Isolation and culture of human BM-MSCs

Human BM-MSCs were isolated and cultured as described previously [14]. After written informed consent and approval of the ethical committee of the University Hospital Tübingen, Germany, BM from patients without metabolic or neoplastic diseases was obtained during orthopedic operations. BM mononuclear cells were isolated by density gradient centrifugation on Lymphoflot (Biotest, Dreieich, Germany), washed twice with PBS (Lonza, Walkersville, MD, USA), counted and seeded at a density of 1 × 105 cells per cm2 in standard culture medium (SCM), composed of α-MEM (Lonza), 1% penicillin-streptomycin (Lonza) and 10% pooled human blood group AB serum (PHS) (ZKT Tübingen, Germany), to tissue culture flasks (Nunc, Roskilde, Denmark). The average concentrations of sex hormones in the PHS (obtained only from male donors) were in the normal ranges for male individuals above 15 years (testosterone: 16.18 nmol/l; estradiol (eE2): 106.8 pmol/l; estrone (E1): 169.4 pmol/l) and the average bFGF concentration was 75.12 pg/ml. The resulting passage (P)0 cultures were kept under standard culture conditions at 37°C in humidified atmosphere with 5% CO 2 . Non-adherent cells were removed after 24 hours and the adherent cells were cultured in SCM. SCM was changed twice a week until cells reached subconfluency, defined as 90% surface coverage by cells corresponding to 15,000 to 20,000 cells per cm2. At this point, the BM-MSCs were detached using Trypsin-EDTA (Lonza), counted using a CASY® cell counter (Roche, Basel, Switzerland) and plated to fresh tissue culture flasks for the next passage (P1) at a density of 1,000 cells per cm2.

Flow cytometry

Flow cytometry analysis of all BM-MSC preparations was performed at the end (subconfluency) of P1 with a FACScan instrument (BD Biosciences, Franklin Lakes, NJ, USA) using BD CellQuest Pro software and the following (secondary) PE-labeled antibodies: anti-CD10, -CD14, -CD19, -CD29, -CD31, -CD34, -CD43, -CD44, -CD45, -CD56, -CD59, -CD71, -CD73, -CD80, -CD86, -CD90, -CD105, -CD106, -CD117, -CD119, -CD130, -CD140a, -CD140b, -CD146, -CD166, -CD273, -CD274, -GD2, SSEA-1, -SSEA-4 and -HLA class I (BD Biosciences); -CD93, -Galectin 1 (R&D Systems, Minneapolis, MN, USA); -CD133 and -CD271 (Miltenyi Biotec, Bergisch Gladbach, Germany); -CD243 (Chemicon (Millipore Corporation), Billerica, MA, USA); -CD173 (AbD Serotec, Puchheim, Germany) and –MSCA-1 (BioLegend, San Diego, CA, USA). PE-conjugated or non-labeled IgG1, -IgG2a, IgG3 and -IgM antibodies (BD Bioscience) were used as isotype matched controls. Secondary antibody was a polyclonal PE-conjugated goat anti-mouse Ig (BD Bioscience). Dead cells were excluded by uptake of 7-Aminoactinomycin D (for gating strategy see Additional file 1: Figure S4, for density plots see Additional file 2: Figure S5). Analysis of percentage of antigen positive cells and fluorescence intensity was performed using FlowJo-7.2.5 software (Tree Star, Ashland, OR, USA). For compensation of unspecific antibody binding, the positivity of the respective matched isotype control was subtracted from all samples.

Analysis of cell size and PDT

Cell count and analysis of cell size was performed at the end of each passage using a CASY cell counter (Roche). Population doubling time (PDT) during P1 was calculated by the equation PDT = (culture time*ln2)/ln(cell number harvested /cell number seeded ). Seeding density was kept constant at 1,000 cells per cm2.

Colony forming assays

For assessment of colony formation capacity, subconfluent primary BM-MSCs (P0) were trypsinized, counted and seeded at a density of 100, 200 and 500 cells per well (for two MSC populations additional data points with 1,000 cells per well were acquired) in six-well plates at P1 (Nunc, Wiesbaden, Germany). To address possible effects of seeding density on colony formation each MSC preparation was seeded at the aforementioned densities. Cells were cultured during 10 days in MesenCult™ Proliferation Kit (human) medium (Stem Cell Technologies, Vancouver, BC, Canada), then fixed and stained with crystal violet containing 4% formalin. Colonies containing >50 cells were counted microscopically. The number of colonies per 100 seeded cells (percentage colony formation) was calculated for each seeding density for each MSC preparation. These percentages were averaged for each MSC preparation (two experiments for each BM-MSC preparation) and used as one CFU-F data point for the respective MSC preparation for statistical analysis.

In vitrodifferentiation assays

Functional characterization of BM-MSCs included induction of adipogenic, osteogenic and chondrogenic differentiation in vitro as described previously [14]. Briefly, for adipogenic and osteogenic differentiation, BM-MSCs were seeded at a density of 1,000 cells per cm2 at P1 and kept under standard culture conditions until reaching subconfluency. Subsequently, either adipogenic differentiation was induced using the hMSC Adipogenic BulletKit (PT-3004, Lonza), or osteogenic differentiation was induced using “osteogenic medium” composed of SCM with 10-8 M dexamethasone, 0.2 mM ascorbic acid and 10 mM β-glycerolphosphate (Sigma). After three weeks under differentiation conditions cells were processed for RNA isolation or for lineage specific staining: lipid vacuoles in adipogenic cultures were stained with Oil Red O and calcium deposits of osteogenic cultures with Alizarin Red S, respectively.

For chondrogenic differentiation, 2.5 × 105 BM-MSCs at P1 were kept in micromass pellet cultures for subsequent staining or in monolayer cultures for RNA isolation respectively. Differentiation was induced using the hMSC Chondrogenic Differentiation BulletKit (PT-3003, Lonza), supplemented with TGF-β 3 (PT-4124, Lonza) as a growth factor. After four weeks of differentiation, cells were processed for RNA isolation or frozen sections of fixed pellets were stained with Safranin O.

Quantitative RT-PCR

For quantitative analysis of lineage specific mRNA indicative for adipogenic, osteogenic and chondrogenic differentiation potential, as well as for expression analysis of octamer-binding transcription factor (Oct4), Nanog, PR domain containing (Prdm)14, sex determining region Y-box (SOX)2, indoleamine 2,3-dioxygenase (IDO)1 and IDO2 mRNA from differentiated and undifferentiated BM-MSCs was isolated and reversely transcribed to cDNA. To compare Oct4, Nanog, Prdm14 and SOX2 mRNA expression in BM-MSCs to pluripotent stem cells, mRNA was isolated from the human pluripotent teratocarcinoma cell line NCCIT (ATCC-CRL-2073, Ch# 5097030, ATCC, Manassas, VA, USA), and from the human pluripotent embryonic stem cell (hESC) line HUES9 (generously provided by the Harvard Stem Cell Institute, Cambridge, MA, USA). Quantitative PCR was performed on resulting cDNA and gene expression was normalized to the expression of the housekeeping gene GAPDH for each sample. Gene induction for differentiation markers was calculated by normalization of the gene expression of differentiated cultures to undifferentiated controls.

RNA isolation

RNA from adipogenic, osteogenic and chondrogenic differentiated MSCs, as well as from undifferentiated BM-MSCs (controls) at P1 was isolated using peqGOLD TriFast according to the manufacturer’s instructions.

RNA for analysis of Oct4, Nanog, Prdm14, SOX2, IDO1 and IDO2 expression was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Remaining genomic DNA was digested using the RNase-Free DNase Set (Qiagen).

RNA concentration was assessed using a NanoDrop photometer (Thermo Scientific, Wilmington, DE, USA. RNA was stored at −80°C for up to three months.

Reverse transcription

For analysis by quantitative PCR, 250 μg RNA from each sample was reversely transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. Resulting cDNA was stored at −20°C for up to six months.

Quantitative PCR

For the adipogenic differentiation markers LPL and PPAR-γ, the osteogenic markers AP and OPN, and the chondrogenic markers SOX9 and COLL2 as well as for GAPDH as housekeeping gene, PCR analysis of cDNA obtained from differentiated and undifferentiated BM-MSC cultures was performed using ready-to-use amplification primer mixes for RT-PCR (search-LC, Heidelberg, Germany) in combination with LightCycler FastStart DNA Master SYBR Green I (Roche) and the LightCycler Instrument (Roche), according to the manufacturer’s instructions. Quantitative PCR assays for Oct4, Nanog, Prdm14 and SOX2 expression of undifferentiated BM-MSCs were performed in the same way.

For the PCR targets IDO1 and IDO2, primers were ordered from Sigma Aldrich (St. Louis, MO, USA) and used in combination with the QuantiTect SYBR Green PCR Kit (Qiagen). Primer sequences were CGGGACACTTTGCTAAAGGCGCT and GGGGGTTGCCTTTCCAGCCAG for IDO1 and CCTGCAGAGGTCCTGCCAAGGAA and ATGCAGGCTCTCTCCCCCAGG for IDO2 cDNA. The primer annealing step was performed at 60°C. PCR specificity for IDO1 and IDO2 was confirmed by product sequencing (4base lab, Reutlingen, Germany).

PCR results were analyzed by normalizing the expression of each target gene to the expression of the housekeeping gene GAPDH in each sample. Gene induction for differentiation markers was calculated by normalization of the gene expression of differentiated cultures to undifferentiated controls.

T cell proliferation assays

The immunosuppressive properties of BM-MSCs were analyzed in co-culture assays with activated allogeneic T cells. BM-MSCs at P2 were mitotically inactivated by treatment with 40 μg/ml mitomycin C for 30 minutes at 37°C in serum free medium. Thereafter, BM-MSCs were washed three times in SCM and seeded into 96-well plates (Nunc, Roskilde, Denmark) in triplicates at a density of 2 × 104 cells per well. After 24 hours, 1 × 105 PBMNCs, freshly isolated by density-gradient centrifugation from heparinized blood of healthy donors and normalized to the lymphocyte number, were added to the appropriate wells of BM-MSC cultures. T cells were stimulated by addition of 10 μg/ml Phytohaemagglutinin-L (Sigma) and cultured for 72 hours under standard culture conditions. During the last six hours, 100 μM Bromodeoxyuridine (BrdU) labeling solution from the Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche) was added to the (co-) cultures and cell proliferation was assessed by the subsequent ELISA, using the anti-BrdU antibody provided in the kit according to the manufacturer’s instructions. Photometric light absorption was measured in a plate reader and absorbance values were averaged and normalized to the PBMNC only control of the respective blood donor. Supernatants of T cell proliferation assays were collected and stored at −80°C for subsequent analysis of cytokine production.

Multiplex analysis of cytokine production

Cytokine production of BM-MSCs alone and of BM-MSC-PBMNC co-cultures was analyzed in supernatants of T cell proliferation assays. Samples of 25 μl cell culture supernatant were analyzed in triplicates using human cytokine/chemokine multiplex kits (Millipore) according to the manufacturer’s instructions. The Kits were composed of beads for detection of IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF-α. Analysis was performed using a Luminex® 200 instrument (Luminex Corporation, Austin, TX, USA).

Analysis of trophic factor secretion

For evaluation of trophic factor secretion, BM-MSCs MSCs from 11 unrelated donors (5 female, 6 male; age: 50.9 y ± 13.3 y) (P2) were seeded to six-well plates at a subconfluent density of 20,000/cm2. The cells were kept under standard culture conditions and the medium was renewed after 24 hours to a precise volume of 3.0 ml per well. After 72 additional hours, culture supernatants were transferred to Eppendorf cups and frozen at −80°C for subsequent cytokine analysis.

Samples were sent to Multimetrix (Heidelberg, Germany) for multiplex analysis of HGF, LIF, VEGF-A, bFGF and NGFB expression in a commercial Luminex® system. Expression of BMP4 and Angiopoietin-1 was analyzed with Quantikine™ ELISA systems (R&D Systems) per the manufacturer’s instructions. ELISA standard curves were aligned using a five-parameter logistic regression model for sample quantification. Background concentration of all factors was measured in complete culture medium and subtracted from the detected concentrations of the respective culture supernatants for both Luminex® analysis and ELISAs. All samples were analyzed in technical duplicates.

Analysis of hormone and growth factor receptor expression

For analysis of androgen receptor and FGFR3 expression, BM-MSCs from 14 unrelated donors (7 female, 7 male; age: 53.7 y ± 13.8 y) (P2) were seeded into 12-well plates in a density of 20,000 cells/cm2 and kept under standard culture conditions for 24 hours. Thereafter, the cell monolayers were washed with ice cold PBS, lysed in 300 μl Procarta™ lysis buffer (Affymetrix, Santa Clara, CA, USA) and frozen at −80°C.

Samples were analyzed for FGFR3 and androgen receptor content with ELISA systems (USCN Life Science, Wuhan, China) per the manufacturer’s instructions. ELISA standard curves were aligned using a five-parameter logistic regression model for sample quantification. Background concentration was measured in pure lysis buffer and subtracted from the detected concentrations of the respective cell lysates. All samples were analyzed in technical duplicates.

Telomerase activity assay

To quantify telomerase activity, the Telo TAGGG Telomerase PCR ELISA kit (Roche Applied Science, Mannheim, Germany), based on the Telomeric Repeat Amplification Protocol (TRAP), was used on 2 × 105 BM-MSCs at P1 according to the manufacturer’s instructions. Samples were classified as telomerase negative when the extinction value was <0.2 after subtracting the negative control.

Statistical analysis

The statistical analyses were performed using ANOVA analysis of variance, the Spearman two-tailed correlation test, or the two-tailed Student’s t-test. Differences were considered significant when P <0.05.