Abstract
Liver diseases are major causes of morbidity and mortality. Dental pulp pluripotent-like stem cells (DPPSCs) are of a considerable promise in tissue engineering and regenerative medicine as a new source of tissue-specific cells; therefore, this study is aimed at demonstrating their ability to generate functional hepatocyte-like cells in vitro. Cells were differentiated on a collagen scaffold in serum-free media supplemented with growth factors and cytokines to recapitulate liver development. At day 5, the differentiated DPPSC cells expressed the endodermal markers FOXA1 and FOXA2. Then, the cells were derived into the hepatic lineage generating hepatocyte-like cells. In addition to the associated morphological changes, the cells expressed the hepatic genes HNF6 and AFP. The terminally differentiated hepatocyte-like cells expressed the liver functional proteins albumin and CYP3A4. In this study, we report an efficient serum-free protocol to differentiate DPPSCs into functional hepatocyte-like cells. Our approach promotes the use of DPPSCs as a new source of adult stem cells for prospective use in liver regenerative medicine.
Highlights
The liver is the largest internal organ providing essential metabolic, exocrine, and endocrine metabolites
Dental pulp pluripotent-like stem cells (DPPSCs) were cultured in precoated flasks with 100 ng/ml fibronectin (Life Technologies, Waltham, MA, USA) in a medium consisting of 60% DMEM-low glucose (Life Technologies) and 40% MCDB-201 (Sigma-Aldrich), supplemented with 1× SITE Liquid Media Supplement (Sigma-Aldrich), 1× linoleic acid-bovine serum albumin (LA-BSA, SigmaAldrich), 10-4 M L-ascorbic acid 2-phosphate (SigmaAldrich), 1× penicillin-streptomycin (Life Technologies), 2% fetal bovine serum (FBS, Sigma-Aldrich), 10 ng/ml hPDGF
Using human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and human umbilical cord Wharton’s jelly mesenchymal stem cells, studies have shown that activin A (Act A) is the main definitive endoderm (DE) induction factor [49, 50, 51, 52]. We tested this concept on DPPSCs using Act A with or without other signaling factors to delineate the optimal conditional media sufficient to mediate DE differentiation
Summary
The liver is the largest internal organ providing essential metabolic, exocrine, and endocrine metabolites. Hepatocytes and cholangiocytes, the bile duct epithelial cells, are parenchymal cells which comprise approximately 70% of the adult liver mass. Both cell types are derived from the embryonic definitive endoderm [1]. The liver is the main body homeostasis regulator; liver diseases cause high morbidity and mortality rates. Orthotopic liver transplantation (OLT) is the current optimal treatment with more than 70% of the overall 5-year survival rate for certain ESLD cases [3]. OLT obstacles include donor availability, surgical risk, high costs, and the requirement for lifelong immunosuppressors [4, 5, 6].
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