The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs) achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications. 1. Introduction Orthotopic liver transplantation has been shown to be an effective treatment for patients with end stage of liver dysfunction. However, this treatment is limited by the shortage of donor organs. Although hepatocytes transplantation has been shown to be successful treatment in some conditions such as liver-based metabolic disorders, the insufficient donor organs and hepatocytes remain obstacles for this technique [1]. Recently, stem cells are a promising tool for using as cell-based therapy because of their superior properties including self-renewal and broad differentiation potential into several cell types. To date, mesenchymal stem cells (MSCs) have been shown to obtain promising capacity not only multilineages differentiation potential but also immunomodulatory properties [2]. In addition, MSCs can be extensively expanded in vitro, due to effective cryopreservation and being easy to access from various sources such as bone marrow, adipose tissue, amniotic fluid, umbilical cord Wharton’s jelly, and placenta [3–6]. These make MSCs become a good stem cell
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