Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

BACKGROUND: The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. RESULTS: Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity. CONCLUSION: hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells

In Vitro Differentiation of Embryonic and Adult Stem Cells into Hepatocytes: State of the Art

Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells

Method for producing adult liver progenitor cells

Novel adult liver progenitor cells (called H2Stem Cells) have been have been characterized on the basis of a series of biological activities and markers. Methods for producing H2Stem Cells allow providing such cells in the form of adherent cells and three-dimensional cell clusters in suspension that can be differentiated into cells having strong liver-specific activities and/or that can be used for treating liver diseases or for evaluating the efficacy, the metabolism, and/or toxicity of a compound

Sequential exposure to cytokines reflecting embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells

Differentiation of adult bone marrow stem cells (BMSC) into hepatocyte-like cells is commonly performed by continuous exposure to a cytokines-cocktail. Here, it is shown that the differentiation efficacy in vitro can be considerably enhanced by sequential addition of liver-specific factors (fibroblast growth factor-4, hepatocyte growth factor, insulin-transferrin-sodium selenite, and dexamethasone) in a time-dependent order that closely resembles the secretion pattern during in vivo liver embryogenesis. Quantitative RT-PCR analysis and immunocytochemistry showed that, upon sequential exposure to liver-specific factors, different stages of hepatocyte differentiation, as seen during liver embryogenesis, can be mimicked. Indeed, expression of the early hepatocyte markers alpha-fetoprotein and hepatocyte nuclear factor (HNF)3beta decreased as differentiation progressed, whereas levels of the late liver-specific markers albumin (ALB), cytokeratin (CK)18, and HNF1alpha were gradually upregulated. In contrast, cocktail treatment did not significantly alter the expression pattern of the hepatic markers. Moreover, sequentially exposed cells featured highly differentiated hepatic functions, including ALB secretion, glycogen storage, urea production, and inducible cytochrome P450-dependent activity, far more efficiently compared to the cocktail condition. In conclusion, sequential induction of the differentiation process, analogous to in vivo liver development, is crucial for in vitro differentiation of adult rat BMSC into functional hepatocyte-like cells. This model may not only be applicable for in vitro studies of endoderm differentiation but it also provides a "virtually unlimited" source of functional hepatocytes, suitable for preclinical pharmacological research and testing, and cell and organ development.status: publishe