Relative genomic stability of adipose tissue derived mesenchymal stem cells: analysis of ploidy, H19 long non-coding RNA and p53 activity
- Orly Ravid†1,
- Ofer Shoshani†1,
- Meirav Sela2,
- Ada Weinstock1,
- Tommy Weiss Sadan2,
- Eyal Gur2,
- Dov Zipori1 and
- Nir Shani2Email author
© Ravid et al.; licensee BioMed Central. 2014
Received: 5 June 2014
Accepted: 12 December 2014
Published: 17 December 2014
Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state.
MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA).
We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs.
The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.
The ability of mesenchymal cells to propagate under tissue culture conditions has been studied since the 1960s. Initially, the capacity of bone marrow (BM)-derived mesenchymal stromal cells to regenerate bone and the BM microenvironment in vivo was demonstrated . Mesenchymal cells were then also derived from the stromal vascular fractions of fat tissue and termed preadipocytes [2, 3]. Cultured mesenchymal cells were later termed mesenchymal stem cells (MSCs) to denote their multipotent differentiation potential, and the use of these cells for regenerative medicine was suggested . Since their identification, multipotent MSCs were isolated from most adult tissues, cord blood and placenta . MSCs usually express a series of cell surface markers . However, their definition lies mainly upon their multipotent nature, adhesiveness and morphology [6, 7]. Although MSCs from different organs share many common features, they may also harbor tissue-specific functions. Characterization of the tissue-specific properties of MSCs may assist the understanding of their physiological functions.
Human MSCs have a relatively stable genome at early passages, but may acquire chromosomal aberrations during extended culture [8, 9] and tend to undergo senescence preventing their long-term propagation . Gaining better understanding of the mechanisms that govern genomic stability of MSCs during passaging may allow one to define improved culture conditions that will prevent these events and the tumorigenic transformation of the MSCs. We previously demonstrated that the majority of murine BM MSCs become polyploid in the early stages of their expansion in culture and that the noncoding RNA H19 plays a major part in these events and predicts their corresponding tumor-forming capacity .
Here we show that, unlike BM MSCs, MSCs from adipose tissue origin retain their normal diploid state during culture. This tendency is accompanied by a reduced H19 expression compared with similar cells from the BM, further suggesting that a restrained H19 expression preserves the normal diploid state. Importantly, adipose-derived mesenchymal stem cells (ASCs) that were prepared from independent mice also demonstrated a significantly more homogeneous expression phenotype of multiple genes compared with BM MSCs under both normal and stress culture conditions. A partial explanation for the higher stability of ASCs may be provided by their increased basal activation of the p53 signaling pathway, which may help preserve their genomic integrity during passaging.
MSCs were derived from the BM of C57Bl mice (Harlan, Jerusalem, Israel). BM MSCs were flushed from femurs and tibias using murine MesenCult Basal Media and supplement (Stem Cell Technologies, Vancouver, Canada). ASCs were isolated from the mice intra-abdominal fat tissue using 0.1% collagenase (Sigma, Rehovot, Israel) and were separated from fat by centrifugation. Both isolates were expanded under tissue culture conditions as detailed below.
To examine the effect of different cell culture media on the ploidy of ASCs, the cells were cultured either in ADSC Basal Media and supplement (Lonza, Walkersville, MD, USA), murine MesenCult Basal Media and supplement (Stem Cell Technologies) or Dulbecco’s modified Eagle’s medium high glucose with 10% fetal calf serum, 60 μg/ml penicillin and 100 μg/ml streptomycin as stated. For comparison of BM and ASCs from the same mouse donor, all cells were cultured in murine MesenCult Basal Media and supplement; the purity and cell number were high enough to be used experimentally for ASCs at passage 4 while BM MSCs were ready for first derivation at passage 7 or higher.
For hypoxia, cells were cultured in 37°C with 10% carbon dioxide in a Thermo Scientific Forma incubator (Thermo Scientific, Waltham, MA USA) monitored to 3% oxygen from the moment of isolation.
The Weizmann and Tel Aviv Medical Center Institutional Animal Care and Use Committee approved all animal experiments.
For adipogenesis, confluent cultured cells in a 24-well plate received adipogenic medium containing 10 μg/ml insulin (Sigma), 1 × 10–6 M dexamethasone (Sigma) and 0.5 mM IBMX (Sigma). ASC adiopogenic medium contained also 50 μM indomethacin (Sigma). The cells were grown for 3 weeks, with medium replacement twice a week. Adipogenesis was detected by Oil red O staining.
For osteogenesis, confluent cultured BM MSC cells in a 24-well plate received osteogenic medium containing 50 μg/ml l-ascorbic acid-2 phosphate, 10 mM glycerol 2-phosphate disodium salt, and 1 × 10–8 M dexamethasone (all from Sigma). For osteogenesis, confluent ASCs were cultured in the StemPro® Osteogenesis Differentiation Kit (Gibco, Grand Island, NY, USA). The cells were grown for 3 weeks with medium replacement twice a week. Osteogenic differentiation was detected by Alizarin red staining. Photographs were taken using an Olympus IX71 microscope equipped with a DP51 camera (Olympus, Tokyo, Japan).
For surface marker analysis, MSCs were harvested and incubated with specific phycoerythrin (PE)-labeled antibodies for 1 hour. Antibodies anti-CD11b–PE, anti-CD45.2–PE, anti-CD31–PE, anti–Ter-119–PE and anti-CD34-PE, rat IgG2b isotype control–PE and rat and mouse IgG2a isotype controls–PE were purchased from eBioscience (eBioscience, San Diego, California, USA), anti–SCA-1–PE was purchased from PharMingen (San Diego, California, USA), and anti–CD34–PE was purchased from Santa Cruz (Santa Cruz, Dallas, Texas, USA). Cells were analyzed using the LSRII flow cytometer (Becton, Dickinson, New Jersey, USA).
For DNA content estimation, cells were fixed with 70% ethanol/phosphate-buffered saline, treated with RNaseA 0.4 mg/ml (Sigma), and stained with propidium iodide 0.1 mg/ml (Sigma). Labeled cells were analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Fresh splenocytes were used as control diploid cells.
Chromosome spreads were prepared from all cells at passage 7 or higher, treated with KCl (0.075 M) and fixed using methanol/acetic acid (3:1), and were then stained with 4′,6-diamidino-2-phenylindole and visualized using a Zeiss Axio Imager Z1 microscope (Carl Zeiss Microscopy, Göttingen, Germany). Chromosomes from at least 10 metaphases were counted from each cell preparation.
BM MSCs or ASCs (200,000 cells) from similar passages were plated in 6 cm plates. The next day, cells were irradiated at UV-C (254 nm) using a low-pressure mercury lamp (TUV 15w G15T8; Philips, Andover, MA, USA). After 24 hours, cells were taken for RNA analysis.
Oxidative stress was induced by adding hydrogen peroxide (H2O2; Sigma) for 1 hour at a concentration of 100 μM. RNA extraction was performed 6 hours after the H2O2 treatment.
DNA double-strand break induction
DNA double-strand breaks were induced by incubating the cells in 0.5 μg/ml doxorubicin (Sigma) for 24 hours. At the end of the 24-hour incubation, cells were washed and RNA was extracted.
p53 knockdown experiments were carried out in the laboratory of Prof. Varda Rotter, (Weizmann Institute of Science, Rehovot, Israel). The p53 short hairpin RNA vector and its human Rb short hairpin RNA control vector were kindly provided by Dr SW Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). Ecotropic Phoenix-packaging cells were transfected with 10 μg DNA of the appropriate retroviral construct by a standard calcium phosphate procedure. Culture supernatants were collected 36 to 48 hours after transfection and filtered. BM MSCs and ASCs were infected with the filtered viral supernatants in the presence of 4 μg/ml polybrene (Sigma) for 12 hours, after which the medium was changed. Fresh viral suspensions were added after a 24-hour interval for an additional 12 hours. RNA was extracted from the cells to verify p53 knockdown. The cells were expanded for an additional three passages and then taken for ploidy analysis.
For immunoblotting, proteins were separated by SDS–PAGE, transferred to a nitrocellulose membrane and detected with the anti-p53 antibody (Santa Cruz) or primary antibody or an anti-glyceraldehyde-3-phosphate dehydrogenase (Sigma) and horseradish peroxidase-conjugated secondary antibody using enhanced chemiluminescence western blotting reagents (Thermo Scientific) and film (Fujifilm, Tokyo, Japan). Densitometry was conducted using ImageJ software ().
Quantitative real-time PCR
RNA from passage 4 ASCs and passage 7 or higher BM MSCs was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany), and cDNA was prepared using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Real-time PCR was carried out using the perfeCTa SYBR mix (Quanta BioSciences, Gaithersburg, MD, USA) and processed using Step One Plus (Applied Biosystems Foster, CA, USA) with normalization to HPRT or 18S.
The real-time primers used are as follows: Hif1α, 5′-ACAAGTCACCACAGGACAG-3′ and 5′-AGGGAGAAAATCAAGTCG-3′ ; VEGFa, 5′-GCGGATCAAACCTCACCAAA-3′ and 5′-TTCACATCTGCTGTGCTGTAGGA-3′ ; H19, 5′-GCTAGGGTTGGAGAGGAATGG-3′ and 5′-AAAAGTAACCGGGATGAATGTCTG-3′ ; p53, 5′-AGAGTATTTCACCCTCAAGATCCG-3′ and 5′-CGGAACATCTCGAAGCGTTT-3′; p21, 5′-CCATGAGCGCATCGCAATC-3′ and 5′-CCTGGTGATGTCCGACCTG-3′; btg2, 5′-ATGAGCCACGGGAAGAGAAC-3′ and 5′-GCCCTACTGAAAACCTTGAGTC-3′; Ercc5, 5′-TGCTGGCCGTGGATATTAGC-3′ and 5′-GCCGGTGGAATAATGTGAGAAGA-3′; Mgmt, 5′-TGCTCTCCATCACCCTGTGTT-3′ and 5′-AACACCTGTCTGGTGAATGAATCTT-3′; Puma, 5′-GCGGCGGAGACAAGAAGA-3′ and 5′-TGTGATGATGGTGAGGATGG-3′; Cycling1, 5′-ACAACTGACTCTCAGAAACTGC-3′ and 5′-CATTATCATGGGCCGACTCAAT-3′; Bax, 5′-TGAAGACAGGGGCCTTTTTG-3′ and 5′-AATTCGCCGGAGACACTCG-3′; Mdm2, 5′-TGTCTGTGTCTACCGAGGGTG-3′ and 5′-TCCAACGGACTTTAACAACTTCA-3′; p19, 5′-GGTCGCAGGTTCTTGGTCAC-3′ and 5′-CGGGATCGCACGAACTTCAC-3′; p16, 5′-TTGGGCGGGCACTGAATCTC-3′ and 5′-AGTCTGTCTGCAGCGGACTC-3′; HPRT, 5′-GCAGTACAGCCCCAAAATGG-3′ and 5′-GGTCCTTTTCACCAGCAAGCT-3′; and Rn18s, 5′-CGAAAGCATTTGCCAAGAAT-3′ and 5′-AGTCGGCATCGTTTATGGTC-3′.
Statistical analysis was done using R statistical software and SigmaPlot, v.11 (SigmaPlot, v.11 (Systat Software, San Jose, CA)). P <0.05 was considered statistically significant using the Mann–Whitney test or t test as stated. The F-variance test was used to compare the variance of two groups, P <0.05 showing statistical difference.
Results and discussion
Unlike BM MSCs, ASCs retain their diploid state under various culture conditions
ASCs ability to retain a diploid state is accompanied by reduced H19 expression
Statistical analysis of RNA expression levels comparing groups of different mesenchymal stem cell preparations
Adipose ( n)
BM ( n)
Normoxic adipose vs. BM
Hypoxic adipose vs. BM
Normoxia ( n )
Hypoxia ( n )
P , Mann–Whitney
Normoxic vs. hypoxic
Diploid vs. polyploid
H19 diploid adipose
H19 polyploid BM
Polyploid BM/diploid adipose
ASCs response to different oxygen conditions is more homogeneous than that of BM MSCs
H19 expression and vascular endothelial growth factor A expression were shown previously to increase under hypoxic conditions in a hypoxia-inducible factor 1α-dependent manner [17–19]. This was therefore used to compare the responses of BM MSCs and ASCs to different oxygen levels. We found a significant increase in H19 and vascular endothelial growth factor A expression, seen in both ASCs and BM MSCs grown under hypoxic conditions (Figure 3D,F respectively and Table 1). A previous report stated that H19 is elevated in hypoxia only in p53 mutant or null settings . Hypoxia-inducible factor 1α mRNA expression remained unchanged under both oxygen conditions (Figure 3E), most probably since its protein levels are increased mainly due to hypoxia-induced stabilization, rather than to increased transcription [17, 18]. Interestingly, H19 mean expression in ASCs under hypoxic conditions was 59-fold lower than in polyploid BM MSCs, indicating again a more stable state of these cells in culture (Table 1). Indeed, ASCs maintain their diploid state during passaging (Figure 1C).
Although the change in RNA expression under different oxygen levels showed a similar trend in both ASCs and BM MSCs (Figure 3D,E,F), the variation in expression was significantly lower in ASCs compared with BM MSCs, as evident from the P (variance) analysis (Table 1), indicating a more homogeneous nature of ASC-independent preparations.
ASCs ability to retain a diploid state is accompanied by a higher basal activity of p53, as indicated by the higher expression of its downstream targets
Lower expression of p16 and p19 in ASCs compared with BM MSCs
The p19ARF and p16ink4a proteins are important tumor suppressors . In contrast to p21 and btg2, both p16 and p19 expression levels were higher in BM MSCs compared with ASCs (Figure 4I,J). The higher p53 activity of ASCs suggests a higher stability of p53 in these cells. Activation of p53 by the p19 pathway occurs through the interaction of p19 with mdm2, thereby preventing p53 degradation . The higher expression of p19 combined with the lower p53 activation in BM MSCs suggests that p53 activation does not occur in these cells in a p19-dependent manner. This is in accordance with reports suggesting p19 activation and growth arrest in response to oncogene-induced proliferation and not tetraploidization or DNA damage [24, 26, 27]. In agreement with our previous findings, p19 and p16 showed a more heterogeneous expression in BM MSC preparations compared with ASCs.
Both BM MSCs and ASCs respond normally to DNA damage, as indicated by an increased expression of p53 targets
ASCs undergo ploidy shifts following p53 depletion
The findings presented in this study clearly demonstrate the higher stability of murine ASCs compared with BM MSCs, as reflected in their ability to maintain a diploid phenotype, together with their uniform nature. Importantly, these markedly different phenotypes do not result from different culture conditions (BM MSCs and ASCs extracted from the same mouse were grown under the exact same conditions) but from a signature that most probably comes from their tissue origin. Consistent with our previous report that demonstrated H19 as a major contributor to BM MSC instability and tumorigenic potential, we find here that the increased stability of ASCs is correlated with their significantly lower H19 expression compared with BM MSCs. This further suggests that a restrained H19 expression serves as a stabilizing parameter in cultured MSCs. In addition to their genomic stability, ASCs from independent preparations demonstrated a far more homogeneous expression pattern of multiple genes under normal and stress conditions compared with BM MSCs, indicating that their transcription state is more stable. Interestingly, the superior uniformity and stability of murine ASCs was also correlated with an increased basal p53 activity. The significant difference in p53 activity between ASCs and BM MSCs was also evident following DNA damage (ultraviolet irradiation, H2O2 and doxorubicin) in which a significantly more robust induction of p53 target genes was seen in BM MSCs. Knockdown of p53 in ASCs resulted in significant ploidy changes, confirming its critical role in maintaining a stable diploid state.
Altogether our results suggest that MSCs isolated from different tissue sources hold distinct properties, which are manifested in vitro. This tissue-specific phenotype may have important bearing on the ability of MSCs to reach any intended clinical purpose. Further emphasis should thus be given to elucidating the origin-specific nature of MSCs.
adipose-derived mesenchymal stem cell
mesenchymal stem cell
The authors express gratitude to Naomi Goldfinger and Varda Rotter for the help in the p53 knockdown experiment.
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