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.