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. 2020 Mar 1;116(3):545-553.
doi: 10.1093/cvr/cvz181.

Expandable human cardiovascular progenitors from stem cells for regenerating mouse heart after myocardial infarction

Verena Schwach  1   2 , Maria Gomes Fernandes  3 , Saskia Maas  1   3 , Sophie Gerhardt  3 , Roula Tsonaka  4 , Louise van der Weerd  5 , Robert Passier  1   2 , Christine L Mummery  1 , Matthew J Birket  1 , Daniela C F Salvatori  3   6
Affiliations

Expandable human cardiovascular progenitors from stem cells for regenerating mouse heart after myocardial infarction

Verena Schwach et al. Cardiovasc Res. .

Abstract

Aims: Cardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner.

Methods and results: CPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors including DOX and basic fibroblast growth factor (bFGF). CPCs in Matrigel were then injected intra-myocardially in mice subjected to MI to assess whether expandable CPCs could mediate cardiac repair. Transplanted CPCs expanded robustly both subcutis and in the myocardium using the same DOX/growth factor inducing regime. Upon withdrawal of these cell-renewal factors, CPCs differentiated with high efficiency at both sites into the major cardiac lineages including CMs, endothelial cells, and smooth muscle cells. After MI, engraftment of CPCs in the heart significantly reduced fibrosis in the infarcted area and prevented left ventricular remodelling, although cardiac function determined by magnetic resonance imaging was unaltered.

Conclusion: Replacement of large areas of muscle may be required to regenerate the heart of patients following MI. Our human/mouse model demonstrated that proliferating hPSC-CPCs could reduce infarct size and fibrosis resulting in formation of large grafts. Importantly, the results suggested that expanding transplanted cells in situ at the progenitor stage maybe be an effective alternative causing less tissue damage than injection of very large numbers of CMs.

Keywords: Human pluripotent stem cells; Expandable human cardiovascular progenitors; Mouse model; Myocardial infarction; Transplantation.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
CPCs expand after subcutis injection in vivo. (A) Schematic of the experimental workflow for CPC expansion in vitro and then after subcutaneous injection in mouse. (B) Representative Ki-67 and human β1-integrin immunostaining in plugs from mouse subcutis showing proliferating CPCs; the mice were treated as indicated for 15 days prior to plug removal. Scale bar = 100 µm.
Figure 2
Figure 2
CPC differentiation in vivo promoted by WNT-inhibition after subcutis injection. (A) Schematic of the experimental workflow for CPC expansion after subcutaneous injection in mouse, followed by directed differentiation by intraperitoneal (i.p.) injection of XAV939. (B) Human cTnT, SMA and human-specific CD31 (huCD31) immunostaining in plugs from mouse subcutis after the CPC expansion and differentiation protocol with or without i.p. injection of XAV939; scale bar = 100 µm in upper panel and lower panel; scale bar = 25 µm in middle panel. (C) Additional immunostaining with human-specific antibodies after CPC differentiation in the subcutis in the presence of XAV939: alpha-actinin is shown to co-label with eGFP in CMs; cTnT+ areas are shown with interspersed huCD31+ endothelial cells (ECs); and SMA+ cTnT-labelling indicates the presence of SMCs; scale bar = 25 µm.
Figure 3
Figure 3
CPCs form large grafts composed of CMs, ECs, and SMCs after intra-myocardial injection post-MI in vivo. (A) Schematic of the experimental workflow for intra-myocardial CPC injection, expansion, and differentiation after MI. (B) Visualization of infarct area by bright field microscopy and intra-myocardial grafts by endogenous GFP (eGFP); infarct area marked in red; scale bar = 500 µm. (C) Representative images of grafts visualized by human β1-integrin staining and DAPI after no treatment and DOX/bFGF treatment, scale bar = 500 µm; Ki-67 expression (green) and cTnT (red) show that DOX/bFGF promotes expansion by proliferation of the CPCs, scale bar = 75 µm. (D) Quantification of graft volume; data are expressed as means ± SEM; one-way ANOVA with Tukey’s multiple comparisons test was applied for differences in means between groups. Statistical significance was defined as P < 0.05; n = 3 for no treatment, n = 10 for DOX/bFGF treatment. (E) Representative confocal immunofluorescent pictures of grafts stained for human β1-integrin (green), cTnT (red), SMA (green), and DAPI (blue); scale bar = 75 µm. High-resolution images of cTnT shows alignment of the sarcomeres in the graft. Scale bar = 25 µm. (F) Representative confocal picture showing the engraftment of the human cells in the mouse heart. Staining for the gap junction proteins GJA1 (connexin 43, red) and N-cadherin (CDH2, green); scale bar = 25 µm. (G) Representative confocal pictures of hearts stained for the human-specific endothelial cell marker CD31 (huCD31, green), cTnT (red), and DAPI (blue); scale bar = 25 µm. (H) Pie chart to illustrate the average cell composition of the grafts after differentiation.
Figure 4
Figure 4
Injection of CPCs did not improve cardiac function, but significantly reduced fibrosis and left ventricular remodelling after MI. (A) Schematic of the experimental workflow highlighting the time points of MRI and histological evaluation. (B) Quantification of cardiac function by ejection fraction; data are expressed as means ± SEM and individual data points of each animal; one-way ANOVA with Tukey’s multiple comparisons test was applied for differences in means between groups. Statistical significance was defined as P < 0.05. Animal groups are described in Supplementary material online, Table S1. (C) High-magnification confocal pictures representative of human cardiomyocytes at different time points during in vivo differentiation in the mouse heart; scale bar = 25 µm. (D) Representative scans of grafts stained for Sirius Red to visualize cardiac fibrosis in mice with MI only (MI) or MI with injected CPCs (MI + CPCs); scale bar = 1000 µm. (E) Quantification of fibrosis volume in percent of left ventricular volume after MI (n = 5) or MI plus injection of CPCs (MI + CPCs) (n = 10). (F) Schematic of the evaluation of ventricular wall thickness by cardiac MRI in a 12-segment model of the left ventricle. (G) Average wall thickness in the fibrotic area in mice with acute MI (n = 6 mice for a total of N = 81 evaluated segments) and mice with MI plus injection of CPCs (n = 4, N = 40 segments) compared to sham (n = 10, N = 120 segments) in segments of the grafted area. Data points represent all segments per animal. (H) Average wall thickness in each segment of the left ventricle. Data are expressed as mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test was applied for differences in means between groups. Statistical significance was defined as P < 0.05.
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