Enhanced cardiac repair by telomerase reverse transcriptase over-expression in human cardiac mesenchymal stromal cells

Figures

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  Figure 1. Human PDGFRα + cMSCs derived from young, adult and diseased hearts express defined cardiac fibroblast and MSC markers. (A) Heat map of RNAseq analysis showing expression of known fibroblast and MSC markers, as well as cardiogenic and pluripotency genes in PDGFRα + cMSCs derived from young, adult and diseased hearts. High expression of genes shown in blue and low expression in white. (B) Gene ontology analysis shows up-regulation of genes associated with dilated cardiomyopathy in diseased compared to non-diseased cells. (C) Gene ontology analysis showing up-regulation of regenerative genes in cells derived from young compared to adult hearts. (D) Growth-curve analysis showing cell number decrease with age/disease in PDGFRα + cMSCs. N = 4 patient samples/group. Data presented as Mean ± SEM; ns, not significant, *p < 0.05, **p < 0.005, using one-way ANOVA with Holm-Sidak post-test.

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  Figure 2. Expression of hTERT in PDGFRα + cMSCs rescues age-related decline of PDGFRα + cMSC growth potential and colony formation. (A) PDGFRα + cMSCs from young, adult and diseased hearts were transduced with lentivirus containing hTERT gene or controls (non-transduced [NT], empty vector [EV] and Green Fluorescent Protein [GFP]). (B,C) Expression of hTERT in PDGFRα + cMSCs confirmed by qRT-PCR and Western blot assays. hESC, human embryonic stem cells. (D) Telomeric repeat amplification protocol (TRAP) assay showing no detectable telomerase in non-transduced cells from diseased, young and adult hearts but induced telomerase activity after hTERT over-expression. HCT116 cell line and buffer only were used as telomerase positive and negative controls, respectively. (E,F) Analysis of telomere length by telomere-qPCR and terminal restriction fragment (TRF) length analysis. (G) hTERT over-expression induces a spindle-shaped morphological change in PDGFRα + cMSCs. (H) Extended long-term growth of PDGFRα + cMSCs from young, adult and diseased hearts transduced with hTERT. (I) Colony-forming unit-fibroblast assays show decreased colony forming ability in PDGFRα + cMSCs from adult and diseased…

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  Figure 3. hTERT over-expression enhances cell proliferation, survival and increases vascular and myocyte protein expression. (A,B) Representative images of BrdU and Ki67 stained cells. Quantification in bar graphs of BrdU and Ki67-positive cells show increased cell cycle activity in hTERT + PDGFRα + cMSCs. (C) Representative flow cytometry analysis of Propidium Iodide/Annexin V and quantification of starvation-induced apoptotic cell death in hTERT-transduced PDGFRα + cMSCs compared to non-transduced PDGFRα + cMSCs and empty vector controls. (D,E) Representative images of PDGFRα + cMSCs subjected to endothelial and smooth muscle cell differentiation assays then stained with CD31 and smooth muscle myosin heavy chain 11 (MYH11). (F) Representative images of GFP-transduced PDGFRα + cMSCs co-cultured with neonatal rat ventricular myocytes (NRVMs) for 14 days then stained for α-actinin. Green = GFP (Human cells); Red = α-actinin (Human cells and NRVMs). (G) Bar graphs show quantification of the CD31, MYH11 and α-actinin positive cells in differentiation assays. N = 4 patient samples/group. Data presented as Mean ± SEM; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, using unpaired…

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  Figure 4. Transcriptome profile of hTERT-expressing PDGFRα + cMSCs. (A) Gene expression changes in hTERT + PDGFRα + cMSCs, non-transduced (NT) and empty vector (EV) controls from young, adult and diseased hearts determined by next-generation RNA-sequencing (RNAseq). Heat map shows up-regulated genes in red and down-regulated genes in blue. Arrangement by gene ontology terms (right). (B–D) qRT-PCR confirmation of selected genes from RNAseq. N = 4 patient samples/group. Data presented as Mean ± SEM; *p < 0.05 versus hTERT, using one-way ANOVA with Holm-Sidak post-test. See also Supplementary Fig. S6.

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  Figure 5. Cardiac functional assessment and histological analysis of PDGFRα + cMSC treated hearts after induced myocardial infarction (MI). (A) Experimental design. (B–G) Representative M-mode images and consecutive echocardiographic data before and four weeks after cell delivery to rats with myocardial infarction. (H) Fibrotic scar area assessed by picroSirius red (purple = fibrotic infarct area; green = non-infarcted area) and (I) Gomori Trichrome staining (blue = fibrotic infarct area; red = non-infarcted area). (J) Analysis of cell engraftment four days after MI. LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; FS, fractional shortening; LV, left ventricle. N = 10 rats/group. Data presented as Mean ± SEM, *p < 0.05, using one-way ANOVA with Holm-Sidak post-test. See also Supplementary Fig. S9.

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  Figure 6. hTERT + PDGFRα + cMSC transplantation decreases myofibroblast activation, increases cardiomyocyte (CM) and non-myocyte proliferation, and enhances angiogenesis. Four weeks post-cell delivery, heart sections were immunostained for (A) Alpha-smooth muscle actin (αSMA), (B) Ki67 and wheat germ agglutinin (WGA) to mark cardiomyocyte border, (C) von Willebrand factor (vWF). Ten random images in different heart regions were selected for quantification in bar graphs. N = 6–10 rats/group. Data presented as Mean ± SEM, *p < 0.05, using one-way ANOVA with Holm-Sidak post-test.

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  Figure 7. hTERT + PDGFRα + cMSC transplantation improves cardiac function and decreases scar by paracrine effects dependent on telomerase function. Expression of hTERT mutant (hTERTmut) in PDGFRα + cMSCs confirmed by (A) Western blot and (B) telomeric repeat amplification protocol (TRAP) assay. HT1080 cell line and buffer only were used as telomerase positive and negative controls, respectively. (C,D) Representative M-mode images and consecutive echocardiographic data before and four weeks after cell delivery to rats with myocardial infarction. (E,F) Fibrotic scar area assessed by picroSirius red and Gomori Trichrome staining. (G) Analysis of cell engraftment four days after MI. (H) Heat map of differentially expressed proteins from supernatant collected from hTERT + PDGFRα + cMSCs, hTERTmut + PDGFRα + cMSCs or non-transduced PDGFRα + cMSCs. High expression of proteins shown in red and low expression in blue. (I) Proteins involved in the regulation of extracellular matrix, angiogenesis, cell proliferation and survival were predominantly found in hTERT + PDGFRα + cMSCs. SNA, sum normalized area; LAMA4, laminin subunit alpha-4; COL12A1, collagen alpha-1(XII) chain; CNN1, calponin-1; CLU,…

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  Figure 8. Schematic summary illustration.