Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro

Figure 1. PC-like cells can be efficiently generated from human iPSCs. (A) Schematic representation of the differentiation process depicting the timeline as well as the used small molecules, growth factors, and media. (B) Pluripotency markers OCT4 and NANOG gradually decrease relative to the D0 of differentiation determined by qRT-PCR. Upregulation of early mesodermal marker T, followed by the upregulation of Kinase Insert Domain Receptor (KDR), indicates the induction of mesoderm. Expression levels of PC markers (PDGFRα, PDGFRβ, and NG2) are expressed relative to human placental pericytes (hPC-PLs), and the EC markers (CD31 and CD144) are shown relative to HUVECs [35] (n = 3–6). (C) Flow cytometric analysis for CD31 and PDGFRβ on D7 and D10 in both iPSC lines used for differentiation (n = 3–6). (D) Representative plot of stained (CD31, PDGFRβ) iPSC6-derived cells on differentiation D10 prior to fluorescence-activated cell sorting.

Figure 2. iPSC-PCs exhibit a PC-like function and cardiac phenotype. (A) Flow cytometric analysis of the mesenchymal surface marker expression post-maturation on D18 (black line—isotype control) and, (B) following expansion, in P4 (n = 3). (C) The level of PDGFRβ and NG2 in iPSC-PCs (P4) compared to hPC-PL (black line—isotype control). (D) Network formation in fibrin matrices (cultured in EGM-2 for 7 days) containing iPSC-EC only, iPSC-EC+hFF, and iPSC-EC+iPSC-PC, and NG2 expression in the fixed co-cultures. Nuclei stained with DAPI, iPSC-ECs express eGFP (iPSC9_eGFP). Scale bars: 100 µm. (E) Higher magnification Z-stack image of iPSC-EC+iPSC-PC networks with orthogonal projections. (F) PCA plot showing the first two principal components (data points represent one sample in the microarray analysis). (G) Significantly enriched gene ontologies (GOs) in iPSC-PC vs. hPSC (selected genes from the GOs marked with blue, pink, and yellow are shown on plot G). (H) Expression of ECM-related structural genes (blue), cardiac transcription factors (yellow), and immunomodulatory cytokines/receptors (pink) in hPSCs, primary PCs and Fbs, and iPSC-PCs. On the balloon plot, the size of the circles…

Figure 3. iPSC-PCs in cardiac tissues initiate a fibrosis-like remodeling process. (A) Schematic representation of BCT cultivation with growing stretch. (B) 1 × 106 iPSC- or hESC-derived CMs were mixed either with 1 × 105 hFFs or 1 × 105 iPSC-PCs for tissue formation. The colors and symbols serve as legends for the other diagrams of the illustration. (C) Tissue diameter measured on D21 (for C and E–G BCT with iPSC-CM n = 3–6; BCT with hESC-CM n = 9–12 tissues per group) (D) Representative spontaneous contractions normalized to peak height (iPSC-CMs—dashed lines, hESC-CMs. continuous lines). PD—peak duration at 50% and 80% peak height. (E) Frequency of spontaneous contraction before and after force measurement (*** p < 0.001 between indicated groups; ## p < 0.01 and ### p < 0.001 L0 vs. L0 + 1 mm). (F) Force of paced isometric contractions recorded over increasing preload measured on day 21. (G) Passive tension development with increasing preload. (H) Transmission Electron Microscopy of longitudinally sectioned BCTs. Left: hESC-CM+Fb; right: hESC-CM+PC. I: Myofibrils (white arrowheads) are visible in both tissues, however are more regularly distributed in CM+PC, with a higher width and…

Figure 4. Fibrosis-related cardiac remodeling in a tri-culture model with ECs. (A) Cell compositions used for tissue preparation. All the BCTs contained 1 × 106 CMs, plus the indicated amount of other cell types. The different colors of the frames serve as legends for the other diagrams of the illustration. (B) Monitoring of tissue remodeling indicated by diameter change over time (n = 3–4). (C) Development of EC networks during cultivation. EC coverage expressed as the percentage of surface area (# p < 0.01 vs. D7). (D) Live imaging of the BCT morphology on D21. CMs are visualized by tetramethylrhodamin-methylester (TMRM, selectively labeling viable mitochondria-rich cells), ECs express endogenous eGFP (scale bar: 100 µm). (E) Immunofluorescent staining for cTnT and VIM to observe cell distribution, nuclei stained by DAPI (scale bar: 100 µm). (F) Directionality analysis of the tissues stained in sub-figure E. The Y-axis label “Amount” represents the proportion of structures with the dominant orientation. (G) Frank–Starling curves recorded on D21. (H) Representative spontaneous contractions normalized to peak height. PD—peak duration at 50% and 80% peak height. (I) Frequency of spontaneous…

Figure 5. BCTs with iPSC-PCs exhibit the molecular signatures of myocardial interstitial fibrosis. (A) PCA plot showing the first two principal components. (B) Vulcano plot showing differences in gene expression between CM+PC+EC and CM+Fb+EC BCTs. The areas above the black curves contain significantly upregulated genes on both sides. Expression of cardiac maturation-related genes (C) in BCTs normalized to hESC-CMs (all genes in C are significant vs. hESC-CMs) and fibrosis-related (D) genes normalized to hRV genes significant vs. hRV are marked by $; ** p < 0.01 *** p < 0.001 between indicated groups; n = 3 in each group. hRV—human right ventricle.