**5. Conclusion**

The ex-vivo expansion of HSC represents a promising approach to obtain large enough quantities for therapeutic intervention in cell and gene therapy protocols. Derivatives of hESCs and iPS cells are also expected to be employed as *de novo* HSC source for therapeutic settings. However, as described in the previous section, practical and ethical issues must be settled before clinical practice can begin. In both cases, the chemical biology approach using small molecules as tools or drugs holds unquestionably greater promise in the outcome of the final goal.

Even though a few molecules are being tested in clinical assays, the ideal soluble factor that enables to increase the number of rare HSC during the *ex vivo* growth culture without limiting their regeneration capacities has yet to be found. Most attempts have been unsuccessful because i) suitable expansion *in vitro* has been mostly correlated with loss of

Searching for the Key to Expand Hematopoietic Stem Cells 231

Abe, S., Lauby, G., Boyer, C., Rennard, S. I. & Sharp, J. G. (2003). *Transplanted BM and BM* 

Aguila, J. R., Liao, W., Yang, J., Avila, C., Hagag, N., Senzel, L. & Ma, Y. (2011). *SALL4 is a robust stimulator for the expansion of hematopoietic stem cells.* Blood *118*, 576-585. Almeida-Porada, G., Zanjani, E. D. & Porada, C. D. (2010). *Bone marrow stem cells and liver* 

Amsellem, S. & Fichelson, S. (2006). *Ex vivo expansion of human hematopoietic stem cells by passive transduction of the HOXB4 homeoprotein.* J Soc Biol *200*, 235-241. Amsellem, S., Pflumio, F., Bardinet, D., Izac, B., Charneau, P., Romeo, P. H., Dubart-

*stem cells by direct delivery of the HOXB4 homeoprotein.* Nat Med *9*, 1423-1427. Antonchuk, J., Sauvageau, G. & Humphries, R. K. (2002). *HOXB4-induced expansion of adult* 

Araki, H., Mahmud, N., Milhem, M., Nunez, R., Xu, M., Beam, C. A. & Hoffman, R. (2006).

Araki, H., Yoshinaga, K., Boccuni, P., Zhao, Y., Hoffman, R. & Mahmud, N. (2007).

*cell divisions while retaining their repopulating potential.* Blood *109*, 3570-3578. Baird, A. (1994). *Fibroblast growth factors: activities and significance of non-neurotrophin* 

Baum, C., Dullmann, J., Li, Z., Fehse, B., Meyer, J., Williams, D. A. & von Kalle, C. (2003).

Becker, P. S., Taylor, J. A., Trobridge, G. D., Zhao, X., Beard, B. C., Chien, S., Adair, J., Kohn,

Bernardo, A. S., Faial, T., Gardner, L., Niakan, K. K., Ortmann, D., Senner, C. E., Callery, E.

Bejsovec, A. (2005). *Wnt pathway activation: new relations and locations.* Cell *120*, 11-14.

*into Embryonic and Extraembryonic Lineages.* Cell Stem Cell *9*, 144-155. Bhardwaj, G., Murdoch, B., Wu, D., Baker, D. P., Williams, K. P., Chadwick, K., Ling, L. E.,

*human hematopoietic cells via BMP regulation*. Nat Immunol *2*, 172-180. Bhatia, M., Bonnet, D., Wu, D., Murdoch, B., Wrana, J., Gallacher, L. & Dick, J. E. (1999). *Bone* 

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

*neurotrophic growth factors.* Curr Opin Neurobiol *4*, 78-86.

*modified lentiviral vector.* Gene Ther *17*, 1244-1252.

*cells.* J Exp Med *189*, 1139-1148.

*111*, 492-503.

*side population cells contribute progeny to the lung and liver in irradiated mice.*

Kupperschmitt, A. & Fichelson, S. (2003). *Ex vivo expansion of human hematopoietic* 

*Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-*

*Chromatin-modifying agents permit human hematopoietic stem cells to undergo multiple* 

*Side effects of retroviral gene transfer into hematopoietic stem cells.* Blood *101*, 2099-2114.

D. B., Wagner, J. E., Shimamura, A. & Kiem, H. P. (2010). *Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-*

M., Trotter, M. W., Hemberger, M., Smith, J. C.*,* et al*.* (2011). *BRACHYURY and CDX2 Mediate BMP-Induced Differentiation of Human and Mouse Pluripotent Stem Cells* 

Karanu, F. N. & Bhatia, M. (2001). *Sonic hedgehog induces the proliferation of primitive* 

*morphogenetic proteins regulate the developmental program of human hematopoietic stem* 

**7. References** 

Cytotherapy 5, 523-533.

*regeneration.* Exp Hematol *38*, 574-580.

*hematopoietic stem cells ex vivo.* Cell *109*, 39-45.

*modifying agents.* Exp Hematol *34*, 140-149.

the regenerative capacities of HSC *in vivo;* ii) no straight forward method allows the association of *in vitro* observations with the *in vivo* outcome; iii) testing the *in vivo* effect of each molecule independently would be costly, time-consuming and would need an imposing number of mice which is ethically inconceivable.

In an attempt to develop new tools that might overcome some of these limitations, we have developed an innovative screening strategy to identify molecules for their potential to improve the *in vitro* HSC self-renewal and proliferation while preserving the HSC regenerative capacities *in vivo* (Sii Felice K, Grosselin J, Leboulch P, Tronik-Le Roux D, manuscript in preparation). Our approach is based on stem cells labeling with specific barcodes before exposure to the molecules (Fig. 7). Then, prior to their infusion in myeloablated mice, all the treated cells are pooled. Several weeks after transplantation, the identification of barcodes present in the blood and the BM of transplanted mice will enable the precise retrospective quantification of the initial effect of the molecule.

Fig. 7. Schematic representation of the strategy developed to simultaneously test dozens of molecules. Each well contains barcoded-HSC (1) treated by a particular molecule. After several days of *in vitro* culture (2), all the cells are pooled, infused in myeloablated mice. The identification of barcodes in blood and BM of transplanted mice will enable the precise retrospective quantification of the initial effect of the molecule.

This strategy might facilitate the development of high-throughput screening for fast and effective identification of small molecules that can be used to burst the production of HSC. This will undoubtedly accelerate the promise of regenerative medicine as a routine therapeutic modality for many blood diseases as well as for gene and cell therapy.
