Comparison of Two Different Methods Induced Human Umbilical Cord Mesenchymal Stem Cells to Differentiate into Chondrocytes In Vitro
ZHANG Quan1#, CHEN Lian2#, CHANG Cheng2, ZHANG Yaqi2, XIAO Cuihong1, RAO Wei1, HAN Bing1, WU Dongcheng1,2*
The purpose of this study is to study the chondrogenic properties capacity of human umbilical cord mesenchymal stem cells (hUC-MSCs) after three-dimensional hanging drop culture method and adherent culture method. The hUC-MSCs were isolated from Wharton’s jelly and cultured by mechanical homogenization in vitro. The morphology of 3D spheroid under optical microscope, and surface antigens were examined by flow cytometric analysis were compared. The hUC-MSCs were induced to differentiate into chondrocytes in vitro, and expression levels of chondrocyte related genes ACAN, MIA, COL1A2, COL2A1 and COL10A1 were detected by fluorescence quantitative PCR on the 3rd, 7th and 14th day. The microspheres were induced for a chondro-like appearance and visualized by Saffron O staining and Alcian blue staining. Immunohistochemistry was used to detect the expression of type II collagen. Flow cytometry data showed the hUC-MSCs at passage 5 are positive for CD105, CD90, CD44, CD73, negative for CD19, CD34, CD45, HLA-DR, which were in line with The morphological characteristics and surface immune markers of mesenchymal stem cells developed by the International Society for Cellular Therapy (ISCT) in 2006. The hUC-MSCs cultured in 3D hanging drop culture can form a dense cell polymer, and the cell morphology of adherent culture can grow into a spindle shape. The results of Saffron O and Alcian blue staining showed that both the hUC-MSCs cultured in 3D hanging drop culture and adherent culture could form chondrocytes, but the results of fluorescence real-time quantitative PCR showed that the number of chondrogenic genes related to the formation of chondrocytes in the hUC-MSCs cultured in 3D hanging drop culture were more than that in the adherent culture. The semi-quantitative analysis result of type II collagen with immunohistochemistry showed that the chondrogenic ability of hUC-MSCs induced by 3D hanging drop culture was stronger than that by adherent culture. 3D hanging drop cultured technology is an ideal method to culture hUC-MSCs.
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