Abstract:

BACKGROUND: Some studies show that basic fibroblast growth factor (bFGF) strongly expresses during the process of embryonic stem cells differentiation into hematopoietic stem cell, yolk sac blooding, and fetal liver hematopoiesis.
OBJECTIVE: To study the regulation of bFGF on the blast-colony-forming cell (BL-CFC) by adding bFGF in the medium of embryoid body generation.
METHODS: The third to fifth generations of the primary mouse embryonic fibroblasts were recovered, and then incubated with the DMEM medium containing mitomycin C for 2.5 hours in order to lose the proliferative capacity. Then cells were suspended into single cell by trypsinization and inoculated in the gelatin-coated bottle at the density of 10×10⁴/cm². After culturing for 24 hours, mouse embryonic stem cells (mESC) of D3 were recovered and placed on the feeder layer cells. According to the composition of medium in embryoid body generation, mESCs were divided into two groups: group A: standard medium + VEGF + SCF; group B: standard medium + VEGF + SCF + bFGF. Each group was cultured for 3 days and 6 days respectively, and the cloning number of BL-CFC was quantified, as well as Flk-1⁺ expression was observed by immunofluorescence staining. Positive number and average absorbance were analyzed using IMAGE-PRO PLUS imaging analysis system.
RESULTS AND CONCLUSION: Adding bFGF in the course of embryoid body growth could significantly increase the number of BL-CFC (P < 0.01), and the positive results of Flk-1 and the average absorbance were also increased significantly (P < 0.01). bFGF effectively promoted embryoid body amplification and proliferation of BL-CFC.