**3. Compounds modifying HSC capacities**

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* maintaining or expanding HSC are reviewed below.

### **3.1 Regulation by cytokines**

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 cytokines that were used to maintain HSC levels in culture.

#### **3.1.1 Thrombopoietin (TPO)**

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

#### **3.1.2 Angiopoietin-like 5 (ANGPLT5) and insulin-like growth factor binding protein 2 (IGFBP2)**

Soluble growth factors, such as ANGPLT5 and IGFBP2, produced by the endothelium may enhance HSC expansion *ex vivo* when used with conventional cytokines. Although the addition of ANGPLT5 and/or IGFBP2 to a 10 days-human CD133+ cord blood cells culture has no effect upon the total nucleated cells number *in vitro*, it significantly enhances *in vivo* repopulation of NOD-SCID mice 2 months post-transplantation as well as secondary transplantation (Zhang et al., 2008a). These results were confirmed recently using human cord blood CD34+CD133+ cells cultured for 10 days in the presence of IGFBP2 and ANGPLT5. Expanded cells were shown to be capable of long-term multi-lineage and multisite hematopoiesis in serial reconstitution in NSG mice (Drake et al., 2011).
