The Effect Of Oxygen Tension On Ex Vivo Expansion Characteristics And Physiological Function Of HSCs

It has been demonstrated that ex vivo expansion of hematopoietic stem cells (HSCs) is a feasible strategy which could overcome the cell dose limitation. However, ex vivo culture environment is quite different from in vivo hematopoietic microenvironment, the physiological function of ex vivo expanded HSCs may be lost. Therefore, it is necessary to optimize ex vivo culture conditions. Oxygen tension (pO2) is proved as an important physicochemical factor in HSCs cultures and it has major effects on the ex vivo expansion and physiological function of HSCs. The oxygen tension in normal culture system is 21%, but the oxygen saturation in bone marrow microenvironment is around 5%. In this research, Cord blood CD34+cells were cultured with media at present of or absence of serum at 21%O2 and 5% O2 with the cytokine combination of SCF+IL-3+IL-6 or SCF+TPO+FL. We studied the effect of oxygen tension on ex vivo expansion of total cells, CD34+cells, CD34+CD38- cells and colony-forming cells (CFCs), the differentiation of HSPCs, re-expansion of CD34+cells and physiological function of ex vivo expanded HSCs by non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice model. We found that ex vivo expansion characteristics of HSCs cultured without serum was not affected by oxygen tension. In culture with serum, the results are as followed:(1) The expansion fold of total cells at 21% O2 was significantly higher than that at 5% O2, which demonstrated that the normoxic oxygen condition favored the expansion of total cells. (2) The percentage of CD34+cells, CD34+CD38- cells and CFCs at 5% O2 was significantly higher than that at 21% O2, indicating that the hypoxic condition was more effective on preserving functional HSCs. (3) The re-expansion of CD34+cells was not affected by oxygen tension. (4) Differentation of HSCs to hematopoietic lineages was not affected by oxygen tension. (5) Hematopoietic cells cultured at 21% O2 and 5% O2 both maintained the engraftment ability, the hematopoietic reconstitution ability and colony forming ability. (6) Hematopoietic cells cultured at 5% O2 showed better engraftment capability, multilineage reconstitution ability and colony-forming ability than those cultured at 21% O2, suggesting that hypoxia preserved better the physiological function of HSCs. Our work provided experimental base for clinical use of transplantation of ex vivo expanded HSCs.