Human iPSC cardiomyocyte patch transplantation modifies extracellular matrix and fibroblast behavior after myocardial infarction

Figure 1. Transplanted hiPSC-derived CM patch engrafted, survived, and aligned in the immunodeficient rat MI model for 12 weeks (A) Experimental scheme using immunodeficient rats (F344/NJcl-rnu/rnu). (B) Diagram of the heart transplanted with the hiPSC-derived CM patch. IZ, infarct zone; BZ, border zone; RZ, remote zone. (C) Engrafted hiPSC-derived CM patch 4 weeks after transplantation is shown. hcTnT, red; human nuclei, green; DAPI, blue. Scale bar, 1 mm. (D) Maturation of the hiPSC-derived CM patch after transplantation. Immunoreactive signal against anti-MLC2a and anti-MLC2v antibodies is shown. The transplanted CMs are shown with immunostaining using an anti-hcTnT antibody or staining with MitoTracker and indicated with yellow dot lines. Scale bar, 200 μm. (E) Sarcomere development of the transplanted hiPSC-derived CM patch. hNA, red; α-actinin, green. Enlarged images of indicated region in the upper part are shown in the lower part. Scale bars, 200 μm or 12.5 μm.

Figure 2. Functional recovery of the heart after hiPSC-derived CM patch transplantation (A) Untransplanted sham control (N = 9) and hiPSC-derived CM patch transplanted rats (N = 10) following MI, and sham control (N = 5) at the indicated time after transplantation. Repeated-measures ANOVA revealed a trend for a group effect (p = 0.054), and post hoc comparisons at each time point with Bonferroni correction are shown. ∗p < 0.05, ∗∗p < 0.01. Data represent mean ± SEM. (B–D) Analysis of LVEDP, LV dP/dt max, and LV dP/dt min in untransplanted control (4 weeks, N = 11; 12 weeks, N = 7) and hiPSC-derived CM patch-transplanted rats (4 weeks, N = 13; 12 weeks, N = 10) following MI at the indicated time after transplantation. Two-tailed unpaired t test, ∗p < 0.05. Data represent mean ± SEM. (E) Lung weight of untransplanted control (4 weeks, N = 10; 12 weeks, N = 7) and hiPSC-derived CM patch-transplanted rats (4 weeks, N = 13; 12 weeks, N = 10) following MI at the indicated time after transplantation. Two-tailed unpaired t test, ∗p < 0.05, ∗∗p < 0.01. Data represent mean ± SEM.

Figure 3. hiPSC-derived CM patch transplantation promoted collagen fiber alignment (A) Ventricular passive pressure-volume relationship of untransplanted control (N = 10) and hiPSC-derived CM patch-transplanted rats (N = 13) following MI at 4 weeks after transplantation is shown. #p < 0.1, ∗p < 0.05. Data represent mean ± SEM. two-tailed unpaired t test. p values adjusted for multiple testing using the Bonferroni method. (B) Visualization of collagen fibers in the infarct zone using a linear-polarizing microscopy. Samples were examined by Pico-Sirius Red staining. The signal corresponding to the collagen type I fiber is shown in orange, while that corresponding to collagen type III is shown in green. The samples were collected from the rats 4 weeks after transplantation. Scale bar, 200 μm. (C) Quantification of the ratio between collagen type I and collagen type III expression at 1 week (N = 5, control; N = 6, patch), 4 weeks (N = 11, control; N = 13, patch), and 12 weeks (N = 9, control; N = 11, patch) after transplantation. Two-tailed unpaired t test, ∗∗p < 0.01, ∗∗∗p < 0.001. Data represent mean ± SEM. (D) Quantification of collagen fiber alignment. Number of collagen fibers within a…

Figure 4. hiPSC-derived CM patch transplantation improved tissue elasticity (A and B) Finite element mesh and first primary strain distribution at LV of control, infarcted, and implanted models. (C) Interstitial fibrosis in the RZ visualized with Masson’s trichrome staining. Representative images from sham control, control, and patch-transplanted rats are shown. Scale bar, 100 μm. (D) Quantification of the interstitial fibrosis is shown. Fibrotic area was measured from the RZ of the rat 4 weeks after transplantation. N = 10 control, N = 13 transplanted rats. Two-tailed unpaired t test, ∗∗p < 0.01. Data represent mean ± SEM.

Figure 5. The hiPSC-derived CM patch secreted TGF-β in vitro and in vivo (A and B) Gene Ontology (GO) analysis of biological process. Metascape gene and functional annotation to produce gene cluster was examined. GO terms corresponding to biological process were extracted. Then, functional cluster analysis was carried out. Among the top 20 clusters, GOs focusing on secreted factors from hiPSC-derived CM patch are listed. (C) Secreted factors from hiPSC-derived CM patch in vitro were measured with ELISA. Top 13 factors identified are listed. Data represent mean ± SD. N = 5.

Figure 6. TGF-β signaling was upregulated in the host rat heart (A) Differentially expressed genes were enriched. Scatterplot showing the relationship between gene expression level with and without hiPSC-derived CM patch on the heart. The red dots and the blue dots represent the upregulated and downregulated genes, respectively. The gray dots represent the genes that were not differentially expressed. (B) Identified genes in (A) were analyzed by Ingenuity Pathway Analysis (IPA) upstream regulators. The summary network depicted the interactions between upstream regulators and downstream genes. (C) Based on the bias-corrected Z score and overlap p value, top 20 predicted upstream factors are shown with the scores. (D) The TGF-β1 pathway was identified with the highest score. All identified genes were overlaid onto the DEGs. (E) RT-qPCR analysis of Acta2, and Cxcl12 in the sample derived from BZ, and Cola1a1 and Col3a1 in the sample derived from IZ in the transplanted heart. The ratio of Cola1a1 and Col3a1 is also shown. Data represent mean ± SEM. N = 6 for the sample derived from BZ, and N = 5 for the sample derived from IZ. The signal was normalized with the value of Actb. Two-tailed…

Figure 7. TGF-β secreted from the hiPSC-derived CM patch induced Col1a1 expression but not Col3a1 (A) Schematic representation of the co-culture system. hiPSC-derived CMs were seeded on the upper chamber, whereas the cardiac fibroblasts isolated from mice heart were cultured at the lower chamber in the presence and absence of SB431542, a TGF-β receptor inhibitor. (B) RT-qPCR analysis of Col1a1, Col3a1, and Pai1 in the cultured cardiac fibroblasts. Two-way ANOVA (co-culture × inhibitor) with interaction; Tukey-adjusted post hoc tests on estimated marginal means. Data represent mean ± SEM. N = 3–6, one-way ANOVA followed by Tukey’s HSD test, p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.