THE EXPANSION AND ORDERLY DIFFERENTIATION OF THE ADULT MESENCHYMAL STROMAL STEM CELL IN VITRO

Li TY & Yang L

Children's Hospital, Chongqing University of Medical Science, Chongqing, China

Objective: To expand and induce the mesenchymal stem cells to differentiate from adult human bone marrow to osteocyte and chondroblast in vitro.

Methods: With normal adult human bone marrow obtained form ribs, a small percentage of mesenchymal stem cells (MSCs) were isolated by using the density interface of 1.073g/ml percoll. Human MSCs were allowed to attach to the surface of the plates for three days, at which time the culture medium was replaced with fresh medium. The culture adherent hMSCs have been allowed to mitotically expand with complete medium changes occurring twice weekly. When hMSC in primary cultures expand to form colonies of several hundred cells that collectively cover 80% of the culture plate, the cells were passaged at 1:3 dilution after being detached from the culture substratum with trypsin. Then they were purified as well as expanded in Mesencult liquid culture system. 10^-7 mol/L dexamethason, 10ng/ml Na-b-glycerophosphate(b-GP) and 50μg /ml vitamin C were added in the culture system to induce osteoblast differentiation, Chondrogenesis was induced by culturing hMSC in micromass pellets in the presence of defined medium that include 10ng/ml TGF-b3. Morphology was observed during cell differentiation. Cytochemistry and immunohistochemistry were used to detect the calcium deposition, alkaline phosphatase activity, cartilage cells and type I, II collagen.

Results: The hMSCs were expanded 15 passages and yielded 7.5Х10¹² MSCs. Some polygonal cells could be observed at about the 7^th day in culture system for inducing osteoblast differentiation and they were dyed AKP positve. By 21 days of induction, calcium deposition and type I collage can be observed. At the same time, 10ng/ml TGF-b in the chemically defined medium induced chondrogenic differentiation was evidenced by the appearance of cartilage cells and the immunohistochemical detection of type II collagen.

Conclusion: Adult human mesenchymal stem cells can be expanded and induced to osteoblast and chondroblast in vitro. HMSCs remain the differentiation potential during expansion.

1839