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First published online February 8, 2007
Stem Cells Vol. 25 No. 5 May 2007, pp. 1166 -1177
doi:10.1634/stemcells.2006-0347; www.StemCells.com
© 2007 AlphaMed Press

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STEM CELL GENETICS AND GENOMICS

Effect of Hypoxia on Gene Expression of Bone Marrow-Derived Mesenchymal Stem Cells and Mononuclear Cells

Shunsuke Ohnishia, Takeshi Yasudab, Soichiro Kitamurac, Noritoshi Nagayaa

Departments of aRegenerative Medicine and Tissue Engineering and
cCardiovascular Surgery, National Cardiovascular Center, Osaka, Japan;
bGeneticLab Company, Ltd., Sapporo, Japan

Key Words. Microarray • Mononuclear cell • Mesenchymal stem cell • Hypoxia • Bone marrow

Correspondence: Noritoshi Nagaya, M.D., Ph.D., Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center, 5-7-1 Fujishirodai, Osaka 565-8565, Japan. Telephone: 81-6-6833-5012; Fax: 81-6-6833-9865; e-mail: nnagaya{at}ri.ncvc.go.jp or Shunsuke Ohnishi, M.D., Ph.D., Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center, 5-7-1 Fujishirodai, Osaka 565-8565, Japan. Telephone: 81-6-6833-5012; Fax: 81-6-6833-9865; e-mail: sonishi{at}ri.ncvc.go.jp

Received June 6, 2006; accepted for publication February 1, 2007.
First published online in STEM CELLS EXPRESS   February 8, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
 References
 
MSC have self-renewal and multilineage differentiation potential, including differentiation into endothelial cells and vascular smooth muscle cells. Although bone marrow-derived mononuclear cells (MNC) have been applied for therapeutic angiogenesis in ischemic tissue, little information is available regarding comparison of the molecular foundation between MNC and their MSC subpopulation, as well as their response to ischemic conditions. Thus, we investigated the gene expression profiles between MSC and MNC of rat bone marrow under normoxia and hypoxia using a microarray containing 31,099 genes. In normoxia, 2,232 (7.2%) and 2,193 genes (7.1%) were preferentially expressed more than threefold in MSC and MNC, respectively, and MSC expressed a number of genes involved in development, morphogenesis, cell adhesion, and proliferation, whereas various genes highly expressed in MNC were involved in inflammatory response and chemotaxis. Under hypoxia, 135 (0.44%) and 49 (0.16%) genes were upregulated (>threefold) in MSC and MNC, respectively, and a large number of those upregulated genes were involved in glycolysis and metabolism. Focusing on genes encoding secretory proteins, the upregulated genes in MSC under hypoxia included several molecules involved in cell proliferation and survival, such as vascular endothelial growth factor-D, placenta growth factor, pre-B-cell colony-enhancing factor 1, heparin-binding epidermal growth factor-like growth factor, and matrix metalloproteinase-9, whereas the upregulated genes in MNC under hypoxia included proinflammatory cytokines such as chemokine (C-X-C motif) ligand 2 and interleukin-1{alpha}. Our results may provide information on the differential molecular mechanisms regulating the properties of MSC and MNC under ischemic conditions.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
 References
 
MSC possess multipotency and terminally differentiate into osteoblasts, chondrocytes, neurons, skeletal muscle cells, endothelial cells, and vascular smooth muscle cells [1, 2]. MSC can be easily isolated from bone marrow-derived mononuclear cells (MNC) and expanded in vitro >1 million-fold. Thus, these features make MSC an attractive therapeutic tool [1]. Although bone marrow-derived MNC have already been established as a tool for cell therapy and have been shown to induce therapeutic neovascularization in critical limb ischemia and myocardial infarction [3–6], MNC transplantation requires harvesting a large number of cells, and some patients are refractory to MNC therapy [7, 8]. We have previously demonstrated that MSC transplantation caused great improvement in rat hind limb ischemia and dilated cardiomyopathy [9, 10]. Recent studies suggest that MSC exert tissue regeneration through paracrine effects, as well as through differentiation into specific cell types [11, 12]. However, the molecular mechanisms that explain the difference between bone marrow-derived MNC and their MSC subpopulation exposed into ischemic conditions are yet to be studied. Thus, the purposes of this study were (a) to compare the gene expression profile of two fractions of clinically applicable bone marrow-derived cells (i.e., freshly isolated MNC versus their cultured MSC subpopulation), and (b) to investigate the effect of hypoxia on gene expression in MSC and MNC.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
 References
 
Expansion of MSC and Isolation of MNC
Isolation and expansion of MSC were performed as described previously [13]. Briefly, bone marrow cells were isolated from male Lewis rats weighing 220–250 g by flushing out the femoral and tibial cavities with phosphate-buffered saline and plated onto 10-cm dishes in complete culture medium: {alpha}-minimal essential medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Five days after plating, nonadherent cells were removed, and adherent cells were further propagated for 4–5 passages. These cells were previously demonstrated to be positive for CD29 and CD90 surface markers and negative for CD34 and CD45 [10]. MNC were isolated from whole bone marrow cells by density gradient centrifugation (Histopaque-1083; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The Animal Care Committee of the National Cardiovascular Center approved the experimental protocol.

Culture of MSC and MNC Under Hypoxia
MSC and MNC (3 x 106 cells) were plated on 10-cm dishes in complete culture medium and incubated under normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) for 24 hours. For time-dependent hypoxia experiments, cells were incubated for the desired time at 1% O2. For the experiments with various O2 levels, cells were incubated under the desired level of O2 (1%, 3%, 10%, and 20%) for 24 hours.

Microarray Analysis
Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's instructions. RNA was quantified by spectrometry, and the quality was confirmed by gel electrophoresis. Double-stranded cDNA was synthesized from 10 µg of total RNA, and in vitro transcription was performed to produce biotin-labeled cRNA using GeneChip One-Cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) according to the manufacturer's instructions. After fragmentation, 10 µg of cRNA was hybridized with GeneChip Rat Genome 230 2.0 Array (Affymetrix) containing 31,099 genes. GeneChips were then scanned in a GeneChip Scanner 3000 (Affymetrix). Normalization, filtering, and Gene Ontology analysis of the data were performed with GeneSpring GX 7.3.1 software (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). The raw data from each array were normalized as follows: each CEL file was preprocessed with robust multichip average, and each measurement for each gene was divided by the 50th percentile of all measurements. Genes with a change of at least threefold were then selected.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from separately prepared cells as described above, and 5 µg of total RNA was reverse-transcribed into cDNA using avian myeloblastosis virus transcriptase (Ambion, Austin, TX, http://www.ambion.com) and oligo(dT) primers. Polymerase chain reaction (PCR) amplification was performed in 50 µl containing 1 µl of cDNA and 2.5 U of Taq DNA polymerase (Takara, Otsu, Japan, http://www.takara.co.jp). The oligonucleotides used in semiquantitative reverse transcription (RT)-PCR analysis are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA amplified from the same samples served as an internal control. PCR mixtures were denatured at 95°C for 5 minutes, and cDNA templates were amplified as follows: 25 cycles (21 cycles for GAPDH) of denaturation at 95°C for 1 minute, annealing at 45–55°C for 45 seconds, and extension at 72°C for 1 minute. At the end of the cycling, the samples were incubated at 72°C for 10 minutes. The amplified DNA products were visualized on 2% agarose gels and photographed under ultraviolet light.


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  		Table 1. Primer pairs designed for semiquantitative and quantitative reverse transcription-polymerase chain reaction

 
Quantitative Real-Time RT-PCR
PCR amplification was performed in 50 µl containing 1 µl of cDNA and 25 µl of Power SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). The oligonucleotides used in quantitative real-time RT-PCR analysis are listed in Table 1. GAPDH mRNA amplified from the same samples served as an internal control. After an initial denaturation at 95°C for 10 minutes, a 2-step cycle procedure was used (denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute) for 40 cycles in a 7700 sequence detector (Applied Biosystems). Gene expression levels were normalized according to that of GAPDH and compared with that at normoxia (20% O2). The data were analyzed with Sequence Detection Systems software (Applied BioSystems).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
 References
 
Reproducibility of Microarray Experiments
Reproducibility in the microarray experiment was assessed by repeated experiments using separately prepared RNAs. The correlation coefficient between two microarray data sets obtained from repeated experiments was greater than 0.98 for all gene probes, indicating that the whole experimental procedure was highly reproducible (data not shown).

Differentially Expressed Genes in Bone Marrow-Derived MSC and MNC Under Normoxia
Of 31,099 genes analyzed, 2,232 genes (7.2%) were highly expressed (>threefold) in MSC (Fig. 1A) and 55 genes (0.18%) were highly expressed more than 100-fold (Table 2), whereas 2,193 genes (7.1%) were highly expressed (>threefold) in MNC, and 69 genes (0.22%) were highly expressed more than 100-fold (Table 3). Noteworthy, the highly expressed genes in MSC (>threefold) included various types of molecules involved in biogenesis of extracellular matrix, such as collagens (I{alpha}1, I{alpha}2, III{alpha}1, IV{alpha}1, IV{alpha}2, V{alpha}1, V{alpha}2, VI{alpha}2, VI{alpha}3, VIII{alpha}1, VIII{alpha}2, XI{alpha}1, XII{alpha}1, XIV{alpha}1, XV, XVI{alpha}1, and XVIII{alpha}1), matrix metalloproteinases (MMP-2, -12, -14, -16, -19, and -23), serine proteases (PRSS9, 11, 23, and 35), and serine protease inhibitors (SERPINE1, SERPINF1, and SERPINH1). To verify the gene expression profile determined by our microarray analysis, the expression levels of serine protease inhibitors (SERPINE1, SERPINF1, and SERPINH1), COL3A1, and MMP-14 were analyzed by semiquantitative RT-PCR, using total RNAs separately obtained from MSC and MNC (Fig. 1B). The results showed that the differential expression pattern was in good agreement with that from the microarray analysis.


Figure 1
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  		Figure 1. Expression profile of bone marrow-derived MSC versus MNC. (A): Normalized microarray data sets of MSC and MNC. All 31,099 gene probes are represented in this plot. The outer lines indicate a threefold difference, whereas the central line represents equality. (B): Semiquantitative reverse transcription-polymerase chain reaction of selected genes from Table 2, including serine protease inhibitors. GAPDH was used as an internal control. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MNC, mononuclear cell.

 


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  		Table 2. Genes upregulated in MSC (>100-fold)

 


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  		Table 3. Genes upregulated in MNC (>100-fold)

 
Functional Classification of Highly Expressed Genes in MSC and MNC Under Normoxia
To evaluate the enriched genes in MSC, a total of 2,232 highly represented genes (>threefold) were classified by functional annotation using gene ontology terms (Table 4). Nineteen terms in the list had a p value of less than .0001, including development (e.g., transgelin, actin-{gamma}1, and short stature homeobox-2), morphogenesis (e.g., bone morphological protein-2, transforming growth factor-β3, and fibrillin-2), cell adhesion (e.g., melanoma cell adhesion molecule, neural cell adhesion molecule-1, and cadherin-11), and cell proliferation (e.g., connective tissue growth factor, fibroblast growth factor-7, and platelet-derived growth factor-A). On the other hand, for MNC, there were 30 listed terms from 2,193 enriched genes with a p value of less than .0001, including hemopoiesis, inflammatory response, and chemotaxis (Table 4).


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  		Table 4. Classification of highly expressed genes in MSC and MNC (>threefold) according to gene ontology terms

 
Differentially Upregulated Genes in Bone Marrow-Derived MSC and MNC in Response to Hypoxia
To investigate the difference in gene expression in response to hypoxia, microarray analysis was performed using total RNAs obtained from MSC and MNC incubated under hypoxia for 24 hours (Fig. 2A; Tables 5Go and 6). The results demonstrated that 135 (0.44%) and 49 (0.16%) genes were upregulated (>threefold) in MSC and MNC under hypoxia, respectively (Fig. 2B), and a significant number of those upregulated genes were involved in glycolysis and metabolism, according to gene ontology classification (data not shown). However, focusing on genes encoding secretory proteins, the upregulated genes in MSC under hypoxia included several molecules involved in cell proliferation and survival, such as vascular endothelial growth factor-D (VEGF-D), placenta growth factor (PGF), pre-B-cell colony-enhancing factor 1 (PBEF1), heparin binding epidermal growth factor-like growth factor (HB-EGF), and matrix metalloproteinase-9 (MMP-9), whereas the upregulated genes in MNC included some proinflammatory cytokines, such as chemokine (C-X-C motif) ligand 2 (CXCL2) and interleukin-1{alpha} (IL-1{alpha}) (Fig. 2B). Pairwise comparison of those upregulated genes from both MSC and MNC revealed that only 29 genes overlapped (21.3% in MSC and 59.2% in MNC), including VEGF-A, adrenomedullin (AM), and macrophage migration inhibitory factor (MIF) (Fig. 2B). Semiquantitative RT-PCR for those upregulated genes encoding secretory proteins confirmed the consistency of microarray data (Fig. 2C). To follow the kinetics of those upregulated genes, cells were cultured under different time points at 1% O2 or different grades of hypoxia, and quantitative real-time RT-PCR was performed. The results demonstrated that the time course and sustainability of gene expression were differently regulated (Fig. 2D). The expression of all genes except AM was gradually increased in MSC under hypoxia, whereas AM expression was peaked at 12 hours and slightly decreased at 24 hours. On the other hand, the expression of MIF, VEGF-A, and AM in MNC were peaked at 6 hours and was sustained up to 24 hours, whereas the expression of IL-1{alpha} and CXCL2 was gradually increased. When cells were cultured at different O2 levels for 24 hours, most of the genes except VEGF-D were upregulated even at 10% O2 in MSC, whereas the expression of three (MIF, IL-1{alpha}, and CXCL2) in MNC was unaffected at 10% O2 and reached a peak at 1% O2 (Fig. 2E).


Figure 2
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  		Figure 2. Expression profiles of MSC and MNC under normoxia versus hypoxia. (A): Normalized microarray data sets. All 31,099 gene probes are represented in this plot. The outer lines indicate a threefold difference, whereas the central line represents equality. (B): Pairwise comparison of upregulated genes from MSC and MNC under hypoxia. The numbers of genes upregulated more than threefold are presented. Gene symbols of selected secretory proteins are provided. (C): Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) of genes encoding secretory proteins listed in (B). (D): Quantitative RT-PCR of genes encoding secretory proteins listed in (B) at different time points of 1% O2. All transcription rates were estimated with reference to expression of each gene at normoxia (20% O2). (E): Quantitative RT-PCR of genes encoding secretory proteins listed in (B) at different O2 levels. All transcription rates were estimated with reference to expression of each gene at normoxia (20% O2). Abbreviations: h, hours; MNC, mononuclear cell.

 


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  		Table 5. Genes upregulated in MSC under hypoxia (>threefold)

 


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  		Table 5. (Continued)

 


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  		Table 6. Genes upregulated in MNC under hypoxia (>threefold)

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosures of Potential...
 Acknowledgments
 References
 
In this study, we focused on differential gene expression of freshly isolated MNC and their cultured MSC subpopulation and the effect of hypoxia on gene expression of those cells. We showed that (a) MSC preferentially expressed a large number of genes involved in development, morphogenesis, cell adhesion, and cell proliferation, whereas MNC expressed various genes involved in inflammatory response and chemotaxis, and (b) MSC and MNC responded to hypoxia mostly in a distinct manner; several genes involved in cell proliferation and survival were upregulated in MSC, whereas some proinflammatory cytokines were upregulated in MNC.

In normoxia, MSC highly expressed various types of molecules that are considered to be essential for development and morphogenesis. Notably, the enriched genes in MSC included a number of molecules involved in biogenesis of extracellular matrix, such as collagens, MMPs, serine proteases, and serine protease inhibitors. MNC, on the other hand, highly expressed a large number of molecules involved in inflammatory response and chemotaxis. This result is largely consistent with a recent report by Silva et al., which compared the gene expression of bone marrow-derived MSC with that of CD34+ hematopoietic precursors by serial analysis of gene expression [14]. Recently, the differential gene expression profile of human umbilical cord blood (UCB)-derived MNC compared with their MSC subpopulation has been reported, and many of the genes listed as highly represented in UCB-derived MSC were identical to our results obtained from adult rat bone marrow [15]. Others have compared the gene expression profile of MSC with that of fibroblasts [16, 17]. However, there is no report regarding the gene expression profile of bone marrow-derived MSC, which are more relevant to clinical settings as a therapeutic tool than UCB-derived MSC, in comparison with bone marrow-derived MNC, which have already been applied for regenerative medicine [3–6]. Our results may provide information on the differential molecular mechanisms regulating the properties of bone marrow-derived MNC and their MSC subpopulation.

We have recently reported that MSC, in comparison with MNC, supplied larger amounts of angiogenic, antiapoptotic, and mitogenic factors such as VEGF, AM, hepatocyte growth factor and insulin-like growth factor-1, and some of the transplanted MSC survived even in an ischemic environment [9, 10]. Recent studies from other groups indicate that MSC mediate pleiotropic effects by secreting a large number of growth factors, antiapoptotic factors, and cytokines [11, 12, 18, 19]. Thus, it is of importance to investigate the difference in gene expression profile between MSC and MNC under hypoxia, which mimics the ischemic environment in vitro. Our observation revealed that MSC had more than five times as many exclusively upregulated genes as did MNC (106 vs. 20 genes), and only 29 genes, including VEGF-A, AM, and MIF, were commonly upregulated. This result is largely consistent with a very recent report by Martin-Rendon et al., which compared the gene expression of human cord blood CD133+ cells with cultured bone marrow-derived MSC in response to hypoxia [20]. Although they cultured MNC in serum-free medium with cytokines such as IL-6, stem cell factor, Flt3-ligand, and thrombopoietin, the commonly upregulated genes in CD133+ cells and MSC included VEGF-A and AM, in agreement with our results. MIF has been reported to be upregulated in response to hypoxia in glial tumor cells and lung fibroblasts [21, 22]. In the present study, a significant number of exclusively upregulated genes were involved in glycolysis and metabolism; however, this might cause the difference in flexibility between MSC and MNC in response to hypoxia. In fact, focusing on genes encoding secretory proteins, the genes upregulated in MSC under hypoxia, but not in MNC, included several growth factors, such as VEGF-D, PGF, PBEF1, and HB-EGF. VEGF-D has angiogenic properties, and its promoter activity has been shown to increase under hypoxic conditions [23, 24]. Moreover, VEGF-D expression is significantly upregulated in response to hypoxia in differentiating embryonic stem cells [25]. PGF, another member of the VEGF family, is transcriptionally activated by hypoxia [26]. PBEF1 was originally identified as a growth factor for early-stage B cells [27] and has been demonstrated to be upregulated by hypoxia in adipocytes and breast cancer cells [28, 29]. HB-EGF, an EGF family member glycoprotein [30], has been implicated in angiogenesis [31, 32] and enhances the proliferation of MSC via interaction with its receptor, HER-1 [33]. HB-EGF expression has been induced in response to hypoxia in cerebral cortical cultures [34]. Interestingly, the present study demonstrated that MSC upregulated the expression of MMP-9 in response to hypoxia. MMP-9 is a member of metalloproteases [35] and is required for angiogenesis associated with bone growth and revascularization of ischemic tissues [36, 37]. MMP-9 releases soluble Kit-ligand from membrane Kit-ligand, causing the release of stromal-derived factor-1 and the recruitment of CXCR4+VEGFR1+ hematopoietic progenitors, "hemangiocytes" [38, 39]. These findings, concurrently with our observations, support that MSC act to promote cell proliferation, including angiogenesis, and cell survival in response to hypoxia.

On the other hand, CXCL2 (macrophage inflammatory protein-2) and IL-1{alpha} were upregulated in MNC, but not in MSC, under hypoxia. Previous reports demonstrated that CXCL2 gene expression is strongly induced in macrophages in response to hypoxia [40], and IL-1{alpha} production is also induced in peripheral blood MNC under hypoxia [41]. However, in contrast to MSC, no growth factors were specifically upregulated in MNC under hypoxia. It has been suggested that monocyte recruitment, as a consequence of an inflammatory response, is important for promoting angiogenesis via paracrine release of cytokines [42, 43]. Moreover, recent studies have suggested that transplantation of MNC, like that of MSC, induces therapeutic angiogenesis mostly through paracrine effects in ischemic disease [7, 44, 45]. Our observations suggest that transplantation of MNC, unlike that of MSC, may induce an inflammatory response under hypoxia, which may induce angiogenesis.

In the present study, we demonstrated that the gene responses to hypoxia at different time courses and different oxygen concentrations were cell-type-specific. In MSC, seven of the eight genes were upregulated even at 10% O2 but responded slowly to hypoxia. On the contrary, three of the five enriched genes in MNC responded rapidly to hypoxia but did not reach a peak up to 1% O2. It remains to be elucidated whether these differences contribute differently to paracrine actions of each type of cells in in vivo situations. Moreover, because bone marrow-derived MNC consists of mixed cell types, such as monocytes, lymphocytes, and erythroblasts, additional studies are needed to clarify which cell types in MNC are responsible for those gene expressions.

Taken together, the difference in gene expression profiles under normoxia and hypoxia, difference in gene expression at various times, and O2 level between MSC and MNC could cause their distinctive paracrine effects in terms of cell proliferation, including angiogenesis, and cell survival.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosures of Potential...
 Acknowledgments
 References
 
Bone marrow-derived MSC highly expressed a number of genes involved in development and morphogenesis compared with bone marrow-derived MNC. MSC and MNC responded to hypoxia mostly in an exclusive manner; this response might cause the difference in paracrine effects between MSC and MNC in ischemic conditions.


   DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
References
 
This work was supported by a research Grant for Cardiovascular Disease (16C-6) and Human Genome Tissue Engineering 009 from the Ministry of Health, Labor and Welfare of Japan.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures of Potential...
 Acknowledgments
 References
 

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