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Directed Differentiation of Adult Liver Derived Mesenchymal Like Stem Cells into Functional Hepatocytes.

ABSTRACT: Shortage of functional hepatocytes hampers drug safety testing and therapeutic applications because mature hepatocytes cannot be expanded and maintain functions in vitro. Recent studies have reported that liver progenitor cells can originate from mature hepatocytes in vivo. Derivation of proliferating progenitor cells from mature hepatocytes, and re-differentiation into functional hepatocytes in vitro has not been successful. Here we report the derivation of novel mesenchymal-like stem cells (arHMSCs) from adult rat hepatocytes. Immunofluorescence and flow cytometry characterization of arHMSCs found expression of mesenchymal markers CD29, CD44, CD90, vimentin and alpha smooth muscle actin. These arHMSCs proliferated in vitro for 4 passages yielding 104 fold increase in cell number in 28 days, and differentiated into hepatocyte-like cells (arHMSC-H). The arHMSC-H expressed significantly higher level of hepatocyte-specific markers (200 fold for albumin and 6 fold for Cyp450 enzymes) than arHMSCs. The arHMSC-H also demonstrated dose response curves similar to primary hepatocytes for 3 of the 6 paradigm hepatotoxicants tested, demonstrating utility in drug safety testing applications.

SUBMITTER: Luo X

PROVIDER: S-EPMC5809507 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Directed Differentiation of Adult Liver Derived Mesenchymal Like Stem Cells into Functional Hepatocytes.

Luo Xiaobei X Gupta Kapish K Ananthanarayanan Abhishek A Wang Zenan Z Xia Lei L Li Aimin A Sakban Rashidah Binte RB Liu Side S Yu Hanry H

Scientific reports 20180212 1

Shortage of functional hepatocytes hampers drug safety testing and therapeutic applications because mature hepatocytes cannot be expanded and maintain functions in vitro. Recent studies have reported that liver progenitor cells can originate from mature hepatocytes in vivo. Derivation of proliferating progenitor cells from mature hepatocytes, and re-differentiation into functional hepatocytes in vitro has not been successful. Here we report the derivation of novel mesenchymal-like stem cells (ar ...[more]

PMID: 29434311

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Project description:Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and "rosette-like structures" from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

Project description:There is a critical need to generate functional hepatocytes to aid in liver repair and regeneration upon availability of a renewable, and potentially personalized, source of human hepatocytes (hHEP). Currently, the vast majority of primary hHEP are obtained from human tissue through cadavers. Recent advances in stem cell differentiation have opened up the possibility to obtain fully functional hepatocytes from embryonic or induced pluripotent stem cells, or adult stem cells. With respect to the latter, human bone marrow mesenchymal stromal cells (hBMSCs) can serve as a source of autogenetic and allogenic multipotent stem cells for liver repair and regeneration. A major aspect of hBMSC differentiation is the extracellular matrix (ECM) composition and, in particular, the role of glycosaminoglycans (GAGs) in the differentiation process. In this study, we examine the influence of four distinct culture conditions/protocols (T1-T4) on GAG composition and hepatic markers. ?-Fetoprotein and hepatocyte nuclear factor-4? were expressed continually over 21 days of differentiation, as indicated by real time quantitative PCR analysis, while albumin (ALB) expression did not begin until day 21. Hepatocyte growth factor (HGF) appears to be more effective than activin A in promoting hepatic-like cells through the mesenchymal-epithelial transition, perhaps due to the former binding to the HGF receptor to form a unique complex that diversifies the biological functions of HGF. Of the four protocols tested, uniform hepatocyte-like morphological changes, ALB secretion, and glycogen storage were found to be highest with protocol T2, which involves both early- and late-stage combinations of growth factors. The total GAG profile of the hBMSC ECM is rich in heparan sulfate (HS) and hyaluronan, both of which fluctuate during differentiation. The GAG profile of primary hHEP showed an HS-rich ECM, and thus, it may be possible to guide hBMSC differentiation to more mature hepatocytes by controlling the GAG profile expressed by differentiating cells.

Dataset Information

Directed Differentiation of Adult Liver Derived Mesenchymal Like Stem Cells into Functional Hepatocytes.

Publications

Directed Differentiation of Adult Liver Derived Mesenchymal Like Stem Cells into Functional Hepatocytes.

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