| Objective:hUC-MSCs were cultured and differentiated into hepatocyte-like cells in a micro-environment provided by a perfusion bioreactor and decellularized whole liver bioscaffold.After inducing differentiation,the expression of hepatocyte-related molecules by hUC-MSCs was analyzed and their functions were evaluated.We aimed at exploring the effect of hydrostatic pressure on hUC-MSCs differentiation into hepatocyte-like cell,and ptimizing culture conditions in which hUC-MSCs differentiation into hepatocyte-like cells.Methods:1.hUC-MSCs were isolated and cultured by tissue adherence method from neonatal umbilical cord.Cells were cultured and passed to the fourth passage.The expression of cell markers by hUC-MSC was detected by flow cytometry and they were cultured in conditional media to evaluate their ability of adipogenic and osteogenic differentiation.2.Decellularized whole liver bioscaffold was prepared by SDS method.Then the quality of decellularized scaffold was controlled.After that,hUC-MSCs were divided into 4 groups:control group(normal culture group),two-dimensional inducing group(cultured in conditional medium),three-dimensional static induction group(cells cultured in decellularized scaffold without pressure)and three-dimensional perfusion inducing group(cells were cultured in decellularized scaffold and applied with a hydrostatic pressure of 10 cmH2O).21 days later,cells were collected and their expression of hepatocyte-associated factors(The expression of ALB,AFP,CK18,CK19,CYP3A5)were detected,as well as their function.Finally,the mRNA and protein expression of Integrin?1,p-FAK and Laminin A/C,were detected.Results:1.Flow cytometry showed that hUC-MSCs obtained at P4 expressed positively CD105(98.9%±1.35%)and CD90(99.17%±0.94%),didn't express CD34(0.06%±0.04%)and CD45(0.19%±0.27%),and about 65%cells expressed OCT4(65.7%±3.25%).These charactors were coincident with MSCs property.After 28 days differentiation into adipocyte,oil red O staining showed lipid droplets inside plasma.Osteogenic differentiation and alizarin red staining showed also calcium nodule formation.All above indicates that the hUC-MSCs can be differentiated into adipocytes and osteoblasts.2.The decellularized liver bioscaffold showed no residual hepatocytes and cell nucleus,either.The residual DNA content(12.79 ± 0.90 ng/mg)in bioscaffold was significantly lower than that of normal liver tissue(4987.60 ± 310.29 ng/mg).No cell residue was detected by H&E staining and SEM.The decellularized scaffold could provide a porus and ideal three-dimensional environment for hUC-MSCs.3.Compared with two-dimensional culture conditions,cells in three-dimensional culture conditions expressed higher hepatocyte-related markers like ALB,AFP,CK18,CK19 and CYP3A5.They also had stronger hepatocyte-related functions as glycogen synthesis,ALB and ALP secretion.In addition,hepatocyte-related markers and hepatocyte-related functions were significantly increased under three-dimensional perfusion inducting conditions4.During the hepatic differentiation of hUC-MSCs,the expressions of the integrin signaling pathway related molecules were reduced such as Integrin?1,p-FAK and LamininA/C.There was a significant reduce under three-dimensional culture conditions compared to two-dimensional culture conditions.Conclusions:1.hUC-MSCs were succefully isolated and obtained by tissue adherent method2.hUC-MSCs can be induced to differentiate into hepatocyte-like cells under 3D static culture conditions and 3D culture conditions with a hydrostatic pressure of 10 cmH2O.3.Decellularized whole liver bioscaffold can provide a good three-dimensional growth environment for in vitro culture and promote hepatocyte-like differentiation of hUC-MSCs.A hydrostatic pressure of 10cmH2O under 3D conditions can promote the hepatic differentiation of hUC-MSCs.Cells are more mature and closer to hepatocyte function under this situation.4.Cell integrin signaling pathway is inhibited during hepatocyte-like differentiation of hUC-MSCs. |
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