Single-cell sequencing reveals the potential oncogenic expression atlas of human iPSC-derived cardiomyocytes

Abstract

Human induced pluripotent stem cells (iPSCs) are important source for regenerative medicine. However, the links between pluripotency and oncogenic transformation raise safety issues. To understand the characteristics of iPSC-derived cells at single-cell resolution, we directly reprogrammed two human iPSC lines into cardiomyocytes and collected cells from four time points during cardiac differentiation for single-cell sequencing. We captured 32,365 cells and identified five molecularly distinct clusters that aligned well with our reconstructed differentiation trajectory. We discovered a set of dynamic expression events related to the upregulation of oncogenes and the decreasing expression of tumor suppressor genes during cardiac differentiation, which were similar to the gain-of-function and loss-of-function patterns during oncogenesis. In practice, we characterized the dynamic expression of the TP53 and Yamanaka factor genes (OCT4, SOX2, KLF4 and MYC), which were widely used for human iPSCs lines generation; and revealed the co-occurrence of MYC overexpression and TP53 silencing in some of human iPSC-derived TNNT2+ cardiomyocytes. In summary, our oncogenic expression atlas is valuable for human iPSCs application and the single-cell resolution highlights the clues potentially associated with the carcinogenic risk of human iPSC-derived cells.

MARVEL: An integrated alternative splicing analysis platform for single-cell RNA sequencing data

“…To demonstrate the utility of MARVEL for single-cell alternative splicing on a dataset from a droplet-based platform, we retrieved scRNA-seq data from iPSC and iPSC- derived cardiomyocytes on days 2, 4, and 10 generated using 10x Genomics Chromium Single Cell 3’ Reagent Kit (version 2) (27). Raw sequencing reads (FASTQ) were downloaded from the Sequence Reads Archive (SRA) and were aligned to the GRCh38 reference genome using Cell Ranger v2.1.1.…”

“…To assess the ability of MARVEL to identify biologically relevant genes that are differentially spliced, we performed differential splicing analysis on a dataset generated from iPSC and iPSC-derived cardiomyocytes on day 10 (27).…”

“…Here, we introduce MARVEL, an R package for integrative single-cell alternative splicing and gene expression analysis. We benchmarked MARVEL against existing computational tools for single-cell alternative splicing analysis and demonstrated its utility by analyzing publicly available datasets generated from the plate- and droplet-based library preparation methods derived from induced pluripotent stem cells (iPSCs) differentiated into endoderm and cardiomyocytes, respectively (26, 27).…”

Cell-Based and Selected Cell-Free Therapies for Myocardial Infarction: How Do They Compare to the Current Treatment Options?

“…Even so, before routine clinical application of stem cells, it is necessary to resolve safety issues regarding the risk of oncogenic transformation, inefficient delivery to the site of injury, problems with cell engraftment due to the lack of microvasculature, cell senescence, and the electric coupling of transplanted CMs within the myocardium. There is also another limitation causing poor cell graft viability in terms of cell adhesion loss driven by the mechanism of anoikis [ 34 , 35 , 36 ]. However, the solution to the mentioned obstacles may be the coordination of cell-based technologies with rapidly developing bioengineering strategies, such as 3D bioprinted cardiac microtissues or patches [ 37 , 38 ].…”

Advances in application of single-cell RNA sequencing in cardiovascular research

“…However, the endothelial cells and cardiomyocytes derived from iPSCs have differences in physiological structure and gene expression compared to mature cardiac cells, which is a barrier to the application of iPSCs in clinical treatment ( 59 ). scRNA-seq can capture the regulatory processes of stem cell differentiation and development with unprecedented sensitivity, providing technical support and a theoretical basis for stem cell transplantation ( 60 ). Paik et al ( 61 ) performed large-scale droplet-based scRNA-seq on thousands of human iPSC-derived endothelial cells.…”