A Preliminary Study Of Stepwise Differentiation Of Monolayer Cultured Human Induced Pluripotent Stem Cells Into Mesenchymal-like Cells Under Chemically Defined Conditions

Mesenchymal stem cells(MSCs)are multilineage cells with the ability to selfrenew and differentiate into a variety of cell types,which play key roles in regenerative medicine and cell therapy.MSCs can be isolated from a variety of tissues,but techniques for extraction may cause damage to the donors and some other disadvantages such as rarity in adult tissues,limited cell proliferative capacity,gradual loss of differentiation during in vitro expansion,cell heterogeneity from different donors or tissues,and decreased performance with increasing age.Human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hiPSCs)emerged in 2006 provided a new idea to solve the limitations of adult MSCs in clinical application.Many researchers have successfully differentiated hPSCs into mesenchymal-like stem cells in vitro.The mesenchymal-like stem cells derived from hPSCs showed similar biological functions and advantages such as unlimited quantity,higher proliferation capacity,and lower immunogenicity.However,most of the published protocols depended on the spontaneous differentiation process and the medium supplemented with FBS,which leads to undefined differentiation conditions,uncontrollable induction,and low differentiation efficiency.This study proposes establishing an efficient culture system in vitro for the differentiation of hiPSCs into mesenchymal-like cells with a defined condition:which can lay the foundation for the mass production of hiPSCs derived mesenchymal-like cells(hiPSCs-MSCs).It was expected to be applied in basic and clinical research-Previously,to efficiently obtain osteoblasts,our laboratory group explored the ideal culture condition that can generte mesodermal progenitor cells from hPSCs,which laid the foundation for this study.Preliminary experiments showed that it was challenging to obtain mesenchymal-like cells by changing to mesenchymal induction medium directly without cell digestion.We have overlooked the importance of cell-cell interactions.Therefore,we first explored the effect of different cell density on the differentiation of mesodermal progenitor cells into mesenchymal-like cells by using the mesenchymal induction medium containing FBS.By detecting the expression of related to MSCs(CD73?CD44?CD90?CD105?CD29)by RT-PCR and flow cytometry and compared the induction efficiency.The results showed that digested into single cells and re-inoculated on the gelatin surface at a proper cell density(200,000 cells·mL-1)were necessary to generate mesenchymal-like cells and obtained CD73+CD44+hiPSCs-MSCs,which was similar to adult MSCs through three serial passages.And then retinoic acid(RA),bone morphogenetic protein-4(BMP4),transforming growth factor-?1(TGF-?1),and basic fibroblast growth(bFGF)were be selected to induce the differentiation of mesenchymal progenitor cells to mesenchymal-like cells.It was found that the treatment of RA for two days could improve the differentiation efficiency at the initial stage.In addition,along with the time extension,the combined use of TGF-?1 and bFGF positively affected the induction efficiency.Unfortunately,whether the medium with FBS or chemically defined medium,the addition of compounds can not effectively improve the induction efficiency,which may be caused by the complex composition of serum or medium and obscured the function of these compounds.These results suggest that mesodermal progenitor cells can easily spontaneously differentiate into mesenchymal-like cells.To improve the induction efficiency of mesenchymal differentiation,we re-explored the culture conditions of mesodermal differentiation of hiPSCs with mesenchymal differentiation as the goal.Following previous research,we divided the mesodermal induction system into two stages containing the first two days(D0-D2)and the last three days(D3-D5),which based on the experience of inducting hPSCs differentiation into cardiomyocytes through mesoderm,and then selected CYC,LY294002,bFGF,BMP4,SB431542,and RA.Moreover,the differentiation efficiency was compared after 14 days of continuous culture in a mesenchymal induction medium.The results showed that SB431542 was the most potent compound in D0-D2,and LY294002 was the most potent compound in D3-D5.The hiPSCs-MSCs with high expression of CD73 and CD44 were obtained after only two passages,which significantly shortened the induction time of mesenchymal differentiation.In addition,the efficiency of cell survival and induction of differentiation was decreased considerably after changing to a chemically defined medium.In the future,further studies will be conducted to determine the role of each component of the induction medium.And then develop a medium suitable for the differentiation of mesodermal progenitor cells into mesenchymal-like cells under the monolayer method.Nevertheless,it laid the foundation for constructing the induction system of directed mesenchymal differentiation of hiPSCs with precise components.