Methods for preparing mesenchymal stem cells

A method for preparing mesenchymal stem cells, comprising:

• a) providing expanded potential stem cells or a first cell culture including the expanded potential stem cells;
• b) culturing the expanded potential stem cells or the first cell culture in a trophoblast stem cell (TSC) differentiation medium to obtain trophoblast stem cells or a second cell culture including the trophoblast stem cells, the TSC differentiation medium including a basal medium, 2-mercaptoethanol, serum replacement, insulin-transferrin-selenium-X supplement (ITS-X supplement), L-ascorbic acid-2-phosphate (L-AA-pi), epidermal growth factor (EGF), glycogen synthase kinase 3 beta (GSK3β) receptor inhibitor CHIR99021, anaplastic lymphoma kinase (ALK) 4/5/7 inhibitor A83-01, ALK5 inhibitor SB431542, valproic acid, Rho-associated protein kinase (ROCK) inhibitor Y27632, and bone morphogenetic protein 4 (BMP4), wherein the basal medium is an essential medium or a medium prepared by mixing dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM/F-12 medium) and iscove's modified dubecco's medium (IMDM medium) at a ratio of 1:1;
• c) culturing the trophoblast stem cells or the second cell culture in a mesenchymal stem cell (MSC) differentiation medium to obtain mesenchymal stem cells or a third cell culture including the mesenchymal stem cells, the MSC differentiation medium including minimum essential medium α (α-MEM) medium, 1×non-essential amino acids (1×NEAA) cell culture supplement, 1×alanyl-glutamine (1×GlutaMax) supplement, 2-mercaptoethanol, fetal bovine serum, and human platelet lysate;
• and
• d) passaging the mesenchymal stem cells or the third cell culture in an MSC expansion medium while maintaining characteristics of the mesenchymal stem cells, the MSC expansion medium including α-MEM medium, 1×NEAA cell culture supplement, 1×GlutaMax supplement, 0.1 mM 2-mercaptoethanol, 5% fetal bovine serum, and 5% human platelet lysate, wherein
• the mesenchymal stem cells obtained in the step c) and the step d) are mesenchymal stem cells expressing cell surface markers CD44, CD73, CD90, CD105, CD166;
• and
• in mesenchymal stem cells at a 16th passage or later, a proportion of the mesenchymal stem cells expressing the cell surface markers CD44, CD73, CD90, CD105, and CD166 is not less than 95%.