In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells

PLoS One. 2022 Nov 15;17(11):e0275600. doi: 10.1371/journal.pone.0275600. eCollection 2022.

Abstract

Cell therapies using human induced pluripotent stem cell (hiPSC)-derived nephron progenitor cells (NPCs) are expected to ameliorate acute kidney injury (AKI). However, using hiPSC-derived NPCs clinically is a challenge because hiPSCs themselves are tumorigenic. LIN28A, ESRG, CNMD and SFRP2 transcripts have been used as a marker of residual hiPSCs for a variety of cell types undergoing clinical trials. In this study, by reanalyzing public databases, we found a baseline expression of LIN28A, ESRG, CNMD and SFRP2 in hiPSC-derived NPCs and several other cell types, suggesting LIN28A, ESRG, CNMD and SFRP2 are not always reliable markers for iPSC detection. As an alternative, we discovered a lncRNA marker gene, MIR302CHG, among many known and unknown iPSC markers, as highly differentially expressed between hiPSCs and NPCs, by RNA sequencing and quantitative RT-PCR (qRT-PCR) analyses. Using MIR302CHG as an hiPSC marker, we constructed two assay methods, a combination of magnetic bead-based enrichment and qRT-PCR and digital droplet PCR alone, to detect a small number of residual hiPSCs in NPC populations. The use of these in vitro assays could contribute to patient safety in treatments using hiPSC-derived cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation / genetics
  • Humans
  • In Vitro Techniques
  • Induced Pluripotent Stem Cells* / metabolism
  • Nephrons
  • Neuroblastoma* / metabolism
  • RNA, Long Noncoding* / metabolism

Substances

  • RNA, Long Noncoding

Grants and funding

This research is supported by RegeNephro Co., Ltd. (150204400001), by the Japan Agency for Medical Research and Development (AMED) through its research grant ‘Research Project for Practical Applications of Regenerative Medicine (22bk0104113h0003)’ to K.O. and by iPS Cell Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.