Age-related changes in rat bone-marrow mesenchymal stem cell plasticity
© Asumda and Chase; licensee BioMed Central Ltd. 2011
Received: 26 July 2011
Accepted: 12 October 2011
Published: 12 October 2011
The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients.
We demonstrated that the expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs.
The results suggest age related differences in the differentiation capacity of BM-MSCs. These changes may affect the efficacy of BM-MSCs for use in stem cell therapy.
Age-related changes in adult stem cells contribute to the decline in tissue regenerative capacity [1, 2]. Adult stem cells are the primary driving force for tissue, and hence organ specific self-renewal. The observed reduction in tissue regenerative capacity suggests diminished stem cell numbers in addition to compromised differentiation and specific lineage commitment ability [2, 3]. In rats and mice, aging compromises the efficacy of MSCs geared towards regeneration of damaged myocardial tissue [4, 5]. The aging micro-environment has also been shown to pose an inhibitory effect on adult stem cell mediated regeneration [3, 6]. Genes involved in stemness, genomic integrity and regulation of transcription are age-repressed in MSCs undergoing replicative senescence in vitro[7, 8]. In this study, MSCs derived from the bone marrow of 4 month old rats (young) were compared to those derived from 15 month old rats (old).
Characterization of young and old BM-MSCs and assessment of changes in pluripotent marker expression
Multilineage differentiation capacity is diminished in BM-MSCs from old rats
Young and old rat BM-MSCs form gap junctions and undergo cardiomyogenesis in co-culture with NVCM
Assessment of DNA damage and telomerase activity in young and old BM-MSCs
We demonstrated here using a rodent model that there were differences in BM-MSCs associated with the age of the animal from which the cells were isolated. We observed rapid expansion in young BM-MSC cultures and striking age-related differences in cell morphology, population size and proliferation. Young BM-MSCs displayed elongated fibroblast-like spindle shaped morphology. Old BM-MSCs displayed spread out, flat enlarged morphology which is consistent with late passage and extensively cultured BM-MSCs [7, 21–23]. Consistent with age-related decreased proliferation, we observed diminished telomerase activity and significantly higher levels of the DNA double strand break marker γ-H2AX in old BM-MSCs. Human fetal bone marrow is known to contain approximately 1 in 10, 000 MSCs in comparison to 1 in 250, 000 in adults . Consistent with our observations here, studies in rats, mice, and rhesus macaque monkeys indicate that there is a correlation between age and declining numbers of BM-MSC [25–27].
There is conflicting data with regard to the effect of aging on the bone forming ability of BM-MSCs [21, 26, 28, 29]. A few studies find no effect, but the majority find age-related decline in differentiation potential . In this study, old BM-MSCs failed to differentiate into osteocytes, chondrocytes and adipocytes. We assessed cardiomyogenic potential by stimulation with a cocktail mixture of BMP-2, FGF-2 and IGF-1, in co-culture with NVCM and with conditioned media derived from the co-culture system. Old BM-MSCs failed to commit to the cardiac lineage when induced with the cocktail mixture and conditioned media. We observed expression of the cardiac phenotype in both young and old BM-MSCs in co-culture with NVCM. When injected into infarcted myocardium, old rat BM-MSCs displayed scant myogenic transdifferentiation and failed to adopt host myocardial tissue architecture in comparison to young MSCs [4, 5]. A potential drawback to co-culture is the fact that fusion of the two different cell types can be misinterpreted as transdifferentiation . Expression of the cardiac phenotype in old BM-MSCs under co-culture conditions in this present study maybe explained in part by cell fusion.
We did not detect striations typical of NVCM in young and old BM-MSCs as evidenced by the lack of α-sarcomeric actinin staining in either cell type. Furthermore, a significant number of GFP positive cells in old BM-MSC cultures failed to express the cardiac proteins analyzed in this study. Positive staining for cardiac proteins was observed in regions of the co-culture system where GFP positive BM-MSCs had fused with NVCM. It is not clear that old BM-MSCs acquired a broader differentiation potential in co-culture with NVCM that enabled commitment to the cardiac lineage. Both young and old BM-MSCs expressed the gap junction protein connexin-43. Therefore, it is more probable that old BM-MSCs fused with NVCM and took on their characteristics. Great interest in BM-MSCs is based in part on their ability to improve function, angiogenesis and neovascularivation via paracrine and cytokine signaling; the same is to be expected of transplanted young and old BM-MSCs. The neovascularization and angiogenesis effects of BM-MSCs are enhanced by the presence of specific growth and cytokine factors [17, 18, 27]. In this study, we observed markedly lower levels of G-CSF, IGF, EGF and VEGF transcripts in old BM-MSCs.
The inability of old BM-MSCs to respond to differentiation media maybe explained in part by the fact that the BM-MSC isolation method typically results in slight heterogeneity in the cell population at early primary isolation due to hematopoietic stem cell contamination which affects the in vitro behavior of the cells. We observed higher contamination by hematopoietic stem cells in old BM-MSC cultures at early passage by flow cytometry (not shown). However, young and old BM-MSCs uniformly expressed CD90, CD73 and CD105, and failed to express CD45 and CD31 at passage 3. It is more likely that the related genes necessary for efficient adipogenesis, chondrogenesis, osteogenesis and cardiomyogenesis were age-repressed in our old BM-MSCs . An Alternative explanation is that our old BM-MSCs had already partially differentiated to another lineage prior to induction with differentiation media. This is highly unlikely; the differentiation experiments in this study were carried out in triplicate (n = 3) on individual cell populations derived from the bone marrow of individual rats (n = 4) for each age group. We induced young and old BM-MSCs under uniform conditions at passage 3.
The uniform expression of CD90, CD105, CD73 and absence of CD31 and CD45 in either age group as observed in this study indicates homogeneity in the BM-MSC population. It does not necessarily correlate with multidifferentiation capacity. Hence, the complete loss of chondrogenic, osteogenic and cardiomyogenic potential may also be due to an age-related loss in multipotency. The combinatorial transcription factor interaction network of Oct-4, NANOG and Sox-2 which confers stemness and maintains pluripotency in embryonic stem cells is well documented [25, 32, 33]. Using an in vitro aging model, Yew et al. recently showed that BM-MSCs show decreased expression of Oct-4 and NANOG at late passage. They also showed that the knockdown of p21 rescues the loss in differentiation capacity and potentiates expression of Oct-4 and NANOG . We observed age-related changes in the expression profile of the three pluripotency markers Oct-4, Sox-2 and NANOG. Old BM-MSCs expressed Oct-4, albeit at a lower intensity in comparison to young BM-MSCs, but failed to express Sox-2 and NANOG. While its presence does not automatically result in pluripotency, precise levels of Oct-4 are required to maintain pluripotency in embryonic stem cells, and its lack thereof results in germ line specific differentiation [34, 35].
Taken together, our observations here present definitive evidence that there are age-related changes in MSCs derived from the bone marrow of old rodents with regard to plasticity and basic stem cell biology. These results support the conclusion that the efficacy of BM-MSCs is compromised as a function of donor age.
All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the Florida State University and with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, publication No. NIH 86-23).
This study investigated among other factors the effect of donor age on BM-MSC differentiation capacity into four mesodermal lineages. BM-MSCs were isolated from four individual "old" and "young" Sprague Dawley rats (15 months; 2 males and 2 females) and (4 months; 2 males and 2 females), respectively. We did not pool bone marrow aspirates from the individual animals; all experiments were performed on the separate cell lines isolated from each individual animal. MSCs are currently being used in several ongoing autologous clinical trials; hence the objective of such a study design was to provide a controlled analysis of the possible effect of individual donor age on molecular predictors of BM-MSC efficacy. Neonatal rats (age, 48 hrs, n = 12) were used for neonatal ventricular cardiomyocyte isolation.
FITC- or phycoerythrin (PE) coupled antibodies against CD45 (559135; 1:100), CD31 (555027; 1:100), CD90 (554897; 1:100) and CD73 (551123; 1:100) were from (BD Pharmingen, San Diego, CA, USA). Mouse IgG1, k Isotype control (551954; 1:100) was from BD Pharmingen. Secondary FITC conjugated anti-goat (705-075-003; 1:200), anti-rabbit (711-095-152; 1:200), and TRITC conjugated anti-mouse (715-025-150; 1:200) antibodies were from (Jackson Immunoresearch, West Grove, PA, USA). Antibodies against CD105 (ab18278; 1:100), Ig2a Isotype control (ab18449; 1:100), cardiac troponin-I (ab19615-500; 1:100), cardiac Myosin Heavy Chain (ab15-100; 1:100) and alpha Sarcomeric actinin (ab9465-500; 1:100) were from (Abcam, Cambridge, MA, USA). Antibodies against p-histone γ-H2A.X (Ser-139) (sc-101696.; 1:200), SOX-2 (sc-17320; 1:100), Oct-3/4 (sc-9081; 1:100) and NANOG (sc-33760; 1:100) were from (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Antibody against connexin-43 (71-0700; 1:100) was from (Invitrogen, Carlsbad, CA, USA). Antibodies against osteocalcin (MAB1419; 1:100), aggrecan (AF1220), and FABP4 (AF1443; 1:100) were from (R&D Systems, Minneapolis, MN, USA).
Bone marrow stem cell isolation
BM-MSCs were isolated from the femur and tibia of Sprague Dawley rats. Briefly, muscle and extra-ostial tissue were trimmed. Bone marrow plugs were flushed out, layered over 15 mL Ficoll solution (Sigma, St. Louis, MO, USA) and centrifuged at 1500 rpm. The cell layer at the interphase was aspirated, and washed with PBS (Fisher, Pittsburgh, PA, USA) in 1% FBS (Innovative Research, Novi, MI, USA). Bone marrow mononuclear cells were transferred to culture flasks with Dulbecco's modified Eagle's Medium-high glucose ([DMEM-HG] (Sigma) supplemented with 20% FBS (Innovative Research) at 37°C with 5% CO2. After 3 days of culture, the media and non-adherent cells were discarded and fresh media added at 3-4 day intervals. Seed culture cells were treated with 0.25% Trypsin-EDTA (Sigma) 7-14 days after the initial plating and labeled as passage 1.
Neonatal heart cell isolation
Neonatal ventricular cardiomyocytes were isolated from 48 hours old Sprague Dawley rat litters (n = 12). Briefly, neonatal rats were anesthetized with halothane, decapitated, and hearts quickly removed and placed in 100 mm Petri dishes (Fisher) with cold phosphate-buffered saline solution (PBS; KCl, 2.7 mmol/L; NaCl, 136.9 mmol/L; KH2PO4, 1.5 mmol/L; Na2HPO4, 8.1 mmol/L [pH 7.3]). Hearts were minced into 1-mm3 pieces, washed with PBS and digested in 0.1% trypsin, 0.3% collagenase and 0.5% DNAse (Worthington, Lakewood, NJ, USA) for 5 minutes at 37°C. The cell suspension was transferred into 30 mL of complete medium and centrifuged at 1500 rpm for 10 minutes. The cell pellet was resuspended in complete medium (F-12 nutrient mixture (Invitrogen) supplemented with 10% FBS and 10% horse serum (Invitrogen), plated in 60 mm dishes (Fisher) and cultured at 37°C in 5% CO2.
BM-MSC morphology, population size and flow cytometry analysis
Primary isolation BM-MSCs derived from individual young and old rats were compared prior to the first passage. BM-MSCs were evaluated under phase contrast (BX61, Olympus; Tokyo, Japan) over 7 days beginning at 24 hours post primary isolation. At passage 3, we analyzed cells by flow cytometry; BM-MSCs from each individual animal were grown to confluency, trypsinized, and 1 × 106 cells were resuspended with phosphate-buffered saline (PBS) in FACS tubes (BD Bioscience). Cells were washed twice with cold PBS and incubated for 30 minutes on ice with the specific conjugated primary antibody, and corresponding isotype control. The immunostained cells were rinsed twice with cold PBS, and analyzed on a FACScan (BD Bioscience). To compare cell size and determine growth cycle, a Cedex HiRes non-flow imaging cytometer was used to enumerate BM-MSCs from each individual animal over 15 passages.
In vitro differentiation
We tested the multilineage differentiation capacity of BM-MSCs by inducing differentiation into adipocytes, osteoblasts, chondrocytes and cardiomyogenic cells using the human stem cell functional identification kit (R&D Systems) and a combination of growth factors, according to the manufacturer's protocol. We induced adipogenesis, osteogenesis, chondrogensis and cardiomyogenesis over 21 day's culture in differentiation medium containing (hydrocortisone, isobutylmethylxanthine and indomethacin [R&D Systems]); (dexamethasone, ascorbate-phosphate, and β-glycerolphosphate [R&D Systems]); (dexamethasone, ascorbate-phosphate, proline, pyruvate and TGF- β3 [R&D Systems]) and DMEM (Sigma) supplemented with 5% FBS (Innovative Research), 50 ng ml-1 FGF-2 (R&D Systems), 5 ng ml-1 IGF-1 (R&D Systems) and 20 ng ml-1 of BMP-2 (R&D Systems) respectively. Differentiation media was refreshed every 3 days.
Neonatal ventricular cardiomyocytes were cultured for three days to subconfluency on glass cover slips placed in 6-well tissue culture treated plates (BD Biosicience). Third passage BM-MSCs were transduced overnight with the BacMam 2.0 GFP transduction control (Invitrogen), trypsinized with 0.25% Trypsin EDTA (Invitrogen), and layered over the subconfluent layer of ventricular cardiomyocytes at a density of 1 × 106 MSCs per plate to make up a co-culture system. The cells were incubated at 37°C in 5% CO2. Cell fusion was determined by dual expression of GFP (FITC) and cardiac specific proteins (TRITC) by GFP positive BM-MSCs in close proximity to NVCMs (TRITC only).
Cells were fixed in 10% formaldehyde at room temperature for 5 minutes, and rinsed with isopropanol. Slides were incubated with Oil-red-O (Fisher) for 20 minutes at room temperature, and rinsed with distilled water. For Alizarin Red staining of mineral deposits, cells were fixed with 10% formaldehyde for 15 minutes, and rinsed with distilled water. Slides were incubated with Alizarin Red staining solution (Fisher) for 20 minutes at room temperature, and rinsed with distilled water. BM-MSCs were viewed under inverted phase-contrast microscope (BX61, Olympus; Tokyo, Japan).
For immunocytochemistry, cells were grown on glass cover slips (Fisher) to subconfluency, washed with PBS, fixed with 2% paraformaldehyde for 10 minutes at room temperature, quenched with 100 mM Glycine (pH7) for 5 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, blocked with 1% BSA in PBS for 20 minutes at room temperature, and sequentially incubated with respective primary antibodies at 4°C overnight and then with appropriate secondary antibodies for 1 hour at 37°C. Between steps, PBS was used for washes and cells were mounted with Prolong Gold Antifade with DAPI (Invitrogen). Confocal images of immunostained cells were obtained using a 40× oil objective on a Zeiss LSM 510 Laser Scanning Confocal Microscope (Zeiss, Frankfurt, Germany). Digitized confocal images were processed with Zeiss LSM Image Browser software, Rel. 4.2 and Adobe Photoshop.
Assessment of telomerase activity
Telomerase activities from young and old rat MSCs were determined using the Quantitative Telomerase Detection Kit (MT3010; Allied Biotech, Inc., Germantown, MD) according to the manufacturer's protocol. Briefly, cell pellets were washed, resuspended in Lysis Buffer and incubated for 30 minutes on ice. Protein concentration was determined for each sample and appropriate volumes of samples, heat inactivated extracts and template controls were dispensed into individual PCR tubes containing a pre-made master mix supplied by the manufacturer. The Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA USA) was used to collect the appropriate Cq values for each sample.
Real-time reverse transcription PCR (qRT-PCR)
Total RNA was extracted from rat BM-MSCs using Trizol Reagent (Invitrogen) according to the manufacturer's protocol. For qPCR, 2 ug of total RNA was reverse transcribed with the RT2 First Strand Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. qPCR was performed using the SsoFast EvaGreen supermix (Bio-Rad) according to the manufacturer's instructions. The cycling profile for real-time PCR (40 cycles) was as follows: 30 seconds at 95°C for enzyme activation, 5 seconds at 95°C for initial denaturation, 5 seconds at 65°C for annealing/extension and a 5 second melt curve step at 65-95°C. Gene analysis was performed using the Bio-Rad CFX Manager software (Bio-Rad). Gene expression is normalized relative to unstimulated cells and fold variation is GAPDH normalized. The primer sequences used have been shown in Additional file 4.
All differentiation and co-culture experiments were carried out in triplicate (n = 3) per animal in each age group (n = 4). qPCR experiments represent three biologically independent experiments realized in duplicate. Data are presented as mean ± SEM. Analyses of variance (ANOVA) were performed using Sigma Plot software from Systat Software, Inc (Chicago, IL, USA) followed by Tukey's multiple comparison tests to establish statistical significance between experimental groups at the p < .05 (*) level.
List of Abbreviations
bone marrow-derived mesenchymal stem cells
bone morphogenetic protein 2
bovine serum albumin
cardiac troponin I
cardiac troponins T
cardiac troponin C: cTrpm: cardiac tropomyosin
Dulbecco's modified Eagle's medium--high glucose
epidermal growth factor
fluorescence-activated cell sorting
fetal bovine serum
fibroblast growth factor 2
granulocyte colony stimulating factor
insulin-like growth factor 1
neonatal ventricular cardiomyocytes
old bone marrow mesenchymal stem cells
phosphate buffered saline
vascular endothelial growth factor
young bone marrow mesenchymal stem cells
The authors gratefully acknowledge Kim Riddle and Tom Fellers for invaluable assistance with confocal microscopy in the Biological Science Imaging Resource (BSIR) at The Florida State University; Dano Fiore in FSU's Biological Science Core Facilities and Dr. Joan Hare in FSU's Institute for Molecular Biophysics Protein Expression Facility for assistance with cell culture and imaging; Dr. Paul Trombley, Mr. John Corthell and members of the Chase laboratory for helpful assistance and critical discussion.
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