Development of genetic quality tests for good manufacturing practice-compliant induced pluripotent stem cells and their derivatives

Hye-Yeong Jo,Soon-Jung Park,Dongho Geum,Sunghwan Moon,Hyemin Kim,Jung-Hyun Kim,Inuk Jung,Glyn N Stacey,Hyo-Won Han,Ji Hyeon Ju,Soo Kyung Koo,Mi-Hyun Park,Sun Kim,Han-Jin Park

doi:10.1038/s41598-020-60466-9

Abstract

Although human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. Accordingly, intensive and relevant quality controls for clinical-grade hiPSCs remain imperative. As a conceptual approach, we performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. We found differences in the degree of single-nucleotide polymorphism (SNP) occurring in cells cultured at three collaborating institutes. However, the cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in early-passage hiPSCs after differentiation. In eSNP karyotyping analysis, none of the predicted copy number variations (CNVs) were identified, which confirmed the results of SNP chip-based CNV analysis. HLA genotyping analysis revealed that each cell line was homozygous for HLA-A, HLA-B, and DRB1 and heterozygous for HLA-DPB type. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. However, time-course analysis identified five clusters showing different patterns of gene expression, which were mainly related to the immune response. In conclusion, RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation.

Highlights

Human induced pluripotent stem cell lines are karyotypically normal, they retain the potential for mutation in the genome
human leucocyte antigen (HLA)-type good manufacturing practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) lines CMC3, CMC9, and CMC11 were distributed to three external institutions, where they were expanded and differentiated into the three germ layers [neuronal cells, cardiomyocytes, and hepatocyte-like cells; Fig. 1a]
We focused in particular on the genomic variations that may affect the genetic stability of hiPSCs and their derivatives, the significant expression levels and expression patterns of lineage-specific markers, and the effects of long-term culture

Summary

Introduction

Human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. We performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. The cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in earlypassage hiPSCs after differentiation. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation. Mary’s Hospital, College of Medicine, Catholic University of Korea, Seoul, South

Methods

Results

Conclusion