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Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine.

ABSTRACT: Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), serve as highly valuable sources for both cell-based therapies and basic research, owing to their abilities to self-renew and differentiate into any cell type of the human body. However, tumorigenic risks of residual undifferentiated stem cells limit the clinical application of hiPSCs, necessitating methods to eliminate undifferentiated hiPSCs from differentiated cells. Here, we found that undifferentiated hiPSCs were more sensitive to the treatment with a medium supplemented with high concentration of L-alanine than human fibroblasts (hFBs), human skeletal muscle cells (hSkMCs), hiPSC-derived cardiomyocytes (iCMs) or hiPSC-derived fibroblast-like cells (iFLCs), which were used as differentiated cells. Undifferentiated hiPSCs co-cultured with differentiated cells were selectively eliminated following treatment. In addition, we found that the medium supplemented with high concentration of D-alanine or ?-alanine also induced cell death of hiPSCs and the treatment at 4?°C didn't induce cell death of hiPSCs. The cell death induced would be associated partly with high osmotic pressure of the medium supplemented with L-alanine. As L-alanine is a component of proteins in human body and popular ingredient of cell culture media, treatment with high concentration of L-alanine may be useful for eliminating tumorigenic residual hiPSCs for stem cell-based therapies.

SUBMITTER: Nagashima T

PROVIDER: S-EPMC6102287 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine.

Nagashima Takunori T Shimizu Kazunori K Matsumoto Ryo R Honda Hiroyuki H

Scientific reports 20180820 1

Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), serve as highly valuable sources for both cell-based therapies and basic research, owing to their abilities to self-renew and differentiate into any cell type of the human body. However, tumorigenic risks of residual undifferentiated stem cells limit the clinical application of hiPSCs, necessitating methods to eliminate undifferentiated hiPSCs from differentiated cells. Here, we found that undifferentiated hiP ...[more]

PMID: 30127448

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Project description:Background & aimsPreclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution.MethodsWe first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform.ResultsHLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis.ConclusionsLiver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

Dataset Information

Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine.

Publications

Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine.

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