**5. Expansion and selection of genetically modified HSCs ex vivo**

Although much hope is currently invested into various schemes aimed at in vivo selection of gene-modified HSCs, a substantially simpler and arguably more elegant solution may be achieved if protocols for long-term culture and robust ex vivo expansion of HSCs could be developed. Very significant expansion of HSCs that occurs during embryonic development indicates that this might be eventually possible.

Over the last two decades, quite a few HSC culture protocols have been developed. The earlier established conditions involved cultivation in the presence of serum and cocktail of "classical" cytokines including SCF, IL3, IL6, FLT3L and TPO. Since bovine serum apparently contains factors that induce differentiation and/or apoptosis of HSCs, recent, more advanced protocols have been developed, which use defined, serum-free conditions that offer better reproducibility and minimize rapid loss of long-term repopulating HSCs during ex vivo culture and transduction with lenti- and retroviral vectors (Mostoslavsky et al., 2005).

In addition to classical cytokines, a number of new growth factors that have pronounced effect on HSC maintenance and expansion were identified in the last years. Among the most important are FGF1 (de Haan et al., 2003), IGFBP2 (Huynh et al., 2008), and several members of angiopoeitin-like family, in particular Angptl3 and 5 (Zhang et al., 2006).

Several major signaling pathways figuring prominently during embryonic development, in particular during specification of hematopoietic lineage, were shown to be important for adult HSC biology. Among those, Notch and Wnt pathways are currently considered as of the most immediate interest as far as HSC-niche interactions and ex vivo expansion are concerned. Stem and progenitor pool-enhancing properties of Notch signaling were demonstrated initially using constitutive Notch1 signaling in murine hematopoietic cells, which produced immortalized, cytokine-dependent stem cell-like cells (Varnum-Finney et al., 2000), and constitutive Notch4 signaling in human cord blood cells, which resulted in significant increase in cells repopulating immunodeficient mice (Vercauteren & Sutherland, 2004). Later on, culture of human CD34+ precursors with the immobilized Notch ligand Delta1 and cytokines was shown to result in a substantial increase in NOD/SCIDrepopulating cells (Delaney et al., 2010); similar results were obtained for mouse cells with immobilized Jagged1 ligand (Toda et al., 2011).

As for Wnt signaling, initial studies indicated that overexpression of activated betacatenin expanded the pool of HSCs in long-term cultures as judged by both phenotype and function. Wnt3a protein induced self-renewal of haematopoietic stem cells, whereas ectopic expression of inhibitors of the Wnt signalling pathway led to suppression of HSC growth in vitro and reduced reconstitution in vivo (Reya et al., 2003; Willert et al., 2003). Later publications demonstrated, though, that inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (Cobas et al., 2004), whereas activation of beta-catenin enforced cell cycle entry of hematopoietic stem cells, thus leading to exhaustion of the long-term stem cell pool (Sheller et al., 2006). Some recent studies demonstrate that it is the noncanonical Wnt signaling promoted by Wnt5a rather than the canonical one, that supports maintenance of competitive repopulating murine HSCs in culture (Buckley et al., 2011; Nemeth et al., 2007).

Although much hope is currently invested into various schemes aimed at in vivo selection of gene-modified HSCs, a substantially simpler and arguably more elegant solution may be achieved if protocols for long-term culture and robust ex vivo expansion of HSCs could be developed. Very significant expansion of HSCs that occurs during embryonic development

Over the last two decades, quite a few HSC culture protocols have been developed. The earlier established conditions involved cultivation in the presence of serum and cocktail of "classical" cytokines including SCF, IL3, IL6, FLT3L and TPO. Since bovine serum apparently contains factors that induce differentiation and/or apoptosis of HSCs, recent, more advanced protocols have been developed, which use defined, serum-free conditions that offer better reproducibility and minimize rapid loss of long-term repopulating HSCs during ex vivo culture and transduction with lenti- and retroviral vectors (Mostoslavsky et

In addition to classical cytokines, a number of new growth factors that have pronounced effect on HSC maintenance and expansion were identified in the last years. Among the most important are FGF1 (de Haan et al., 2003), IGFBP2 (Huynh et al., 2008), and several members

Several major signaling pathways figuring prominently during embryonic development, in particular during specification of hematopoietic lineage, were shown to be important for adult HSC biology. Among those, Notch and Wnt pathways are currently considered as of the most immediate interest as far as HSC-niche interactions and ex vivo expansion are concerned. Stem and progenitor pool-enhancing properties of Notch signaling were demonstrated initially using constitutive Notch1 signaling in murine hematopoietic cells, which produced immortalized, cytokine-dependent stem cell-like cells (Varnum-Finney et al., 2000), and constitutive Notch4 signaling in human cord blood cells, which resulted in significant increase in cells repopulating immunodeficient mice (Vercauteren & Sutherland, 2004). Later on, culture of human CD34+ precursors with the immobilized Notch ligand Delta1 and cytokines was shown to result in a substantial increase in NOD/SCIDrepopulating cells (Delaney et al., 2010); similar results were obtained for mouse cells with

As for Wnt signaling, initial studies indicated that overexpression of activated betacatenin expanded the pool of HSCs in long-term cultures as judged by both phenotype and function. Wnt3a protein induced self-renewal of haematopoietic stem cells, whereas ectopic expression of inhibitors of the Wnt signalling pathway led to suppression of HSC growth in vitro and reduced reconstitution in vivo (Reya et al., 2003; Willert et al., 2003). Later publications demonstrated, though, that inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (Cobas et al., 2004), whereas activation of beta-catenin enforced cell cycle entry of hematopoietic stem cells, thus leading to exhaustion of the long-term stem cell pool (Sheller et al., 2006). Some recent studies demonstrate that it is the noncanonical Wnt signaling promoted by Wnt5a rather than the canonical one, that supports maintenance of competitive repopulating murine HSCs in culture (Buckley et al., 2011;

of angiopoeitin-like family, in particular Angptl3 and 5 (Zhang et al., 2006).

**5. Expansion and selection of genetically modified HSCs ex vivo** 

indicates that this might be eventually possible.

immobilized Jagged1 ligand (Toda et al., 2011).

Nemeth et al., 2007).

al., 2005).

Yet another line of evidence indicates that activation of beta-catenin in the niche components rather than in HSCs may produce support of LTR cells ex vivo (Nemeth et al., 2009). Currently, there is little doubt that Wnt signaling plays important role in HSC biology, but the issue is apparently more complex than was implied by initial publications and remains highly controversial.

Other embryonic signaling pathways also might be exploited in HSC culture. Morphogens of the hedgehog family, namely Sonic and Indian hedgehogs, are able to support ex vivo expansion of human NOD/SCID repopulating cells (Bhardwaj et al., 2001; Kobune et al., 2004), despite the fact that in vivo Hedgehog signaling seems to not be necessary for adult murine hematopoietic stem cell function (Hofmann et al., 2009). BMP4, a member of BMP superfamily, is a critical component of the hematopoietic niche that regulates both HSC number and function (Goldman et al., 2009), and is able to expand NOD/SCID-repopulating cells in culture (Hutton et al., 2006).

In addition to the use of secreted proteins to for ex vivo HSC culture, one apparent trend of the last years is the application of low-molecular weight chemicals, in particular agonists or inhibitors of particular intracellular signaling pathways, for ex vivo culture. Thus, specific inhibitor of p38 kinase induces self-renewal and ex vivo expansion of HSCs as shown by the in vitro cobblestone area forming cell assay and serial transplantation (Wang et al., 2011). GSK-3β inhibitors, which stimulate Wnt signaling, were shown to promote engraftment of cultured HSCs (Ko et al., 2011; Trowbridge et al., 2006). Of significant clinical interest is the finding that ex vivo treatment with stabilized prostaglandin E2 enhances frequency of both hematopoietic progenitors and long-term repopulating HSCs present as analyzed by competitive transplantation (North et al., 2007). According to other data, only the short-term repopulating HSCs are expanded by this treatment, though (Frisch et al., 2009).

The initial studies demonstrating substantial degree of expansion of HSCs ex vivo relied the use of stromal cells as feeder layers (Moore et al., 1997). Based on the substantial progress in identification of HSC niches in bone marrow, there is currently a revival of interest in development of protocols for co-culture of HSC with stromal cell layers (Chou & Lodish, 2010; De Toni et al., 2011). These stromal cells produce a range of factors that significantly improve the maintenance and expansion of HSCs in culture, most likely by mimicking more or less successfully niche conditions. Very prominent components of the HSC niche are cell surface proteins, in particular cell adhesion molecules. The importance of cell-cell interactions was highlighted by the study by Wagner et al., 2007, indicating that maintenance of primitive hematopoietic progenitors by stromal lines is associated with expression of cell adhesion proteins rather than with secretory profiles of these lines. In particular, N-cadherin was shown to be an important component of the osteoblastic HSC niche (Zhang et al., 2003). However, importance of N-cadherin for HSC-niche interactions was later questioned (Kiel et al., 2007), thus rising substantial controversy. In an elegant in vitro study Lutolf et al. (2009) have shown that N-cadherin, as well as Wnt3a, are the only proteins among those tested that were capable of supporting self-renewal divisions of HSCs in vitro. N-cadherin expression was also shown to be important for maintenance of long-term repopulating cells in culture (Hosokawa et al., 2010). Ability of stromal cell line FMS/PA6-P to support primitive murine hematopoietic cells was found to depend critically on N-CAM expression (Wang et al., 2005). Yet another cell adhesion protein, namely mKirre, plays a prominent role in hematopoietic supportive capacity of OP9 stromal cells (Ueno et al., 2003).

Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives 449

Factor Observed effects References

Proteins of angiopoeitin-like family provide 20- to 30-fold net expansion of long-term HSCs according to reconstitution

IGFBP2 IGFBP2 enhances ex vivo expansion of mouse HSCs. Huynh et al., 2008

Culturing murine or human cells with surface-immobilized

Wnt3a protein induces self-renewal of haematopoietic stem cells. Wnt10b enhances growth of hematopoietic precursors.

Wnt5a inhibits canonical Wnt signaling and supports maintenance of competitive repopulating murine HSCs in

expansion of human NOD/SCID repopulating cells.

in the percentage of human cells repopulating

Bmp4 BMP4 expands NOD/SCID-repopulating cells in culture. Hutton et al., 2006

TAT-HOXB4 protein produces significant ex vivo expansion

TAT-NF-Ya protein treatment produces several-fold increase

TAT-SALL4B fusion protein rapidly expands long-term

Ex vivo incubation with PGE2 increases the frequency of long-term repopulating HSCs as measured by competitive

SR1, aryl hydrocarbon receptor antagonist, provides substantial increase in cells engrafting into immunodeficient

Pretreatment with GSK-3 inhibitors (BIO or CHIR-911) promotes engraftment and repopulation of ex vivo-

Cord blood CD34+ cells cultured in presence of zVADfmk or zLLYfmk (inhibitors of caspases and calpains, respectively) have a higher ability for engraftment in NOD/SCID mice.

Copper chelator tetraethylenepentamine increases long-term ex vivo expansion and engraftment capabilities of blood

Table 3. Proteins and compounds affecting ex vivo maintenance and expansion of HSCs

of murine HSCs. Krosl et al., 2003

NOD/SCID repopulating cells. Aguila et al, 2011

self-renewal and ex vivo expansion. Wang et al., 2011

rapamycin demonstrate enhanced engraftment. Rohrabaugh et al., 2011

maintenance of long-term repopulating cells in culture. Hosokawa et al., 2010

murine hematopoietic cells. Wang et al., 2005

of OP9 stromal cells. Ueno et al., 2003

Notch ligands resulted in expansion of primitive

serially transplantable, long-term repopulating HSCs. de Haan et al., 2003

Zhang C et al., 2006

Moldenhauer et al.,

Delaney et al., 2010; Toda et al., 2011;

Willert et al., 2003; Congdon et al., 2010

Nemeth et. al, 2007; Buckley et al., 2011

Bhardwaj et al., 2001; Kobune et al., 2004

Domashenko et al.,

North et al., 2007

Boitano et al., 2010

Imai et al., 2010; Sangeetha et al, 2010;

Ko et al., 2011; Trowbridge et al., 2006

Peled et al., 2004

2010

2011

FGF1 FGF1 under serum-free conditions stimulates expansion of

IL32 IL-32 significantly induces the proliferation of HSCs in

Shh, Ihh Sonic hedgehog and Indian hedgehog support ex vivo

SB203580 SB203580, specific p38 inhibitor, leads to increase in HSC

Rapamycin HSCs cultured in vitro in the presence of mTOR inhibitor

N-cadherin N-cadherin expression on stromal cells is important for

N-CAM N-CAM expression on stromal cells supports primitive

mKirre mKirre is responsible for hematopoietic supportive capacity

immunodeficient mice.

transplantation.

expanded HSCs.

progenitors.

("classical" cytokines not listed)

mice.

hematopoietic population.

analysis.

culture.

culture.

Angptl2, 3 and 5

Delta 1, Jagged1 (Notch ligands)

Wnt3a, Wnt10b (Wnt canonical pathway)

Wnt5a (Wnt noncanonical pathway)

TAT-HOXB4 fusion

TAT-NF-Ya fusion

TAT-SALL4B fusion

Prostaglandin E2

StemRegenin 1

GSK-3 inhibitors

Copper helators

zVADfmk, zLLYfmk

protein

protein

protein

Quite promising developments occur currently in the field of 3-D culture (Yuan et al., 2011; Tan et al., 2010; Miyoshi at el., 2011). Despite a relative paucity of data related to the 3-D culture of HSCs, available publications demonstrate significant advantages of this technique and indicate that in combination with correctly chosen or gene-modified stromal cell layers, 3-D culturing may eventually lead to creation of artificial niche that will be able to support substantial expansion of human HSCs ex vivo.

A question of paramount importance for the field is whether specific combinations of soluble factors will be able to attain a bone fide ex vivo expansion of HSCs, or this goal can only be achieved if specific cell surface proteins produced by the niche cells are also employed in the process, or perhaps the only way to the eventual success is the use of supporting stromal cell layers for ex vivo culture? As a number of molecules that contribute to the maintenance of HSCs in vitro and in vivo continues to rise, and there is a steady improvement in techniques for culturing HSCs, chances are that within a matter of a few years, key combination(s) of specific factors and modes of their application that can produce robust self-renewal and expansion of human HSC ex vivo will be identified. Table 3 provides a list, albeit incomplete, of factors and chemicals that, in addition to "classical" cytokines, are being used for maintenance and expansion of HSCs ex vivo.
