In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells

Daniel P Collins, 1 Joel H Hapke, 1 Rajagopal N Aravalli, 2 Clifford J Steer 3, 4 1Cytomedical Design Group, LLC, Saint Paul, MN 55127, USA; 2Department of Electrical and Computer Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA; 3Department of...

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Autores principales:	Collins DP, Hapke JH, Aravalli RN, Steer CJ
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2020
Materias:	cord blood tert mlpc differentiation hepatocyte-like cells Diseases of the digestive system. Gastroenterology RC799-869
Acceso en línea:	https://doaj.org/article/d6287c9e598a4f8db1f33c346a012c88
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spelling	oai:doaj.org-article:d6287c9e598a4f8db1f33c346a012c882021-12-02T09:11:28ZIn vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells1179-1535https://doaj.org/article/d6287c9e598a4f8db1f33c346a012c882020-06-01T00:00:00Zhttps://www.dovepress.com/in-vitro-differentiation-of-tert-transfected-multi-lineage-progenitor--peer-reviewed-article-HMERhttps://doaj.org/toc/1179-1535Daniel P Collins, 1 Joel H Hapke, 1 Rajagopal N Aravalli, 2 Clifford J Steer 3, 4 1Cytomedical Design Group, LLC, Saint Paul, MN 55127, USA; 2Department of Electrical and Computer Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA; 3Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA; 4Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USACorrespondence: Daniel P CollinsCMDG, LLC, Saint Paul, MN 55127, USAEmail dc@cmdgllc.comBackground: Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.Methods: E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.Results: E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.Conclusion: The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.Keywords: cord blood, TERT, MLPC, differentiation, hepatocyte-like cellsCollins DPHapke JHAravalli RNSteer CJDove Medical Pressarticlecord bloodtertmlpcdifferentiationhepatocyte-like cellsDiseases of the digestive system. GastroenterologyRC799-869ENHepatic Medicine: Evidence and Research, Vol Volume 12, Pp 79-92 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	cord blood tert mlpc differentiation hepatocyte-like cells Diseases of the digestive system. Gastroenterology RC799-869
spellingShingle	cord blood tert mlpc differentiation hepatocyte-like cells Diseases of the digestive system. Gastroenterology RC799-869 Collins DP Hapke JH Aravalli RN Steer CJ In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
description	Daniel P Collins, 1 Joel H Hapke, 1 Rajagopal N Aravalli, 2 Clifford J Steer 3, 4 1Cytomedical Design Group, LLC, Saint Paul, MN 55127, USA; 2Department of Electrical and Computer Engineering, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, USA; 3Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA; 4Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455, USACorrespondence: Daniel P CollinsCMDG, LLC, Saint Paul, MN 55127, USAEmail dc@cmdgllc.comBackground: Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.Methods: E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.Results: E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.Conclusion: The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.Keywords: cord blood, TERT, MLPC, differentiation, hepatocyte-like cells
format	article
author	Collins DP Hapke JH Aravalli RN Steer CJ
author_facet	Collins DP Hapke JH Aravalli RN Steer CJ
author_sort	Collins DP
title	In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
title_short	In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
title_full	In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
title_fullStr	In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
title_full_unstemmed	In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells
title_sort	in vitro differentiation of human tert-transfected multi-lineage progenitor cells (mlpc) into immortalized hepatocyte-like cells
publisher	Dove Medical Press
publishDate	2020
url	https://doaj.org/article/d6287c9e598a4f8db1f33c346a012c88
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In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells

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