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

ABSTRACT: A limitation in the application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. The mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, remain unclear. Here, we generate a single-cell RNA sequencing (scRNA-seq) reference of mouse in vivo CM maturation with extensive sampling of previously difficult-to-isolate perinatal time periods. We subsequently generate isogenic embryonic stem cells to create an in vitro scRNA-seq reference of PSC-CM-directed differentiation. Through trajectory reconstruction, we identify an endogenous perinatal maturation program that is poorly recapitulated in vitro. By comparison with published human datasets, we identify a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study can be leveraged toward improving the clinical viability of PSC-CMs.

SUBMITTER: Kannan S

PROVIDER: S-EPMC10545814 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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

Kannan Suraj S Miyamoto Matthew M Zhu Renjun R Lynott Michaela M Guo Jason J Chen Elaine Zhelan EZ Colas Alexandre R AR Lin Brian Leei BL Kwon Chulan C

Cell reports 20230403 4

A limitation in the application of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. The mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, remain unclear. Here, we generate a single-cell RNA sequencing (scRNA-seq) reference of mouse in vivo CM maturation with extensive sampling of previously difficult-to-isolate perinatal time periods. We subsequ ...[more]

PMID: 37014753

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Project description:Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including ?-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.

Project description:The timing of lung maturation is controlled precisely by complex genetic and cellular programs. Lung immaturity following preterm birth frequently results in Respiratory Distress Syndrome (RDS) and Broncho-Pulmonary Dysplasia (BPD), which are leading causes of mortality and morbidity in preterm infants. Mechanisms synchronizing gestational length and lung maturation remain to be elucidated. In this study, we designed a genome-wide mRNA expression time-course study from E15.5 to Postnatal Day 0 (PN0) using lung RNAs from C57BL/6J (B6) and A/J mice that differ in gestational length by ∼30 hr (B6<A/J). Comprehensive bioinformatics and functional genomics analyses were used to identify key regulators, bioprocesses and transcriptional networks controlling lung maturation. We identified both temporal and strain dependent gene expression patterns during lung maturation. For time dependent changes, cell adhesion, vasculature development, and lipid metabolism/transport were major bioprocesses induced during the saccular stage of lung development at E16.5-E17.5. CEBPA, PPARG, VEGFA, CAV1 and CDH1 were found to be key signaling and transcriptional regulators of these processes. Innate defense/immune responses were induced at later gestational ages (E18.5-20.5), STAT1, AP1, and EGFR being important regulators of these responses. Expression of RNAs associated with the cell cycle and chromatin assembly was repressed during prenatal lung maturation and was regulated by FOXM1, PLK1, chromobox, and high mobility group families of transcription factors. Strain dependent lung mRNA expression differences peaked at E18.5. At this time, mRNAs regulating surfactant and innate immunity were more abundantly expressed in lungs of B6 (short gestation) than in A/J (long gestation) mice, while expression of genes involved in chromatin assembly and histone modification were expressed at lower levels in B6 than in A/J mice. The present study systemically mapped key regulators, bioprocesses, and transcriptional networks controlling lung maturation, providing the basis for new therapeutic strategies to enhance lung function in preterm infants.

Dataset Information

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

Publications

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

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