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

We have previously reported a subpopulation of mesenchymal stromal cells (MSCs) within the platelet-derived growth factor receptor-alpha (PDGFRα)/CD90 co-expressing cardiac interstitial and adventitial cell fraction. Here we further characterise PDGFRα/CD90-expressing cardiac MSCs (PDGFRα + cMSCs) and use human telomerase reverse transcriptase (hTERT) over-expression to increase cMSCs ability to repair the heart after induced myocardial infarction. hTERT over-expression in PDGFRα + cardiac MSCs (hTERT + PDGFRα + cMSCs) modulates cell differentiation, proliferation, survival and angiogenesis related genes. In vivo, transplantation of hTERT + PDGFRα + cMSCs in athymic rats significantly increased left ventricular function, reduced scar size, increased angiogenesis and proliferation of both cardiomyocyte and non-myocyte cell fractions four weeks after myocardial infarction. In contrast, transplantation of mutant hTERT + PDGFRα + cMSCs (which generate catalytically-inactive telomerase) failed to replicate this cardiac functional improvement, indicating a telomerase-dependent mechanism. There was no hTERT + PDGFRα + cMSCs engraftment 14 days after transplantation indicating functional improvement occurred by paracrine mechanisms. Mass spectrometry on hTERT + PDGFRα + cMSCs conditioned media showed increased proteins associated with matrix modulation, angiogenesis, cell proliferation/survival/adhesion and innate immunity function. Our study shows that hTERT can activate pro-regenerative signalling within PDGFRα + cMSCs and enhance cardiac repair after myocardial infarction. An increased understanding of hTERT’s role in mesenchymal stromal cells from various organs will favourably impact clinical regenerative and anti-cancer therapies.


Immunofluorescence staining
Immunofluorescence staining of the cells was performed as previously described. 3 Briefly, the cells were fixed with 4% paraformaldehyde (PFA), washed, permeabilised and incubated with primary antibodies (Table S3) for 1 hour at room temperature. The cells were then incubated with appropriate fluorochrome-conjugated secondary antibodies, washed and stained with DAPI (1 µg/mL, Sigma-Aldrich). Slides were analysed using an Olympus FV 1000 Confocal Laser Scanning microscope with FV10-ASW 1.7c software (Olympus, Japan).
For BrdU staining, the cells were incorporated with 20 µM BrdU (Sigma-Aldrich) for 2 hours and then fixed with 4% (wt/vol) PFA for 15 min. After that, cells were incubated with 1M HCl for 10 min on ice followed by 2M HCl for 10 min at room temperature before moving them to an incubator for 20 min at 37 o C. Immediately after the acid washes, 0.1M sodium borate buffer (pH9.0) was added to the cells for 12 minutes at room temperature. After the cells were washed with PBS/Tween-20 (0.05%), immunofluorescence was performed using anti-BrdU antibody (1:300, BioLegend), as described above.

Flow cytometry analysis
Cells were harvested, washed and stained with conjugated primary antibodies (Table S3) in 2% FBS in PBS for 60 minutes in the dark. Isotype controls were performed concurrently. The analysis was performed using flow cytometry (BD FACS Canto II; BD Biosciences).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted using Isolate II RNA mini kit (Bioline). cDNA templates were synthesised from 1 µg RNA using the M-MLV Reverse Transcription System (Promega) with random hexamers (Promega) following manufacturer's instructions. Quantitative PCR (qPCR) was performed on the Biorad CFX384 using SensiFAST SYBR No-ROX Mix (Bioline) with 0.5 µM gene-specific primers listed in Table S2. RT-minus controls were included. PCR conditions were 95°C for 2 minutes and 40 cycles of 95°C for 5s, 60°C for 10s and 72°C for 15s, followed by melt curve analysis. Relative gene expression was calculated using the 2 −ΔΔCt method, which was normalised against the housekeeping gene GAPDH.

Western blot
Protein extraction and western blot were performed as previously described. 4 Protein samples were mixed with Laemmli sample buffer containing 10% β-mercaptoethanol prior to boiling at 95°C for 5 minutes. Samples were resolved by SDS-PAGE and transferred onto PVDF membrane (Immobilon-P, Merck Millipore). Membranes were blocked with Odyssey blocking buffer (LICOR Biosciences) and then incubated with hTERT antibody (Rockland, 1:500) overnight at 4°C. Bound antibodies were detected with IRDye 800CW-conjugated secondary antibody (1:5000, LICOR Biosciences). Membranes were scanned using an Odyssey Infrared Imaging system (LICOR Biosciences).

Long-term growth analysis
Long-term growth was performed as previously described. 3,5 Cells were expanded after plating 20,000 cells per T75 flask. Resulting cells were counted and passaged every ten days or when cells reached 80-90% confluence. Cumulative cell numbers were calculated and plotted (log10 scale).
Colony-forming unit fibroblast assay 500 cells were plated into six-well plates (triplicates for each sample). The cells were cultured in completed MEMα media containing 20% FBS for 12 days. The colonies were then fixed with 4% (wt/vol) PFA and stained with 0.05% (wt/vol) crystal violet. Numbers of colonies were quantified according to their size: large (>2 mm), small (<2 mm) and micro (~25-50 cells) colonies. 3,5 Cell apoptosis assay The cells were plated in six-well plates below 80% confluence, then 24 hours later were replaced with serum-free medium and incubated for 24 hours. The Annexin V-FITC Apoptosis kit (Abcam) was used to detect cell apoptosis following the manufacturers' instructions. Cells were analysed using flow cytometry (BD FACS Canto II; BD Biosciences).

In vitro vascular cell and myocyte differentiation assays
The cells were seeded onto gelatin-coated glass coverslips in 24-well plates and cultured in complete MEMα media containing 20% FBS. To determine the differentiation potential, the basal medium was removed and specific differentiation medium was added for 14 days with changes every 3-4 days, as described previously. 3,6 For cardiomyocyte differentiation, the cells were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). Co-cultures were maintained with M199 medium containing 2% FBS. For endothelial cell differentiation, cells were cultured in IMDM medium (Invitrogen) supplemented with 10 ng/mL VEGF (R&D Systems), 10 ng/mL bFGF and 2% FBS. For smooth muscle cell differentiation, cells were cultured in DMEM-HG medium supplemented with 50 ng/mL PDGF-BB (R&D Systems) and 2% FBS. Cells were fixed after 14 days of differentiation. Immunofluorescence staining was performed as described above. Primary antibodies for cTnT, αactinin, von Willebrand Factor (vWF) and smooth muscle myosin heavy chain 11 (MYH11) were used (Table S3). Samples were analysed using an Olympus FV 1000 Confocal Laser Scanning microscope with FV10-ASW 1.7c software (Olympus, Japan). Ten fields per sample were randomly selected for quantification.

Immunostaining of heart sections
Immunostaining was performed on paraffin-embedded sections. Heart sections were deparaffinized using histoclear and series of ethanol. Antigen retrieval was performed using sodium citrate (10 mM) in PBS/Tween-20 (0.05%) followed by blocking with 5% goat serum (Sigma-Aldrich). The sections were incubated with primary antibodies (Table S3) for 1 hour at room temperature followed by secondary antibodies staining. Blood vessels were identified using von Willebrand Factor (vWF). Myofibroblasts were stained using alpha-smooth muscle actin (α-SMA). Cardiomyocytes (CM) and non-CM proliferation were identified using Ki67 and wheat germ agglutinin (WGA) antibodies. Nuclei were identified with DAPI (1 µg/mL, Sigma-Aldrich). Slides were analysed using a slide scanner (Hamamatsu Nanozoomer, Japan) and an Olympus FV 1000 Confocal Laser Scanning microscope with FV10-ASW 1.7c software (Olympus, Japan). Ten fields per sample were selected for quantification.

Active force study
Human pluripotent stem cells. Ethical approval for the use of human embryonic stem cells (hESCs) was obtained from The University of Queensland's Medical Research Ethics Committee (2014000801) and was carried out in accordance with the National Health and Medical Research Council of Australia (NHMRC) regulations. HES3 (WiCell) were maintained as TypLE (ThermoFisher Scientific) passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore). Karyotyping and DNA fingerprinting were performed as a quality control. Cardiac differentiation. Cardiac cells were produced using recently developed protocols where cardiomyocytes and stromal cells are produced in the same differentiation culture; 7-10 multi-cellular cultures are critical for function. 11,12 Based on flow cytometry the cells generated and used for tissue engineering were ~70% α-actinin + /cTnT + hPSC-CMs with the rest being predominantly CD90 + stromal cells. 8 Heart-Dyno hCO fabrication. Heart-dyno culture inserts were fabricated using standard SU-8 photolithography and PDMS molding practices. 7 hCO were fabricated as per. 7 hTERT+GFP+PDGFRα+cMSCs hCO and GFP+PDGFRα+cMSCs hCO were fabricated by incorporating 3,000 cMSCs per hCO (5% total cell number). hCO were cultured in CTRL medium: α-MEM GlutaMAX (ThermoFisher Scientific), 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (ThermoFisher Scientific) with changes every 2-3 days. Force analysis of hCO in Heart-Dyno. The pole deflection was used to approximate the force of contraction as per. 7 A Leica DMi8 inverted high content Imager was used to capture a 10s time-lapse of each hCO contracting in real time at 37°C. Custom batch processing files were written in Matlab R2013a (Mathworks) to convert the stacked TIFF files to AVI, track the pole movement (using vision.PointTracker), determine the contractile parameters, produce a force-time figure, and export the batch data to an Excel (Microsoft) spreadsheet.          There are no significant differences in IVSd (left) and PWd (right) in non-hTERT (PDGFRα+cMSCs) and hTERT-transduced cells (hTERT+PDGFRα+cMSCs) treated animals compared to vehicle controls.

Figure S8. hTERT+PDGFRα+cMSC transplantation does not cause tumorgenicity.
Representative images of hematoxylin and eosin (H&E) staining of heart, lung, spleen, kidney and liver cross sections are shown. Representative H&E images demonstrate that both PDGFRα+cMSC and hTERT+PDGFRα+cMSC treatment did not change the normal structure or produce any tumours. Figure S9.

Figure S9. Engraftment capacity of non-hTERT and hTERT transduced PDGFRα+cMSCs in non-injured (no myocardial infarction [no MI]) and infarcted (MI) hearts. (A) Human
PDGFRα+cMSCs and hTERT+PDGFRα+cMSCs were injected into non-injured athymic rat hearts (no MI). Immunostaining against human nuclei was used to identify human cells. Representative confocal immunofluorescence images demonstrating engrafted cells at 2, 7 and 14 days after transplantation. A higher number of human cells was seen at 2 days and minimal human cells were seen at 14 days. The area enclosed by the white box is magnified in the right panel: Pink = human nuclei (colocalised with DAPI), Blue = DAPI-stained nuclei. (B) Representative confocal immunofluorescence images demonstrating engrafted PDGFRα+cMSCs and hTERT+PDGFRα+cMSCs into infarcted area at 4 days after transplantation. See also Figure 5J for engraftment ratio.