Primary study on inductive methods of hematopoietic stem cellfrom embryonic stem cells in vitro / 中国病理生理杂志

ABSTRACT

AIM: To explore inductive methods of hematopoietic stem cell (HSC) from embryonic stem cells (ESC) in vitro. METHODS: Using mice E14 line, the first step was the primary differentiation in which the ESC form embryoid bodies (EB) in methylcellulose-based cultures with SCF and VEGF. The second step involved the plating of cells originating from the EB into three different system of cultures containing SCF, VEGF,IL-3, IL-6 and EPO for HSC. And identifying HSC by flow cytometry analysis, colonogenic cells assay and Wright-Giemsa stain were also used. RESULTS: By two-step differentiating, it showed that HSC differentiated slowly in methylcellulose medium, percent age of CD34+/Sca-1+ cells slight increased about(31.5?4.7)% after day 14 induction. However, EBs were induced after 10 days to fast differentiate for HSC with more cells population by coculture on bone marrow stromal cells feeder. Flow cytometry analysis showed that percentages of CD34+/Sca-1+ cells might reached to (47.8?6.3)%. The more optimistic system of differentiation was bone marrow stromal cells feeder (BMSCF) in combination with supernatants of stromal cells from mice fetal liver(SSCFL), it significant supported differentiation of ESC into HSC with higher percent (53.6?7.2)%. Colonogenic cell assay and Wright-Giemsa stain confirmed that it possessed character of hematopoietic progenitors. CONCLUSION: Using methods of two-step differentiation, mice ESC were induced to differentiate into HSC by coculturing with BMSCF and SSCFL in combination of SCF,VEGF,IL-3,IL-6 and EPO.