Abstract
Background: Small molecule compounds have been well recognized for their promising power in generation, expansion and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood derived-CD34+ cells. Methods: Considering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pμ and NAM. Eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model. Results: Our data shows that simultaneous use of SB431542 (TGF-β inhibitor), Chir9901 (GSK3 inhibitor) and Bpv (PTEN inhibitor), resulted in a 50-fold increase in the number of CD34+CD38- cells. This was further reflected in approximately 3 times increase in clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of NMRI model of in utero transplantation. These results are in total conformity with up-regulation of HOXB4, GATA2 and CD34 marker gene as well as CXCR4 homing gene. Conclusion: Taken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.
Small molecule–mediated ex vivo expansion of long-term repopulating progenitor cells in cord blood–derived hematopoietic stem cells (HSCs)