In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice / 中国病理生理杂志

ABSTRACT

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.