Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Soluble Factors from Human Mesenchymal Stem Cells

Abstract

In this study, we proposed that the functionality or phenotype of differentiated cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) might be modified by co-culture with mesenchymal stem cells (MSCs), resulting in an improved therapeutic potential for failing myocardial tissues. Structural, motility, electrophysiological, and metabolic analyses revealed that iPSC-CMs co-cultured with MSCs displayed aligned myofibrils with A-, H-, and I-bands that could contract and relax quickly, indicating the promotion of differentiation and the establishment of the iPSC-CM structural framework, and showed clear gap junctions and an electric pacing of >2 Hz, indicating enhanced cell-cell interactions. In addition, soluble factors excreted by MSCs, including several cytokines and exosomes, enhanced cardiomyocyte-specific marker production, produced more energy under normal and stressed conditions, and reduced reactive oxygen species production by iPSC-CMs under stressed condition. Notably, gene ontology and pathway analysis revealed that microRNAs and proteins in the exosomes impacted the functionality and maturation of iPSC-CMs. Furthermore, cell sheets consisting of a mixture of iPSC-CMs and MSCs showed longer survival and enhanced therapeutic effects compared with those consisting of iPSC-CMs alone. This may lead to a new type of iPSC-based cardiomyogenesis therapy for patients with heart failure.

Keywords: induced pluripotent stem cells; maturation of cardiomyocytes; mesenchymal stem cells.

Graphical abstract

Figure 1

Human Mesenchymal Stem Cells Increase the Population of Cardiac Troponin T-Positive Cells and Promote the Molecular Development of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells (A) Cardiomyogenic differentiation protocol and co-culture with human mesenchymal stem cells (hMSCs). (B) Representative flow cytometry data of differentiated human induced pluripotent stem cells (hiPSC-CMs) stained with anti-cardiac troponin T (cTnT) antibodies at day 16. (C) Representative flow cytometry data of differentiated hiPSC-CMs with and without hMSC-derived soluble factors stained with anti-cTnT antibodies at day 19 (CM+SF and CM, respectively). (D) Percentage of cTnT-positive cells in the CM and CM+SF groups as determined by flow cytometry (n = 5 for each group). **p < 0.01, Student t test. (E) Number of cTnT-positive or -negative cells in the CM and CM+SF groups (n = 3 for each group). n.s., not significant, Student t test. (F) Expression of cardiac cell-specific genes (GATA binding protein 4 [GATA4], NK2 homeobox 5 [NKX2-5], and myosin heavy chain 6 [MYH6] and MYH7) in CM or CM+SF cells, normalized against GAPDH expression (n = 7 for each group). *p < 0.05, Student t test. (G) Western blot of CM or CM+SF cells using anti-myosin heavy chain alpha (MHC-α) antibody, anti-MHC-β antibody, and anti-GAPDH antibodies. (H) Ratio of MHC-β to MHC-α in CM or CM+SF cells as determined by western blotting (n = 4 for each group). *p < 0.05, Student t test. For all experiments, results are shown as mean + SEM. bFGF, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; VEGF, vascular endothelial growth factor.

Figure 2

hMSCs Promote Structural Development in hiPSC-CMs (A) Immunohistochemistry of cardiac troponin T (cTnT; green), myosin heavy chain (MHC; red), and nuclei (Hoechst33258; blue) in differentiated cardiomyocytes (CM), cardiomyocytes co-cultured with mesenchymal stem cells (CM+MSC), and cardiomyocytes cultured with MSC-derived soluble factors (CM+SF). Scale bars: 30 μm. (B–D) Cell sphericity (B), cell size (C), and filament length (D) in the CM, CM+MSC, and CM+SF groups (n = 7 for each group). *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with post hoc Tukey’s honestly significant difference (HSD) test. (E) Upper panels display immunohistochemistry of cTnT (white) in the CM, CM+MSC, or CM+SF groups through super-resolution microscopy. Lower panels show the intensity of cTnT at the white lines in the above images. Scale bars: 10 μm. (F) Upper panels show immunohistochemistry of connexin 43 (Cx43; green) and Hoechst33258 (blue) in the CM and CM+SF groups. Lower panels show immunohistochemistry of N-cadherin (green) and nuclei (Hoechst33258; blue) in the CM and CM+SF groups. Scale bars: 20 μm. (G) Percent of fluorescence area, which was stained with Cx43 and N-cadherin, in the CM and CM+SF groups (n = 4 for each group). *p < 0.05, Student t test. (H) Transmission electron microscopy images of cardiomyocytes in the CM, CM+MSC, and CM+SF groups. For all experiments, results are shown as mean + SEM.

Figure 3

hMSCs Promote Contractile and Electrophysiological Development in hiPSC-CMs (A) Representative velocity data in differentiated cardiomyocytes (CM; left panel) and cardiomyocytes cultured with MSC-derived soluble factors (CM+SF; right panel) by a motion analysis system. Red and blue represent high and low velocity, respectively. (B–E) Percentage of beating area (B), acceleration (C), contraction velocity (D), and relaxation velocity (E) in the CM and CM+SF groups (n = 4 for each group). **p < 0.01; ***p < 0.001, Student t test. (F) Representative wave forms associated with Ca²⁺ transients in CM, CM with soluble factors secreted from 25% total percentage hMSCs (CM+SF 25%), or CM with soluble factors secreted from 50% total percentage hMSCs (CM+SF 50%), using the FDSS/μCELL system. (G–J) Beating rate (G), peak ratio (H), rising slope (I), or peak width duration (PWD) (J) of cells in the CM, CM+SF 25%, and CM+SF 50% groups as analyzed by FDSS software U8524-12 (n = 6 for each group). *p < 0.05; **p < 0.01, one-way ANOVA with post hoc Tukey’s HSD test. (K) Representative wave forms associated with Ca²⁺ transients in CM, CM+SF 25%, and CM+SF 50% cells with a pacing rate of 0.5, 1, 1.5, 2, 2.5, or 3 Hz. For all experiments, results are shown as mean + SEM.

Figure 4

hMSCs Promote Metabolic Development in hiPSC-CMs (A) Representative mitochondrial respiration in differentiated cardiomyocytes (CM), CM with soluble factors secreted from 25% of hMSCs (CM+SF 25%), or CM with soluble factors secreted from 50% of hMSCs (CM+SF 50%) after incubation with the ATP synthase inhibitor oligomycin, the respiratory uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and the respiratory chain blockers rotenone and antimycin A. (B–D) Basal respiration (B), ATP production (C), and spare respiratory capacity (D) of cells in the CM, CM+SF 25%, and CM+SF 50% groups (n = 9 for each group). *p < 0.05; ***p < 0.001, one-way ANOVA with post hoc Tukey’s HSD test. (E) Cell energy phenotype of cells in the CM, CM+SF 25%, and CM+SF 50% groups under normal (outlined shapes) and stressed (solid shapes) conditions (n = 9 for each group). (F) Metabolic potential of glycolysis or mitochondrial respiration in the CM, CM+SF 25%, and CM+SF 50% groups (n = 9 for each group). *p < 0.05. (G) Ratio of reactive oxygen species (ROS) levels in cells undergoing oxidative stress compared with normal cells in the CM, CM+MSC, and CM+SF groups (n = 7 for each group). ***p < 0.001, one-way ANOVA with post hoc Tukey’s HSD test. (H and I) Quantitative analysis of mitochondrial genes (NADH, H; COX3, I) from media containing CM, CM+MSC, or CM+SF cell culture (n = 4 for each group). ***p < 0.001, one-way ANOVA with post hoc Tukey’s HSD test. (J) Concentration of stanniocalcin 1 (STC-1) in media containing CM or CM+SF cell culture (n = 3 for each group). *p < 0.05, Student t test. (K) Expression of the STC1 gene in the CM and CM+SF groups, normalized against GAPDH expression (n = 7 for each group). ***p < 0.001, Student t test. For all experiments, results are shown as mean + SEM. ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

Figure 5

Soluble Factors Derived from hMSCs (A–D) Concentration of vascular endothelial growth factor (VEGF) (A), basic fibroblast growth factor (bFGF) (B), stromal cell-derived factor (SDF-1) (C), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (D) in differentiated cardiomyocytes (CM) and cardiomyocytes cultured with MSC-derived soluble factors (CM+SF; n = 8 for each group). **p < 0.01; ***p < 0.001, Student t test. (E) Distribution of particle size after ultracentrifugation of media with cultured hMSCs using the qNano system. (F) Representative western blotting data of the exosomes of hMSCs using anti-CD63 antibody. (G) Transmission electron microscopy images of an exosome derived from hMSCs stained with anti-CD63 antibody. Scale bar: 50 nm. (H) RNA (stained green, left) and sphingolipids (stained red, middle) are shown; particles containing both (right) RNA and sphingolipids were considered to be exosomes derived from hMSCs. Scale bars, 2 μm. (I) Exosomes derived from hMSCs stained with RNA cargo (green) were incubated with hiPSC-CMs stained with phalloidin (red) and nuclei (Hoechst33258; blue) 12 hr after addition of the exosomes into the culture media. Scale bar, 10 μm. For all experiments, results are shown as mean + SEM.

Figure 6

Impact of the Soluble Factors Derived from hMSCs on the Maturity of hiPSC-CMs Recombinant vascular endothelial growth factor (rVEGF), recombinant basic fibroblast growth factor (rbFGF), recombinant stromal cell-derived factor 1 (rSDF-1), recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), all four recombinant proteins (all rProteins), or hMSC exosomes (MSC exosome) were added to culture media containing hiPSC-CMs (CM). Anti-VEGF neutralizing antibody (anti-VEGF), anti-bFGF neutralizing antibody (anti-bFGF), anti-SDF-1 neutralizing antibody (anti-SDF-1), anti-GM-CSF neutralizing antibody (anti-GM-CSF), all four neutralizing antibodies (all antibodies), or GW4879 were also added to culture media containing hiPSC-CMs with hMSC-derived soluble factors. (A) Ratio of myosin heavy chain (MHC)-β to MHC-α in all groups (n = 4 for each group). *p < 0.05, one-way ANOVA with post hoc Tukey’s HSD test. (B) Heatmap regarding expression of the cardiac genes in all groups, normalized against GAPDH expression (n = 9 for each group). (C) Representative mitochondrial respiration rates in the CM, rVEGF, rbFGF, rSDF-1, rGM-CSF, all rProteins, and CM+SF groups. (D) Representative mitochondrial respiration rates in the CM, anti-VEGF, anti-bFGF, anti-SDF-1, anti-GM-CSF, all antibodies, and CM+SF groups. (E) Representative mitochondrial respiration rates in the CM, MSC exosome, GW4879, and CM+SF groups. (F and G) Contraction velocity (F) or relaxation velocity (G) in all groups (n = 5 for each group). *p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA with post hoc Tukey’s HSD test. For all experiments, results are shown as mean + SEM. OCR, oxygen consumption rate.

Figure 7

MicroRNAs and Proteins in hMSC-Derived Exosomes (A and B) Expression of microRNAs reported to promote the maturation of cardiomyocytes (let7 family, microRNA134, microRNA145, microRNA296) or to be specific to cardiomyocytes (microRNA1, microRNA133, microRNA208, microRNA499) in exosomes from culture media containing differentiated cardiomyocytes (CM), cardiomyocytes co-cultured with MSCs (CM+MSC), or MSC (A); and in CM or cardiomyocytes cultured with MSC-derived soluble factors (CM+SF) (B). (C and D) Gene ontology (C) and pathway analysis (D) of the microRNAs associated with the maturation of cardiomyocytes. The vertical axes show the gene ontology category and pathway category, respectively, and the horizontal axes show the extent of enrichment of each gene ontology category or pathway, respectively. (E and F) Gene ontology (E) and pathway analysis (F) of proteins in hMSC exosomes. The vertical axes show the gene ontology category and the pathway category, respectively, and the horizontal axes show the extent of enrichment of each gene ontology category or pathway, respectively.

Figure 8

Combination of hiPSC-CMs and hMSCs Enhanced Therapeutic Effects In Vivo (A) Immunohistochemistry of human troponin T (hTnT; green), human nuclei (HNA; red), isolectin B4 (IB4; white), and nuclei (DAPI; blue) in a rat transplanted with a cell sheet containing both differentiated cardiomyocytes and MSCs (MIX) 4 weeks after cell sheet transplantation. Scale bars: 200 μm (left); 100 μm (right, top); 10 μm (right, bottom). (B) Expression of human DNA in the whole hearts of rat given no transplant (sham) or transplanted with a CM sheet, a MIX sheet, or an MSC sheet, normalized against rat β-actin expression. (C) Serial changes in the relative left ventricular ejection fraction in each group compared with sham rats, analyzed by transthoracic echocardiography (n = 10 for each group). pre-Tx, pre-transplantation; w, weeks after transplantation. (D–G) The dP/dt max (D), dP/dt min (E), end-systolic elastance (F), and end-diastolic elastance (G) in each group, analyzed by cardiac catheterization. (H) Left panels display representative images of myocardial fibrosis in each group, as assessed by Sirius Red staining. Right graph shows the percentage of fibrotic to myocardial tissue in each group (n = 8 for each group). (I) Left panels display representative images of immunohistochemistry of von Willebrand factor in each group. Right graph shows the capillary density per unit area in each group (n = 8 for each group). (J–L) Expression of the hepatocyte growth factor (HGF) (J), stromal cell-derived factor 1 (SDF-1) (K), or vascular endothelial growth factor (VEGF) (L) genes in each group, normalized against GAPDH expression (n = 8 for each group). For all experiments, one-way ANOVA with post hoc Tukey’s HSD test is used, and results are shown as mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001.