Hepatic cell-sheet fabrication of differentiated mesenchymal stem cells using decellularized extracellular matrix and thermoresponsive polymer

Purpose: Liver tissue engineering via cell sheet technology would open new doors for treatment of patients with liver failure. Decellularized tissues could provide sufficient extracellular matrix (ECM) to support development of hepatocytes in in vivo niches. Besides, with the potential of temperature responsive polymer (pNIPAAm) as an intelligent surface for controlling the attachment/detachment of cell, we set out to generate three in vitro microenvironments models including I: pNIPAAm hydrogel (pN hydrogel), II: decellularized ECM incorporated into pNIPAAm hydrogel (dECM + pN hydrogel) and III: decellularized ECM scaffold (dECM scaffold) to investigate the structural and function cues of hepatocyte-like cells after differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) on the surface of these models. Method: dECM scaffold was obtained after decellularization of rat liver, and its efficiency was analyzed. pN hydrogel and dECM + pN hydrogel (1:3 and 2:3 ratios) of were fabricated, and scaffold architecture was characterized. Each well of culturing plates was coated separately with these three constructs and AT-MSCs were instructed to differentiate into hepatocyte-like cells (HLCs). After recellularization, patterns of differentiation, and expression of hepatogenic markers were investigated via biochemical assays and qRT-PCR at different time points. Results: Multipotency of AT-MSCs, after their ability for osteogenesis and adipogenesis was documented. Production of dense and intact cell sheets was reported in dECM + pN hydrogel, as opposed to pN hydrogel and dECM scaffold. Also, statistically significant difference of HLCs functionality in dECM + pN hydrogel was confirmed after evaluation of the expression of hepatocyte markers including, alpha-fetoprotein, cytokeratin 18, cytochrome P450-2E1 and phosphoenolpyruvate carboxykinase. Conclusion: Our results proved dECM + pN hydrogel were able to preserve hepatocyte function in cell sheets owing to the high level of albumin, urea, hepatogenic markers, and glycogenesis potential of HLCs. Accordingly, dECM incorporated in pN hydrogel could remodel microenvironments to guide the AT-MSCs into conducive differentiation and proliferation to give rise to multilayer sheets of cells in their own ECM.