The Epigenetic Alterations Of Human Umbilical Cord Mesenchymal Stern Cells During In Vitro Culture

Mesenchymal stem cells (MSCs) are multi-potential cells harboring immunosuppressive properties and able to differentiate into multiple mesodermal tissues including fat, bone, cartilage, muscle, ligament, stroma and tendon that make MSCs an ideal candidate for immunomodulation and regenerative medicine. As a member of MSC family, Human umbilical cord mesenchymal stem cells (hUC-MSCs) also have biological characteristics of MSCs, but are more easily accessible. hUC-MSCs have a stronger proliferation and differentiation ability, lower immunogenicity, and not express HLA-type Ⅱ surface antigen, and not produce the teratoma.In order to isolate and culture hUC-MSCs and study the epigenetic alterations during in vitro culture, We separated hUC-MSCs from Warton’s jelly of human umbilical cord via manual mechanical combined with multi-enzyme digestion. MTT was conducted to test the growth activity of hUC-MSCs. Flow cytometry was conducted to test the hUC-MSCs surface markers. Differentiation ability of hUC-MSCs was tested. Soft agar cloning method was conducted to test the tumorigenicities of hUC-MSCs of different passage.β-galactosidase was conducted to test senescent associated activity of hUC-MSCs. Methylation specific PCR (MSP) was used to test methylation status of targeted genes during in vitro culture of hUC-MSCs. Western Blot was conducted to test MGMT protein expression. In this study3-6×106hUC-MSCs could be isolated from30cm-long umbilical cords. hUC-MSCs expressed high level of CD29, CD73, CD90, CD105, CD44and low level of CD45, CD34, HLA-DR. hUC-MSCs had the ability to differentiate to adipocytes, osteoblasts and chondrocytes. Soft agar assay did not detected anchorage-independent growth of hUC-MSCs even in the late stage of culture. During the late phase of in vitro culture, hUC-MSCs became senescent and DNA repair gene MGMT protein was absent due to DNA methylation.In conclusion, hUC-MSCs have biological characteristics of mesenchymal stem cell. During in vitro culture, MGMT appeared methylated with cell passaging. More studies need to be done to explore the relativity of MGMT methylation and cell senescence.