Human epicardial organoids from pluripotent stem cells resemble fetal stage with potential cardiomyocyte- transdifferentiation

Abstract

Epicardium, the most outer mesothelium, exerts crucial functions in fetal heart development and adult heart regeneration. Here we use a three-step manipulation of WNT signalling entwined with BMP and RA signalling for generating a self-organized epicardial organoid that highly express with epicardium makers WT1 and TCF21 from human embryonic stem cells. After 8-days treatment of TGF-beta following by bFGF, cells enter into epithelium-mesenchymal transition and give rise to smooth muscle cells. Epicardium could also integrate and invade into mouse heart with SNAI1 expression, and give birth to numerous cardiomyocyte-like cells. Single-cell RNA seq unveils the heterogeneity and multipotency exhibited by epicardium-derived-cells and fetal-like epicardium. Meanwhile, extracellular matrix and growth factors secreted by epicardial organoid mimics the ecology of subepicardial space between the epicardium and cardiomyocytes. As such, this epicardial organoid offers a unique ground for investigating and exploring the potential of epicardium in heart development and regeneration.

Keywords: Epicardial organoid; Epicardial-derived cells; Epithelial-mesenchymal transition; Heterogeneity; Paracrine.

Fig. 1

Generation of epicardial organoid from hPSCs. (A) Up, bright field images captured with time course showed gradually enlarged chamber. Middle, scheme of the protocol used to differentiate hPSCs toward the epicardium lineage highlighting four main stages of development: (i) mesoderm induction, (ii) cardiac progenitor cells canalization, (iii) PE induction, (iv) Epicardium specification. Key chemicals used in each step are list under the arrow. Down, heatmap of representative genes on time course bulk RNA sequencing at d0, d1.5, d3.5, d6.5, d10.5, d16.5 and d31 with two replicates each timepoint. (B) Quantification qRT-PCR analysis on epicardium markers TCF21 at d6.5, TBX18 at d10.5 and WT1 at critical knots. Immunofluorescence of mesodermal marker BRACHYURY at d1.5 (C) and epicardium marker WT1 at d12.5 and d40.5 (D). (E) Expression level (log2FC) of other reported epicardium markers, PDGFRA, SEMA3D, UPK3B and ALDH1A2. (F) GSEA score of enriched genes at d10.5 showed ECM receptor pathways and epicardium development are specifically activated. Scale bar, 500 μm. Significance analysis uses a standard unpaired Student t-test (2-tailed; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance)

Fig. 2

Epicardial organoid underwent EMT to give rise to SMCs and supported with paracrine factors. (A) Scheme of the protocol for inducing EMT and qRT-PCR analysis of WT1, SMC marker SMTN and EMT marker TWIST1 and SNAI1.Bright field images of organoid at 17.5 with or without treatment (B, upper, scale bar 500 μm). Immunofluorescence of WT1 (B, lower), mesenchymal marker VIM and SMC marker α-SMA at d17.5 (C) showed organoid can transit from epithelium to mesenchymal type leading to increase of SMCs. (D) Polarity validation by staining ZO-1 (marker of epicardium tight junction) and CDH1 (marker of epicardium adherens junction). (E) and (F) Transmission electron microscopy image of epicardial organoid at day 108. Desmosomes (arrow), adherens junctions (arrowhead), tight junctions (asterisks) of epicardium, ciliary tract (dash arrow) of EC. Venn diagram of ECM (G) and GF (H) by comparing fresh culturing medium, inner-organoid fluid (InFlu) and extra-organoid fluid (ExFlu) enriched by mass spectrum. (I) KEGG and GO analysis of GFs secreted by epicardial organoid. Significance analysis uses a standard unpaired Student t-test (2-tailed; *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Epicardial organoid with inner heterogeneity. (A) UMAP dimensional reduction plot showing the 17 cell clusters obtained by scRNA-seq at d7.5, d9.5 and d18.5; main cell types are annotated. (B) Violin plots showing the expression levels of different cell type markers. (C) Stream flow plot for cell number percent of different cell type. (D) Immunofluorescence label for TNNT2 (CM), CDH5 (EC), CDX2, AFP, and HNF4α (pF/H Epithe). (E) Feature plot of canonical epicardial markers. (F) Nine subclusters analyzed from c5 and c12. (G) Heatmap of differential TF sets between EPDC (CM diff.), EPDC (CM diff.) and Epi (EMT)

Fig. 4

Epicardium exerts migration and EMT features both in vitro and in vivo. (A) Scheme of coculture of epicardium organoid and cardioid. (B) Epicardium wrapped outside the myocardium after coculturing epicardial organoid with cardioid for more than 1 month. (C) Enlarged image from white box of (B) showed sarcomere (arrowhead) and CM maturation marker MYL2. Scale bar, 100 μm. (D) Implantation procedure of epicardial organoid. Mouse heart function detected by ultrasonic test from parasternal long-axis view (PLAX), parasternal short-axis view (PSAX) and apical four-chamber view (AFC) (E), as well as electrocardiograph (ECG) test (F) before and after transplantation. Whole-heart staining showed human epicardial organoid integrated with mouse heart with WT1 + cells lining the most outside (G) and tdTomato + SNAI1 + cells spreading LV, RV, IVS and LVPM (H) and (I). (J) Enlarged picture of LVPM showed colocation of tdTomato + and SNAI1 + cells. (K) Cell number of tdT⁺ human cells distributed different parts of mouse heart. EF, ejection fraction. FS, fractional shortening. CO, cardiac output. HR, heart rate. RR-I, RR interval. R-H, R peak height. LV, left ventricle. RV, right ventricle. IVS, interventricular septum. LVPM, left ventricle papillary muscle. Significance analysis uses one-way ANOVA with the Tukey’s or Dunnett’s post-test correction was applied when appropriate (*p < 0.05, **p < 0.01, ***p < 0.001)