Research Of Ex Vivo Expansion And Osteogenesis Of Human Umbilical Cord Mesenchymal Stem Cells

Human umbilical cord mesenchymal stem cells(hUMSCs)have attracted extensive attention in clinical applications due to their wide availability,strong proliferative capacity and low immunogenicity.Due to the limited number of hUMSCs isolated from tissues,ex vivo culture and expansion are required.The cells obtained by traditional two-dimensional culture are limited,and the use of fetal bovine serum(FBS)during the culture process fails to meet clinical requirements.Therefore,development of a clinical-grade system for large-scale expansion of mesenchymal stem cells(MSCs)is essential.In addition,hUMSCs hold low osteogenic differentiation capacity,which limits their further application in clinical treatment.Accordingly,it is necessary to identify therapeutic strategies to enhance hUMSCs osteogenesis.In the present study,the effect of human platelet lysates(hPL),a human-derived serum substitute,on the biological characteristics of hUMSCs was investigated.By optimizing the operating parameters of microcarrier culture,an expansion technology based on xeno-free medium and microcarrier system was established.The effect of small molecule compound β-mercaptoethanol(BME)on osteogenic differentiation of hUMSCs and its regulatory mechanism were explored to promote hUMSCs osteogenesis.Moreover,in order to optimize the SFM medium suitable for hUMSCs proliferation,RNA-seq technology was used to analyze the differences in gene expression of hUMSCs cultured in hPL,FBS supplemented medium and serum-free medium(SFM)obtained from previous laboratory work.The differentiation ability,surface antigen expression,cell senescence and apoptosis of hUMSCs cultured to passage P10 in hPL-containing medium did not significantly change.By comparing with FBS-containing medium,hUMSCs cultured in hPL-containing medium displayed stronger proliferative and anti-apoptosis ability,and the expression of CD 105 decreased more slowly in long-term subculture.By optimizing the operating parameters including microcarrier type,microcarrier concentration and feeding strategy,a higher cell density of(83.7±8.9)±104 cells/mL(16.7 expanded fold)was obtained when hUMSCs were seeded at a cell density of 5×104 cells/mL on 3.0 mg/mL Cultispher S microcarriers and glucose was added during the culture,higher than that in the FBS-containing medium.In addition,the effect of BME on osteogenic differentiation of hUMSCs was investigated.It was showed that the addition of 50 μM BME significantly promoted the ALP activity,calcium deposition and the expression of osteogenic markers(ALP,OCN,OPN and COLI).Further research found that BME promoted Runx2 expression by activating the sirt1-ERK1/2 signaling pathway,thereby enhancing the osteogenic differentiation of hUMSCs.By comparing with cells cultured in hPL,FBS supplemented medium,the differential expressed genes cultured in SFM medium were enriched in GO terms such as proteins binding,nucleus,cytoplasm,cell cycle and cell division.KEGG enrichment analysis indicated the differential expressed genes were associated with cancer pathogenesis,MAPK signaling pathway,cell adhesion and cell aging.These findings provide a theoretical basis for understanding the mechanism of osteogenic differentiation of hUMSCs and developing a chemically defined SFM medium to eventually establish a clinical-grade expanded MSCs system.