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

Daniel P Collins,Rajagopal N Aravalli,Clifford J Steer,Joel H Hapke,Gianpaolo Papaccio

doi:10.1371/journal.pone.0234002

Daniel P Collins, Rajagopal N Aravalli + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0234002

Copy DOI

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.

Highlights

Human primary hepatocytes (PH) play a crucial role in the study of liver diseases, the development of new therapies, and are the standard for toxicological studies of drug metabolism
The morphology of the various cells in this study is shown in phase contrast photos of the individual E12 cells, primary hepatocytes and the fused progeny of the two cell types (Fig 1)
Primary hepatocytes are characterized by a roundish to square shaped morphology that appears like a cobblestone when cultured at high culture densities

Summary

Introduction

Human primary hepatocytes (PH) play a crucial role in the study of liver diseases, the development of new therapies, and are the standard for toxicological studies of drug metabolism. Limitations of the use of PH include (i) brief in vitro viability with diminishing enzymatic activity over time; and (ii) large variability between donor hepatocytes with regards to plate-ability, enzymatic activity, and toxicological reactivity. A stable cell line with the functionality of hepatocytes and the proliferative capacity to be greatly expanded in vitro, in combination with the potential for largescale applications could be a useful tool for in vitro hepatocyte studies. In this study we report a novel methodology to create a long-lived cell line with the functional characteristics of PH by the in vitro fusion of an immortalized cord blood-derived stem cell with a primary human hepatocyte

Methods

Results

Discussion

Conclusion