ABSTRACT
BACKGROUND:Very smal embryonic-like stem cells are a kind of non-hemopoietic stem cells, which have similar biological characteristics to embryonic stem cells. But the method of its culture and in vitro proliferation is rarely reported. Studies have speculated that human embryonic fibroblasts can provide a good microenvironment for in vitro culture and proliferation of very smal embryonic-like stem cells. OBJECTIVE:To isolate and cultivate human embryonic fibroblasts derived from human embryonic trunks and to establish a feeder layer culture system of human embryonic fibroblasts for culturing very smal embryonic-like stem cells derived from human bone marrow. METHODS:The human embryonic fibroblasts were isolated from the subcutaneous connective tissue of human embryos at pregnant 5-9 weeks using trypsin digestion method. Different concentrations of mitomycin C were used to pretreat feeder layers, which were used for cultivating very smal embryonic-like stem cells derived from human bone marrow. The effects of human embryonic fibroblasts and feeder layers were assessed by cel morphology and growth curves. RESULTS AND CONCLUSION:The human embryonic fibroblasts were successful y isolated and cultivated from human embryos, and they could be passaged beyond the 24th generation. The biologic characteristics of the cells had no changes after passage and cryopreservation. The optimal concentration of mytomcin C to inhibit proliferation of human embryonic fibroblasts was l2 mg/L for 3 hours. The human embryonic fibroblasts derived from human embryos are successful y isolated and cultivated and to produce feeder layers for very smal embryonic-like stem cells derived from human bone marrow.