Primary bone marrow stromal cells associated with cytokines induce ES-D_3 cells to differentiate to hematopoietic cells in vitro

Objective:To study the efficiency of primary bone marrow stromal cells associated with cytokines, i.e. vascular endothelial growth factor (VEGF), stem cell factor (SCF) and thrombopoietin (TPO) inducing ES-D_3 cells to differentiate to hematopoietic cells in vitro. and to explore the method of inducing ES to differentiate to hematopoietic progenitors in vitro.Method:BM samples of 6~8 weeks old Kunming mice were used to prepare primary bone marrow stromal cells (BMSC) feeder layer. Embryoid body (EB) were derived from ES-D_3 cells, and then induced under four different system: spontaneous differentiation control group, cytokines group (VEGF+SCF+TPO), BMSC group and BMSC+VEGF+SCF+TPO group. Some 7-day differentiated cells were used to carry through hematopoietic colonies assay. Flow cytometry were used to observe the positive rate and indirect immunofluorescence cell staining were used to detect the positive cells of CD34、c-kit、CD11b、Ter119 in 7/14-day differentiated cells. Hematopoietic transcription factors (Flk-1, GATA-2, SCL, β-H1 and β-major) were detected by RT-PCR.Result:7-day differentiated cells expressed hematopoietic progenitors antigen (CD34 and c-kit), 14-day differentiated cells expressed monocyte and granulocyte antigen (CD11b), red cell antigen (Ter119). Differentiated cells also expressed transcription factors (Flk-1, GATA-2, SCL, β-H1 and β-major) and could generate hematopoietic colonies. The BMSC+ VEGF+SCF+TPO group got the highest inducing rate according to the numbers of hematopoietic progenitors and colonies (P 0.05 or 0.01 ).Conclusion:Bone marrow feeder layer associated with cytokines strongly promotes hematopoietic differentiation in vitro. Those induced hematopoietic progenitors could form hematopoietic colonies or differentiate mature cells. They also expressed genestromal cells associated with hematotopoietic transcription and differentiation.