Studies on the biology of ex vivo expanded hematopoietic stem/progenitor cells: prevention of Apoptosis to improve the efficiency of expansion, cryopreservation and engraftment

Abstract

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. The pancaspase inhibitor zVADfmk and calpain 1 inhibitor, zLLYfmk are well known for exerting anti apoptosis, and hence used in various studies including the cryopreservation of cells and cell lines where apoptosis is a major concern. Apart from their biochemical role of reducing the apoptosis, these two proteases are implicated in the regulation of cell proliferation and motility. Our earlier consistent observation of higher incidence of apoptosis in the cytokine cultured cord blood derived CD34+ cells, prompted us to assess the role of these two proteases in the CD34+ cell expansion. The present study was undertaken to assess whether the transient regulation (using pharmacological inhibitors) of apoptosis could improve the outcome of expansion. Here we expanded the CB CD34+ cells with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. Apart from the reduction in cellular apoptosis, a novel role of apoptotic protease inhibitors was observed in increasing the CD34+ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. Interestingly, we also noticed a higher gene expression of signaling molecules like Notch-jagged, Wnt5a, and Foxo3a which are directly involved in stem cell renewal and maintenance. The cytokine expanded graft is known to exhibit reduced/altered adhesion molecules/integrin profile. The presence of either zVADfmk/zLLYfmk enhanced the homing molecule CXCR4 expression and increased the CD34+CXCR4+ subset in the expansion culture. The in vitro migration as well as adhesion properties was significantly increased in these sets. The expression of major integrins and adhesion molecules like VLA-4 and ICAM1 and HCAM1 were also up regulated.