| Objective: In this study,we propose to add 2-mercaptoethanol(2-ME)in the osteogenic medium at specific stages during human embryonic stem cells(h ESCs)and human induced pluripotent stem cells(hi PSCs)osteogenic differentiation to investigate its effect on the process of osteogenic differentiation,and ultimately to improve the efficiency of osteogenic differentiation by adding 2-ME at specific time periods.Methods: Firstly,the effect of different concentrations of 2-ME on the proliferation of human pluripotent stem cells(h PSCs)was evaluated by CCK-8 assay to clarify the 2-ME concentration to be used in subsequent studies.Afterwards,h ESCs and hi PSCs were subjected to osteogenic induction using the monolayer method.Experimental studies were carried out according to the grouping method using osteogenic induction medium supplemented with 2-ME on days 0-7,7-14,14-21 and21-28 of induction,with control groups without and with addition of 2-ME throughout.At different times of culture(7,14,21,28 days),changes in cell activity were detected by CCK-8,changes in expression levels of osteogenesis-related genes(ALP,RUNX2,COL1A1,OPN)were detected by RT-PCR,changes in expression levels of osteogenesis-related proteins(RUNX2,COL1A1,OPN)were evaluated using immunofluorescence,and calcium nodules formation was evaluated using an Alizarin red staining assay.Through the above experiments and assays,the effect of adding 2-ME at different stages of osteogenic differentiation of h ESCs and hi PSCs on the efficiency of osteogenic differentiation was evaluated.Results: The addition of 2-ME at concentrations lower than 0.1 m M had less effect on the cellular activity of h PSCs and was therefore applied to regulate the osteogenic differentiation of cells in vitro.The addition of 2-ME on days 0-7 of osteogenic differentiation of h ESCs significantly increased cell activity during osteogenic differentiation,promoting the expression of RUNX2 at 14 th day of differentiation and ALP,RUNX2 and COL1A1 at 21 st day of differentiation.The addition of 2-ME at days7-14 helped to promote the expression of ALP and OPN at 28 th day of differentiation and increased calcium nodule deposition.There was no significant promotion of cell activity,expression of osteogenesis-related markers and calcium nodule deposition by addition 2-ME at days 14-21 and 21-28 compared to control groups without 2-ME addition throughout.The effect of 2-ME on osteogenic differentiation of hi PSCs was different from that of h ESCs,probably due to cell lineage differences.Addition 2-ME at days 0-7 did not significantly promote the osteogenic differentiation process of hi PSCs.Addition 2-ME at 7-14 days promoted the expression of ALP at 21 st day and OPN at 28 th day of differentiation.14-21 days of 2-ME addition contributed to the expression of COL1A1 at 28 th day of differentiation.Full or 21-28 days addition of 2-ME contributed to increased cell activity.Addition of 2-ME at different time stages did not significantly promote calcium nodule deposition compared to the control group.Conclusion: Based on the above results,the following conclusions were tentatively drawn from this study: 1)The main time of action of 2-ME on the osteogenic differentiation of h ESCs monolayer method is 0-14 days of osteogenic differentiation,the addition of 2-ME on days 0-7 helps to improve the cell activity,and the addition on days 7-14 can promote osteogenic differentiation.2)The main time of function of 2-ME on the osteogenic differentiation of hi PSCs is on days 7-21,the addition of 2-ME on days 7-14 and 14-21 are beneficial to promote expression of OPN and COL1A1.The present work clarifies the necessity and optimal time phase for the addition of 2-ME during the osteogenic differentiation of h PSCs by monolayer method,which will lay the foundation for the subsequent clarification of the mechanism of action and the construction of an efficient in vitro osteogenic differentiation system for hPSCs. |
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