Study of stem cell homing & self-renewal marker gene profile of ex vivo expanded human CD34+ cells manipulated with a mixture of cytokines & stromal cell-derived factor 1

Background & objectives: Next generation transplantation medicine aims to develop stimulating cocktail for increased ex vivo expansion of primitive hematopoietic stem and progenitor cells (HSPC). The present study was done to evaluate the cocktail GF (Thrombopoietin + Stem Cell factor + Flt3-ligand) and homing-defining molecule Stromal cell-derived factor 1 (SDF1) for HSPC ex vivo expansion. Methods: Peripheral blood stem cell (n= 74) harvests were analysed for CD34(hi)CD45(lo) HSPC. Immunomagnetically enriched HSPC were cultured for eight days and assessed for increase in HSPC, colony forming potential in vitro and in vivo engrafting potential by analyzing human CD45+ cells. Expression profile of genes for homing and stemness were studied using microarray analysis. Expression of adhesion/homing markers were validated by flow cytometry/confocal microscopy. Results: CD34(hi)CD45(lo) HSPC expansion cultures with GF+SDF1 demonstrated increased nucleated cells (n= 28, P< 0.001), absolute CD34+ cells (n= 8, P= 0.021) and increased colony forming units (cfu) compared to unstimulated and GF-stimulated HSPC. NOD-SCID mice transplanted with GF+SDF1-HSPC exhibited successful homing/engraftment (n= 24, P< 0.001). Microarray analysis of expanded HSPC demonstrated increased telomerase activity and many homing-associated genes (35/49) and transcription factors for stemness/self-renewal (49/56) were significantly upregulated in GF+SDF1 stimulated HSPC when compared to GF-stimulated HSPC. Expression of CD44, CXCR4, CD26, CD14, CD45 and soluble IL-6 in expanded cultures were validated by flow cytometry and confocal microscopy. Interpretation & conclusions: Cocktail of cytokines and SDF1 showed good potential to successfully expand HSPC which exhibited enhanced ability to generate multilineage cells in short-term and long-term repopulation assay. This cocktail-mediated stem cell expansion has potential to obviate the need for longer and large volume apheresis procedure making it convenient for donors.