Repair of senescent myocardium by mesenchymal stem cells is dependent on the age of donor mice
Sheikh RIAZUDDIN, Ph.D., National Center of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan.
Myocardial infarction is one of the leading causes of mortality in aged people. Whether age of donors of mesenchymal stem cells (MSCs) affects its ability to repair the senescent heart tissue is unknown. In the present study, MSCs from young (2 months) and aged (18 months) green fluorescent protein expressing C57BL/6 mice were characterized with p16INK4a and β-gal associated senescence. Myocardial infarction was produced in 18-month-old wild-type C57BL/6 mice transplanted with MSCs from young and aged animals in the border of the infarct region. Expression of p16INK4a in MSCs from aged animals was significantly higher (21.5%± 1.2, P < 0.05) as compared to those from young animals (9.2%± 2.8). A decline in the tube-forming ability on Matrigel was also observed in aged MSCs as well as down-regulation of insulin-like growth factor-1, fibroblast growth factor (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) compared to young cells. Mice transplanted with young MSCs exhibited significant improvement in their left ventricle (LV) systolic and diastolic function as demonstrated by dp/dtmax, dp/dtmin, Pmax. Reduction in the LV fibrotic area was concomitant with neovascularization as demonstrated by CD31 and smooth muscle actin (SMA) expression. Real-time RT-PCR analysis for VEGF, stromal cell derived factor (SDF-1α) and GATA binding factor 4 (GATA-4) genes further confirmed the effect of age on MSC differentiation towards cardiac lineages and enhanced angiogenesis. These studies lead to the conclusion that repair potential of MSCs is dependent on the age of donors and the repair of senescent infarcted myocardium requires young healthy MSCs.
Mesenchymal stem cells (MSCs) have been shown to differentiate into cells of many organs [1, 2] including cardiomyocytes [3, 4] and can therefore effectively repair infarcted myocardium resulting in an improved cardiac function [5, 6]. Since the regenerative potential of the body declines with age, stem cells like other cells of the body are also prone to the adverse effects of aging. Nevertheless, there are no clear indications as to what happens within the cellular microenvironment causing the progenitor cells to shift from the quiescent to the senescent state . Cyclin-dependent kinase inhibitors such as p16INK4a a marker for cellular senescence have been shown to be involved in haematopoietic stem cell (HSC) regulation [8, 9]. The expression of p16INK4a is undetectable in human and rodent tissues and is associated with replicative senescence in cell lines [10, 11]. Down-regulation of p16INK4a mitigates defects in HSC repopulation and apoptosis thus improving stress tolerance and animal survival .
Several studies have been carried out to elucidate the effect of aging on stem cell function. Recently, it was demonstrated that a decline in the size of the progenitor pool hampers the ability of stem cells to repair aged muscle . Circulating HSCs and endothelial progenitor cell (EPCs) numbers have also shown to be lower in aged as compared to young individuals . Furthermore, aging can cause a decline in the osteoprogenitors due to decreased proliferative capacity of bone marrow stem cells (BMSCs)  and also reduce number of mesenchymal progenitor cells .
It is pointed out that incidence of cardiac disorder predominates in old age whereas most studies have focused on resurrecting young mouse heart [6, 17]. These studies have been used as the basis of treatment in human beings; however, source of stem cells for aged patients with myocardial infarction in most clinical trials has been autologous [18, 19] thereby inherently reducing the effectiveness of the therapy. The aim of the present study was to investigate the ability of MSCs obtained from young and aged animals to repair senescent mouse heart. An important finding of the study is the age-induced expression of p16INK4a in MSCs and its effect on cellular function. The results of the present study indicate a role of p16INK4a induced decline in cardiac differentiation potential and functional incapacitation of aged MSCs for the treatment of myocardial infarction.
Materials and methods
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985). All animals were treated according to procedures approved by the Institutional Review Board (IRB) at the National Center of Excellence in Molecular Biology, Lahore, Pakistan (protocol FWA00001758).
Cell isolation and culture
MSCs were isolated according to the procedure described previously . The tibias and femurs were removed from C57BL/6 transgenic green fluorescent protein (GFP) expressing 2 and 18 months old mice. Cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with 20% foetal bovine serum), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in humid air and 5% CO2. After being seeded for 2 days the MSCs adhered to the bottom of the culture flasks and the HSCs remain suspended in the medium. Medium was changed after every 3 days. Cells were grown for a week till 90% confluent.
For flow cytometry analysis, MSCs from experimental animals were washed with phosphate buffered solution (pH = 7.4) and incubated in the dark for 30 min. at room temperature with CD45FITC (BD Pharmingen, San Diego, CA, USA), CD34PE (BD Pharmingen), CD44PE (BD Pharmingen), CD90FITC (BD Pharmingen) and CD105FITC (BD Pharmingen) antibodies. The specific fluorescence of 10,000 cells was analysed on FACScalibur (Becton Dickinson, Franklin Lakes, NJ, USA) using Cell Quest Pro software.
Gene profiling of MSCs from young and aged animals
RNA was extracted from MSCs isolated from young and aged animals using trizole reagent (Invitrogen Corporation, Carlsbad, CA, USA) and quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). cDNA synthesis was carried out from 1 μg of RNA sample using M-MLV reverse transcriptase (Invitrogen). RT-PCR analysis for IGF-1, FGF-2, vascular endothelial growth factor (VEGF) and HGF was carried out using a GeneAmp PCR system 9700 (Applied Biosystems, Inc., Foster City, CA, USA) with GAPDH as internal control.
Analysis of β-galactosidase associated senescence
Senescence associated β-galactosidase staining was performed as described by Dimri et al.. Cells were cultured to 80–90% confluence, fixed in 2% formaldehyde and incubated overnight at 37°C with fresh β-gal staining solution (1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside (Sigma-Aldrich, St Louis, MO, USA), 40 mM citric acid, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2.
In vitro tube-forming assay
Matrigel was thawed on ice to prevent premature polymerization, aliquots of 50 μl were plated into individual wells of a 48-well tissue culture plate (Corning Incorporated, Corning, NY, USA) and allowed to polymerize at 37°C for at least 30 min. To investigate the tube-forming potential of MSCs from young and aged animals, cells were subjected to hypoxia by treatment with 100 μM H2O2 for 60 min. MSCs were removed with trypsin/ethylenediaminetetraacetic acid after normoxic and hypoxic treatments and plated on Matrigel coated wells in a concentration of 1 × 105 cells/well. The formation of tubular structures in the respective wells were examined at 6, 24 and 48 hrs after incubation of cells at 37°C in 5% CO2 with a phase contrast microscope (IX-51–Olympus, Tokyo, Japan).
MSCs and neonatal cardiomyocytes co-culture model
Neonatal rat cardiomyocytes were isolated from Sprague-Dawley rat pups (1–2 days old, n= 15 per isolation). The cells were separated with neonatal cardiomyocyte isolation system (Worthington Biomedical Corporation, Lakewood, NJ, USA) as described by Xu et al.. MSCs from young and aged animals were separately co-cultured with neonatal cardiomyocytes in a ratio of 1:40. Cells were then allowed to grow for a week followed by immunocytochemical analysis.
Immunocytochemistry was done as described by Kudo et al. on both the cell types after passage 1 (day 4). Primary antibodies used for MSC mouse monoclonal anti-p16INK4a (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies. Expression of early cardiac transcription factors was assessed by staining with rabbit polyclonal anti-Myosin enhancing factor-2 (MEF; Santa Cruz) and goat polyclonal anti-GATA binding protein-4 (GATA-4; Santa Cruz) antibodies. Secondary antibodies of donkey anti-rabbit IgG, anti-goat IgG and antimouse IgG (Jackson ImmunoResearch Laboratories, Inc., Westgrove, PA, USA) conjugated with rhodamine were applied. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescent images were obtained with an Olympus BX-61 microscope equipped with DP-70 digital camera (Olympus).
Myocardial infarction and cell transplantation
Myocardial infarction was produced in female wild-type C57BL/6 aged (18 months old) mice under anaesthesia with sodium pentobarbital (40 mg/kg) as described by Lanza et al.. In brief, the heart was exposed by left side limited thoracotomy and left anterior descending artery (LAD) was ligated with 6–0 silk suture 1 mm from the tip of the normally positioned left auricle. Animals with myocardial infarction were divided into three groups (n= 8) each. Group I mice were injected with normal saline and represented the control (sham) group. Groups II and III mice were transplanted with MSCs from aged and young animals, respectively, in the border of the infarct area. MSC concentration used in the experiments was 1 × 106/ml and animals were given intramyocardial injections of MSCs (50 μl ∼ 50,000 cells/animal) at the time of LAD ligation.
Assessment of heart function
Mice were anaesthetized with sodium pentobarbital (40 mg/kg) by intraperitoneal injection. The right carotid artery was cannulated with a micro tip pressure transducer catheter (SPR-839, Millar Instruments, Inc., Houston, TX, USA) connected to MPCU-400 P-V signal conditioning hardware for data acquisition. The inferior vena cava was exposed and inferior vena cava occlusion was performed by external compression. Haemodynamic parameters and PV loops were recorded during steady state. Left ventricle (LV) systolic function was evaluated by Pmax, dp/dtmax and arterial elastance. LV diastolic function was evaluated by end-diastolic pressure and dp/dtmin. Haemodynamic parameter analysis was carried out using Millar’s PVAN software (Version 3.3).
Measurement of infarct size
Fixed hearts were embedded in paraffin and sections were cut from the mid-LV and base. All sections were stained with Masson’s Trichrome. Images of the LV area of each slide were taken by an Olympus BX-61 microscope equipped with Digital Camera DP-70 (Olympus). Fibrosis and total LV area of each image were measured using the Image Pro Plus and the percentage of the fibrotic area was calculated as (fibrosis area/LV area) × 100.
Mice were killed at 2 and 8 weeks after MSC transplantation. Hearts were fixed in 4% paraformaldehyde and then snap frozen in liquid nitrogen. Sections were cut at 5–8 μm in a Cryostat (Microm International GmbH, Walldorf, Germany) at –20°C. Sections were stained with mouse monoclonal anti-Ki67 antibody (BD Pharmingen) for cell proliferation, mouse monoclonal anti-p16INK4a antibody (Santa Cruz Biotechnology, Inc.) for the identification of senescent myocytes, α-sarcomeric actin (Sigma-Aldrich) that stains the cardiac muscle and mouse anti-CD31 (Chemicon International, Temecula, CA, USA) and α-smooth muscle actin (Sigma-Aldrich) antibodies to check evidence for angiogenesis. β-galactosidase associated senescence was also measured in heart sections from all the experimental groups. Secondary antibodies of Donkey anti-rabbit IgG, anti-goat IgG and antimouse IgG (Jackson ImmunoResearch Laboratories, Inc.) conjugated with rhodamine and FITC were used. Capillary density was measured by identifying CD31 and α-SMA expressing capillaries and small arterioles per five random fields within two separate sections of each group.
RNA was isolated using RNA isolation Midi kit (Qiagen, Inc., Germantown, MD, USA) from the hearts of control and treated groups. Isolated RNA was used for cDNA synthesis using c-DNA synthesis kit (Fermentas International Inc., Burlington, ON, Canada). Serial dilutions of control mouse c-DNA starting from 10pg were made and used as a standard. Real-time RT-PCR was carried out by using SYBR Green PCR Super Mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 8 μM of each primer, and 100–500 ng/μl of template DNA on BioRad System iQ5. The relative ratio and standard deviation between the normal and treated samples were calculated using comparative Cτ method (ΔΔCτ value), as recommended by the BioRad iQ5system.
Terminal dUTP nick end-labelling (TUNEL) assay was performed to determine the level of apoptosis within the MSC transplanted hearts. Heart sections 5 μm thick were stained with MEBSTAIN Apoptosis Kit II (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) as described by Takashi et al.. For each heart, the percentage of apoptotic myocyte nuclei in peri-infarcted area was calculated by counting the number of TUNEL+ myocyte nuclei per total number of nuclei in six sections. Apoptotic myocytes in the infarcted region were excluded from counting.
Quantitative data were obtained from two cover slips each from three separate experiments for MSC characterization. Five random fields per cover slip were analysed and the data were expressed as mean ± S.E.M. Haemodynamic parameters were assessed by one-way analysis of variance anova (P-value of less than 0.05 was considered statistically significant). Analysis of differentiation markers MEF-2 and GATA-4, percentage of fibrosis, comparison of vascular density and measurements of senescent and apoptotic myocytes between groups was performed by Student’s unpaired t-test (P-value of less than 0.05 was considered statistically significant).
The aim of the study was to determine the effect of aging on MSCs and for this purpose cells from young and aged animals were characterized with p16INK4a, a marker for cellular senescence . The results show a significantly high expression of p16INK4a in MSCs from aged animals and provide evidence for a possible role for p16INK4a induced aging in MSCs. There have been reports relating p16INK4a activation with age and a consequent decline in forebrain progenitors  and islet regenerative potential . Recent data also suggests that expression of p16INK4a induces an age-dependent decrease in the proliferative capacity of certain tissue-specific stem cells and unipotent progenitors . We also observed a significantly high expression of β-galactosidase in MSCs from aged animals after 7 days of culture and this has been shown as an effective way for the identification of senescent cells in culture . This finding is in concordance with previous reports  and further validates senescent characteristics of the p16INK4a expressing MSCs from aged animals.
MSCs from young and aged animals were co-cultured with rat neonatal cardiomyocytes to assess the expression of MEF-2 and GATA-4. MSCs were easily distinguished by the expression of GFP and the presence of multiple nucleoli. MEF-2 transcription factor is involved in cardiac morphogenesis  and its expression level was found to be significantly higher in MSCs from young animals. GATA-4 has been shown to control cardiac structural and regulatory gene expression . Our studies showed that MSCs from young animals were able to express high level of GATA-4 suggesting the superior ability of these cells to form cardiac phenotypes. MSCs from aged animals were positive for GATA-4 but their number was significantly low. Our results demonstrate a decline in commitment of MSCs from aged animals towards cardiac lineages and can be compared to previous reports that show a loss in the ability of MSCs to differentiate into osteogenic lineages with age [33, 34].
One of the important aspects of the study was to determine whether aging limits the ability of MSCs for the repair of senescent infarcted heart. Our results demonstrated that MSCs from young animals were able to improve function of the senescent heart significantly as compared to MSCs from aged animals. Our results show a possible association between improvement in senescent heart function and enhanced expression of paracrine mediators IGF-1, FGF-2, VEGF and HGF in young MSCs. Our finding concurs with previous report showing decline in expression of various paracrine factors  and also points towards susceptibility of paracrine capability of MSCs to advanced age. There have been reports documenting a diminished role for aged MSCs in the repair of infarcted myocardium . Nevertheless, there is no report documenting the impact of aging on MSC mediated repair of the senescent heart. Myocardial repair potential of aged bone marrow stromal cells, skeletal muscle cells and smooth muscle cells has been compared previously . However, this work does not document differences in the ability of young and aged MSCs for the repair of senescent infarcted heart. We also observed that the cardiac function declined in the senescent myocardium with age and this is in unison with previous reports [37, 38].
The ability of cells to reduce apoptotic levels is an important factor in the MSC mediated improvement of the senescent heart function and this was demonstrated by TUNEL assay and immunohistochemistry with p16INK4a. Our decision to employ p16INK4a as a marker for the identification of senescent myocytes is in concordance with recent reports correlating p16INK4a and myocyte aging [39, 40]. Myocyte apoptosis in animals and human beings is restricted to p16INK4a+ cells which do not express telomerase and typically show severe telomere shortening .
It is known that the senescent heart is prone to cardiac failure mainly due to weakened angiogenesis . Angiogenic response within the heart involves different cytokines and growth factors. Among them, VEGF has been shown to be essential for the improvement of angiogenesis in the senescent heart . Our data provided a direct comparison between MSCs from young and aged animals highlighting the poor angiogenic response of MSCs from aged animals. These findings are in concordance with previous reports demonstrating the attenuation of the angiogenic ability of stem cells with age .
The ability of MSCs from aged animals to repair the senescent infarcted heart as demonstrated by our results is severely hampered with age and is in concordance with studies demonstrating declined stem cell function with advanced age [15, 16, 44]. Our mouse model for heart repair mimics the clinical pathology and points towards the incapacity of autologous MSCs isolated from aged patients. Therefore our results strongly support the use of MSCs from young individuals for the treatment of infarcted myocardium in aged patients. Moreover, there may be a pivotal role for p16INK4a in the functional impairment of MSCs with age and requires further investigations to validate this assumption. Therefore, it would be beneficial to transplant cells from young healthy donors and at the same time identify various factors involved in the impaired stem cell function with age.
We are grateful to Ms. Saba Tasneem and Mr. Irfan for their technical assistance. This work was supported by Higher Education Commission, Islamabad, Pakistan; Ministry of Science and Technology Government of Pakistan and the Pakistan Academy of Sciences, Islamabad.