archives of oral biology 60 (2015) 29–36
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Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells Thanaphum Osathanon a,b, Philaiporn Vivatbutsiri a,b, Waleerat Sukarawan c,d, Wannakorn Sriarj c,e, Prasit Pavasant a, Sireerat Sooampon e,f,* a
Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand Developing Research Unit in Genetic and Craniofacial Analyses of Craniofacial Structures, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand c Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand d Developing Research Unit in Tissue Engineering, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand e Developing Research Unit in Cell Signaling and Protein Function, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand f Department of Pharmacology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand b
article info
abstract
Article history:
Objective: Low oxygen tension is one of the crucial factors of the stem-cell niche. However,
Accepted 30 August 2014
the long-term hypoxic culture of stem cells is difficult and requires special equipment. In
Keywords:
maintain human periodontal ligament (HPDL) cell stemness.
Cobalt chloride
Methods: HPDL cells were treated with either 50 or 100 mM CoCl2. Cell proliferation was
this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could
Stem cells
determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic
Human periodontal ligament cells
associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation
Osteoblastic differentiation
was determined by assaying alkaline phosphatase activity and in vitro mineralization. Results: The results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1a inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1a- dependent mechanism. Conclusion: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Pharmacology, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Pathumwan, Bangkok, 10330, Thailand. Tel.: +66 2 218 8882; fax: +66 2 218 88882. E-mail address: [email protected] (S. Sooampon). http://dx.doi.org/10.1016/j.archoralbio.2014.08.018 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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1.
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Introduction
Stem-cell-based therapy is a promising approach for the treatment of various degenerative diseases. Embryonic and adult stem cells are the main cell types currently used in regenerative medicine. Because of the ethical concerns in obtaining embryonic stem cells,1 adult stem cells have been intensively studied in the field of stem-cell research. Adult stem cells can be isolated from various tissues, including adipose tissue, bone marrow, and teeth.2–4 Among adult tissues, the tooth is considered as one of the easily accessible sources of stem cells. Different types of healthy teeth such as exfoliated deciduous teeth, impacted teeth, and teeth extracted for orthodontic purposes can be easily obtained. Dental stem cells can be isolated from different parts of the tooth such as the dental pulp and periodontal ligament.4,5 The PDL contains cell populations capable of differentiating into cementoblasts or osteoblasts.6,7 Periodontal ligament stem cells (PDLSCs) have been successfully isolated from the periodontal ligament of extracted human third molars.4 PDLSCs are mesenchymal stem cells that are able to selfrenew, with multilineage differentiation potential, including osteoblasts, adipocytes, chondrocytes, and neurons.4,8–10 Human PDLSCs transplanted into immuno-compromised mice generated cementum-like tissue and dense type I collagen-positive PDL-like tissue.4 The use of autologous PDLSCs, obtained from the extracted teeth of mini-pigs, could regenerate periodontal tissues in a porcine periodontitis model.11 These data indicate the potential role of PDLSCs in stem-cell-based periodontal therapy. However, one of the limitations of adult stem cells is the limited and low amount of cells. Thus, finding laboratory methods to increase their proliferation, while maintaining the stem-cell state, is a challenge for the clinical use of stem cells. Stem cells are resided in a special microenvironment known as the stem-cell niche. The niche is defined as an anatomical compartment, including cellular and acellular components, that provides signals controlling stem-cell behavior.12,13 One of the critical components of the stem-cell niche is oxygen concentration.13 Measuring the oxygen content of tissues known as stem-cell sources revealed a wide range of tissue oxygen concentrations that were lower than the inhaled oxygen tension of 21%. For example, hematopoietic stem cells were found to reside in a hypoxic microenvironment with an oxygen gradient between 1 and 6%.14 The dental pulp, which contains dental pulp stem cells, has an oxygen profile of 3 and 4.5% in rats and rabbits, respectively.15,16 However, cells are usually cultured under normoxic conditions, which might not be a suitable environment for the maintenance of the stem-cell state. These findings led to investigation into the effect of hypoxia on stemcell behavior. Hypoxia plays an important role in stem-cell proliferation and the maintenance of pluripotency. Exposing mouse bone marrow stromal cells to 3% O2 enhanced cell proliferation and increased the expression of the embryonic stem-cell markers OCT4 and REX1.17 Similar studies on human dental pulp cells indicated that culturing with 3% O2 promoted cell proliferation and increased the number of cells stained for the early
mesenchymal stem-cell marker STRO-1.18,19 Moreover, hypoxia was also found to maintain the stemness of bone marrow stromal cells and human dental pulp cells by suppressing their differentiation.17,19 To establish and maintain physical hypoxia, special equipment such as gas cylinders (containing a gas mixture of 95% nitrogen and 5% carbon dioxide), hypoxic chambers, and an oxygen analyzer are generally required. The use of this method to maintain a hypoxic environment in long-term cell culture, which usually requires media changes, faces the technical difficulty. The mimicking of hypoxia using a chemical reagent, so-called chemical hypoxia, is much more convenient and easier to establish. Cobalt chloride (CoCl2) is a hypoxia-mimicking agent that is commonly used in hypoxic culture studies. CoCl2 mimics the hypoxic response by inhibiting the activity of prolyl hydroxylase, a key enzyme in the oxygen sensing pathway.20 In the present study, we tested whether CoCl2 could maintain the stemness of human PDL (HPDL) cells as was found in physical hypoxic studies. The effect of CoCl2 on cell proliferation, stem-cell marker expression, and osteogenic differentiation was examined.
2.
Material and methods
2.1.
Cell isolation and culture
The protocol for HPDL cell isolation was approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University. HPDL cells were isolated and cultured as previously described.21 The isolated cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) containing 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 100 unit/ mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco), and 5 mg/mL amphotericin B (Gibco) at 37 8C, in a humidified atmosphere containing 5% carbon dioxide. The medium was changed every 48 h. After reaching confluence, the cells were passaged at a 1:3 ratio. Cells from passages 3–6 were used in the experiments. When treating the cells with CoCl2, CoCl2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was added to the culture medium at the designated concentration (50 or 100 mM). To examine osteoblast differentiation, cells were seeded at a density of 2.5 104 cells/well in 24-well-plates and cultured in osteogenic medium (growth medium supplemented with 50 mg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM b-glycerophosphate). The medium was changed every 48 h. Cells cultured in normal growth medium were used as the control.
2.2.
Flow cytometry
Cells were harvested with trypsin-EDTA and resuspended in the wash buffer. For cell surface staining, cells were incubated with FITC-conjugated anti-CD44 antibody (BD Biosciences Pharmingen, San Diego, CA, USA), FITC-conjugated anti-CD73 antibody (BD Biosciences Pharmingen), PerCP-CyTM5.5-conjugated anti-CD90 antibody (BD Biosciences Pharmingen), PE-conjugated anti-CD105 antibody (BD Biosciences Pharmingen), PerCP-conjugated anti-CD45 antibody (BD Biosciences
archives of oral biology 60 (2015) 29–36
Pharmingen). The isotype antibody was utilized as the negative controls. After staining, cells were washed in the wash buffer and then fixed with 1% paraformaldehyde. Data analysis was performed using CellQuest software (BD Bioscience).
2.3.
Cell proliferation assay
Cell proliferation was analyzed using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT; USB Corporation, Cleveland, USA) assay. Briefly, cells were seeded in 24-well-plates at a density of 12,500 cells/well. At the end of the culture period, the medium was replaced with MTT solution and the cells were incubated at 37 8C for 30 min. The purple formazan crystals that formed were dissolved in dimethylsulfoxide. The absorbance was measured at 570 nm using a microplate reader (Biotek Instruments, Winooski, VT, USA). Cell number was calculated using a standard curve and the percentage of cells compared to the control was calculated at each time point.
2.4. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (real-time PCR) Total cellular RNA was extracted with TRI reagent (Molecular Research Center, Cincinnati, OH, USA). RNA samples (1 mg) were reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA). RTPCR was performed using Tag polymerase (Invitrogen, NY, USA). The amplified DNA was then electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide staining. For Real-time PCR, the reaction was performed in a LightCycler Nano (Roche, USA) with a LightCycler_480 SYBR Green I Master kit (Roche). The primer sequences used for RT-PCR were: VEGF forward 50 -ATGAGGACACCGGCTCTGACCA-30 , reverse 50 -AGGCTCCTGAATCTTCCAGGCA-30 ; REX1 forward 50 -AGAATTCGCTTGAGTATTCTGA-30 , reverse 50 -GGCTTTCAGGTTATTTGACTGA-30 ; OCT4 forward 50 -AGACCCAGCAGCCTCAAAATC-30 , reverse 50 -GCAACCTGGAGAATTTGTTCCT-30 ; ALP forward 50 -CGAGATACAAGCACTCCCACTTC-30 , reverse 50 -CTGTTCAGCTCGTACTGCATGTC-30 ; OCN forward 50 -ATGAGAGCCCTCACACTCCTC-30 , reverse 50 -GCCGTAGAAGCGCCGATAGGC-30 ; RUNX2 forward 50 -CCCCACGACAACCGCACCAT-30 , reverse 50 -CACTCCGGCCCACAAATC-30 ; 18S forward 50 -GTGATGCCCTTAGATGTCC30 , reverse 50 -CCATCCAATCGGTAGTAGC-30 . The primer sequences used for Real-time PCR were the same as RT-PCR except RUNX2. The primer sequences were: RUNX2 forward 50 -ATGATGACACTGCCACCTCTGA-30 and reverse 50 -GGCTGGATAGTGCATTCGTG-30 .
2.5.
Western blotting
Cells were washed twice in PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail. Whole cell lysates were separated by SDSPAGE and transferred onto nitrocellulose membranes. The membranes were subsequently probed with rabbit anti-HIF-1a immunoglobulin G (Millipore, USA), mouse anti-HIF-2a immunoglobulin G (R&D systems, USA), or mouse anti-b-actin
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immunoglobulin G (Millipore). The membranes were then treated with goat anti-rabbit immunoglobulin G (Millipore) or goat anti-mouse immunoglobulin G (Invitrogen, USA). The bands were visualized by adding SuperSignal1 West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
2.6.
Alkaline phosphatase (ALP) enzymatic activity assay
ALP activity was analyzed using p-nitrophenol phosphate as the assay substrate. The cells were lysed in alkaline lysis buffer. Aliquots were incubated at 37 8C in a solution containing 2 mg/mL p-nitrophenol phosphate (Invitrogen), 0.1 M 2amino-2methyl-1-propanol (Sigma, St. Louis, MO, USA), and 2 mM MgCl2 for 30 min. The reaction was stopped by adding 50 mM NaOH. The presence of p-nitrophenol was measured at an absorbance of 410 nm. The enzyme activity was normalized to cellular protein concentration which had been measured by BCA assay (Thermo Scientific, IL, USA).
2.7.
Mineralization assay
The cells were fixed with cold methanol for 10 min, washed with deionized water, and stained with 1% Alizarin Red S solution (Sigma, USA) at room temperature on a shaker for 3 min. We quantified the amount of calcium deposition by destaining with 10% cetylpyridinium chloride monohydrate (Sigma, USA) in 10 mM sodium phosphate at room temperature for 15 min. The absorbance was measured at 570 nm.
2.8.
Statistical analyses
The data are presented as mean standard deviation. The two-tail Student’s t test was used to compare two groups of samples. One-way analysis of variance (ANOVA) followed by the Dunnett test was used to compare multiple groups of samples. Values of p < 0.05 were considered to be statistically significant. All experiments were done in triplicate and were repeated using cells from three different donors.
3.
Results
3.1. HPDL cells exhibited mesenchymal stem-cell characteristics According to criteria proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy,22 the multipotent mesenchymal phenotype of HPDL cells was analyzed by flow cytometry and in vitro ostegenic differentiation. As shown in Fig. 1A, flow cytometry analyses revealed that the isolated HPDL cells were positive for CD44, CD73, CD90, and CD105, the cell surface markers of mesenchymal stem cells. In contrast, cells were negative for CD45, the cell surface marker of hematopoietic stem cells. To study the potential of osteogenic differentiation, cells were cultured in osteogenic differentiation medium for 14 days. Alizarin red staining showed the marked increase of mineral deposition (Fig. 1B and C).
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Fig. 1 – Mesenchymal stem-cell phenotype of HPDL cells. (A) Cell surface markers of mesenchymal stem cells were analyzed by flow cytometry. (B) HPDL cells were cultured in normal growth medium or osteogenic medium for 14 days and the cultures were stained with Alizarin red. (C) The quantitative analysis of Alizarin red staining was performed. The results were expressed as fold change compared to the control. (*p < 0.05 vs. control).
3.2.
CoCl2 had no effect on cell proliferation
The effect of CoCl2 on HPDL cell proliferation was further examined. CoCl2 at the concentration of 50 mM was shown to induce the proliferation of umbilical cord blood-derived CD133+ cells.23 Thus, the effect of CoCl2 on HPDL cell proliferation was tested at the concentration of 50 and 100 mM for 1, 3, and 7 days. The results of the MTT assay indicated that the rate of cell proliferation was not significantly different between the control and the CoCl2 treated groups (50 and 100 mM) upon 7 days of treatment (Fig. 2). This data indicated that CoCl2 had no effect on the proliferation of HPDL cells.
3.3.
was used as a positive control (Fig. 3A and B). In addition, western blot analysis was performed to confirm the effect of CoCl2 on HIF-1a protein expression. As shown in Fig. 3C, HIF1a protein level was increased in the presence of CoCl2, whereas HIF-2a protein level was not changed upon the treatment.
CoCl2 induced stem-cell marker expression
Next, we examined whether CoCl2 could induce the expression of REX1 and OCT4, which are widely used stem-cell markers. RT-PCR and Real-time PCR analysis indicated that the mRNA expression of REX1 was markedly induced by CoCl2. Though, the statistical significance was not observed (Fig. 3A and B). The OCT4 mRNA expression was significantly increased when the cells were treated with 100 mM CoCl2 (Fig. 3A and B). Because CoCl2 is known to induce vascular endothelial growth factor (VEGF) expression through HIF-1a stabilization, the up-regulation of VEGF, which we observed,
Fig. 2 – The effect of CoCl2 on cell proliferation. HPDL cells were left untreated or treated with CoCl2 (50 and 100 uM) for 1, 3, and 7 days. The number of cells was measured using the MTT assay. Data was expressed as the percentage of cell proliferation relative to control (untreated, day 1).
archives of oral biology 60 (2015) 29–36
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Fig. 3 – The effect of CoCl2 on stem-cell marker expression. HPDL cells were left untreated or treated with CoCl2 (50 and 100 mM) for 7 days. (A and B) The mRNA expression of VEGF, REX1, and OCT4 was examined by RT-PCR (A) and Real-time PCR (B). Graphs demonstrated the relative mRNA expression compared to the control. (C) Representative pictures illustrated the HIF-1a and HIF-2a protein expression as determined by western blotting. (*p < 0.05 vs. control).
3.4.
CoCl2 inhibited osteogenic differentiation
To study the effect of CoCl2 on osteogenic differentiation, HPDL cells were cultured in osteogenic differentiation medium supplemented with either 50 or 100 mM CoCl2. After 7 days of osteogenic induction, 100 mM CoCl2 could significantly inhibit alkaline phosphatase (ALP) activity, which is an early
marker of osteogenic differentiation (Fig. 4A).24 The effect of CoCl2 on the expression of osteogenic associated genes was also determined by RT-PCR and Real-time PCR. Corresponding to the ALP activity assay, the mRNA expression of ALP was markedly decreased by CoCl2 treatment (Fig. 4B and C). The mRNA expression of osteocalcin (OCN), a non-collagenous matrix protein secreted during the late stage of osteoblast
Fig. 4 – CoCl2 decreased ALP activity and osteogenic associated gene expression. HPDL cells were cultured in osteogenic medium with or without CoCl2 (50 and 100 mM) for 7 days. (A) ALP activity assay was examined. Data were expressed as fold change compared to the control. (B and C) The mRNA expression of ALP, OCN, and RUNX2 was examined by RT-PCR (B) and Real-time PCR (C). Graphs demonstrated the relative expression compared to the control. (*p < 0.05 vs. control).
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Fig. 5 – Apigenin abolished CoCl2-suppressed osteogenic differentiation. HPDL cells were cultured in osteogenic medium. In some conditions, CoCl2 (100 mM) and/or Apigenin (40 mM) were added in the culture condition. (A) Graph demonstrated the ALP activity at day 7. (B) Representative pictures illustrated the mineral deposition determined by Alizarin red staining at day 14. (C) Graph showed the quantitative analysis of Alizarin red staining. The results shown in ‘‘A’’ and ‘‘C’’ were expressed as fold change compared to the control. (*p < 0.05 vs. control).
differentiation,25 was significantly decreased when cells were treated with 100 mM CoCl2 (Fig. 4B and C). Notably, the mRNA expression of RUNX2, an essential transcription factor for osteoblast differentiation,26 was also suppressed by CoCl2 treatment (Fig. 4B and C). Taken together, these findings suggest that CoCl2 might maintain the stemness of HPDL cells by the suppression of osteogenic differentiation.
3.5. Apigenin reversed the effect of CoCl2 on osteogenic differentiation Because CoCl2 is known to mimic hypoxia through the stabilization of HIF-1a, which is a key transcription factor responsible for the hypoxic response, we hypothesized that the inhibitory effect of CoCl2 was mediated by HIF-1a. Thus, we used Apigenin, which inhibits HIF-1a expression, to test this hypothesis.27,28 ALP activity was assayed after the cells were cultured in osteogenic medium for 7 days. As shown in Fig. 5A, treatment with Apigenin alone had no effect on ALP activity. However, the decrease in ALP activity induced by CoCl2 was abolished when the cells were co-treated with Apigenin. To study the calcification capability of HPDL cells, Alizarin red staining was performed after 14 days of osteogenic medium induction. Although, the statistical significance was not observed, CoCl2 treatment resulted in less mineral deposits than the control group (Fig. 5B and C). When co-cultured with Apigenin, CoCl2 was unable to inhibit mineral deposition (Fig. 5B and C). Therefore, these findings suggest that CoCl2 may inhibit osteogenic differentiation through a HIF-1a dependent mechanism.
4.
Discussion
One of the challenges of adult stem-cell investigation is to determine the optimal conditions for expanding cell number while maintaining the stem-cell state. Hence, the effects of growth factors and culture conditions on stem-cell behavior are now the focus of many researchers. It is now known that hypoxia
is common in the various stem-cell niches. Thus, the addition of a hypoxia-mimicking agent to culture media might be a method to maintain the stem-cell state in the laboratory setting. In the present study, we found that CoCl2, a demonstrated hypoxiamimicking reagent, could induce the expression of stem-cell markers and inhibit the osteoblastic differentiation of HPDL cells. Therefore, our study demonstrates that media supplementation with a hypoxia-mimicking agent such as CoCl2 might be alternative approach for stem-cell culture. In the present study, the effect of CoCl2 on HPDL cell proliferation was evaluated. While hypoxia has been shown to promote the proliferation of many types of stem cells,17,18,29 the effect of CoCl2 on cell proliferation varies depending on the cell type. For example, CoCl2 treatment at the concentration of 10–100 mM had no effect on the proliferation of human neuroprogenitor cells.30 CoCl2 treatment, at the range of 50– 200 mM, was shown to induce the proliferation of umbilical cord blood-derived 133+ cells.23,30 In our study, we found that the number of HPDL cells was not changed upon 7 days of treatment with 50–100 mM CoCl2. However, from this data, we cannot conclude that CoCl2 treatment had no effect on the proliferation of PDLSCs because the HPDL cells used in this study are a pool of heterogeneous cells. Other direct methods to quantify the number of stem cells using specific markers such as STRO-1 are further required. Also, it is interesting to study whether the longer period of CoCl2 supplementation could affect the population proportion of stem cells. Currently, a specific PDLSC stem-cell marker has not been identified. We investigated the expression of REX1 and OCT4, which are recognized stem-cell markers.31 REX1, also known as zinc-finger protein-42 (zfp42), and OCT4, a member of POU domain transcription factor family, are expressed in embryonic and adult stem cells.32,33 The levels of REX1 and OCT4 expression decreases during cell differentiation,34,35 indicating their essential role in maintaining pluripotency. It has been shown that specific stimuli such as basic fibroblast growth factor or hypoxia could up-regulate the expression of REX1 and OCT4.17,36 Our results indicate that CoCl2 treatment, which mimics hypoxia, could enhance the expression of both
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REX1 and OCT4 in human periodontal ligament cells. These results imply that CoCl2-supplemented media may increase the stem-cell population. The ability of CoCl2 to maintain HPDL cell stemness was studied by investigating their differentiation potential. It has been shown that hypoxia inhibited the osteogenic differentiation of adult stem cells isolated from marrow and human dental pulp cells.19,37 Correspondingly, we found that CoCl2 could inhibit alkaline phosphatase activity and the expression of osteogenic-associated genes in HPDL cells. The inhibitory effect of CoCl2 on osteoblastic differentiation was confirmed by Alizarin red staining that showed CoCl2 treatment attenuated the mineral deposition. These findings suggest that CoCl2 may maintain stemness by inhibiting osteoblastic differentiation. The ability of CoCl2 to influence the differentiation of PDLSCs to other cell fates such as chondrocytes or neurons is an interesting topic for future study. The involvement of HIF-1a in the CoCl2 mediated inhibition of osteogenic differentiation was elucidated using Apigenin, a flavonoid shown to inhibit HIF-1a expression. We found that Apigenin could reverse the effect of CoCl2 treatment on alkaline phosphatase activity and mineral deposition. These data suggest that HIF-1a might be involved in the inhibitory effect of CoCl2. However, Apigenin is not a specific inhibitor of HIF-1a. It can also inhibit a number of hypoxia responsive genes such as VEGF and glucose transporter 1 (GLUT-1),27 as well as non-hypoxia target genes such as cyclin A and cyclin B.38 To ensure that HIF-1a is involved in this mechanism, future study using an siRNA approach is necessary. In conclusion, this study shows that CoCl2 treatment can induce stem-cell marker expression and maintain the stemness of HPDL cells. These findings imply that the usage of hypoxiamimicking agents such as CoCl2 may be an alternative approach for maintaining the stem-cell state in the laboratory. Comparing to physical hypoxia, CoCl2 supplementation is much more convenient to mimic a hypoxic condition. However, the response of cell to CoCl2 may not absolutely identical to the real hypoxia. As CoCl2 mimics hypoxia by stabilizing HIF-1a, the hypoxic signal through HIF-1a-independent pathway will not occur when using CoCl2. Future study comparing the effect of CoCl2 and hypoxia on stem-cell behavior is needed to confirm the possible use of CoCl2 in maintaining stem cell.
Acknowledgements This study was supported by Young Investigator Research Funding, Faculty of Dentistry, Chulalongkorn University and in part by ‘‘Integrated Innovation Academic Center: IIAC’’ Chulalongkorn University Centenary Academic Development Project. PP was supported by the Research Chair Grant 2012, the National Science and Technology Development Agency (NSTDA), Thailand.
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factor alpha (HIF-alpha): relevance in neural progenitor/stem cells. Neurotox Res 2009;15(4):367–80. Calloni R, Cordero EA, Henriques JA, Bonatto D. Reviewing and updating the major molecular markers for stem cells. Stem Cells Dev 2013;22(9):1455–76. Abeyta MJ, Clark AT, Rodriguez RT, Bodnar MS, Pera RA, Firpo MT. Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum Mol Genet 2004;13(6):601–8. Baal N, Reisinger K, Jahr H, Bohle RM, Liang O, Munstedt K, et al. Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood. Thromb Haemost 2004;92(4):767–75. Rogers MB, Hosler BA, Gudas LJ. Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes. Development 1991;113(3):815–24. Nichols J, Zevnik B, Anastassiadis K, Niwa H, KleweNebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95(3):379–91. Osathanon T, Nowwarote N, Pavasant P. Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCgamma signaling pathway. J Cell Biochem 2011;112(7):1807–16. D’Ippolito G, Diabira S, Howard GA, Roos BA, Schiller PC. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone 2006;39(3):513–22. Ujiki MB, Ding XZ, Salabat MR, Bentrem DJ, Golkar L, Milam B, et al. Apigenin inhibits pancreatic cancer cell proliferation through G2/M cell cycle arrest. Mol Cancer 2006;5:76.
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Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells Thanaphum Osathanon a,b, Philaiporn Vivatbutsiri a,b, Waleerat Sukarawan c,d, Wannakorn Sriarj c,e, Prasit Pavasant a, Sireerat Sooampon e,f,* a
Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand Developing Research Unit in Genetic and Craniofacial Analyses of Craniofacial Structures, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand c Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand d Developing Research Unit in Tissue Engineering, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand e Developing Research Unit in Cell Signaling and Protein Function, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand f Department of Pharmacology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand b
article info
abstract
Article history:
Objective: Low oxygen tension is one of the crucial factors of the stem-cell niche. However,
Accepted 30 August 2014
the long-term hypoxic culture of stem cells is difficult and requires special equipment. In
Keywords:
maintain human periodontal ligament (HPDL) cell stemness.
Cobalt chloride
Methods: HPDL cells were treated with either 50 or 100 mM CoCl2. Cell proliferation was
this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could
Stem cells
determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic
Human periodontal ligament cells
associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation
Osteoblastic differentiation
was determined by assaying alkaline phosphatase activity and in vitro mineralization. Results: The results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1a inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1a- dependent mechanism. Conclusion: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory. # 2014 Elsevier Ltd. All rights reserved.
* Corresponding author at: Department of Pharmacology, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Pathumwan, Bangkok, 10330, Thailand. Tel.: +66 2 218 8882; fax: +66 2 218 88882. E-mail address: [email protected] (S. Sooampon). http://dx.doi.org/10.1016/j.archoralbio.2014.08.018 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
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1.
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Introduction
Stem-cell-based therapy is a promising approach for the treatment of various degenerative diseases. Embryonic and adult stem cells are the main cell types currently used in regenerative medicine. Because of the ethical concerns in obtaining embryonic stem cells,1 adult stem cells have been intensively studied in the field of stem-cell research. Adult stem cells can be isolated from various tissues, including adipose tissue, bone marrow, and teeth.2–4 Among adult tissues, the tooth is considered as one of the easily accessible sources of stem cells. Different types of healthy teeth such as exfoliated deciduous teeth, impacted teeth, and teeth extracted for orthodontic purposes can be easily obtained. Dental stem cells can be isolated from different parts of the tooth such as the dental pulp and periodontal ligament.4,5 The PDL contains cell populations capable of differentiating into cementoblasts or osteoblasts.6,7 Periodontal ligament stem cells (PDLSCs) have been successfully isolated from the periodontal ligament of extracted human third molars.4 PDLSCs are mesenchymal stem cells that are able to selfrenew, with multilineage differentiation potential, including osteoblasts, adipocytes, chondrocytes, and neurons.4,8–10 Human PDLSCs transplanted into immuno-compromised mice generated cementum-like tissue and dense type I collagen-positive PDL-like tissue.4 The use of autologous PDLSCs, obtained from the extracted teeth of mini-pigs, could regenerate periodontal tissues in a porcine periodontitis model.11 These data indicate the potential role of PDLSCs in stem-cell-based periodontal therapy. However, one of the limitations of adult stem cells is the limited and low amount of cells. Thus, finding laboratory methods to increase their proliferation, while maintaining the stem-cell state, is a challenge for the clinical use of stem cells. Stem cells are resided in a special microenvironment known as the stem-cell niche. The niche is defined as an anatomical compartment, including cellular and acellular components, that provides signals controlling stem-cell behavior.12,13 One of the critical components of the stem-cell niche is oxygen concentration.13 Measuring the oxygen content of tissues known as stem-cell sources revealed a wide range of tissue oxygen concentrations that were lower than the inhaled oxygen tension of 21%. For example, hematopoietic stem cells were found to reside in a hypoxic microenvironment with an oxygen gradient between 1 and 6%.14 The dental pulp, which contains dental pulp stem cells, has an oxygen profile of 3 and 4.5% in rats and rabbits, respectively.15,16 However, cells are usually cultured under normoxic conditions, which might not be a suitable environment for the maintenance of the stem-cell state. These findings led to investigation into the effect of hypoxia on stemcell behavior. Hypoxia plays an important role in stem-cell proliferation and the maintenance of pluripotency. Exposing mouse bone marrow stromal cells to 3% O2 enhanced cell proliferation and increased the expression of the embryonic stem-cell markers OCT4 and REX1.17 Similar studies on human dental pulp cells indicated that culturing with 3% O2 promoted cell proliferation and increased the number of cells stained for the early
mesenchymal stem-cell marker STRO-1.18,19 Moreover, hypoxia was also found to maintain the stemness of bone marrow stromal cells and human dental pulp cells by suppressing their differentiation.17,19 To establish and maintain physical hypoxia, special equipment such as gas cylinders (containing a gas mixture of 95% nitrogen and 5% carbon dioxide), hypoxic chambers, and an oxygen analyzer are generally required. The use of this method to maintain a hypoxic environment in long-term cell culture, which usually requires media changes, faces the technical difficulty. The mimicking of hypoxia using a chemical reagent, so-called chemical hypoxia, is much more convenient and easier to establish. Cobalt chloride (CoCl2) is a hypoxia-mimicking agent that is commonly used in hypoxic culture studies. CoCl2 mimics the hypoxic response by inhibiting the activity of prolyl hydroxylase, a key enzyme in the oxygen sensing pathway.20 In the present study, we tested whether CoCl2 could maintain the stemness of human PDL (HPDL) cells as was found in physical hypoxic studies. The effect of CoCl2 on cell proliferation, stem-cell marker expression, and osteogenic differentiation was examined.
2.
Material and methods
2.1.
Cell isolation and culture
The protocol for HPDL cell isolation was approved by the Ethics Committee of the Faculty of Dentistry, Chulalongkorn University. HPDL cells were isolated and cultured as previously described.21 The isolated cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) containing 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 100 unit/ mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco), and 5 mg/mL amphotericin B (Gibco) at 37 8C, in a humidified atmosphere containing 5% carbon dioxide. The medium was changed every 48 h. After reaching confluence, the cells were passaged at a 1:3 ratio. Cells from passages 3–6 were used in the experiments. When treating the cells with CoCl2, CoCl2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was added to the culture medium at the designated concentration (50 or 100 mM). To examine osteoblast differentiation, cells were seeded at a density of 2.5 104 cells/well in 24-well-plates and cultured in osteogenic medium (growth medium supplemented with 50 mg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM b-glycerophosphate). The medium was changed every 48 h. Cells cultured in normal growth medium were used as the control.
2.2.
Flow cytometry
Cells were harvested with trypsin-EDTA and resuspended in the wash buffer. For cell surface staining, cells were incubated with FITC-conjugated anti-CD44 antibody (BD Biosciences Pharmingen, San Diego, CA, USA), FITC-conjugated anti-CD73 antibody (BD Biosciences Pharmingen), PerCP-CyTM5.5-conjugated anti-CD90 antibody (BD Biosciences Pharmingen), PE-conjugated anti-CD105 antibody (BD Biosciences Pharmingen), PerCP-conjugated anti-CD45 antibody (BD Biosciences
archives of oral biology 60 (2015) 29–36
Pharmingen). The isotype antibody was utilized as the negative controls. After staining, cells were washed in the wash buffer and then fixed with 1% paraformaldehyde. Data analysis was performed using CellQuest software (BD Bioscience).
2.3.
Cell proliferation assay
Cell proliferation was analyzed using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT; USB Corporation, Cleveland, USA) assay. Briefly, cells were seeded in 24-well-plates at a density of 12,500 cells/well. At the end of the culture period, the medium was replaced with MTT solution and the cells were incubated at 37 8C for 30 min. The purple formazan crystals that formed were dissolved in dimethylsulfoxide. The absorbance was measured at 570 nm using a microplate reader (Biotek Instruments, Winooski, VT, USA). Cell number was calculated using a standard curve and the percentage of cells compared to the control was calculated at each time point.
2.4. Reverse transcriptase polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (real-time PCR) Total cellular RNA was extracted with TRI reagent (Molecular Research Center, Cincinnati, OH, USA). RNA samples (1 mg) were reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA). RTPCR was performed using Tag polymerase (Invitrogen, NY, USA). The amplified DNA was then electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide staining. For Real-time PCR, the reaction was performed in a LightCycler Nano (Roche, USA) with a LightCycler_480 SYBR Green I Master kit (Roche). The primer sequences used for RT-PCR were: VEGF forward 50 -ATGAGGACACCGGCTCTGACCA-30 , reverse 50 -AGGCTCCTGAATCTTCCAGGCA-30 ; REX1 forward 50 -AGAATTCGCTTGAGTATTCTGA-30 , reverse 50 -GGCTTTCAGGTTATTTGACTGA-30 ; OCT4 forward 50 -AGACCCAGCAGCCTCAAAATC-30 , reverse 50 -GCAACCTGGAGAATTTGTTCCT-30 ; ALP forward 50 -CGAGATACAAGCACTCCCACTTC-30 , reverse 50 -CTGTTCAGCTCGTACTGCATGTC-30 ; OCN forward 50 -ATGAGAGCCCTCACACTCCTC-30 , reverse 50 -GCCGTAGAAGCGCCGATAGGC-30 ; RUNX2 forward 50 -CCCCACGACAACCGCACCAT-30 , reverse 50 -CACTCCGGCCCACAAATC-30 ; 18S forward 50 -GTGATGCCCTTAGATGTCC30 , reverse 50 -CCATCCAATCGGTAGTAGC-30 . The primer sequences used for Real-time PCR were the same as RT-PCR except RUNX2. The primer sequences were: RUNX2 forward 50 -ATGATGACACTGCCACCTCTGA-30 and reverse 50 -GGCTGGATAGTGCATTCGTG-30 .
2.5.
Western blotting
Cells were washed twice in PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail. Whole cell lysates were separated by SDSPAGE and transferred onto nitrocellulose membranes. The membranes were subsequently probed with rabbit anti-HIF-1a immunoglobulin G (Millipore, USA), mouse anti-HIF-2a immunoglobulin G (R&D systems, USA), or mouse anti-b-actin
31
immunoglobulin G (Millipore). The membranes were then treated with goat anti-rabbit immunoglobulin G (Millipore) or goat anti-mouse immunoglobulin G (Invitrogen, USA). The bands were visualized by adding SuperSignal1 West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
2.6.
Alkaline phosphatase (ALP) enzymatic activity assay
ALP activity was analyzed using p-nitrophenol phosphate as the assay substrate. The cells were lysed in alkaline lysis buffer. Aliquots were incubated at 37 8C in a solution containing 2 mg/mL p-nitrophenol phosphate (Invitrogen), 0.1 M 2amino-2methyl-1-propanol (Sigma, St. Louis, MO, USA), and 2 mM MgCl2 for 30 min. The reaction was stopped by adding 50 mM NaOH. The presence of p-nitrophenol was measured at an absorbance of 410 nm. The enzyme activity was normalized to cellular protein concentration which had been measured by BCA assay (Thermo Scientific, IL, USA).
2.7.
Mineralization assay
The cells were fixed with cold methanol for 10 min, washed with deionized water, and stained with 1% Alizarin Red S solution (Sigma, USA) at room temperature on a shaker for 3 min. We quantified the amount of calcium deposition by destaining with 10% cetylpyridinium chloride monohydrate (Sigma, USA) in 10 mM sodium phosphate at room temperature for 15 min. The absorbance was measured at 570 nm.
2.8.
Statistical analyses
The data are presented as mean standard deviation. The two-tail Student’s t test was used to compare two groups of samples. One-way analysis of variance (ANOVA) followed by the Dunnett test was used to compare multiple groups of samples. Values of p < 0.05 were considered to be statistically significant. All experiments were done in triplicate and were repeated using cells from three different donors.
3.
Results
3.1. HPDL cells exhibited mesenchymal stem-cell characteristics According to criteria proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy,22 the multipotent mesenchymal phenotype of HPDL cells was analyzed by flow cytometry and in vitro ostegenic differentiation. As shown in Fig. 1A, flow cytometry analyses revealed that the isolated HPDL cells were positive for CD44, CD73, CD90, and CD105, the cell surface markers of mesenchymal stem cells. In contrast, cells were negative for CD45, the cell surface marker of hematopoietic stem cells. To study the potential of osteogenic differentiation, cells were cultured in osteogenic differentiation medium for 14 days. Alizarin red staining showed the marked increase of mineral deposition (Fig. 1B and C).
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Fig. 1 – Mesenchymal stem-cell phenotype of HPDL cells. (A) Cell surface markers of mesenchymal stem cells were analyzed by flow cytometry. (B) HPDL cells were cultured in normal growth medium or osteogenic medium for 14 days and the cultures were stained with Alizarin red. (C) The quantitative analysis of Alizarin red staining was performed. The results were expressed as fold change compared to the control. (*p < 0.05 vs. control).
3.2.
CoCl2 had no effect on cell proliferation
The effect of CoCl2 on HPDL cell proliferation was further examined. CoCl2 at the concentration of 50 mM was shown to induce the proliferation of umbilical cord blood-derived CD133+ cells.23 Thus, the effect of CoCl2 on HPDL cell proliferation was tested at the concentration of 50 and 100 mM for 1, 3, and 7 days. The results of the MTT assay indicated that the rate of cell proliferation was not significantly different between the control and the CoCl2 treated groups (50 and 100 mM) upon 7 days of treatment (Fig. 2). This data indicated that CoCl2 had no effect on the proliferation of HPDL cells.
3.3.
was used as a positive control (Fig. 3A and B). In addition, western blot analysis was performed to confirm the effect of CoCl2 on HIF-1a protein expression. As shown in Fig. 3C, HIF1a protein level was increased in the presence of CoCl2, whereas HIF-2a protein level was not changed upon the treatment.
CoCl2 induced stem-cell marker expression
Next, we examined whether CoCl2 could induce the expression of REX1 and OCT4, which are widely used stem-cell markers. RT-PCR and Real-time PCR analysis indicated that the mRNA expression of REX1 was markedly induced by CoCl2. Though, the statistical significance was not observed (Fig. 3A and B). The OCT4 mRNA expression was significantly increased when the cells were treated with 100 mM CoCl2 (Fig. 3A and B). Because CoCl2 is known to induce vascular endothelial growth factor (VEGF) expression through HIF-1a stabilization, the up-regulation of VEGF, which we observed,
Fig. 2 – The effect of CoCl2 on cell proliferation. HPDL cells were left untreated or treated with CoCl2 (50 and 100 uM) for 1, 3, and 7 days. The number of cells was measured using the MTT assay. Data was expressed as the percentage of cell proliferation relative to control (untreated, day 1).
archives of oral biology 60 (2015) 29–36
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Fig. 3 – The effect of CoCl2 on stem-cell marker expression. HPDL cells were left untreated or treated with CoCl2 (50 and 100 mM) for 7 days. (A and B) The mRNA expression of VEGF, REX1, and OCT4 was examined by RT-PCR (A) and Real-time PCR (B). Graphs demonstrated the relative mRNA expression compared to the control. (C) Representative pictures illustrated the HIF-1a and HIF-2a protein expression as determined by western blotting. (*p < 0.05 vs. control).
3.4.
CoCl2 inhibited osteogenic differentiation
To study the effect of CoCl2 on osteogenic differentiation, HPDL cells were cultured in osteogenic differentiation medium supplemented with either 50 or 100 mM CoCl2. After 7 days of osteogenic induction, 100 mM CoCl2 could significantly inhibit alkaline phosphatase (ALP) activity, which is an early
marker of osteogenic differentiation (Fig. 4A).24 The effect of CoCl2 on the expression of osteogenic associated genes was also determined by RT-PCR and Real-time PCR. Corresponding to the ALP activity assay, the mRNA expression of ALP was markedly decreased by CoCl2 treatment (Fig. 4B and C). The mRNA expression of osteocalcin (OCN), a non-collagenous matrix protein secreted during the late stage of osteoblast
Fig. 4 – CoCl2 decreased ALP activity and osteogenic associated gene expression. HPDL cells were cultured in osteogenic medium with or without CoCl2 (50 and 100 mM) for 7 days. (A) ALP activity assay was examined. Data were expressed as fold change compared to the control. (B and C) The mRNA expression of ALP, OCN, and RUNX2 was examined by RT-PCR (B) and Real-time PCR (C). Graphs demonstrated the relative expression compared to the control. (*p < 0.05 vs. control).
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Fig. 5 – Apigenin abolished CoCl2-suppressed osteogenic differentiation. HPDL cells were cultured in osteogenic medium. In some conditions, CoCl2 (100 mM) and/or Apigenin (40 mM) were added in the culture condition. (A) Graph demonstrated the ALP activity at day 7. (B) Representative pictures illustrated the mineral deposition determined by Alizarin red staining at day 14. (C) Graph showed the quantitative analysis of Alizarin red staining. The results shown in ‘‘A’’ and ‘‘C’’ were expressed as fold change compared to the control. (*p < 0.05 vs. control).
differentiation,25 was significantly decreased when cells were treated with 100 mM CoCl2 (Fig. 4B and C). Notably, the mRNA expression of RUNX2, an essential transcription factor for osteoblast differentiation,26 was also suppressed by CoCl2 treatment (Fig. 4B and C). Taken together, these findings suggest that CoCl2 might maintain the stemness of HPDL cells by the suppression of osteogenic differentiation.
3.5. Apigenin reversed the effect of CoCl2 on osteogenic differentiation Because CoCl2 is known to mimic hypoxia through the stabilization of HIF-1a, which is a key transcription factor responsible for the hypoxic response, we hypothesized that the inhibitory effect of CoCl2 was mediated by HIF-1a. Thus, we used Apigenin, which inhibits HIF-1a expression, to test this hypothesis.27,28 ALP activity was assayed after the cells were cultured in osteogenic medium for 7 days. As shown in Fig. 5A, treatment with Apigenin alone had no effect on ALP activity. However, the decrease in ALP activity induced by CoCl2 was abolished when the cells were co-treated with Apigenin. To study the calcification capability of HPDL cells, Alizarin red staining was performed after 14 days of osteogenic medium induction. Although, the statistical significance was not observed, CoCl2 treatment resulted in less mineral deposits than the control group (Fig. 5B and C). When co-cultured with Apigenin, CoCl2 was unable to inhibit mineral deposition (Fig. 5B and C). Therefore, these findings suggest that CoCl2 may inhibit osteogenic differentiation through a HIF-1a dependent mechanism.
4.
Discussion
One of the challenges of adult stem-cell investigation is to determine the optimal conditions for expanding cell number while maintaining the stem-cell state. Hence, the effects of growth factors and culture conditions on stem-cell behavior are now the focus of many researchers. It is now known that hypoxia
is common in the various stem-cell niches. Thus, the addition of a hypoxia-mimicking agent to culture media might be a method to maintain the stem-cell state in the laboratory setting. In the present study, we found that CoCl2, a demonstrated hypoxiamimicking reagent, could induce the expression of stem-cell markers and inhibit the osteoblastic differentiation of HPDL cells. Therefore, our study demonstrates that media supplementation with a hypoxia-mimicking agent such as CoCl2 might be alternative approach for stem-cell culture. In the present study, the effect of CoCl2 on HPDL cell proliferation was evaluated. While hypoxia has been shown to promote the proliferation of many types of stem cells,17,18,29 the effect of CoCl2 on cell proliferation varies depending on the cell type. For example, CoCl2 treatment at the concentration of 10–100 mM had no effect on the proliferation of human neuroprogenitor cells.30 CoCl2 treatment, at the range of 50– 200 mM, was shown to induce the proliferation of umbilical cord blood-derived 133+ cells.23,30 In our study, we found that the number of HPDL cells was not changed upon 7 days of treatment with 50–100 mM CoCl2. However, from this data, we cannot conclude that CoCl2 treatment had no effect on the proliferation of PDLSCs because the HPDL cells used in this study are a pool of heterogeneous cells. Other direct methods to quantify the number of stem cells using specific markers such as STRO-1 are further required. Also, it is interesting to study whether the longer period of CoCl2 supplementation could affect the population proportion of stem cells. Currently, a specific PDLSC stem-cell marker has not been identified. We investigated the expression of REX1 and OCT4, which are recognized stem-cell markers.31 REX1, also known as zinc-finger protein-42 (zfp42), and OCT4, a member of POU domain transcription factor family, are expressed in embryonic and adult stem cells.32,33 The levels of REX1 and OCT4 expression decreases during cell differentiation,34,35 indicating their essential role in maintaining pluripotency. It has been shown that specific stimuli such as basic fibroblast growth factor or hypoxia could up-regulate the expression of REX1 and OCT4.17,36 Our results indicate that CoCl2 treatment, which mimics hypoxia, could enhance the expression of both
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REX1 and OCT4 in human periodontal ligament cells. These results imply that CoCl2-supplemented media may increase the stem-cell population. The ability of CoCl2 to maintain HPDL cell stemness was studied by investigating their differentiation potential. It has been shown that hypoxia inhibited the osteogenic differentiation of adult stem cells isolated from marrow and human dental pulp cells.19,37 Correspondingly, we found that CoCl2 could inhibit alkaline phosphatase activity and the expression of osteogenic-associated genes in HPDL cells. The inhibitory effect of CoCl2 on osteoblastic differentiation was confirmed by Alizarin red staining that showed CoCl2 treatment attenuated the mineral deposition. These findings suggest that CoCl2 may maintain stemness by inhibiting osteoblastic differentiation. The ability of CoCl2 to influence the differentiation of PDLSCs to other cell fates such as chondrocytes or neurons is an interesting topic for future study. The involvement of HIF-1a in the CoCl2 mediated inhibition of osteogenic differentiation was elucidated using Apigenin, a flavonoid shown to inhibit HIF-1a expression. We found that Apigenin could reverse the effect of CoCl2 treatment on alkaline phosphatase activity and mineral deposition. These data suggest that HIF-1a might be involved in the inhibitory effect of CoCl2. However, Apigenin is not a specific inhibitor of HIF-1a. It can also inhibit a number of hypoxia responsive genes such as VEGF and glucose transporter 1 (GLUT-1),27 as well as non-hypoxia target genes such as cyclin A and cyclin B.38 To ensure that HIF-1a is involved in this mechanism, future study using an siRNA approach is necessary. In conclusion, this study shows that CoCl2 treatment can induce stem-cell marker expression and maintain the stemness of HPDL cells. These findings imply that the usage of hypoxiamimicking agents such as CoCl2 may be an alternative approach for maintaining the stem-cell state in the laboratory. Comparing to physical hypoxia, CoCl2 supplementation is much more convenient to mimic a hypoxic condition. However, the response of cell to CoCl2 may not absolutely identical to the real hypoxia. As CoCl2 mimics hypoxia by stabilizing HIF-1a, the hypoxic signal through HIF-1a-independent pathway will not occur when using CoCl2. Future study comparing the effect of CoCl2 and hypoxia on stem-cell behavior is needed to confirm the possible use of CoCl2 in maintaining stem cell.
Acknowledgements This study was supported by Young Investigator Research Funding, Faculty of Dentistry, Chulalongkorn University and in part by ‘‘Integrated Innovation Academic Center: IIAC’’ Chulalongkorn University Centenary Academic Development Project. PP was supported by the Research Chair Grant 2012, the National Science and Technology Development Agency (NSTDA), Thailand.
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