Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes

PLoS One. 2020 Jun 4;15(6):e0234002. doi: 10.1371/journal.pone.0234002. eCollection 2020.

Abstract

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Fusion*
  • Cells, Cultured
  • Hepatocytes / cytology*
  • Hepatocytes / metabolism
  • Humans
  • Hybrid Cells / cytology*
  • Hybrid Cells / metabolism
  • Stem Cells / cytology*
  • Stem Cells / metabolism
  • Telomerase / genetics
  • Transfection

Substances

  • TERT protein, human
  • Telomerase

Grants and funding

CytoMedical Design Group, LLC (CMDG) provided funding for this study in the form of salaries for DPC and JHH. Additionally, funding was provided by BioE, LLC. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.