Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture

Characteristics of OS patients and healthy stem cell donors can be found in Table 1. Bone marrow cells of OS patients were harvested under general anesthesia prior to start of the chemotherapeutic treatment. The site of MSC harvest (iliac crest) was different from the location of the primary tumor (metaphyseal ends of the long bones) in all cases. Healthy donors were either identical sibling donors for patients with malignant or benign disease requiring hematopoietic stem cell transplantation; or haploidentical donors for either hematopoietical stem cell transplantation or the therapeutic infusion of MSCs for steroid-resistant graft-versus-host disease. Written informed consent was obtained from all patients and donors prior to bone marrow harvesting. The study was approved by the Institutional Review Board on Medical Ethics [LUMC Medical Ethics Committee (CME), P06.152].

Bone marrow derived mononuclear cells were obtained from 5 to 15 mL of heparinized bone marrow aspirate by density gradient centrifugation on Ficoll. Cells were plated on non-coated 75 cm² polystyrene flasks at a cell density of 160,000/cm² in complete culture medium (LG-DMEM; Invitrogen, Paisley, UK) supplemented with penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (FBS; HyClone, Verviers, Belgium). We used a characterized and defined FBS batch preselected for its potential to support MSC expansion and continued to use this specific batch throughout the culture period. MSCs were plastic adherent and had spindle shaped morphology. Chondrogenic, adipogenic and osteoblastic differentiation were performed as described earlier [29]. Medium was refreshed twice a week and cells were replated when reaching 80–90% confluence at a density of 4,000/cm². The first nine OS patient and first five healthy donor MSC samples that were obtained were cultured long-term, the subsequent samples were used for confirmatory mRNA expression analysis (Table 1). Morphology of the cells was recorded and population doublings per passage (PD) were calculated using the log ratio of the harvesting cell count (N) to the starting (baseline) count (X0), divided by the log of 2 (PD = [log (N/X0)]/log 2).

Expression of the cell surface markers CD73, CD90 and CD105 and absence of hematopoietic markers on MSCs was determined using flow cytometry, according to the statement by the International Society for Cellular Therapy [30]. Cells were detached using Trypsin/EDTA and washed in PBS/0.05% bovine serum albumin. Antibodies used were (FITC) conjugated anti-CD86-fluorescein isothiocyanate (FITC) (cat. no. 555657), anti-HLA-DR-FITC (cat. no. 347400), anti-CD31-phycoerythrin (PE) (cat no 555446), anti-CD34-PE (cat no 348057), anti-CD73-PE (cat no 550257), anti-CD90-PE (cat no 555596), anti-CD3-peridinin chlorophyll protein(PerCP)-Cy5.5 (cat no 332771), anti-CD45-PerCPCy5.5 (cat no 332784), all from BD (San Diego, CA, USA) and anti-CD105-PE (cat no SN6), from Ancell (Bayport, MN, USA). Flow cytometry was performed on a FACScalibur, and analyzed using Cellquest software (both Becton–Dickinson). Mean fluorescence intensity (MFI) ratio was calculated by determining the MFI of the specific staining relative to the MFI of the appropriate isotype control staining.

MSCs were grown on coverslips. Coverslips were washed with PBS and fixed in 4% PFA. Coverslips were incubated for 10 min in 1 μg/mL acridine orange (Sigma-Aldrich, Zwijndrecht, the Netherlands) to stain cytoplasm and 1 μg/mL wheat-germ agglutinin with an Alexa Fluor^® 594 conjugate (Invitrogen) in PBS to stain cell membranes. Following washes in PBS, coverslips were mounted using Vectashield with DAPI (Vector laboratories, Burlingame, CA, USA) to stain nuclei. Images were acquired using a COHU 4910 series monochrome CCD camera (COHU, San Diego, CA, USA) attached to a DM fluorescence microscope (Leica, Wetzlar, Germany) and analyzed using ImageJ software with the Cell Counter plug-in (National Institute of Mental Health, Bethesda, MD, USA).

RNA was isolated from frozen cell pellets of at least 1*10^e6 undifferentiated MSCs at passage 2–5 using TRIzol reagent (Invitrogen). Cells were lysed in TRIzol, followed by phase separation in chloroform, precipitation using 2-propanolol and washing in 75% ethanol. RNA clean-up was performed using the QIAGEN Rneasy mini kit (Venlo, the Netherlands) with on-column DNAse treatment. RNA quality and concentration were measured using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) and Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA), respectively.

Gene expression profiling was performed using Human-6 v2 Expression BeadChips (San Diego, CA, USA) containing >48,000 transcript probes. Synthesis of cDNA, cRNA amplification, and hybridization of cRNA onto the Illumina Human v2.0 Expression BeadChips were performed as described previously [31, 32].

cDNA synthesis for qPCR was performed as described earlier [32]. qPCR was performed using the iQ™ SYBR^® Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacture’s instructions, reverse transcriptase negative (RT−) and H₂O controls were taken along for each sample. For normalization, three genes with stable expression in the microarray experiments were chosen (CPSF6, GPR108 and CAPNS1). Data were normalized by geometric mean expression levels of the three reference genes using geNorm (http://​medgen.​ugent.​be/​jvdesomp/​genorm/​). Primer sequences can be found in Additional file 1: Table S1. A standard curve was taken along for each primer set and used for quantification, PCR efficiencies ranged from 93 to 104%.

Six early passage samples (three derived from OS patients, three derived from healthy donors) were subjected to a multicolor FISH based karyotyping test (COBRA-FISH) as described earlier [33]. From each sample at least 20 metaphase cells were recorded and analyzed.

Microarray data were normalized using the Cubic Spline normalization method with the Illumina BeadStudio Gene Expression Module. Microarray data are available at GEO using the accession no. GSE42572. Statistical analysis of microarray was performed using Significance Analysis for Microarrays, using a false discovery rate of 20% (SAM, http://​www-stat.​stanford.​edu/​~tibs/​SAM/​) [34]. Univariate statistical analyses were performed using GraphPad Prism software (version 5.01, La Jolla, CA, USA). Two-sided P values lower than 0.05 were determined to be significant.

We tested the MSC cultures from OS patients and healthy donors for phenotypic markers and functionality. Samples from 16/16 patients and 9/9 controls were able to differentiate into chondrogenic (Additional file 2: Figure S1A), adipogenic (Additional file 2: Figures S1B and C) and osteoblastic lineages (Additional file 2: Figures S1D and E). Also, all MSC cultures expressed CD73, CD90 and CD105 and lacked expression of hematopoietic markers (Additional file 2: Figure S1F). Level of expression of CD73, CD90 and CD105 as determined by MFI-ratios (specific staining/isotype control) did not differ between MSC cultures derived from OS patients and healthy donors. Early passage samples from three OS patient derived MSCs and three healthy donor derived MSCs were karyotyped; no structural or numerical aberrations were observed in the analyzed samples (Additional file 3: Figure S2): MSC001: 46,XY (26 metaphases), MSC003: 46,XX (27 metaphases), MSC008:46,XX (22 metaphases), MSC-HB: 46,XY (21 metaphases), MSC-MH: 46,XY (20 metaphases), MSC-TD: 46,XX (21 metaphases). Late passage cells could not be karyotypically analyzed due to the low numbers of dividing cells.

MSCs of nine OS patients and five healthy donors were long term cultured (average number of days in culture 649, range 601–679 days). From each individual sample, duplicate cultures were established. There were no significant differences between growth rate, cumulative population doublings (median cumulative population doublings OS patients 34 vs. healthy donors 39; P value Mann–Whitney U test 0.70), passage number at termination of culture (mean passage number OS patients 21 vs. healthy donors 23; P value Mann–Whitney U test 0.74) or time to growth arrest (median days to growth arrest OS patients 441 days vs. healthy donors 222 days; P value Mann–Whitney U test 0.15) between MSCs of OS patients and healthy donors. The cumulative population doublings are shown in Figure 1 (averages of the duplicate cultures are shown per sample). All cultures exhibited rapid exponential growth in the first few passages. Later, proliferation slowed down and eventually stopped, characteristic of cultured cells in crisis. At termination of the cultures, viable cells were present in all samples. Cultures were terminated at the end of the growth curves. At this point, in none of the cultures there was evidence of cells escaping the crisis by an increase in proliferation and corresponding morphological changes indicative for spontaneous in vitro malignant transformation.

Morphology of the cells was inspected with every passage. Low passage cells were spindle-shaped, higher passage cells were larger. Upon increasing passage number, increasing frequencies of binucleate cells were noted using phase contrast microscopy. To quantify binucleation, cells were grown on coverslips and cytoplasm, cell membranes and nuclei were stained. Mono- and binucleate cells were counted (Figure 2a–d). There was no difference in percentage of binucleate cells between MSCs derived from OS patients and healthy donors (Figure 2e).

Using microarray gene expression analysis, five genes were found to be differentially expressed between early passage healthy donor derived MSCs (n = 5) and OS patient derived MSCs (n = 7), with a false discovery rate of 20%. Expression of HCLS1, ADM, EEF1A1, LOC644739 (or WASF4) and LOC441155 was lower in patient derived MSCs as compared to healthy donor derived MSCs. To perform simultaneous technical and biological validation of the microarray results, the original low passage MSC samples were recultured and the original series expanded to now include four additional healthy donor derived MSCs (total of n = 9) and seven additional OS patient derived MSCs (total of n = 14). Four of the differentially expressed genes were validated using qPCR: hematopoietic cell specific Lyn substrate 1 (HCLS1), adrenomedullin (ADM), eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) and LOC644739 (or WAS protein family, member 4; WASF4). LOC441155 is a pseudogene for which we did not succeed in designing a sufficiently specific and efficient primer pair. In this second, newly cultured and expanded series, expression of HCLS1 was significantly lower in OS patient derived MSCs as compared to healthy donor derived MSCs (Figure 3, fold change 0.25, P value 0.0005). The other genes were not differentially expressed in this series, perhaps because this was a newly expanded batch of cells. We tried to determine if the observed difference in HCLS1 expression was also true at the protein level. Although HCLS1 protein could be clearly detected in the positive control cell line Jurkat, we were unable to reproducibly detect HCLS1 in MSCs. For five OS patients we had mRNA available from both bone-marrow derived MSCs and the primary tumor. In these samples, expression of HCLS1 was higher in tumor tissue as compared to MSCs, but there was no correlation between level of expression of HCLS1 in low passage MSCs and in the corresponding tumor tissue.