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. Author manuscript; available in PMC: 2016 Jul 13.
Published in final edited form as: Stem Cells. 2009 Feb;27(2):375–382. doi: 10.1634/stemcells.2008-0546

Epigenetic Reprogramming of IGF1 and Leptin Genes by Serum Deprivation in Multipotential Mesenchymal Stromal Cells

Cecilia Sanchez a, Adam Oskowitz a, Radhika R Pochampally a,b
PMCID: PMC4943331  NIHMSID: NIHMS764915  PMID: 19038795

Abstract

Recent studies on the therapeutic effect of multipotential mesenchymal stem cells (MSCs) in various models of injury have shown that paracrine factors secreted by MSCs are responsible for tissue repair with very little engraftment. In this study we tested the hypothesis that MSCs under stress undergo epigenetic modifications that direct secretion of paracrine factors responsible for tissue repair. Microarray assays of MSCs that had been deprived of serum (SDMSCs), to induce stress, demonstrated an increase in the expression of several angiogenic, prosurvival, and antiapoptotic factors, including insulin-like growth factor 1 (IGF1) and leptin. Real-time polymerase chain reaction assays demonstrated a >200-fold increase in the expression of IGF1 and leptin in SD-MSCs. Chromatin immunoprecipitation of SD-MSCs revealed histone tail modifications consistent with transcriptional activation of IGF1 and leptin promoters in a reversible manner. To identify the functional significance of the epigenetic changes in stressed MSCs, we tested the prosurvival properties of SD-MSCs and the ability of conditioned medium from SD-MSCs to enhance survival of apoptotic cancer cells. First, we showed that SD-MSCs are more resistant to oxidative damage than MSCs using alkaline comet assays. Next, we demonstrated that conditioned medium from SD-MSCs decreased staurosporin-induced cell death in the KHOS osteosarcoma cell line, and that this effect was partially reversed by immunodepletion of IGF1 or leptin from the conditioned medium. In conclusion, we demonstrate that serum deprivation induces epigenetic changes in MSCs to upregulate the expression of the proangiogenic and antiapoptotic factors IGF1 and leptin.

Keywords: Stem cells, MSCs, IGF1, Leptin, Serum starvation, Epigenetic

Introduction

Epigenetic mechanisms involving changes in DNA methylation, histone modification, and noncoding RNA expression are now broadly acknowledged as critical for the development of improved strategies to reprogram differentiated cells or direct the differentiation of stem cells with therapeutic applications [1–5]. On the other hand, little is known about the epigenetic regulation of paracrine factors secreted by stem cells that could lead to tissue repair. Among the best studied cells being explored for therapeutic applications are the stem/progenitor cells from bone marrow, referred to originally as fibroblastic colony-forming units, then as mesenchymal stem cells, and most recently, as multipotent mesenchymal stromal cells (MSCs) [6, 7]. MSCs are isolated from bone marrow by their adherence to tissue culture plastic, and the cells can be differentiated both in vitro and in vivo into multiple cellular phenotypes [7, 8]. In addition, studies have demonstrated MSCs’ ability to survive adverse conditions, including serum deprivation and hypoxia [9, 10].

The therapeutic potential of MSCs is attributed to both direct cellular engraftment and secretion of cytokines that can activate endogenous wound-healing mechanisms [11–13]. Direct evidence of cell engraftment consists of observations that demonstrate homing of MSCs to sites of tissue damage and MSC differentiation. One of the first reports demonstrating that MSCs home to sites of tissue damage came from the work of Ferrari et al. [14], who demonstrated that after cardiotoxin-induced muscle damage in mice, prelabeled MSCs appeared in wounded and regenerating tissues. In addition to the studies on homing and reparative differentiation of MSCs in injured tissue, recent observations have demonstrated that MSCs can repair tissues without extensive engraftment or differentiation [11, 12]. The secretion of cytokines that suppress inflammatory and immune reactions [15–17] and the enhancement of differentiation of tissue endogenous stem/progenitor cells have been linked to tissue repair [18, 19].

A large spectrum of studies on insulin-like growth factor 1 (IGF1) and leptin implicated their role in the promotion of cell survival [20, 21], angiogenic potential [22–25], immunoregulation [26, 27], and tissue repair [28–31]. A variety of injury models were used in the above studies, including cirrhosis [20, 32, 33], neuron damage [29], cardiomyopathy [21, 34], atherosclerosis [25, 35], diabetic wound [30], and cartilage injury [31].

We recently discovered that MSCs utilize autophagy to survive under stress, using prolonged serum deprivation (Sanchez C et al., unpublished data). In this paper, we propose a model in which serum-deprived culture conditions could lead to reversible changes in the epigenotype of MSCs, specifically directing the synthesis and secretion of prosurvival cytokines, such as IGF1 and leptin. In addition, we demonstrate that serum-deprived MSCs (SD-MSCs) are more resistant to oxidative damage than MSCs grown in the presence of serum, and SD-MSCs are able to repair DNA after oxidative damage more efficiently than MSCs. Also, the prosurvival properties of conditioned medium from SD-MSCs (Cd-SD medium) are in part due to paracrine effects of secreted IGF1 and leptin.

Materials and Methods

Human MSC Culture

Human MSCs were prepared as described previously [36]. In brief, nucleated cells were isolated with a density gradient (Ficoll-Paque; Pharmacia, Uppsala, Sweden, http://www.pfizer.com/products/) from 2 ml human bone marrow aspirated from the iliac crests of healthy volunteers under a protocol approved by the institutional review board of Tulane University Health Sciences Center. All of the nucleated cells (30–70 million) were plated in a 145-cm2 dish in 20 ml complete culture medium (CCM): [H9251]-modified minimum essential media (α-MEM) (Gibco-BRL, Carlsbad, CA, http://www.gibcobrl.com), 17% fetal bovine serum (FBS) (lot-selected for rapid growth of MSCs; Atlanta Biologicals, Norcross, GA, http://www.atlantabio.com), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Gibco-BRL). After 24 hours at 37°C in humidified 5% CO2, nonadherent cells were discarded and adherent cells were incubated in fresh medium for 4 days. The cells were lifted with 0.25% trypsin and 1 mM EDTA for 5 minutes at 37°C and replated at 50 cells/cm2 in an interconnecting system of culture flasks (6,320 cm2, Cell Factory; Nunc, Rochester, NY, http://www.nuncbrand.com). After 7–9 days, the cells were lifted with trypsin/ EDTA, frozen in 1-ml aliquots at about 106 cells/ml in 5% dimethyl sulfoxide and 30% FBS in α-MEM, and stored in liquid nitrogen as passage 1 cells. To prepare SD-MSCs and the MSC control, passage 2 cells were plated at two densities—one 15-cm diameter plate at 500 cells/cm2 and another 15-cm diameter plate at 50 cells/cm2. When the first set of plates reached 70% confluency, they were incubated with α-MEM without serum (SD-medium) or growth factors to prepare SD-MSCs. The second set of plates was incubated with CCM as a parallel control set. The appropriate medium was replaced every 4 days for 2–4 weeks.

Culture of KHOS Osteosarcoma Cell Line

Frozen vials of passage 136 KHOS cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org) and were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL), 10% FBS (Atlanta Biologicals), 100 units/ml penicillin, and 100 μg/ml streptomycin. KHOS cells were treated with 1 μg/ml staurosporin either in the presence of serum-deprived conditioned medium, conditioned medium depleted for either IGF1 or leptin, or control medium. After 72 hours, the nonadherent (dead) cells were removed and the surviving cells were quantified by the cyquant DNA quantitation method (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com).

Western Blotting

Briefly, cells were prepared and lysed in buffer (cell lysis buffer; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) supplemented with Halt protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, http://www.piercenet.com), and the protein concentration was determined using the Micro BCA Kit (Pierce Biotechnology). The cell lysate (20 μg of protein) was fractionated by SDS-PAGE (NuPAGE, 4%–12% Bis-Tris gels; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and transferred to Immobilon-P polyvinylidene difluoride membrane (Gen-Hunter, Nashville, TN, http://www.genhunter.com) by electroblotting (XCell II Blot Module; Invitrogen). The filter was blocked for 2 hours with 5% nonfat dry milk in TBS (phosphate-buffered saline [PBS] containing 0.05% Tween 20) and then incubated overnight at 4°C with the primary antibody. The filter was washed three times for 10 minutes each with TBS, and antibody binding was detected by incubating for 1 hour with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Chemicon, Temecula, CA, http://www.chemicon.com). The filter was washed and developed using a chemiluminescence assay (Visualizer Spray & Glow ECL detection system; Upstate Biotechnology, Lake Placid, NY, http://www.upstate.com). The primary antibodies used correspond to leptin (ab 9828), IGF1 (ab 40789), α-tubulin (ab7291–100), proliferating cell nuclear antigen (PCNA) (ab18197–100), DNA methyltransferase 1 (DNMT1) (ab5208–100) β-actin loading control (ab8227) (Abcam, Cambridge, MA, http://www.abcam.com), and histone deacetylase 1 (HDAC1) (2E10) (Upstate Biotechnology).

Cytokine Blot Analysis

Standard immunoblotting was performed using a RayBio human cytokine antibody array C series 2000 (Raybiotech Inc., Norcross, GA, http://www.raybiotech.com; catalog no. AAH-CYT-2000–8), which can detect 174 proteins, using the manufacturer's recommended protocol, and blots were developed with Kodak X-omat AR film.

Quantitative Reverse Transcription-Polymerase Chain Reaction

cDNA was synthesized using 1.25 μg RNA with AMV-RT enzyme and random hexamers (Promega, Madison, WI, http://www.promega.com). A Bio-Rad iCycler system (Bio-Rad, Hercules CA, http://www.bio-rad.com) was used for quantitative analysis. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using a SYBR Green supermix kit (Bio-Rad). The level of each gene transcript was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels. All primers showed 80%–100% efficiency. The PCR was performed in a total volume of 25 μl on 30 ng cDNA with iQ SYBR Green Supermix (Bio-Rad) and 400 nM forward and reverse primer. The following primers were used for this study. DNMT1: forward, 5′-CCGAGTTGGTGATGGTGTGTAC-3′; reverse, 5′-AGGTTGATGTCTGCGTGGTAGC-3′; DNMT3a: forward, 5′-AGGGCTCCTGGTGCTGAAG-3′; reverse, 5′-AGGGCTCCTGGTGCTGAAG-3′; and GAPDH: forward, 5′-TCCCATCACCATCTTCCA-3′; reverse, 5′-CATCACGCCACAGTTTCC-3′. For IGF1 and leptin expression, we used the Taqman gene expression assay Hs00153126 and Hs01084494 (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) following the manufacturer's instructions. This real-time-RT-PCR was done in an ABI 7900 System (Applied Biosystems).

Chromatin Immunoprecipitation

The chromatin immunoprecipitation (ChIP) assay was performed with the ChIP Assay Kit (Upstate Biotechnology) using the manufacturer's protocol with minor adjustments. In the first set of plates, MSCs were grown in CCM to subconfluence and incubated in air or hypoxia (1% O2) for 4 hours. In a second set of plates, MSCs were maintained under serum deprivation (SD-medium) for 2 weeks. Formaldehyde was added directly to the culture medium for a final concentration of 1% followed by incubation for 15 minutes at 37°C. The cells were washed at 4°C in PBS and lysed on ice for 10 minutes in lysis buffer (10 mM Tris HCl, pH 8.0, 1% SDS) containing phosphatase and protease inhibitors. The lysates were sonicated six times for 30 seconds (Branson Sonifier 450; Branson, Danbury, CT, http://www.bransonultrasonics.com/contact_us.asp), and debris was removed by centrifugation. The supernatant was split into several aliquots. Sonication was verified and optimized to produce average DNA fragments of 1 kb. One aliquot of the soluble chromatin was collected and stored at −20°C for preparation of input DNA. The remainder was diluted 1:10 in IP buffer (10 mM Tris HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 1 mM EDTA, and 150 mM NaCl) containing phosphatase and protease inhibitors and incubated overnight (4°C) without antibody (control). For the others, 10 μl (1:100) of each antibody for H4tetraAc, H3methyl K4, H3methyl K9, and Pol II (Upstate Biotechnology) was added. DNA–protein complexes were isolated on protein A agarose beads, previously incubated with salmon sperm DNA to eliminate background, and eluted with 1% SDS and 0.1 M NaHCO3. Crosslinking was reversed by incubation at 65°C for 5 hours. Proteins were removed with proteinase K, and DNA was extracted with phenol/chloroform, dissolved, and PCR-amplified with IGF1, leptin, and β-actin promoter primers. PCR signals from the immunoprecipitated material were first calculated as a percentage of the input for all primers. To correct for the variation in the quality of different batches of chromatin from different cells and for varying background signals generated by different antibodies, signals were normalized to those obtained with the coding region of the β-actin gene. The following oligonucleotides were used for the PCR on the IGF1 and leptin promoters. IGF1: forward, 5′-TGTTAGTGACAGGGTTCGCAGACA-3′; reverse, 5′-AGACAGTGCCCTAAAGGGACCAAT-3′; leptin: forward, 5′-TGGGAGGTACCCAAGG-3′; reverse, 5′-GGCCCGATCACAACTT-3′. The control primers were GAPDH (Upstate Biotechnology, catalog no. 22–004): forward, 5′-TACTAGCGGTTTTACGGGCG-3′; reverse, 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′ and Chr4 satellite α (heterochromatin control): forward, 5′-GATGGTTCAACACTCTTACA-3′; reverse, 5′-CTGCACTACCTGAAGAGGAC-3′. The primers for the PCR were designed after promoter analysis using Genomatix software (Genomatic Software, Inc., Ann Arbor, MI, http://www.genomatix-software.com), followed by primer generation using the National Center for Biotechnology Information Basic Local Assignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to yield an amplification product of 100–300 bp.

DNA Bisulfite Transformation

DNA was treated with bisulfite, which selectively deaminates cytosine but not 5-methylcytosine to uracil. In brief, 2.0 μg of DNA was denatured in 5.5 μl of 0.2 M NaOH for 10 minutes at 37°C, followed by incubation with 30 μl of freshly prepared 10 mM hydroquinone (Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com), and 520 μl of 3 M sodium bisulfite (Sigma-Aldrich) at pH 5.0 was added and mixed. The samples were overlaid with mineral oil to prevent evaporation and incubated at 50°C for 16 hours. Bisulfite-treated DNA was isolated using the Wizard DNA Clean-Up System (Promega). DNA was eluted in 50 μl of warm water and 5.5 μl of 3 M NaOH was added for 5 minutes. DNA was ethanol precipitated with glycogen as a carrier and resuspended in 100 μl water. Bisulfite-treated DNA was stored at −20°C.

Methylation Analysis of Long Interspersed Nucleotide Elements 1 Promoter by Pyrosequencing

Methylation at the long interspersed nucleotide elements (LINE)-1 was quantitated by pyrosequencing using the primers and conditions described previously [37]. The LINE-1 assay was performed using 10 pmol of forward primer, 5′-TTTTGAGTTAGGTGTGGG-3′, and 10 pmol of reverse biotinylated primer, 5′-TCTCACTAAAAAATACCAAACAA-3′. The LINE-1 methylation analysis was performed in a 50 μl reaction containing 50 ng of bisulfite-treated genomic DNA, 60 mM Tris-HCl, pH 8.8, 15 mM ammonium sulfate, 0.5 mM MgCl2, 1 mM deoxynucleotide triphosphate (dNTP) mix, and 1U of Taq polymerase. The PCR cycling conditions were 95°C for 30 seconds, 47°C for 30 seconds, and 72°C for 30 seconds for 47 cycles. The PCR product was purified and quantitated using the PSQ HS Pyrosequencing System (EpigenDx, Inc, Worcester, MA, http://www.epigendx.com). The ratio of C to T nucleotides was evaluated for LINE-1 methylation. The experiments were repeated twice and the mean values were taken from these separate experiments.

LINE-1 Promoter Methylation Analysis by PCR, Cloning, and Sequencing

A 50 l PCR was carried out in 60 mM Tris-HCl, pH 8.8, 15 mM ammonium sulfate, 0.5 mM MgCl2, 1 mM dNTP mix, and 1 unit of Taq polymerase using bisulfite-transformed DNA (above). The PCR cycling conditions were 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds for 28 cycles. Fifty picomoles of each PCR primer was used—L1 forward, 5′-TTGAGTTGTGGTGGGTTTTATTTAG-3′; L1 reverse, 5′-TCATCTCACTAAAAAATACCAAACA-3′—to amplify fragments of 413 bp [38]. PCR products were cloned using the TOPO-TA cloning kit (Stratagene, La Jolla, CA, http://www.stratagene.com) as per the manufacturer's protocol. Minipreps were prepared using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA, http://www1.qiagen.com). The Tulane Health Science Center Sequencing Core Facility performed all DNA sequencing.

Alkaline Comet Assay

To assess for the ability of serum depletion to prevent peroxide-induced DNA damage, alkaline comet assays were performed on MSCs and SD-MSCs at 75% confluency that were treated with 200 mM H2O2 for 18 hours in the presence of Cd-SD medium diluted 1:2 in α-MEM or in the presence of α-MEM alone (control). After 18 hours, the medium was changed to CCM for 4 hours. Cells were collected in cold PBS. The alkaline comet assay was performed using the Comet Assay HT Reagent Kit for single cell gel electrophoresis (Trevigen, Gaithersburg, MD, http://www.trevigen.com) following the manufacturer's instructions. Cells were analyzed using an epifluorescence microscope (Nikon, Melville, NY, http://www.nikonusa.com). Digital images were acquired with a Sensi-Cam QE digital camera (Cooke Corporation, Romulus, MI, http://www.cookecorp.com). Tail lengths and tail moments were quantified for at least 75 random cells per sample using the Comet Assay IV (Perceptive Instruments, Suffolk, U.K., http://www.perceptive.co.uk) comet scoring software. The tail moment is defined as the ratio of DNA in the comet tail to total DNA.

Immunodepletion

Cd-SD medium from 2 weeks was incubated with the antibodies anti-IGF1 or anti-leptin or isotype for 1 hour at room temperature. IGF1 and leptin were immunoprecipitated with Sepharose A/G beads (Invitrogen). After centrifugation at 1,000 rpm for 2 minutes, the supernatant was used for prosurvival tests in osteosarcoma cells.

Results

Changes in Expression of Epigenetic Regulators

We investigated the role of epigenetic changes in the survival of SD-MSCs by studying the changes in chromatin-remodeling protein expression. Microarray analysis, confirmed by real-time RT-PCR and Western blotting, demonstrated that culture conditions have an effect on the expression of epigenetic regulators (Fig. 1, supporting information Fig. S1). Gene expression assays indicated the upregulation and downregulation of specific epigenetic regulators, whereas some remained unchanged. For example, HDAC1 and DNMT1 and DNMT3b were downregulated by serum starvation. Although the variation in expression could be due to changes in the cell cycle, we demonstrated that SD-MSCs are replicating using the expression of PCNA, a marker of active cell cycling (Fig. 1B). Data from our laboratory indicate that approximately 1% of SD-MSCs are replicating (Sanchez C et al., unpublished data). Furthermore, we observed an increase in expression of histone variants such as H6 by microarray analysis (supporting information Fig. S1). These preliminary data indicate active chromatin remodeling in response to serum starvation and without overall cell cycle-associated expression variation.

Figure 1.

Figure 1

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Differential expression of specific epigenetic regulators in MSCs and SD-MSCs. (A): Relative levels of DNMT1 and DNMT3B mRNA in MSCs and SD-MSCs determined by real-time RT-PCR as-says. Values are normalized to GAPDH mRNA levels. The average of three replicates is displayed. (B): Western blot assays of DNMT1, HDAC1, and PCNA with β-actin as a loading control. Abbreviations: DNMT, DNA methyl-transferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; MSC, mesenchymal stem cell; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcription-polymerase chain reaction; SD-MSC, serum-deprived mesenchymal stem cell.

MSCs Maintain Constant Global DNA Methylation Under Prolonged Serum Starvation

Next, we tested if changes in expression of epigenetic regulators affect the global epigenome. Methylation analysis of repetitive elements, as used in this study (Fig. 2), can serve as a surrogate marker for global genomic DNA methylation [37]. Twenty-two CpGs in the LINE-1 promoter were analyzed by sequencing of amplified bisulfite-transformed DNA, and four CpGs were analyzed by direct pyrosequencing (Fig. 2). The results indicate that there are no significant differences in the levels of global DNA methylation between MSCs and SD-MSCs (Fig. 2A, 2B).

Figure 2.

Figure 2

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Global DNA methylation analysis of MSCs and SD-MSCs. (A): Bisulfite transformation assay on DNA extracted from cells; 22 CpGs were analyzed in the LINE-1 promoter region. Four clones from SD-MSCs and MSCs are presented; black circles represent methylated CpGs and white circles represent unmethylated CpGs. (B): Pyrosequencing assays on bisulfite-converted DNA from cells. The figure shows the percentage of methylation at four CpGs on two MSC samples from donors A and B. The data do not show a significant difference in global methylation between MSCs and SD-MSCs. Abbreviations: LINE-1, long interspersed nucleotide elements 1; MSC, mesenchymal stem cell; SD-MSC, serum-deprived mesenchymal stem cell.

MSCs Express and Release IGF1 and Leptin During Serum Starvation

We thus hypothesized that the changes in SD-MSC chromatin are not global, but specific to some genes. Microarray assays, confirmed by real-time RT-PCR, indicated an upregulation of IGF1 and leptin in SD-MSCs versus MSCs grown in the presence of serum, for two different MSC donors (Fig. 3A, 3B). The microarray analysis demonstrated a consistent increase in IGF1 and leptin transcripts with time in serum deprivation. Using real-time RT-PCR for confirmation, we demonstrated a >200-fold increase in the expression of IGF1 and leptin in SD-MSCs (Fig. 3C). Cytokine analysis of Cd-SD medium demonstrated high levels of cytokines secreted with antiapoptotic, anti-inflammatory, and angiogenic potential, including IGF1 and leptin (Fig. 3D).

Figure 3.

Figure 3

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IGF1 and leptin expression is upregulated in SD-MSCs. (A): Microarray data for IGF1 expression in SD-MSCs and MSCs. (B): Microarray data for leptin expression in SD-MSCs and MSCs. (C): Real-time RT-PCR assays for leptin and IGF1 expression in SD-MSCs and MSCs (A and B represent cells from two different donor MSCs). (D): Cytokine blot for Cd-SD medium shows the release of a variety of cytokines, including leptin. (a): angiogenin; (b): brain-derived neurotrophic factor; (c): eotaxin-3; (d): fibroblast growth factor 6; (e): fractalkine; (f): insulin growth factor binding protein 2; (g): IL-1ra; (h): IL-3; (i): IL-6; (j): leptin; (k): macrophage chemoattractant protein-1. Abbreviations: Cd-SD medium, conditioned medium from SDMSCs; IGF1, insulin-like growth factor 1; IL, interleukin; MSC, mesenchymal stem cell; RT-PCR, reverse transcription-polymerase chain reaction; SD-MSC, serum-deprived mesenchymal stem cell.

Epigenetic Regulation of IGF1 and Leptin in MSCs Under Serum Starvation

ChIP assays of the promoter regions of leptin and IGF1 using Pol II antibody were performed with MSCs and SD-MSCs. The results indicate that Pol II was bound to the promoter region of IGF1 and leptin under serum starvation but not under serum-containing culture conditions. Furthermore, when SD-MSCs were cultured in CCM for 5 days, the binding of Pol II at the promoter of IGF1 and leptin was undetectable (Figs. 4A, 5A, supporting information Table 1).

Figure 4.

Figure 4

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Epigenetic analysis of IGF1 promoter: ChIP assay for SD-MSCs, MSCs, and SD-MSCs after 5 days in CCM. The data are presented as the relative abundance of immunoprecipitated protein in the IGF1 promoter. (A): ChIP assay of Pol II followed by quantitative PCR for IGF1 promoter region. (B): ChIP assay of acetylated H4 followed by quantitative PCR for IGF1 promoter region. (C): Ratio of dimethylated H3K4 and trimethylated H3K9 distribution on IGF1 promoter. (D): Gel electrophoresis of the amplification product of the ChIP assay with H3K4Me and H3K9Me for SD-MSCs and MSCs. Abbreviations: CCM, complete culture medium; ChIP, chromatin immunoprecipitation; IGF1, insulin-like growth factor 1; MSC, mesenchymal stem cell; PCR, polymerase chain reaction; SD-MSC, serum-deprived mesenchymal stem cell.

Figure 5.

Figure 5

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Epigenetic analysis of leptin promoter: ChIP assay for SD-MSCs, MSCs, and SD-MSCs after 5 days in complete culture medium. The data are presented as the relative abundance of immunoprecipitated protein in the leptin promoter. (A): ChIP assay of Pol II followed by quantitative PCR. (B) ChIP assay of acetylated H4 followed by quantitative PCR. (C): Ratio of relative abundance of dimethylated H3K4 to trim-ethylated H3K9 on leptin promoter. (D): Gel electrophoresis of the amplification product of the leptin promoter on the tetracetylated H4 ChIP assay versus chromatin input (diluted 1/100) in MSCs, SD-MSCs, and MSCs cultured under hypoxia. Abbreviations: ChIP, chromatin immunoprecipitation; MSC, mesenchymal stem cell; PCR, polymerase chain reaction; SD-MSC, serum-deprived mesenchymal stem cell.

In order to identify the particular changes in the epigenetic “marks” for “open” and “closed” chromatin status in the promoter of IGF1 and leptin, we assessed the histone modifications associated with transcriptionally active chromatin states, including tetracetylated H4 [38–40]. SD-MSCs had significantly higher levels of tetracetylated H4 at the promoter of IGF1 and leptin than MSCs grown in the presence of serum; an increase of >200% in the abundance of this histone modification was shown at the promoter for IGF1 and leptin in SD-MSCs compared with MSCs (Figs. 4B, 5B). In addition, the increase in tetracetylated H4 under serum starvation was reversed by the addition of serum to SD-MSCs (Figs. 4B, 5B). To test if this effect is serum specific or a stress response exhibited by MSCs, we cultured MSCs under hypoxic versus normoxic conditions. ChIP assays again showed a dramatic increase in the frequency of tetracetylated H4 under hypoxia, a known regulator of leptin gene expression (Fig. 5D) [41].

We also used ChIP to assess the abundance of histone modifications associated with transcriptionally repressed chromatin states, such as H3 methylated at lysine 9. The ratio between H3K4 methylation and H3K9 methylation was used as a measurement of changes (open/close) in chromatin structure. A semiquantitative analysis confirmed by real-time PCR revealed that the average ratio of H3K4Me to H3K9Me in MSCs for IGF1 and leptin was <0.5. After serum starvation, the average ratio showed a 30-fold increase for IGF1 and a 12-fold increase for leptin. The changes observed were reversible; the addition of serum to the medium of SD-MSCs decreased the H3K4Me–H3K9Me ratio significantly (Figs. 4C, 4D, 5C, supporting information Table 1). The results above indicated the “open” chromatin state of the promoter of these two cytokines.

SD-MSCs Are More Resistant to Oxidative Damage than MSCs

To assess the role of these survival factors, we exposed MSCs and SD-MSCs to 200 μM H202 for 18 hours; cells were tested for DNA damage immediately with or without a 4-hour recovery period in CCM. The DNA damage was analyzed using the comet assay [42], and the tail length was calculated in 70 comets for each group. The results showed that SD-MSCs were more resistant to oxidative damage than MSCs, as indicated by significantly lower tail migration in the assay (Fig. 6A, 6B). In addition, SD-MSCs had a significant (p < .005) decrease in tail migration after a 4-hour recovery period; in contrast, no difference was observed with MSCs (Fig. 6A). These results indicate that SD-MSCs are able to repair DNA more efficiently than MSCs.

Figure 6.

Figure 6

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SD-MSCs are resistant to oxidative DNA damage: analysis of DNA damage and DNA repair capacity by comet assay. MSCs and SD-MSCs were exposed to 200 μM H202 for 18 hours and cells were tested for DNA damage with or without a 4-hour recovery time in complete culture medium. The DNA damage was analyzed by comet assay. The tail momentum was calculated in 70 comets per sample. (A): Two representative photographs of each experiment. (B): Graphical representation of average tail lengths of MSCs and SD-MSCs under oxidative stress as described in (A); statistical analysis, t-test. Abbreviations: Cd-SD medium, conditioned medium from SD-MSCs; MSC, mesenchymal stem cell; SD-MSC, serum-deprived mesenchymal stem cell.

IGF1 and Leptin Are Necessary to Preserve the Prosurvival Properties of Cd-SD Medium

To test the hypothesis that Cd-SD medium can promote cell survival, we treated KHOS cells (osteosarcoma cell line from the ATCC) with staurosporin to induce apoptosis in either the presence or absence of Cd-SD medium. When treated with staurosporin in the presence of Cd-SD medium, the survival rate of KHOS cells was 40%, versus 20% in the control medium (Fig. 7). Next, we tested the role of IGF1 and leptin in the antiapoptotic property of Cd-SD medium. We performed cytokine depletion experiments in Cd-SD medium in the above model. The data demonstrate that depletion of leptin and IGF1 decreased the protective role of the conditioned medium. This effect was higher with leptin depletion than with IGF1 depletion (Fig. 7).

Figure 7.

Figure 7

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Antiapoptotic property of Cd-SD medium. KHOS cells were treated with 1 μg/ml staurosporine in the presence of Cd-SD, conditioned medium depleted for either IGF1 or leptin, or control medium. After 72 hours, the surviving cells were quantified by the cyquant DNA quantitation method. Depletion of leptin and IGF1 decreased the protective role of Cd-SD medium. Abbreviations: Cd-SD medium, conditioned medium from SD-MSCs; IGF1, insulin-like growth factor 1; MSC, mesenchymal stem cell; SD-MSC, serum-deprived mesenchymal stem cell.

Discussion

We previously showed that MSCs survive prolonged serum deprivation and use autophagy as a survival mechanism to synthesize antiapoptotic factors [9] (Cecilia Sanchez C et al., unpublished data). Here, we used the same model to identify the role of epigenetic regulation in the activation and expression of secreted prosurvival factors by MSCs under stressed conditions.

First, we tested if complete serum deprivation induces global methylation changes. Using methylation at LINE-1 as a surrogate marker for global methylation, we showed that long-term serum deprivation does not have a global effect on the genomic stability of SD-MSCs. However, we identified changes in gene expression of general chromatin remodeling factors in SD-MSCs. The changes in gene expression could be, in part, associated with changes in the cell cycle [43], and also with changes in the epigenotype of MSCs. Previous studies have shown that serum deprivation in the culture leads to a cell-cycle arrest that also initiates broad and complex alterations at the transcriptional level of individual genes that may or may not affect global methylation [44, 45]. The specific changes in DNMT1 and DNMT3b, with little or no change in the expression of other DNA methylation regulators (data not shown), suggest insignificant global methylation changes with significant changes in the expression of specific genes (IGF1 and leptin).

SD-MSC chromatin seems to be extremely active. We observed upregulation in the expression of histone variants such as H3.3, which has been linked to transcriptionally active chromatin [46–48]. Interestingly, it has been suggested that deposition of H3.3 may be associated with major chromatin remodeling, perhaps as a way to “reset” histone modifications at the level of different promoters [49, 50].

We investigated the epigenetic changes associated with serum deprivation conditions in MSC culture in specific genes. IGF1 and leptin were good candidate genes for this study because of their upregulated expression in SD-MSCs, their secretion by SD-MSCs, their high spectrum of implications in tissue repair, and the recently established prosurvival and angiogenic potential of Cd-SD medium (Oskowitz A et al., unpublished data). Our epigenetic approach consisted of the analysis of changes in histone tail modifications at the promoter of IGF1 and leptin during serum starvation. It is known that a large number of different histone modifications influence the transcriptional activity of a gene by two general mechanisms—they can dictate the higher-order chromatin structure and they provide context-dependent recruitment of regulatory proteins to chromatin [41, 51, 52]. In active genes, there is a strong correlation among histone acetylation, high levels of trimethylation of H3-lysine 4, and RNA Pol II occupancy [53]. The H3K4Me3 mark has been shown to lead to further chromatin modification that can facilitate transcriptional activation [53]. We used ChIP assays to analyze acetylated H4, H3K9me3, and H3K4Me3, histone marks of IGF1 and leptin. We found enrichment of H3K4Me3, tetracetylated H4, and Pol II in the promoter of IGF1 and leptin in SD-MSCs compared with MSCs; these changes were reversible with the addition of serum to the medium. Our results are consistent with a chromatin structure of the IGF1 and leptin genes in SD-MSCs, which is conducive to their active transcription.

Consistent with the data on gene expression, we found that levels of H3K9me3 were low in the IGF1 and leptin promoter in SD-MSCs. Methylation of histone H3-lysine 9 has been linked to heterochromatin formation and gene silencing; H3K9me3 provides a binding site for the chromodomain of heterochromatin protein 1 and is, in general, enriched at the 5′ end of silenced genes [41, 52]. Together, these results indicate that the chromatin status changed from a “closed” conformation to an “open” conformation during serum starvation, with the re-establishment of the epigenetic “marks” associated with the transcriptional status of the specific gene, and these changes were reversible.

To address the role of these survival factors in SD-MSCs, we demonstrated that IGF1 and leptin are, in part, responsible for the prosurvival potential of the Cd-SD. In addition, we showed that SD-MSCs are more resistant to oxidative damage and that they are able to repair DNA damage more efficiently than MSCs. This last observation is particularly interesting for cell therapy, because it is well known that oxidative damage can impair cell survival and the proliferation of transplanted cells [54]. IGF1 and leptin have been found to relieve oxidative damage [21] and to increase mitochondrial protection in different models such as aging rats and cardiomyocytes after hypoxia injury [21, 55–57]. In addition, we recently found that Cd-SD medium has angiogenic properties important for tissue repair (Oskowitz A et al., unpublished data), and leptin is a cytokine with well-known angiogenic properties [22–25]. With the above results, we speculate that the epigenetic regulation of IGF1 and leptin may lead to improvement in cell survival and tissue repair by MSCs. Further studies with SD-MSCs and Cd-SD medium in animal models will need to be performed to address this hypothesis.

In conclusion, we demonstrated that survival factors such as IGF1 and leptin are epigenetically regulated under serum deprivation. Changes in epigenetic “marks,” indicating a change in chromatin structure, are followed by the accessibility to Pol II to initiate transcription in SD-MSCs. Our data support a model whereby stressful conditions, such as serum deprivation, can reprogram survival genes such as IGF1 and leptin to be expressed in MSCs. Secretion of these cytokines will have an important impact on the survival of SD-MSCs and probably on the survival of neighboring cells.

Supplementary Material

Supp data

Acknowledgments

We would like to thank Dr. Darwin J. Prockop for being an excellent mentor to C.S. Some of the materials employed in this work were provided by the Tulane Center for Gene Therapy through a grant from NCRR of the NIH, Grant # P40RR017447, and grants NIH AR 47796 and AR 48323, the Oberkotter Foundation, the HCA, the Health Care Company, and the Louisiana Gene Therapy Research Consortium to Dr. Darwin J. Prockop. Some of the materials for comet assays were provided by Tulane Cancer Centre through a grant from NCRR of the NIH, Grant # P20RR020152. This project was funded, in part, with federal funds from the NHLBI, NIH, under contract No. N01-HV-28186 to Dr. Diane Krause, Yale University, CT, for support to C.S., immediately after Hurricane Katrina. Our thanks to Dr. Prockop, Dr. Krause, and Patrice Penfornis for critical review of the manuscript.

Footnotes

Author contributions: C.S.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; A.O.: collection and/or assembly of data; R.P.: conception and design, data analysis and interpretation, manuscript writing, financial support, final approval of manuscript.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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