compared to myeloid progenitors

HOXC4, another member of the *Hox* family, is also expressed in proliferating hematopoietic cells suggesting a role in the control of normal proliferation. Using retroviral gene transfer in human CD34+ cells, Daga et *al.* showed that HOXC4 induced an *in vitro* expansion of committed cells and early hematopoietic progenitors, with the most striking effect on LTC-IC (13-fold expansion) (Daga et al., 2000). These results are consistent with those of Amsellem and Fichelson who showed a more efficient expansion of human CD34+/CD38low cells on MS-5 cell line secreting HOXC4 compared to those secreting HOXB4. The simultaneous presence of HOXB4 and HOXC4 seems synergize to improve expansion (Amsellem and Fichelson, 2006). However, the *in vivo* effect of HOXC4 still remains to be

All these observations clearly implicated Hox family proteins in HSC self renewal but further studies are required to determine if the use of these compounds could be suitable for

The low efficiency obtained with purified proteins and the safety concerns when attempting to expand HSC with viral vector-mediated gene transfer (Baum et al., 2003) lead to searching for alternative and safer approaches. One of these promising strategies involved

Chemical molecules constitute a particularly useful tool for modifying biological signaling pathways since they can be arrayed in chemical libraries for high-throughput analysis, and they can be withdrawn from the biological system once the desired effect is obtained. The use of a small molecule allows the study of the kinetics of a response in a more subtle and graduated way that is not possible with gene disruption techniques. These molecules may be further transposed into drugs for therapeutic use. Their use is rapid and cost-

LTC-IC (15-fold), and a 1.5- to 2.7-fold increase in SRC (Tang et al., 2009).

**3.2.2 Other HOX family proteins** 

(Fournier et al., 2011).

established.

effective.

clinical applications.

**3.3 Chemical compounds** 

the use of chemical compounds.

What are the sources of molecules available?

enforced high level of HOXB4 expression in human hematopoietic cord blood cells cultured for 24 hours induced a 5-10-fold increase in LTC-IC and a 4-fold increase in SRC (Buske et al., 2002). However, this HOXB4 overexpression markedly impaired the lymphoid and myeloerythroid differentiation (Schiedlmeier et al., 2003). Altogether these studies demonstrated that high levels of HOXB4 perturbed the myeloid differentiation program both *in vivo* and *in vitro* and are consistent with a dose dependant activity of HOXB4 to control the differentiation or self-renewal of HSC (Klump et al., 2005).

To increase the effect of HOXB4, a *NUP98-HOXB4* fusion gene was engeeniered since the fusion of *Hox* genes with the nucleoporine gene *NUP98* is often reported in leukemia. Ohta et *al.* observed, in a murine transplantation model, a 300-fold increase in CRUs among NUP98-HOXB4-overexpressing cells compared to only 80-fold increase with HOXB4 alone. An even higher increase (2000-fold) was observed using the *NUP98-HOXA10* fusion gene that, in contrast to HOXB4, blocks terminal differentiation and leads to a sustained output of cells with a "primitive" phenotype (Pineault et al., 2005; Pineault et al., 2004). The authors did not observe any long-term hematological defect in recipients repopulated with NUP98- HOXA10 expanded HSC (Ohta et al., 2007). However, these results contrast with those obtained by Watts et al. in a nonhuman primate stem cell transplantation model. Transplantation of comparable doses of HOXB4- and NUP98-NUP98-HOXA10 overexpressing cells revealed that HOXB4 contributed more to early hematopoiesis whereas NUP98-HOXA10 contributed more to later hematopoeisis. The emergence of a deleterious effect, such as leukaemia, could not be monitored due to the short survey period of the study (Watts et al., 2011).

In 2006, Zhang et al. investigate the ability of HOXB4 to expand HSC in a clinically relevant nonhuman primate competitive repopulation model. They found an initial 56-fold advantage for the *HOXB4*-transduced cells which decline significantly over time (Zhang et al., 2006). In addition, the first appearence of myeloid leukemia linked to HOXB4 expression were observed two years later, both in the original group of monkeys (1 out of 2) and in dogs (2 out of 2) that received cells transduced with a HOXB4 expressing vector (Zhang et al., 2008b). None of the 40 dogs and monkeys that received cells transduced with control vectors developed leukemia. Besides, a profound growth inhibition and a rapid cell differentiation was induced by siRNA knocking down HOXB4 using a cell line derived from the leukemic cells of one animal. The direct implication of HOXB4 in the development of leukemia can not be certify since analysis of the vector insertion sites in the genome of all tumors revealed insertion of the transgene near or within protooncogenes, such as *c-myb* and *PRDM16* (Zhang et al., 2008b).

To avoid the use of retroviral vectors, Amsellem et al. generate an MS-5 stromal cell line secreting HOXB4 to expand human cord blood hematopoietic cells. Using a 5-week long term culture system, they show a 4-fold increase in LTC-IC and 2.5-fold increase in SRC in NOD-SCID mice. This expansion did not appear to interfere with myeloid or lymphoid differentiation. However, the coculture system might not be suitable for clinical applications (Amsellem et al., 2003). To avoid this issue, Krosl et al. used a soluble recombinant HOXB4 protein fused to a small peptide derived from the HIV TAT protein. TAT-HOXB4 treatment of murine HSC for 4 days expanded approximately 4- to 6-fold and were 8-20 times more numerous than non treated HSC. This TAT-HOXB4 expanded population retained its normal *in vivo* potential for differentiation and long-term repopulation (Krosl et al., 2003). The capacity of soluble HOXB4 to expand human HSC was verified using several recombinant human HOXB4 proteins. The N-terminal-tat and C-terminal histidine-tagged version of HOXB4 (T-HOXB4-H) had the highest activity in expanding CFC (10-fold) and LTC-IC (15-fold), and a 1.5- to 2.7-fold increase in SRC (Tang et al., 2009).

#### **3.2.2 Other HOX family proteins**

218 Advances in Hematopoietic Stem Cell Research

enforced high level of HOXB4 expression in human hematopoietic cord blood cells cultured for 24 hours induced a 5-10-fold increase in LTC-IC and a 4-fold increase in SRC (Buske et al., 2002). However, this HOXB4 overexpression markedly impaired the lymphoid and myeloerythroid differentiation (Schiedlmeier et al., 2003). Altogether these studies demonstrated that high levels of HOXB4 perturbed the myeloid differentiation program both *in vivo* and *in vitro* and are consistent with a dose dependant activity of HOXB4 to

To increase the effect of HOXB4, a *NUP98-HOXB4* fusion gene was engeeniered since the fusion of *Hox* genes with the nucleoporine gene *NUP98* is often reported in leukemia. Ohta et *al.* observed, in a murine transplantation model, a 300-fold increase in CRUs among NUP98-HOXB4-overexpressing cells compared to only 80-fold increase with HOXB4 alone. An even higher increase (2000-fold) was observed using the *NUP98-HOXA10* fusion gene that, in contrast to HOXB4, blocks terminal differentiation and leads to a sustained output of cells with a "primitive" phenotype (Pineault et al., 2005; Pineault et al., 2004). The authors did not observe any long-term hematological defect in recipients repopulated with NUP98- HOXA10 expanded HSC (Ohta et al., 2007). However, these results contrast with those obtained by Watts et al. in a nonhuman primate stem cell transplantation model. Transplantation of comparable doses of HOXB4- and NUP98-NUP98-HOXA10 overexpressing cells revealed that HOXB4 contributed more to early hematopoiesis whereas NUP98-HOXA10 contributed more to later hematopoeisis. The emergence of a deleterious effect, such as leukaemia, could not be monitored due to the short survey period of the

In 2006, Zhang et al. investigate the ability of HOXB4 to expand HSC in a clinically relevant nonhuman primate competitive repopulation model. They found an initial 56-fold advantage for the *HOXB4*-transduced cells which decline significantly over time (Zhang et al., 2006). In addition, the first appearence of myeloid leukemia linked to HOXB4 expression were observed two years later, both in the original group of monkeys (1 out of 2) and in dogs (2 out of 2) that received cells transduced with a HOXB4 expressing vector (Zhang et al., 2008b). None of the 40 dogs and monkeys that received cells transduced with control vectors developed leukemia. Besides, a profound growth inhibition and a rapid cell differentiation was induced by siRNA knocking down HOXB4 using a cell line derived from the leukemic cells of one animal. The direct implication of HOXB4 in the development of leukemia can not be certify since analysis of the vector insertion sites in the genome of all tumors revealed insertion of the transgene near or within protooncogenes, such as *c-myb*

To avoid the use of retroviral vectors, Amsellem et al. generate an MS-5 stromal cell line secreting HOXB4 to expand human cord blood hematopoietic cells. Using a 5-week long term culture system, they show a 4-fold increase in LTC-IC and 2.5-fold increase in SRC in NOD-SCID mice. This expansion did not appear to interfere with myeloid or lymphoid differentiation. However, the coculture system might not be suitable for clinical applications (Amsellem et al., 2003). To avoid this issue, Krosl et al. used a soluble recombinant HOXB4 protein fused to a small peptide derived from the HIV TAT protein. TAT-HOXB4 treatment of murine HSC for 4 days expanded approximately 4- to 6-fold and were 8-20 times more numerous than non treated HSC. This TAT-HOXB4 expanded population retained its normal *in vivo* potential for differentiation and long-term repopulation (Krosl et al., 2003).

control the differentiation or self-renewal of HSC (Klump et al., 2005).

study (Watts et al., 2011).

and *PRDM16* (Zhang et al., 2008b).

Surveys of *Hox* gene expression in HSC enriched populations showed dominancy of the *Hox*A cluster. In d14.5 fetal liver populations enriched for HSC, the expression of HOXA4 is a log higher than that of HOXB4. The fact that during this phase of development HSC undergo their major expansion, combined with the high homology and functional redundancy found within *Hox* paralog groups, suggests a putative role of HOXA4 to expand HSC with negligible or null oncogenic potential. HOXA4 overexpressing HSC expanded 6.6-fold after a week of culture. Although HOXA4 expressing HSC produced mature myeloid and lymphoid progeny in irradiated recipient mice, B-cell progenitors were preferentially expanded compared to myeloid progenitors (Fournier et al., 2011).

HOXC4, another member of the *Hox* family, is also expressed in proliferating hematopoietic cells suggesting a role in the control of normal proliferation. Using retroviral gene transfer in human CD34+ cells, Daga et *al.* showed that HOXC4 induced an *in vitro* expansion of committed cells and early hematopoietic progenitors, with the most striking effect on LTC-IC (13-fold expansion) (Daga et al., 2000). These results are consistent with those of Amsellem and Fichelson who showed a more efficient expansion of human CD34+/CD38low cells on MS-5 cell line secreting HOXC4 compared to those secreting HOXB4. The simultaneous presence of HOXB4 and HOXC4 seems synergize to improve expansion (Amsellem and Fichelson, 2006). However, the *in vivo* effect of HOXC4 still remains to be established.

All these observations clearly implicated Hox family proteins in HSC self renewal but further studies are required to determine if the use of these compounds could be suitable for clinical applications.

#### **3.3 Chemical compounds**

The low efficiency obtained with purified proteins and the safety concerns when attempting to expand HSC with viral vector-mediated gene transfer (Baum et al., 2003) lead to searching for alternative and safer approaches. One of these promising strategies involved the use of chemical compounds.

Chemical molecules constitute a particularly useful tool for modifying biological signaling pathways since they can be arrayed in chemical libraries for high-throughput analysis, and they can be withdrawn from the biological system once the desired effect is obtained. The use of a small molecule allows the study of the kinetics of a response in a more subtle and graduated way that is not possible with gene disruption techniques. These molecules may be further transposed into drugs for therapeutic use. Their use is rapid and costeffective.

What are the sources of molecules available?

Searching for the Key to Expand Hematopoietic Stem Cells 221

In 2008, using a two step culture system, Seet et al. showed that VPA induced a 2-fold expansion of human cord blood CD34+CD45+ cells. Higher numbers of treated cells resided in the S phase compare to controls. VPA-treated cells reconstituted hematopoiesis in NOD-SCID mouse with a 6-fold higher efficiency compare to control cells. The advantage of using VPA resides on the fact that this molecule is clinically well-known since it has been used for more than 25 years to treat neurologic disorders (Seet et al., 2009). Chlamydocin, was showed to enhance Thy-1 expression on human CD34+ cells and to display a 4-fold increase

Fig. 4. A diagrammatic representation of an experimental design typology to test the effect of molecules on HSC expansion. Each molecule is added individually to the *in vitro* culture of HSC and the expansion capacities are then measured. However, infusion of the treated cells in myeloablated mice is essential to answer the question (**?**) on whether the HSC treated with the selected molecule have still the capacity to regenerate blood cells in transplanted animals.

Another HDAC inhibitor, trichostatin A (TSA), and 5-aza-2'-deoxycytidine (5azaD), a DNA methyl transferase inhibitor where shown to act in synergy to yield a 12.5-fold increase of human CD34+CD90+ cells after 9 days of culture in comparison to the input cell numbers, a 9.8-fold increase in the numbers of CFU and a 9.6-fold increase in SRC. Several genes implicated in HSC self-renewal including *HOXB4*, *BMI1*, *GATA2*, *P21*, and *P27* were up-

Several clinical observations have suggested that copper plays a role in regulating HSC development. Peled et al. reported that modulation of cellular copper content might shift the balance between self-renewal and differentiation (Peled et al., 2005; Peled et al., 2002). This group cultured human CD34+ cord blood cells with the copper chelator TEPA during extended periods of time and showed a higher percentage of early progenitors (CD34+CD38-

) in the TEPA-treated cultures compared with controls and a 1- to 3-log-fold

,

regulated in the 5azaD/TSA-treated cells (Araki et al., 2006; Araki et al., 2007).

**3.3.2 Copper chelator tetraethylenepentamine (TEPA)** 

CD34+CD38-

Lin-

in SRC in NOD-SCID (Young et al., 2004).

Historically, the pharmaceutical companies gathered the collections of molecules accumulated during the year in-house companies. These molecules can come from two different sources, one from natural origin and the other from chemically-synthesized compounds. Several companies have pooled their collections through partnerships to increase the size and diversity. At present, a large collection of oriented chemical libraries is available. In the milieu of academia, access to these collections is almost impossible unless a very restrictive partnership is framed. The number of screenable drug candidates have dramatically increased in the last years, and might account for 10 000 to 1 000 000 compounds. The difficulty to use these large collections resides in the ability to order millions of natural products, many of which are available in only limited amounts and are not yet completely characterized or even purified. Further, to identify a molecule producing the desired biological effect, different concentrations covering several orders of magnitude should be initially screened. This is why their widespread use has not yet been generalized and most discoveries to date are mainly available through the pharmaceutical industry. During the past ten years, various companies have specialized in the provision of allpurpose or targeted libraries. ChemBridge, ChemDiv, Asinex, Prestwick, Maybridge, enamine, Interbioscreen, TimTec can be mentioned as examples of commercially available collections. These libraries are relatively diverse and oriented "drug-like" (Kugawa et al., 2007). Small-molecule compounds approved for use as drugs may also be "repurposed" for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Major efforts are now underway to produce comprehensive collections of these small molecules amenable to high-throughput screening (Huang et al., 2011).

During the last ten years, cell-based phenotypic and pathway-specific screens using synthetic small molecules have provided new insights into stem cell biology and help to identify a number of small molecules that can be used to selectively (a) control self-renewal of embryonic and adult stem cells; (b) expand therapeutically desirable mature cell types; (c) control lineage commitment; and (d) enhance the reversion of lineage-restricted cells back to the multipotent or pluripotent state. All four practices are beginning to find application in therapeutic settings.

In this section we will focus on chemical compounds that were used to expand HSC. However, the most important question to keep in mind is whether the *in vitro* expanded cells preserve their capacities to regenerate hematopoiesis *in vivo* (Fig. 4).

#### **3.3.1 Chromatin-modifying agents**

Valproic acid (VPA) and chlamydocin are histone deacetylase (HDAC) inhibitors that exert their activity by interacting with the catalytic site of HDACs.

VPA was first studied by De Felice et al. on human CD34+ cells isolated from cord blood, mobilized peripheral blood and BM. They showed that VPA preserves the CD34+ population after 1 week (40-89%) or 3 weeks (21-52%) of culture with cytokines and VPA increases H4 acetylation levels at specific sites on *HOXB4* and AC133 (De Felice et al., 2005).

Historically, the pharmaceutical companies gathered the collections of molecules accumulated during the year in-house companies. These molecules can come from two different sources, one from natural origin and the other from chemically-synthesized compounds. Several companies have pooled their collections through partnerships to increase the size and diversity. At present, a large collection of oriented chemical libraries is available. In the milieu of academia, access to these collections is almost impossible unless a very restrictive partnership is framed. The number of screenable drug candidates have dramatically increased in the last years, and might account for 10 000 to 1 000 000 compounds. The difficulty to use these large collections resides in the ability to order millions of natural products, many of which are available in only limited amounts and are not yet completely characterized or even purified. Further, to identify a molecule producing the desired biological effect, different concentrations covering several orders of magnitude should be initially screened. This is why their widespread use has not yet been generalized and most discoveries to date are mainly available through the pharmaceutical industry. During the past ten years, various companies have specialized in the provision of allpurpose or targeted libraries. ChemBridge, ChemDiv, Asinex, Prestwick, Maybridge, enamine, Interbioscreen, TimTec can be mentioned as examples of commercially available collections. These libraries are relatively diverse and oriented "drug-like" (Kugawa et al., 2007). Small-molecule compounds approved for use as drugs may also be "repurposed" for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Major efforts are now underway to produce comprehensive collections of these small molecules amenable to high-throughput screening

During the last ten years, cell-based phenotypic and pathway-specific screens using synthetic small molecules have provided new insights into stem cell biology and help to identify a number of small molecules that can be used to selectively (a) control self-renewal of embryonic and adult stem cells; (b) expand therapeutically desirable mature cell types; (c) control lineage commitment; and (d) enhance the reversion of lineage-restricted cells back to the multipotent or pluripotent state. All four practices are beginning to find application in

In this section we will focus on chemical compounds that were used to expand HSC. However, the most important question to keep in mind is whether the *in vitro* expanded

Valproic acid (VPA) and chlamydocin are histone deacetylase (HDAC) inhibitors that exert

VPA was first studied by De Felice et al. on human CD34+ cells isolated from cord blood, mobilized peripheral blood and BM. They showed that VPA preserves the CD34+ population after 1 week (40-89%) or 3 weeks (21-52%) of culture with cytokines and VPA increases H4 acetylation levels at specific sites on *HOXB4* and AC133 (De Felice et al., 2005).

cells preserve their capacities to regenerate hematopoiesis *in vivo* (Fig. 4).

their activity by interacting with the catalytic site of HDACs.

(Huang et al., 2011).

therapeutic settings.

**3.3.1 Chromatin-modifying agents** 

In 2008, using a two step culture system, Seet et al. showed that VPA induced a 2-fold expansion of human cord blood CD34+CD45+ cells. Higher numbers of treated cells resided in the S phase compare to controls. VPA-treated cells reconstituted hematopoiesis in NOD-SCID mouse with a 6-fold higher efficiency compare to control cells. The advantage of using VPA resides on the fact that this molecule is clinically well-known since it has been used for more than 25 years to treat neurologic disorders (Seet et al., 2009). Chlamydocin, was showed to enhance Thy-1 expression on human CD34+ cells and to display a 4-fold increase in SRC in NOD-SCID (Young et al., 2004).

Fig. 4. A diagrammatic representation of an experimental design typology to test the effect of molecules on HSC expansion. Each molecule is added individually to the *in vitro* culture of HSC and the expansion capacities are then measured. However, infusion of the treated cells in myeloablated mice is essential to answer the question (**?**) on whether the HSC treated with the selected molecule have still the capacity to regenerate blood cells in transplanted animals.

Another HDAC inhibitor, trichostatin A (TSA), and 5-aza-2'-deoxycytidine (5azaD), a DNA methyl transferase inhibitor where shown to act in synergy to yield a 12.5-fold increase of human CD34+CD90+ cells after 9 days of culture in comparison to the input cell numbers, a 9.8-fold increase in the numbers of CFU and a 9.6-fold increase in SRC. Several genes implicated in HSC self-renewal including *HOXB4*, *BMI1*, *GATA2*, *P21*, and *P27* were upregulated in the 5azaD/TSA-treated cells (Araki et al., 2006; Araki et al., 2007).

#### **3.3.2 Copper chelator tetraethylenepentamine (TEPA)**

Several clinical observations have suggested that copper plays a role in regulating HSC development. Peled et al. reported that modulation of cellular copper content might shift the balance between self-renewal and differentiation (Peled et al., 2005; Peled et al., 2002). This group cultured human CD34+ cord blood cells with the copper chelator TEPA during extended periods of time and showed a higher percentage of early progenitors (CD34+CD38- , CD34+CD38- Lin- ) in the TEPA-treated cultures compared with controls and a 1- to 3-log-fold

Searching for the Key to Expand Hematopoietic Stem Cells 223

The continual production of ROS in the *in vitro* culture (Iiyama et al., 2006) might be overcome by the addition of antioxidants. These molecules will maintain the ROS at a low level, thereby regulating the proliferation, growth, signal transduction, and gene expression

Antioxidants are classified into enzyme and non-enzyme antioxidants. Enzyme antioxidants include superoxide dismutase, catalase, and glutathione peroxidase. Non-enzyme

The application of enzyme antioxidants is limited because of the poor stability and ease of inactivation (Wojcik et al., 2010). However, when culturing mouse HSC in the presence of catalase, the number of short-term or long-term HSC with LSK immune markers was significantly increased and the stem cells begin to degenerate as the catalase is removed

Ascorbic acid (vitamin C) is a natural water-soluble antioxidant but under some conditions such as the air, heat, light, alkaline substances, enzymes and trace amount of copper oxide and iron, oxidation of vitamin C could be accelerated and the oxidative products lead to the damage of cellular DNA. The ascorbic acid 2-phosphate (AA2P), one derivative of vitamin C, is stable at 37°C in cell culture media and has no cytotoxic effect; therefore it might constitute an advantageous antioxidant (Duarte et al., 2009). Reducing oxidative stress by N-acetyl-L-cysteine (NAC) may enhance the viability and engraftment of HSC as treatment of gene corrected BM mononuclear cells or purified CD34(+) cells from FANCA patients with the reducing agent NAC showed increased CFC (Becker et

Although the current amplification under normal oxygen can expand a certain number of HSC, the application of glutathione for stem cell mobilization and re-infusion as well as the application of AA2P in the *in vitro* amplification culture of cells may become effective methods for protecting the hematopoietic reconstitution capacity of HSC (Hao et al., 2011). Moreover, *in vitro* culturing HSC-enriched samples under O2 concentrations that more closely resemble the BM environment (low O2 concentrations, 1–3%) might also improve

Prostaglandin E2 (PGE2) was first identified as capable of enhancing HSC formation in zebrafish, following a high-toughput chemical screen. This effect was also tested using murine transplantation assays. When murine BM cells where briefly treated *ex vivo* by PGE2, a 3-fold increase in the CFU number and a 3.3-fold increase of SRC 6 weeks post transplantation were observed (North et al., 2007). Hoggatt et al. confirmed enhanced murine HSC engraftment following PGE2 exposure as they observed a 4-fold increase in HSC 20 weeks after transplantation. The increase in chimerism was still present in primary recipient 32 weeks post-transplant and in secondary recipients without additional PGE2 treatment. Several studies were performed to determine whether the action of PGE2 on HSC could be the result of an increase in HSC numbers, homing capability, proliferation, survival, or a combination thereof. Hoggatt et al. observed a significant increase in homing of PGE2-treated LSK cells. This was partially attributed to an increase in CXCR4 expression, a SDF1α specific receptor. This effect also occurs in

their expansion and preserve proper stem cell functions for engraftment.

of the cells (Chen et al., 2008).

antioxidant includes vitamin C.

(Gupta et al., 2006).

al., 2010).

**3.3.4 PGE2** 

expansion of CD34+ cells compare with that of controls. They cultured human CD133+ cord blood cells during 3 weeks, in order to use a clinically suitable protocol, and found that the median output value of CD34+ cells increased by 89-fold, CD34+CD38 by 30-fold and CFU by 172-fold over the input values. Moreover, the CD34+ cells expanded with TEPA appeared to show improved NOD-SCID engraftment compare to control cells (Peled et al., 2004a; Peled et al., 2004b). Based on these data, a phase 1 trial was initiated. In this study, a portion of a single cord blood unit was cultured with TEPA and cytokines for 21 days and co-infused with the remainder of the untreated cell fraction. Although this methodology showed a 219-fold expansion of total nucleated cells *in vitro*, it did not improve the time to neutrophil or platelet recovery (de Lima et al., 2008). A phase 2/3 study is under way in more than 28 centers in the United States, Europe, and Israel, to evaluate the safety and efficacy of this approach ("StemEx") in 100 patients with advanced hematologic malignancies (http://clinicaltrials.gov/ct2/show/NCT00469729).

#### **3.3.3 Oxygen, reactive oxygen species and antioxidants**

Low oxygen levels were also described to play a beneficial role on HSC expansion *in vitro*. This is consistent with the observation that protection of HSC *in vivo* is achieved by a predominantly low-oxygen environment of the stem-cell niche (Cipolleschi et al., 1993; Eliasson and Jonsson, 2010).

The positive effect of hypoxia on the survival and/or self-renewal of the HSC population *in vitro* was demonstrated quantitatively on human marrow cells with Lin-CD34+CD38 phenotype which are enriched in SRC. A significant increase in SRC after 4 days was found in cultures under 1.5% O2 compared to normoxic conditions. The positive effect of hypoxia on SRCs is short-lived but their engraftment into immmunocompromised mice was to some extent improved (Danet et al., 2003).

Similar studies have been performed with cord blood cells (Hermitte et al., 2006). The authors reported preferential survival of primitive HSC among cord blood CD34+ cells in cultures under 0.1% O2. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2. At 0.1% O2, 46.5%+/-19.1% of divided cells returned to G0 compared with 7.9%+/-0.3% at 20%. This shows a return of the cycling CD34+ cells into G0, a quiescent state that characterizes steady-state HSC.

During the process of HSC purification or mobilization from the BM to the peripheral blood, the cells go across different levels of oxygenation until reach maxima in culture assays. Furthermore, cell factors added to these cultures can lead to an abnormal increase in reactive oxygen species (ROS) in the HSC and to a ROS stress that might change their properties and functions (Hao et al., 2011; Ito et al., 2006; Pervaiz et al., 2009). These ROS are unstable reactive molecular species possessing an unpaired electron that are produced continuously in cells as a byproduct of metabolism. They participate in vital signal transduction pathways but they can also oxidize DNA, proteins, and lipids leading to cell differentiation, senescence, and apoptosis. Notably, the mouse long-term repopulating HSC capacities were found in a Roslow population (Jang and Sharkis, 2007). This cell population has a higher self-renewal activity than a Roshigh population both *in vitro* and *in vivo*. Moreover, distinct metabolic profiles of HSC reflect their location in the hypoxic niche (Simsek et al., 2010; Takubo et al., 2010).

The continual production of ROS in the *in vitro* culture (Iiyama et al., 2006) might be overcome by the addition of antioxidants. These molecules will maintain the ROS at a low level, thereby regulating the proliferation, growth, signal transduction, and gene expression of the cells (Chen et al., 2008).

Antioxidants are classified into enzyme and non-enzyme antioxidants. Enzyme antioxidants include superoxide dismutase, catalase, and glutathione peroxidase. Non-enzyme antioxidant includes vitamin C.

The application of enzyme antioxidants is limited because of the poor stability and ease of inactivation (Wojcik et al., 2010). However, when culturing mouse HSC in the presence of catalase, the number of short-term or long-term HSC with LSK immune markers was significantly increased and the stem cells begin to degenerate as the catalase is removed (Gupta et al., 2006).

Ascorbic acid (vitamin C) is a natural water-soluble antioxidant but under some conditions such as the air, heat, light, alkaline substances, enzymes and trace amount of copper oxide and iron, oxidation of vitamin C could be accelerated and the oxidative products lead to the damage of cellular DNA. The ascorbic acid 2-phosphate (AA2P), one derivative of vitamin C, is stable at 37°C in cell culture media and has no cytotoxic effect; therefore it might constitute an advantageous antioxidant (Duarte et al., 2009). Reducing oxidative stress by N-acetyl-L-cysteine (NAC) may enhance the viability and engraftment of HSC as treatment of gene corrected BM mononuclear cells or purified CD34(+) cells from FANCA patients with the reducing agent NAC showed increased CFC (Becker et al., 2010).

Although the current amplification under normal oxygen can expand a certain number of HSC, the application of glutathione for stem cell mobilization and re-infusion as well as the application of AA2P in the *in vitro* amplification culture of cells may become effective methods for protecting the hematopoietic reconstitution capacity of HSC (Hao et al., 2011). Moreover, *in vitro* culturing HSC-enriched samples under O2 concentrations that more closely resemble the BM environment (low O2 concentrations, 1–3%) might also improve their expansion and preserve proper stem cell functions for engraftment.

#### **3.3.4 PGE2**

222 Advances in Hematopoietic Stem Cell Research

expansion of CD34+ cells compare with that of controls. They cultured human CD133+ cord blood cells during 3 weeks, in order to use a clinically suitable protocol, and found that the

172-fold over the input values. Moreover, the CD34+ cells expanded with TEPA appeared to show improved NOD-SCID engraftment compare to control cells (Peled et al., 2004a; Peled et al., 2004b). Based on these data, a phase 1 trial was initiated. In this study, a portion of a single cord blood unit was cultured with TEPA and cytokines for 21 days and co-infused with the remainder of the untreated cell fraction. Although this methodology showed a 219-fold expansion of total nucleated cells *in vitro*, it did not improve the time to neutrophil or platelet recovery (de Lima et al., 2008). A phase 2/3 study is under way in more than 28 centers in the United States, Europe, and Israel, to evaluate the safety and efficacy of this approach ("StemEx") in 100 patients with advanced hematologic malignancies

Low oxygen levels were also described to play a beneficial role on HSC expansion *in vitro*. This is consistent with the observation that protection of HSC *in vivo* is achieved by a predominantly low-oxygen environment of the stem-cell niche (Cipolleschi et al., 1993;

The positive effect of hypoxia on the survival and/or self-renewal of the HSC population *in vitro* was demonstrated quantitatively on human marrow cells with Lin-CD34+CD38 phenotype which are enriched in SRC. A significant increase in SRC after 4 days was found in cultures under 1.5% O2 compared to normoxic conditions. The positive effect of hypoxia on SRCs is short-lived but their engraftment into immmunocompromised mice was to some

Similar studies have been performed with cord blood cells (Hermitte et al., 2006). The authors reported preferential survival of primitive HSC among cord blood CD34+ cells in cultures under 0.1% O2. After 72 hours, cells were 1.5 and 2.5 times more in quiescence (G0) at 3% and 0.1% O2. At 0.1% O2, 46.5%+/-19.1% of divided cells returned to G0 compared with 7.9%+/-0.3% at 20%. This shows a return of the cycling CD34+ cells into G0, a quiescent

During the process of HSC purification or mobilization from the BM to the peripheral blood, the cells go across different levels of oxygenation until reach maxima in culture assays. Furthermore, cell factors added to these cultures can lead to an abnormal increase in reactive oxygen species (ROS) in the HSC and to a ROS stress that might change their properties and functions (Hao et al., 2011; Ito et al., 2006; Pervaiz et al., 2009). These ROS are unstable reactive molecular species possessing an unpaired electron that are produced continuously in cells as a byproduct of metabolism. They participate in vital signal transduction pathways but they can also oxidize DNA, proteins, and lipids leading to cell differentiation, senescence, and apoptosis. Notably, the mouse long-term repopulating HSC capacities were found in a Roslow population (Jang and Sharkis, 2007). This cell population has a higher self-renewal activity than a Roshigh population both *in vitro* and *in vivo*. Moreover, distinct metabolic profiles of HSC reflect their location in the hypoxic niche

by 30-fold and CFU by

median output value of CD34+ cells increased by 89-fold, CD34+CD38-

(http://clinicaltrials.gov/ct2/show/NCT00469729).

Eliasson and Jonsson, 2010).

extent improved (Danet et al., 2003).

state that characterizes steady-state HSC.

(Simsek et al., 2010; Takubo et al., 2010).

**3.3.3 Oxygen, reactive oxygen species and antioxidants** 

Prostaglandin E2 (PGE2) was first identified as capable of enhancing HSC formation in zebrafish, following a high-toughput chemical screen. This effect was also tested using murine transplantation assays. When murine BM cells where briefly treated *ex vivo* by PGE2, a 3-fold increase in the CFU number and a 3.3-fold increase of SRC 6 weeks post transplantation were observed (North et al., 2007). Hoggatt et al. confirmed enhanced murine HSC engraftment following PGE2 exposure as they observed a 4-fold increase in HSC 20 weeks after transplantation. The increase in chimerism was still present in primary recipient 32 weeks post-transplant and in secondary recipients without additional PGE2 treatment. Several studies were performed to determine whether the action of PGE2 on HSC could be the result of an increase in HSC numbers, homing capability, proliferation, survival, or a combination thereof. Hoggatt et al. observed a significant increase in homing of PGE2-treated LSK cells. This was partially attributed to an increase in CXCR4 expression, a SDF1α specific receptor. This effect also occurs in

Searching for the Key to Expand Hematopoietic Stem Cells 225

The transcription factor SALL4 was reported to play a role in maintaining ES cell pluripotency through interaction with Oct4 and Nanog (Wu et al., 2006; Yang et al., 2010). It was recently showed that overexpression of SALL4 can expand *ex vivo* human mobilized HSC from peripheral blood (Aguila et al., 2011). SALL4-transduced cells seemed capable of *ex vivo* expansion of both, CD34+CD38- and CD34+CD38+ cells and showed enhanced stem cell engraftment and long term repopulation capacity in NOD-SCID mice. Moreover, human CD34+ cells cultured 3 to 4 days with a soluble SALL4 fusion protein (TAT-SALL4B) showed a 10-fold increase in total mononuclear cells, a 8-fold increase in CD34+ cells and a 10-fold increase in the CFU number compare to controls (Aguila et al., 2011). However, *in vivo* studies with this fusion protein still have to be conducted to validate that these expanded

Considering the interest in HSC expansion for treatment of both malignant and nonmalignant diseases as well as their use in gene therapy and the difficulty to obtain *ex vivo* expansion of HSC without loss of their regeneration capacities, relevant methods to produce

One of these methods was initiated 20 years ago when ESC could be cultivated *in vitro* and directed to generate hematopoietic cells (Wiles and Keller, 1991). Since then, culture conditions were constantly optimized and allowed the differentiation into specific hematopoietic lineages such as erythroid and myeloid lineages, T and B lymphocytes and megakaryocytes (for review see Sakamoto et al., 2010). These protocols were then adapted to human (h) ESC. These cells like their murine counterparts, are karyotypically stable, capable of prolonged self-renewal, and might differentiate into most cell types. These properties might be exploited for therapeutic benefits to cure many human degenerative diseases and

Different methods were established to generate hematopoietic progenitors and specific lineages from mouse ESC including embryoid bodies formation, coculture with stromal cells, and direct differentiation in coated plates using a mixture of cytokines and growth factors without stromal cells (Tian and Kaufman, 2008). These protocols were then optimized for efficient differentiation of hESC into early mesodermal cells (Bernardo et al., 2011) and for obtaining defined hematopoietic precursors from ES cells (Chiang and Wong,

The ultimate goal of these strategies is to produce HSC capable of robust, long-term, multilineage engraftment to alleviate blood cells diseases; however the numbers and the capacities of the *de novo* cells generated are not quite sufficient to fulfill the clinical challenge. At present, multipotent hematopoietic progenitors (short-term HSC) with limited engrafting ability in transplanted mice were obtained (Woods et al., 2011). Other groups reported efficient generation of cells that mostly produce the myeloid lineage following long term engraftment or produce CD34+ hematopoietic precursors that have phenotype similar

cells are still able to reconstitute hematopoiesis in transplanted recipients.

**3.3.6 SALL4** 

**4.** *De novo* **generation of HSC** 

*de novo* HSC have emerged.

**4.1 Obtaining HSC from ESC** 

resulted in intense biomedical studies.

2011; Salvagiotto et al., 2011).

human HSC, since PGE2-treated cord blood cells transplanted into NOD-SCID mice displayed an enhanced homing to marrow. In addition, PGE2 treatment increased survivin expression, reduced intracellular active caspase-3 that lead to enhanced HSC survival and increased the percentage of cycling cells (Hoggatt et al., 2009). Frish et al. treated mice *in vivo* with PGE2 by intraperitoneal injection twice a day for 16 days. They observed a significant increase of the LSK population without inhibiting their differentiation. The treatment expands preferentially the short-term-HSC/MPP subpopulation since this advantage was lost 6 weeks post-transplant in primary recipients and in secondary transplants. The disparities between these studies may be the result of the extended exposure of mice to PGE2 compared with a short term pulse used hitherto (Frisch et al., 2009).

Goessling et al. briefly treated human cord blood CD34+ cells *in vitro* with dimethyl-PGE2 (dmPGE2). They showed that dmPGE2 treatment decreased apoptosis, increased 1.4-fold the CFU number and enhanced engraftment of unfractionated and CD34+ cord blood cells after xenotransplantation in NOD-SCID mice. Using a non-human primate transplantation model, they found no significant enhancement of CD34+-treated cells engraftment but showed that dmPGE2 treatment had no negative impact on HSC function, including multilineage repopulation, even 1 year post-transplantation. They suggested that these results reflect suboptimal compound dosing and anticipate the use of 50µM rather than 10µM of dmPGE2 in future transplantation assays (Goessling et al., 2011). Based on these data, this brief *ex vivo* incubation with dmPGE2 is currently being tested in a phase 1 clinical trial in which adults with hematologic malignancies receive a non-myeloablative conditioning treatment followed by double-unit cord blood transplantation in which 1 of the 2 cord blood units has been incubated with dmPGE2 before infusion (http://clinicaltrials.gov/ct2/show/ NCT00890500).

#### **3.3.5 Aryl Hydrocarbon receptor (AhR) antagonists**

Using a high-throughput screen based on CD34/CD133 expression, Boitano et al identified a purine derivative (StemRegenin1 or SR1) capable of *in vitro* enhancing the levels of a CD34+ cell population derived from blood of mobilized donors. SR1 added to human CD34+ cells cultured for 5 weeks led to a 10-fold increase in total nucleated cells, a 47-fold increase in CD34+ cells and a 65-fold increase in CFU. CD34+ cord blood cells cultured in the presence of SR1 for 3 weeks revealed a 17-fold increase in SRC content in NOD-SCID Gamma (NSG) primary recipient and a 12-fold increase in the number of secondary SRC compared to input (Boitano et al., 2010). Additional screens followed by a quantitative structure-activity relationship identified three novel compounds (i.e SR2, SR3 and SR4), structurally distinct from SR1, that expand the number of human CD34+ cells. Experiments that aimed to determine the ability of cord blood derived human HSC expanded with these molecules to engraft NSG mice are still undergoing (Bouchez et al., 2011). SR1, SR2, SR3 and SR4 were showed to act as antagonists of AhR signaling. Indeed, this receptor has been implicated in HSC biology and hematopoietic disease through numerous factors including c-MYC, HES-1, PU.1, C/EBP, -catenin, CXCR4, and STAT-5 (Singh et al., 2009). However, the precise mechanism whereby an AhR inhibitor might induce HSC self-renewal remains unknown.

#### **3.3.6 SALL4**

224 Advances in Hematopoietic Stem Cell Research

human HSC, since PGE2-treated cord blood cells transplanted into NOD-SCID mice displayed an enhanced homing to marrow. In addition, PGE2 treatment increased survivin expression, reduced intracellular active caspase-3 that lead to enhanced HSC survival and increased the percentage of cycling cells (Hoggatt et al., 2009). Frish et al. treated mice *in vivo* with PGE2 by intraperitoneal injection twice a day for 16 days. They observed a significant increase of the LSK population without inhibiting their differentiation. The treatment expands preferentially the short-term-HSC/MPP subpopulation since this advantage was lost 6 weeks post-transplant in primary recipients and in secondary transplants. The disparities between these studies may be the result of the extended exposure of mice to PGE2 compared with a short term pulse used

Goessling et al. briefly treated human cord blood CD34+ cells *in vitro* with dimethyl-PGE2 (dmPGE2). They showed that dmPGE2 treatment decreased apoptosis, increased 1.4-fold the CFU number and enhanced engraftment of unfractionated and CD34+ cord blood cells after xenotransplantation in NOD-SCID mice. Using a non-human primate transplantation model, they found no significant enhancement of CD34+-treated cells engraftment but showed that dmPGE2 treatment had no negative impact on HSC function, including multilineage repopulation, even 1 year post-transplantation. They suggested that these results reflect suboptimal compound dosing and anticipate the use of 50µM rather than 10µM of dmPGE2 in future transplantation assays (Goessling et al., 2011). Based on these data, this brief *ex vivo* incubation with dmPGE2 is currently being tested in a phase 1 clinical trial in which adults with hematologic malignancies receive a non-myeloablative conditioning treatment followed by double-unit cord blood transplantation in which 1 of the 2 cord blood units has been incubated with dmPGE2 before infusion

Using a high-throughput screen based on CD34/CD133 expression, Boitano et al identified a purine derivative (StemRegenin1 or SR1) capable of *in vitro* enhancing the levels of a CD34+ cell population derived from blood of mobilized donors. SR1 added to human CD34+ cells cultured for 5 weeks led to a 10-fold increase in total nucleated cells, a 47-fold increase in CD34+ cells and a 65-fold increase in CFU. CD34+ cord blood cells cultured in the presence of SR1 for 3 weeks revealed a 17-fold increase in SRC content in NOD-SCID Gamma (NSG) primary recipient and a 12-fold increase in the number of secondary SRC compared to input (Boitano et al., 2010). Additional screens followed by a quantitative structure-activity relationship identified three novel compounds (i.e SR2, SR3 and SR4), structurally distinct from SR1, that expand the number of human CD34+ cells. Experiments that aimed to determine the ability of cord blood derived human HSC expanded with these molecules to engraft NSG mice are still undergoing (Bouchez et al., 2011). SR1, SR2, SR3 and SR4 were showed to act as antagonists of AhR signaling. Indeed, this receptor has been implicated in HSC biology and hematopoietic disease through numerous factors including c-MYC, HES-1, PU.1, C/EBP, -catenin, CXCR4, and STAT-5 (Singh et al., 2009). However, the precise mechanism whereby an AhR inhibitor might

hitherto (Frisch et al., 2009).

(http://clinicaltrials.gov/ct2/show/ NCT00890500).

**3.3.5 Aryl Hydrocarbon receptor (AhR) antagonists** 

induce HSC self-renewal remains unknown.

The transcription factor SALL4 was reported to play a role in maintaining ES cell pluripotency through interaction with Oct4 and Nanog (Wu et al., 2006; Yang et al., 2010). It was recently showed that overexpression of SALL4 can expand *ex vivo* human mobilized HSC from peripheral blood (Aguila et al., 2011). SALL4-transduced cells seemed capable of *ex vivo* expansion of both, CD34+CD38- and CD34+CD38+ cells and showed enhanced stem cell engraftment and long term repopulation capacity in NOD-SCID mice. Moreover, human CD34+ cells cultured 3 to 4 days with a soluble SALL4 fusion protein (TAT-SALL4B) showed a 10-fold increase in total mononuclear cells, a 8-fold increase in CD34+ cells and a 10-fold increase in the CFU number compare to controls (Aguila et al., 2011). However, *in vivo* studies with this fusion protein still have to be conducted to validate that these expanded cells are still able to reconstitute hematopoiesis in transplanted recipients.
