人诱导多能干细胞来源心肌细胞的低致瘤风险及亚型

The Low Tumorigenic Risk and Subtypes of Cardiomyocytes Derived from Human-induced Pluripotent Stem Cells.

作者信息

Lu Jizhen, Zhang Lu, Cao Hongxia, Ma Xiaoxue, Bai Zhihui, Zhu Hanyu, Qi Yiyao, Zhang Shoumei, Zhang Peng, He Zhiying, Yang Huangtian, Liu Zhongmin, Jia Wenwen

机构信息

National Stem Cell Translational Resource Center/GMP Laboratory of Stem Cell Transformation Medicine Industry Base, Shanghai East Hospital (East Hospital Affiliated to Tongji University), Tongji University School of Life Sciences and Technology, Shanghai, People's Republic of China.

Translational Medical Center for Stem Cell Therapy & Institute for Heart Failure and Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine and Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, People's Republic of China.

出版信息

Curr Stem Cell Res Ther. 2025;20(3):317-335. doi: 10.2174/011574888X318139240621051224.

DOI:10.2174/011574888X318139240621051224

PMID:40351082

Abstract

BACKGROUND

Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a promising approach for the treatment of heart diseases. However, the tumorigenicity of hiPSC-CMs remains a concern for their clinical applications and the composition of the hiPSC-CM subtypes need to be clearly identified.

METHODS

In the present study, hiPSC-CMs were induced from hiPSCs via modulation of Wnt signaling followed by glucose deprivation purification. The structure, function, subpopulation composition, and tumorigenic risk of hiPSC-CMs were evaluated by single-cell RNA sequencing (scRNAseq), whole exome sequencing (WES), and integrated molecular biology, cell biology, electrophysiology, and/or animal experiments.

RESULTS

The high purity of hiPSC-CMs, determined by flow cytometry analysis, was generated. ScRNAseq analysis of differentiation day (D) 25 hiPSC-CMs did not identify the transcripts representative of undifferentiated hiPSCs. WES analysis showed a few newly acquired confidently identified mutations and no mutations in tumor susceptibility genes. Further, no tumor formation was observed after transplanting hiPSC-CMs into NOD-SCID mice for 3 months. Moreover, D25 hiPSC-CMs were composed of subtypes of ventricular-like cells (23.19%) and atrial-like cells (66.45%) in different cell cycle stages or mature levels, based on the scRNAseq analysis. Furthermore, a subpopulation of more mature ventricular cells (3.21%) was identified, which displayed significantly up-regulated signaling pathways related to myocardial contraction and action potentials. Additionally, a subpopulation of cardiomyocytes in an early differentiation stage (3.44%) experiencing nutrient stress-induced injury and heading toward apoptosis was observed.

CONCLUSIONS

This study confirmed the biological safety of hiPSC-CMs and described the composition and expression profile of cardiac subtypes in hiPSC-CMs which provide standards for quality control and theoretical supports for the translational applications of hiPSC-CMs.

摘要

背景

人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）的临床应用是治疗心脏病的一种有前景的方法。然而，hiPSC-CMs的致瘤性仍然是其临床应用中的一个问题，并且hiPSC-CM亚型的组成需要明确鉴定。

方法

在本研究中，通过Wnt信号通路的调节，随后进行葡萄糖剥夺纯化，从人诱导多能干细胞（hiPSCs）诱导生成hiPSC-CMs。通过单细胞RNA测序（scRNAseq）、全外显子组测序（WES）以及综合分子生物学、细胞生物学、电生理学和/或动物实验，对hiPSC-CMs的结构、功能、亚群组成和致瘤风险进行评估。

结果

通过流式细胞术分析确定生成了高纯度的hiPSC-CMs。对分化第25天（D25）的hiPSC-CMs进行scRNAseq分析，未发现代表未分化hiPSCs的转录本。WES分析显示有一些新获得的经可靠鉴定的突变，且肿瘤易感基因无突变。此外，将hiPSC-CMs移植到NOD-SCID小鼠体内3个月后，未观察到肿瘤形成。而且，基于scRNAseq分析，D25 hiPSC-CMs由处于不同细胞周期阶段或成熟水平的心室样细胞亚型（23.19%）和心房样细胞亚型（66.45%）组成。此外，鉴定出一个更成熟的心室细胞亚群（3.21%），其与心肌收缩和动作电位相关的信号通路显著上调。另外，观察到一个处于早期分化阶段的心肌细胞亚群（3.44%），其经历营养应激诱导的损伤并趋向凋亡。