In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

AIM: To investigate the potential of hematopoietic stem cells (HSCs) derived from mouse embryonic stem cells (ESCs) to reconstruct hematopoiesis in vivo. METHODS: Using a three-step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34+/ Sca-1+ were identified by flow cytometry analysis, then HSCs (1×109 cells/L) from third-step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS: The method of three-step differentiation, combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells (as high as 58.64%±4.20%) with more CFU-E, CFU-GM and CFU-GEMM populations. The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining. Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% (5/7) successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION: The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.