Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy

Abstract

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases.

Keywords: ARVC; T-tubule; calcium transient; cardiac progenitor; cardiomyocyte; cardiomyopathy; disease modeling; iPS; maturation; neonatal; sarcomere shortening.

Figure 1. In Vivo-Matured PSC-CMs Show Adult CM Morphology

(A) α-Actinin (cyan) staining of mESC-CMs matured in vitro for 10 or 60 days (top) and endogenous mouse CMs at postnatal day 1 and 2 months (adult) (bottom). DAPI (blue) was used to counterstain nuclei. (B) 3D image of mESC-CMs matured in the rat heart for 2 months (Movie 1). (C) CX43 staining (red) of mESC-CMs matured in the rat heart. (D) Adult mouse heart section stained with Rat cTnT (top) and mESC-CMs (GFP⁺) matured in the rat heart (bottom). The red dotted line indicates mESC-CMs. Inset (bottom right) shows a magnified image of the white box. (E) In vivo-matured mESC-CM (GFP⁺) isolated from the rat heart (top) and adult rat CMs (bottom). (F) Average cell length and width of mouse CMs and in vivo-matured mESC-CMs at indicated stages. Data are mean ± SD; n=7 per group; *p<0.05; ***p<0.001; ns, not significant (p>0.05). p values were determined using the paired Student t test. (G) Binucleation % of adult rat CMs (n=3 hearts) and in vivo-matured mESC-CMs (n=3 hearts). (H) In vivo-matured mESC-CM (GFP⁺) in rat heart (top, left). WGA binary image and selected t-tubule network excluding surface membrane of rat-CM (yellow line) and mESC-CMs (green line) (top, middle and right). Segmentation and particle analysis of rat CMs and in vivo-matured mESC-CMs (bottom). (I) Transmission electron micrographs of adult rat CM and in vivo-matured mESC-CM. D, day; M, month; p, postnatal day; .H, H band; M, mitochondria; S, sarcomere; Tt, t-tubule; Z, Z-line. Student’s t test and one way-ANOVA were used for statistical analyses.

Figure 2. In Vivo-Matured mESC-CMs Show Adult CM Function

(A) Definitions for Ca²⁺ transient analysis. (B) Representative trace and quantification of Ca²⁺ transients, time to peak and baseline 50% and 90% for in vitro-matured mESC-CMs at day 10 (n=13) and 1 month (n=10) and in vivo-matured mESC-CMs at 1 month (n=14). (C) Representative Ca²⁺ transients and sarcomere shortening of endogenous mouse CMs and in vivo-matured mESC-CMs at indicated stages, stimulated at 0.5 Hz with pulse. (D) Quantifications of peak amplitude of Ca²⁺ transients and sarcomere shortening, time to peak, and time to baseline 50% and 90% measured with endogenous mouse CMs at 1 month (n=10) or adult stage (n=7) and with in vivo-matured mPSC-CMs at 1 month (n=8–14) or 2 months (n=13). Data are mean ± SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant (p>0.05). p values were determined using the One-way ANOVA (B) or Two-way ANOVA (D) with non-parametric multiple comparison.

Figure 3. Single-cell RNA-Seq Analysis

(A) Outline of RNA-Seq pipeline for data analysis. (B) Heatmap visualization of hierarchically clustered samples showing high (red) and low (blue) expression of 8 in vitro-matured mESC-CMs, 8 in vivo-matured mESC-CMs and 8 adult mouse CMs. (C) PCA of gene expression of in vitro (red), in vivo (blue), and adult (green) CMs. (D) Treemap plot of gene ontology (GO) analysis of differentially expressed genes showing superclusters of related terms.

Figure 4. In Vivo-Matured hiPSC-CMs Become Adult-like CMs

(A) hiPSC-CMs (GFP) engrafted in the rat heart for 1 month. (B) High resolution images of adult rat CMs (two CMs) and in vivo-matured hiPSC-CM showing well-organized sarcomeric structure. Boxed regions are enlarged in the bottom. (C) Representative sarcomere shortening and Ca²⁺ transients for in vivo-matured hiPSC-CMs compared to adult human CMs. (D) Quantifications of time to peak, time to baseline 50% and 90% of sarcomere shortening and Ca²⁺ transients of adult human CMs (n=10, red) and in vivo-matured hiPSC-CMs (n=12, black). (E) Binucleation % of in vivo-matured hiPSC-CMs. Data are mean ± SEM; ns, not significant (p>0.05). p values were determined using the non-parametric Mann-Whitney test.

Figure 5. in Vivo-Matured ARVC-hiPSC-CMs Exhibit Human ARVC Disease Phenotype

(A) Adult WT/ARVC mouse heart sections stained with antibodies against Perilipin (yellow), cTnT (green) and DAPI. Perilipin is a lipid droplet-associated protein. (B) Adult WT/ARVC mouse heart sections stained with TUNEL (red), showing apoptotic cells. (C) In vitro-matured GFP-labeled ARVC hiPSC-CMs stained with Perilipin (red), GFP (green), and α-Actinin (cyan) antibody. (D) In vivo-matured GFP labeled control hiPSC-CMs (left) and ARVC hiPSC-CMs (right) stained with Perilipin (red) and human-specific mitochondria (cyan) antibodies. DAPI (blue) was used to counterstain nuclei. (E) TUNEL staining of control hiPSC-CMs and ARVC hiPSC-CMs matured in vivo. (F) Quantification of TUNEL positive CMs. Data are mean ± SD; section number=3; hiPSC CMs (n=391), ARVC hiPSC-CMs (n= 430); *p<0.05; p values were determined using the paired Student t test. (G) Transmission electron micrographs of human control, ARVC patient CMs, and in vivo-matured ARVC hiPSC-CMs. n=10 Rats. Blue arrows indicate intercalated disc abnormalities (widening of intercellular space).