IMR Press / FBL / Volume 27 / Issue 8 / DOI: 10.31083/j.fbl2708239
Open Access Original Research
Universal Markers for hiPSCs Residue Detection
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1 Molecular Pharmacology Laboratory, Institute of Molecular Medicine, Peking University, 100871 Beijing, China
2 Allife Medical Science and Technology Co., Ltd. Economic and Technological Development Zone, 100176 Beijing, China
*Correspondence: wldpaper@pku.edu.cn (Lida Wu); ycgu@pku.edu.cn (Yuchun Gu)
Academic Editor: Graham Pawelec
Front. Biosci. (Landmark Ed) 2022, 27(8), 239; https://doi.org/10.31083/j.fbl2708239
Submitted: 27 May 2022 | Revised: 8 July 2022 | Accepted: 25 July 2022 | Published: 11 August 2022
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

Background: Residual undifferentiated induced pluripotent stem cells (iPSCs) detection is essential for both Embryonic Stem Cells (ESCs) and iPSCs application in final cell therapy products. However, specific differentiated cells require specific genes for residual detection; identifying the suitable marker is costly and time-consuming. Thus, a universal marker for iPSCs residue detection for all three germline cells would greatly benefit PSC-derived cellular therapies. Methods: Next-generation sequencing (NGS) was performed on total RNAs isolated from the iPSC cell lines and embryonic stem cells (H9), the top 30 expressed genes were selected as candidates. By analysis expression fold change comparing iPSC cells to the differentiated cells, seven genes were highly expressed in iPSCs but showed minimal background expression in differentiated cells. Tissue expression pattern of the candidate genes were explored in the Genotype-Tissue Expression (GTEx) project database, candidate genes were narrowed down to two genes. Spike-in experiments were performed to determine the detection limit and correlation with the number of iPSCs and gene expression by ddPCR. Results: By next-generation sequencing (NGS), we identified two marker genes (ESRG and ZSCAN10) suitable for universal undifferentiated iPSC detection. Both ESRG and ZSCAN10 are highly expressed in iPSCs. ZSCAN10 is slightly expressed in the testis, pituitary, and cerebellum; ESRG is highly expressed in the vagina and scarcely expressed in the other tissues. Furthermore, the ddPCR method with a probe and primers for ESRG and ZSCAN10 detected a trace of undifferentiated hiPSCs to a spiked level of 0.0001%. Conclusions: These results suggest that targeting ESRG/ZSCAN10 transcripts is highly sensitive, quantitative, and could be broadly applied to quality control of almost all iPSC-derived cell therapy products.

Keywords
human pluripotent stem cell
cell therapy
ddPCR
ESRG
ZSCAN10
Figures
Fig. 1.
Funding
2013CB531206/ 973 Project
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