[Sequentially inducting murine embryonic stem cells into hematopoietic stem cells in vitro by hematopoietic development procedure for reconstitution of hematopoiesis in vivo].

This study was purposed to directly induce murine embryonic stem cells (ESC) into hematopoietic stem cells (HSC) by simulating the spatial and temporal hematopoietic microenvironment changes in embryonic development, and to investigate the function of in vivo hematopoietic reconstitution of these HSC. E14 ESC were induced into embryoid body (EB) firstly. Then the cells from EB were further co-cultured with human aorta-gonad-mesonephros (AGM) region, fetal liver (FL) and bone marrow (BM) stromal cells in Transwell non-contact system in sequential orders. After 6 days of each co-cultured stage, the induced cells derived from EB were collected to analyze the Sca-1(+)c-Kit(+) cells by flow cytometry, check teratoma formation and transplant to BALB/C female mice exposed to lethal dose (60)Co γ-ray. The recipient mice were divided randomly into 5 groups: AGM, AGM + FL, AGM + FL + BM, irradiation control and normal control groups. The survival rates, hematopoietic reconstitution and engraftment of donor cells in the different groups were monitored. The results showed that Sca-1(+)c-Kit(+) cell level in EB cells co-cultured with human AGM region and FL stromal cells reached to peak value (21.96 ± 2.54)%. Teratomas could be found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region stromal cells, while there was no teratoma in the mice after subcutaneous injection of EB cells induced by human AGM region and FL stromal cells. The recipients in AGM group and irradiation control group all died. The survival rate was 77.8% in AGM+FL group, and 66.7% in AGM+FL+BM group. The peripheral blood cell count was near normal at day 21 after transplantation, and Sry gene copies from donor could be detected in recipient mice of these two groups. It is concluded that sequentially inductive system with feeder cells from human AGM region, fetal liver and bone marrow simulating embryonic defined hematopoiesis procedures can enhance the directed differentiation of ESC into HSC which can safely reconstitute hematopoiesis in vivo.