Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

Abstract

A primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

Integrative transcriptomics and cell systems analyses reveal protective pathways controlled by Igfbp‐3 in anthracycline‐induced cardiotoxicity

“…We assessed general pathways enriched in NRVM and iPSC-CM by gene ontology, which further supports our findings that iPSC-CM are in a maturation pseudotime between P7 and P14 CM, whereas NRVM are in P0 pseudotime, with ensuing distinct transcriptomic profiles. Our results are corroborated by a recent single-cell study characterizing the maturation of iPSC-CM and P0-P84 CM 74 that also suggested an overlap between iPSC-CM and P11-P14 CM.…”

scInTime: A Computational Method Leveraging Single-Cell Trajectory and Gene Regulatory Networks to Identify Master Regulators of Cellular Differentiation

“…The data set can be found on the GEO database using accession GSE137524. Data set 3: Cardiomyocyte data set from the study of [ 9 ]. The data were downloaded from the GEO database using accession GSE164591.…”

“…Data set 2 is from the human SCC6 cell line treated with cetuximab and PBS (control) [ 8 ]. Data set 3 is from differentiating mouse cardiomyocytes across fifteen real timepoints [ 9 ]. In all three cases, we set out to examine whether the top-ranking gene candidates reported by scInTime will be aligned well with those obtained from functional studies, in which corresponding master regulators are reported.…”

Using human induced pluripotent stem cell-derived cardiomyocytes to understand the mechanisms driving cardiomyocyte maturation

“…According to a recent study, NrF2 encoded by the NFE2L2 gene is highly expressed during the early stages of mouse in vivo CM differentiation which aligns well with its role in enhancing mitochondrial respiration as part of early CM metabolic maturation. However, the time-course of NFE2L2 activity in vivo is not recapitulated in in vitro PSC-CM differentiation and was posited to be one of the key factors responsible for maturation arrest of in vitro CM differentiation ( 93 ). It is complicated to interpret the significance of NFE2L2 stage-specific activity in CM maturation; however, increased ROS levels is an important inducer of DNA damage response which is implicated in CM proliferation arrest as part of CM maturation process ( 94 ).…”

“…The results of recent studies suggest that current maturation protocols produce CMs that are still far from the adult CM state; this failure is partly due to the fact that none of the methods are sufficient to fully activate all major TFs involved in CM maturation ( 93 ). Another major limitation of current methodologies is the absence of a global metric for the assessment of the degree of CM maturation reported by different studies.…”