iPSC-derived human cardiac progenitor cells improve ventricular remodelling via angiogenesis and interstitial networking of infarcted myocardium

Abstract

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors, cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 μl, P < 0.05) and cardiomyocyte (19.52 ± 3.97 μl, P < 0.05) groups, but not in progenitor group (25.65 ± 3.61 μl), significantly deteriorated as compared to sham control group (28.41 ± 4.41 μl). Consistently, pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 μl, P < 0.05; ESV: 17.08 ± 5.82 μl, P < 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 μl, P < 0.05; ESV: 18.03 ± 6.58 μl, P < 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 μl; ESV: 8.41 ± 2.94 μl). In contrast, cardiac progenitors (EDV: 20.09 ± 7.76 μl; ESV: 13.98 ± 6.74 μl) persistently protected chamber geometry against negative cardiac remodelling. Similarly, as compared to sham control (54.64 ± 11.37%), LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%, P < 0.05) and saline (35.34 ± 11.86%, P < 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue, P < 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone.

Keywords: cardiac progenitors; cardiac repair; cardiomyocytes; interstitial cells; telocytes.

Figure 1

In vitro cardiac developmental ontogeny of human induced pluripotent stem cells. (A) Heat map representing temporal gene expression kinetics of MSnviPSNF3 hiPSC line. Normalized Ct values were plotted, where lower Ct values represent higher expression (green). Note the expression of progenitor markers at Day 8 (notably c‐kit, Sirpa, Isl1 & Nkx2.5) and cardiomyocyte markers post‐day 14 (notably MLC2v, cTNT, HCN4, Cacna1c) of differentiation. Data represent mean ±S.E.M. of three independent experiments. (B) Flow cytometry analysis of progenitors and cardiomyocytes showing high expression of Nkx2.5 and SIRPA in the progenitor stage and expression of Nkx2.5 and cTNT in the cardiomyocyte stage.

Figure 2

Characterization of integrin and laminin expression of progenitors and cardiomyocytes. (A) Increased cellular expression of major integrins for collagen and laminin matrices in the differentiated cardiomyocytes. (B) Distinct expression profile of laminin subunits during cardiac differentiation with laminin‐411/421 matrices pre‐dominant early in progenitors and a switch to pre‐dominantly laminin‐211/221 matrices later in cardiomyocytes. Data represent mean ± S.D. of three independent experiments.

Figure 3

Infarct size estimation with Masson's trichrome staining. (A) Infarcted mouse heart received progenitor and cardiomyocyte injection showed thinning of anterior myocardial wall and transmural fibrotic remodelling. (B) Saline‐injected mouse heart showing thinned ventricular wall and extension of fibrosis into adjacent peri‐infarct regions; scale bar: 1 mm.

Figure 4

Localization of transplanted human iPSC‐derived progenitors and cardiomyocytes in myocardium. (A) Human‐specific nuclear staining of Ku80 in transplanted progenitors (arrows) and cardiomyocytes (arrows) in the interstitial space of α‐actinin (green) stained cardiac muscle at 2‐week post‐injection in the peri‐infarcted myocardium. Progenitor: Peri‐vascular (boxed) and interstitial (demarcated line) localization of Ku80 positive progenitors (arrows) in the infarct and peri‐infarct zone at 4‐week post‐injection. Magnified view of boxed region, showing Ku80 stained progenitors (arrows) located in proximity of vascular structures. Cardiomyocyte: Engraftment of Ku80 positive human cardiomyocytes (arrows) into the cardiac muscle fibers (boxed) of the peri‐infarcted myocardium. Magnified view of boxed region, showing Ku80 stained nuclei of human cardiomyocytes (arrows) revealing sarcomeric cardiac cross‐striations of α‐actinin. (B) Saline‐injected groups show no Ku80 staining in the myocardium of peri‐infarct or infarct region (demarcated line) at 2‐week or 4‐week post infarction. (C) Control group shows no infarction and no Ku80 staining. Dotted lines demarcates intact myocardium form infarcted zone; scale bar: 100 μm.

Figure 5

Presence of telocytes in the infarct region of the myocardium. (A) Human‐specific nuclear staining of Ku80 shows transplanted human progenitors (arrows) in close physical proximity (boxed) with c‐kit (green) stained (but human Ku80 negative) resident cardiac telocytes (asterisks). (B) Dual positive staining of human‐specific Ku80 and c‐kit in the transplanted human progenitors (arrows) that intermixed with c‐kit (green) positive (but Ku80 negative) resident cardiac telocytes (asterisks) in the infarcted region of the myocardium; scale bar: 50 μm (for A), 20 μm (for B).

Figure 6

Microvascular neoangiogenesis at 2‐week and 4‐week post‐myocardial infarction. Immunofluorescent staining for microvasculature positive for α‐smooth muscle actin (SMA) in left ventricle post‐infarction. (B) Microvascular density counts of α‐SMA stained microvessels in the left ventricle. *P < 0.05 versus control; #P < 0.05 progenitor (2‐week versus 4‐week); scale bar: 20 μm.