The effect of decellularized liver matrix on stem cell differentiation into hepatocyte-like cells

Liver transplantation is a commonly accepted life saving surgical, the major challenge facing the transplant community is a consequence of their great success; more organs for transplantation are now needed. The severe discrepancy between livers available for donation and the candidates' waiting list is a problem. Hepatocyte transplantation has been demonstrated to have a positive therapeutic effect. However, the application of this technology has been hindered by the inability to secure a reliable and healthy cell source(s), shortage of organ donors, problems related to isolation of high quality hepatocytes. Furthermore, hepatocytes do not proliferate and de-differentiate in vitro. Faced with that problem, stem cells (MSCs) had been considered as a candidate to substitute hepatocytes transplants. However, despite published results showing hepatocyte-like cells derived from different mesenchymal stem cells, only a limited group of unique molecules/functions are shown to be up-regulated. In order to improve the differentiation of hepatocyte-like cells, we developed a substrate made of solubilized decellularized porcine liver tissue and differentiated stem cells from various sources (adipose, bone marrow, amnion) into hepatocyte-like cells. We effectively decellularized liver tissue and successfully optimized the solubilization of the decellularized liver matrix (pLEACM-AA) to be used as a substrate for in vitro studies. The substrate had low immunogenicity and no cytotoxicity. A series of hepatocyte sequential differentiation protocols were evaluated on ASC, then we selected the protocol that yielded the best hepatocyte-like cell based on relevant functions (urea, P450 metabolism, LDL-uptake, glycogen storage, gene expression. Finally, we demonstrated that the liver tissue substrate (pLECM-AA) enhances the hepatocyte differentiation by doing RNAseq analysis of human ASC, BMMSC and AEC differentiated into hepatocyte cells while cultured on the pLECM-AA. These results highlight the importance of the native extracellular bioactivity and their impact on stem cell differentiation.