Differentiation of Wharton's jelly-derived mesenchymal stromal cells into hepatocyte-like cells using a refined method.

Abstract

Background: The production of functional hepatocyte cells in enough quantities is of paramount importance for the replacement of lost hepatocytes. In this investigation, a series of 7-mimic microRNAs was harnessed to induce the differentiation of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) into hepatocyte-like cells (HLC) through the application of two distinct techniques: transfection agents and electroporation. The results were then compared with those of HLCs differentiated through the consumption of chemical compounds.

Results: Different time points (48 h, 72 h, and 96 h), unlike concentrations of mimic miRNAs (100 pM, and 200 pM), and dissimilar combinations of mimic-miRNAs (4-mimic and 7-mimic miRNAs) were selected to assess the stage of differentiated cells through electroporation and lipofection methods. For chemical differentiation, a two-step chemical hepatic differentiation protocol was used (for 21 days). The expression level of eleven key genes that were selected to estimate the stage of produced HLCs by each method were tested at different time points, concentrations and combination of mimic-miRNA. Results demonstrated that the 7-miR-mimics/72 h culture method by electroporation, then the 7-miR-mimics/72 h culture method by lipofection, and finally the chemical differentiation (72 h culture) showed the best result for differentiation. Furthermore, the period in which HLCs are maintained under culture conditions is important, as prolonged culture (more than 72 h) leads to cell loss.

Conclusion: In conclusion, the results demonstrated that the 7-miR cocktail delivered by electroporation after 72 h effectively promoted the acquisition of hepatocyte-like characteristics which was evidenced by a significant decrease in the Oct4 stemness factor and an increase in the expression of ALB, TAT, AAT, CYP, G6P and HNF4A.