Experimental Study Of In Vitro Chondrogenic Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells

Purpose The purpose of this study is to establish a method of culturing human umbilical cord mesenchymal stem cell(hUCMSC)and investigate the feasibility of in vitro chondrogenic differentiation of hUCMSC in monolayer culture as potential seeding cells in cartilage tissue engineering.Methods The original hUCMSC derived from The Nation Research Center of Cell Production,hUCMSC cultured in the Dulbecco's Modified Eagle's Medium/F-12 Nutrient Mixtur(DMEM/F-12)with low glucose containing 10%fetal bovine serum,flow cytometric analysis was performed to examine the expression of cell surface molecules on hUCMSC. HUCMSC from the third passage were grown in complete medium(A),inductive serum-free medium(B),inductive medium with 5%fetal bovine serum(C),inductive medium with 10%fetal bovine serum(D)respectively.The growth of the cultured hUCMSC was observed.The synthetic proteoglycans were detected by toluidine blue staining.Immunohistochemistry and in situ hybridization were applied to detect the expression of collagen typeâ…¡.The proliferation of the cells was examined by MTT test.Results After proliferation in vitro,The adherent cells showed a spindle-like appearance and expressed positive CD44,but negative CD34,CD38.After 9 days induction by inductive serum-free medium or inductive medium with 5%fetal bovine serum,the cells were positively stained by toluidine blue,immunohistochemistry and in situ hybridization analysis proved typeâ…¡collagen expression.The sequences of the MTT test were D＞C＞A＞B.Conclusion HUCMSC cultured in vitro grew stably and were a homogenous population;10ng/ml TGF-β₁ can induce the chondrogenic differentiation of the hUCMSC,but it can not facilitate the proliferation of the cells in serum-free medium;the combination of TGF-β₁ and fetal bovine serum in low concentration(5%)can not only facilitate,the proliferation of hUCMSC,but also induce the chondrogenic differentiation.