Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells Can Be Enriched by a Combination of Mitochondrial Content and Activated Leukocyte Cell Adhesion Molecule.

Abstract

Introduction: Non-genetic purification methods for pluripotent stem cell-derived hepatocyte-like cells are useful for liver regenerative therapy and pharmaceutical applications. Methods: Fluorescent activated cell sorting (FACS) was used to separate cells by combining two parameters: cellular mitochondrial content evaluated by the mitochondrial membrane potential-dependent fluorescent probe (TMRM) and immunocytochemical detection of activated leukocyte cell adhesion molecule (ALCAM). This method was applied to murine fetal, human embryonic stem cell (ESC)-derived, and human induced pluripotent stem cell (iPSC)-derived cell-mixtures. Separately sorted cell fractions were evaluated by quantitative PCR, immunohistochemistry, and cytochemistry for HNF4a, AFP, and albumin mRNA and/or protein expression. Results: Hepatocyte-like cells were segregated into the high TMRM signal and ALCAM-positive population. The purity of hepatocyte-like cells derived from human iPSCs was 97 ± 0.38% (n = 5). Conclusions: This hepatocyte-like cell purification method may be applicable to the quality control of cells for liver regenerative cell therapy and pharmaceutical development.