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ISSN : 1226-7155(Print)
ISSN : 2287-6618(Online)
International Journal of Oral Biology Vol.38 No.3 pp.111-119
DOI : https://doi.org/10.11620/IJOB.2013.38.3.111

Effects of CoCl₂ on Osteogenic Differentiation of Human Mesenchymal Stem Cells

Min-Seok Kim*, Yeon-Hee Moon, Jung-Wan Son, Jung-Sun Moon, Jee-Hae Kang, Sun-Hun Kim
Dental Science Research Institute, School of Dentistry, Chonnam National University
(received July 15, 2013 ; revised August 7, 2013 ; accepted August 10, 2013)

Abstract

Objective. To investigate the effects of the hypoxia induciblefactor-1 (HIF-1) activation–mimicking agent cobalt chloride(CoCl2) on the osteogenic differentiation of human mesenchymalstem cells (hMSCs) and elucidate the underlying molecularmechanisms. Study design. The dose and exposureperiods for CoCl2 in hMSCs were optimized by cell viabilityassays. After confirmation of CoCl2-induced HIF-1α and vascularendothelial growth factor expression in these cells byRT-PCR, the effects of temporary preconditioning with CoCl2on hMSC osteogenic differentiation were evaluated by RTPCRanalysis of osteogenic gene expression, an alkaline phosphatase(ALP) activity assay and by alizarin red S staining.Results. Variable CoCl2 dosages (up to 500 μM) and exposure times(up to 7 days) on hMSC had little effect on hMSC survival.After CoCl2 treatment of hMSCs at 100 μM for 24 or 48hours, followed by culture in osteogenic differentiatingmedia, several osteogenic markers such as Runx-2, osteocalcinand osteopontin, bone sialoprotein mRNA expressionlevel were found to be up-regulated. Moreover, ALP activitywas increased in these treated cells in which an acceleratedosteogenic capacity was also verified by alizarin red Sstaining. Conclusions. The osteogenic differentiation potentialof hMSCs could be preserved and even enhanced byCoCl2 treatment.

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Introduction

 Stem cells are generally defined as cells that have the capacity to self-renew as well as to give rise to differentiated progeny [1]. Stem cells are present in small numbers in many vertebrate adult and fetal tissues, including the hematopoietic system, nervous system, gut, skin and tooth. They are responsible for normal tissue renewal and regeneration following damage. It was traditionally thought that only embryonic stem cells are pluripotent, while adult stem cells are restricted in their differentiative and regenerative potential to the tissues in which they reside [2]. However, this view of adult stem cell potential has been challenged by the recent discoveries that mesenchymal stem cells (MSCs), when stimulated by differentiated microenvironment cues, have the capacity to differentiate into a range of cell types of different organs, including chondrocytes, osteoblasts, adipocytes, cardiac and skeletal muscle cells, neurons and astrocytes [3-10]. MSCs, also known as bone marrow stromal stem cells (BMSSCs) or colony-forming units fibroblastic (CFU-F), are a population of non-circulating bone marrow-derived cells with remarkable plasticity. They can be isolated based on their adhesive properties, and are capable of clonal expansion in culture [11,12]. MSCs share characteristics with other multipotent stem cells, and give rise to differentiated progeny, including both mesenchymal and nonmesenchymal lineages. The properties of rapid expansion in vitro and multipotential of differentiation make MSCs one of the most important adult stem cell sources for potential therapeutic use and tissue engineering.

Differences of oxygen levels between standard in vitro cell culture conditions and physiological environments in vivo such as bone marrow cavity [13-17] or insufficient supply of oxygen to cells after MSC transplantation must be assumed [18-20] have led many researches for MSC biology and application on hypoxia. Although several studies for the effects of hypoxia on MSC survival, proliferation and differentiation have performed in order to determine the definite effects on MSC behavior [21-23], huge discrepancies of results have existed depending on sources of used cells such as primary cells, cell lines, donor ages, species, different experimental conditions including different serum concentration, the presence of preconditioning and duration of hypoxia [24-31]. 

 Cobalt chloride (CoCl2) can mimic hypoxic conditions through transcriptional changes of some genes such as hypoxia inducible factor (HIF-1α), and p53, p21 by stimulating reactive oxygen species generation via mitochondriaindependent mechanism [32,33]. Although CoCl2 appeared cytotoxic and especially CoCl2-induced apoptosis may serve as a simple and convenient in vitro model to elucidate molecular mechanism in hypoxia-linked cell death [34], it has rarely been used in experiments to reveal the beneficial effects such as enhancement of osteogenic potential of MSCs. Based on these results, it is hypothesized that differentiation of MSCs into osteoblast, one of the most essential steps in bone regeneration strategies, can be affected by temporary CoCl2 pre-treatment and possibly acquire improved osteogenic potential of MSCs when proper culture conditions are provided.

 The aims of this study was to elucidate potential role of temporary CoCl2 pretreatment in bone regeneration by examining the effects of CoCl2 on osteogenic differentiation of hMSC and understanding its possible molecular mechanisms in vitro culture conditions.

Materials and Methods

Cell culture

 Human fetal mesenchymal stem cell line (hTERT-hfMSC), kindly gift from Dr. Glackin (City of Hope and Beckman Research Institute, Duarte, CA, USA), which was derived from primary human fetal bone marrow stromal cells and immortalized by manipulating human telomerase reverse transcriptase (hTERT) was used in this study [35-37]. hTERThfMSCs were maintained in growth medium containing alpha Minimum Essential Medium (αMEM, Gibco BRL, MD, USA) with 15% fetal bovine serum (FBS, Gibco BRL, MD, USA), 2 mM L-glutamine (Gibco BRL, MD, USA), 100 mM L-ascorbic acid 2-phosphate (Sigma Aldrich, St Luis, MO, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco BRL, MD, USA). When hfMSCs reached 60-70% confluence, they were used for subsequent experiments.

Multipotency of hTERT-hfMSCs

 hTERT-hfMSCs were manipulated by varying culture conditions to commit to mesenchymal lineages using standard protocols.

1. Induction of osteogenic differentiation

 hTERT-hfMSCs were cultured to approximately 90% confluence in the growth medium and then replaced with osteogenic medium consisting of αMEM with 15% FBS, 2 mM Lglutamine, 100 mM L-ascorbic acid 2-phosphate, 1.8 mM KH2PO4 (Gibco BRL, MD, USA), 10 nM dexamethasone (Gibco BRL, MD, USA), 100 U/mL penicillin and 100 mg/mL streptomycin. After 3 weeks of culture, the cells were fixed in PBS containing 1% paraformaldehyde (PFA, (Merck, Darmstadt, Germany) and stained with 1% Alizarin red S solution (Sigma Aldrich, St Luis, MO, USA) to evaluate mineralization.

2. Induction of adipogenic differentiation

 hTERT-hfMSCs with 90% confluence were exposed to the adipogenic medium containing αMEM with 10% FBS, 5 μg/mL insulin, 10-7 M dexamethasone, 0.5 mM isobutylmethylxanthine (Sigma Aldrich, St Luis, MO, USA), and 60 μM indomethacin (Sigma Aldrich, St Luis, MO, USA). After 21 days of culture, the cells were fixed in PBS containing 1% PFA mand stained with Oil Red-O (Sigma Aldrich, St Luis, MO, USA).

3. Induction of chondrogenic differentiation

 hTERT-hfMSCs were cultured to 90% confluence in the growth medium and then were replaced with chondrogenic medium consisting of the α-MEM supplemented with transforming growth factor 1 (TGF-1; 10 μg/mL, Invitrogen, Carlsbad, CA, USA), IGF-I (10 μg/mL, Invitrogen, Carlsbad, CA, USA), Vitamin C (50 μg/mL; Invitrogen, Carlsbad, CA, USA). Pellet culture was performed to evaluate the chondrogenic differentiation. 2×105 cells were centrifuged in a 15 mL polypropylene tube, and the pellets were cultured in chondrogenic medium for 3 weeks. Then, the pellets were embedded in paraffin, cut into 5 μm-thick-sections and stained with alcian blue.

Cell viability assay

 The MTT assay was used to provide an indirect measurement of the cell viability. The hTERT-hfMSCs were plated onto 96 well plates and exposed to various durations (100 μM or 250 μM CoCl2 for 1, 2, 3 and 7 days) and different concentration of CoCl2 (100, 500, 1000 and 5000 μM CoCl2 for 48 h). After the treatments, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Invitrogen, Carlsbad, CA, USA) was added to the culture medium at a final concentration of 0.1 mg/ml for respective times and incubated at 37℃ for 4 h. The reaction product of MTT was extracted in dimethylsulfoxide (DMSO, Invitrogen, Carlsbad, CA, USA) and optical density (OD) was spectrophotometrically measured at 570 nm using an ELISA reader (BIO Tek Instruments, Winooski, USA) with DMSO as the blank.

Detection of HIF-1α and VEGF after exposure of CoCl₂ to hTERT-hfMSCs

 hTERT-hMSCs were plated at 5000 cells/cm2 and allowed to adhere overnight, followed by the exposure of hTERThfMSCs to 100 and 200 μM CoCl2 in growth medium for 24 and 48 hours respectively. Total mRNA was extracted and then RT-PCR was performed to assess the transcription levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial cell growth factor (VEGF).

hTERT-hfMSCs osteogenic differentiation after exposure of CoCl₂

 hTERT-hfMSCs were plated at 5000 cells/cm2 and allowed to adhere overnight. After exposure of hTERT-hfMSCs to 100 μM CoCl2 in growth medium for 24 and 48 h respectively, medium was replaced by osteogenic medium and the cells were cultured in control conditions for 0, 7, 14, 21 days. At each time point, total mRNA was extracted and then RTPCR were performed to assess the transcription levels of osteogenic markers; osteocalcin (OC), ALP, type I collagen (collagen I), osteopontin (OP), bone sialoprotein (BSP) and Runx-2.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

 The total mRNA extraction was performed using a Trizol® Reagent (Gibco BRL, MD, USA). Before PCR was done, contaminated DNA, if any, was removed by treating extracted DNA with DNase I (Gibco BRL, MD, USA). RNA samples were quantitated using UV spectrophotometer and qualified by obtaining OD 260/280 ratios > 1.8. AccPower® RT PreMix (Bioneer, Daejeon, Korea) was used for reverse transcription. Briefly describing the PCR, mixtures of total RNA and Oligo dT18 were added to AccPower RT PreMix tube (Bioneer, Daejeon, Korea). cDNA synthesis was performed by incubating these mixtures at 42℃, 60 min. For PCR reaction, AccPower® PCR PreMix (Bioneer, Daejeon, Korea) was used. Briefly describing, after the cDNA and primers were added to AccPower® PCR PreMix, PCR was performed in a Perkin-Elmer GeneAmp PCR system 2400 (Applied Biosystems/Perkin Elmer, Foster City, CA, USA) with the following profile: denaturation for 30 sec at 95℃, annealing for 30 sec at 57℃ and 30 sec extension step at 72℃. Preliminary experiments were performed to determine the optimum number of PCR cycles. Products were resolved on a 1% agarose gel and visualized using ethidium bromide. The product size was confirmed using 100 bp (Takara, Otsu, Shiga, Japan). DNA template was omitted for the negative control in PCR. Primer sequences used are shown Table 1.

Table 1. Primer sequences for PCR

Alkaline phosphatase activity assay

 To assay ALP activity, cells were cultured in osteogenic media for 1, 3, 5, 7, 14, 21 and 28 days at an initial seeding density of 5 × 104 cells/well. The total cellular ALP activity in cell lysates was measured in 0.1M diethanolamine (pH 8.3) with p-nitrophenyl phosphate (Sigma Aldrich, St Luis, MO, USA) as the substrate. The absorbance change at 405 nm was measured using an ELISA reader (BIO Tek Instruments, Winooski, USA).

Alizarin red S staining

 After 4 weeks of osteogenic differentiation under CoCl2 treatment, the cells were assessed for calcium depositions using Alizarin red S staining. The cells were washed with PBS and then fixed with 60% isopropanol (Sigma Aldrich, St Luis, MO, USA) for 1 min. After fixation, the cells were rehydrated with excess distilled water for 3 min. and stained with 1% Alizarin red S solution (Sigma Aldrich, St Luis, MO, USA) for 3 min. Then, they were washed with distilled water and air-dried for phototaking under a microscope. To quantify Alizarin red S staining results, after the incubation with 10% cetylpyridinium chloride (Sigma Aldrich, St Luis, MO, USA) for 20 min, optical density (OD) was measured spectrophotometrically at 570 nm using an ELISA reader (BIO Tek Instruments, Winooski, USA).

Statistical analysis

 Data are expressed as mean standard deviation. Statistic analysis was performed using an ANOVA test. The results were taken to be significant at a probability level of p<0.05. For all experiments, three independent experiments were performed.

Results

Verification of multipotency of hTERT-hfMSCs

 In order to verify the multipotency of hTERT-hfMSCs used in this study, cells were cultured in either osteogenic, adipogenic or chondrogenic differentiation medium and characterized for biomarker expression profiles. First, alizarin red S staining was observed after incubation of hTERThfMSCs with osteogenic medium for 3 weeks (Fig. 1B). Next, after culture of hTERT-hfMSCs in adipogenic or chondro genic medium for 3 weeks, the adipogenic Oil Red Opositive (Fig. 1C), or chondrogenic alcian blue-positve reactivities were also appeared (Fig.1D), respectively.

Fig. 1. Multipotency of hTERT-hfMSCs. hTERT-hfMSC cell line was subjected to growth(A) and differentiating media conditions and stained for osteogenic alizarin red S (B), adipogenic Oil Red O (C), or chondrogenic alcian blue staining (D).

Variable dosages and time of CoCl₂ exposure on hTERThfMSCs

 To assess the effects of CoCl2 on cell viability of hTERThfMSCs, cells were exposed to1, 2, 3, and 7 days at 100 or 250 μM concentrations. The MTT results showed that CoCl2 did not cause any significant loss of cell viability at all conditions (Fig. 2A). The exposure of CoCl2 at different concentrations from 100 to 5000 μM for 48 h caused a loss of cell viability in a dose-dependent manner (Fig. 2B). The results of the MTT assay showed that CoCl2 exposure of hTERThfMSCs( at the considerable dosages; up to 500 μM and time; 7 days) had little effects on hTERT-hfMSCs survival. The valuable dosages and time of CoCl2 exposure on hTERThfMSCs were determined as a 100 μM for 24 or 48 h and for the next study.

Fig. 2. Effects of CoCl2 exposure on viability of hfMSCs. (A) After cells were exposed to 100 or 250 μM CoCl2 (for 0, 1, 2, 3, 7 days), the viability was estimated by the MTT method. (B) After the hTERT-hfMSCs were incubated in the media containing different CoCl2 concenturations (0, 0.1, 0.5, 1, 5 mM) for 48 h, MTT assay was also carried out. The data are represented as a mean±S.D. from triplicate independent experiments.

HIF-1α and VEGF expression after exposure of CoCl₂ to hTERT-hfMSCs

 To verify that exposure of hTERT-hfMSCs to CoCl2 induces a hypoxia-mimicking response, the expression of HIF-1α mRNA, a marker of hypoxia, was examined and elevated in CoCl2-treated cells (Fig. 3A). In addition, changes of VEGF mRNA expression were observed in CoCl2-treated groups and the increased expression compared to the control continued even after osteogenic differentiation (Fig. 3B).

Fig. 3. CoCl2 increased expression of HIF-1α and VEGF mRNA in hTERT-hfMSCs. (A-1) Cells were incubated in the growth media containing the 0, 100 or 250 μM CoCl2 for 6, 24, or 48 h, respectively. RT-PCR analysis for HIF-1α was performed. (A-2) Obtained results from (A-1) were scaled by densitometry (B) Cells were incubated in the absence or the presence of 100 μM CoCl2 for 24 or 48 h. Cells were subjected to the RT-PCR analysis for VEGF.

Effects of exposure of CoCl₂ on osteogenic differentiation of hTERT-hfMSCs

 To investigate the effects of temporary exposure of CoCl2 on hTERT-hfMSCs osteogenic potential, after 24 or 48 h exposure to CoCl2 or control conditions, cells were transferred to osteogenic medium and osteogenic differentiation was estimated by RT-PCR assays to detect the expression of several osteogenic markers. The levels of osteogenic gene expression were determined by performing semi-quantitative RTPCR assays. The levels of expression of OC, OP, ALP, Runx2 and BSP were up-regulated by temporary CoCl2 exposure. However, the levels of type I collagen expressions were similar to those exposed to the control conditions (Fig. 4). To verify the osteogenic gene expression results by RTPCR after temporary CoCl2 exposure, ALP activity assay was carried out at each time point during osteogenic differentiation of hTERT-hfMSCs. ALP activities increased in CoCl2-treated groups compared to control. ALP activities were at peaks after 2 weeks osteogenic differentiation and later those were reduced in both CoCl2-treated and control cells (Fig. 5). Finally, to confirm the positive effects of temporary preconditioning using CoCl2 on osteogenic differentiation of hfMSCs, alizarin red S was stained after either CoCl2-treated prior to osteogeic differentiation. As expected, CoCl2-treated groups displayed stronger alizarin red S staining than the control (Fig. 6 A, B).

Fig. 4. Effects of temporary exposure of CoCl2 on the OC, OP, ALP, Runx2, Type I collagen and BSP expression by hTERThfMSCs. hTERT-hfMSCs were exposed to 100 μM CoCl2 for 24 or 48 h. After exposure, the media were replaced with osteogenic medium and hTERT-hfMSCs were cultured for 0, 7, 14, 21 days. At the end of each time period, mRNA expression levels of osteogenic markers were determined by semiquantitive RTPCR. β-actin was used as the endogenous reference gene.

Fig. 5. Effects of temporary exposure of CoCl2 on alkaline phosphatase activiy of hTERT-hfMSCs. hTERT-hfMSCs were exposed to 100 μM CoCl2 for 0, 24 or 48 hours. After exposure, the media were replaced with osteogenic medium and hTERThfMSCs were cultured for 0, 1, 3, 5, 7, 14 and 28 days. At the end of each time periods, ALP activity assay was performed. The data are represented as a mean±S.D. from triplicate independent experiments.

Fig. 6. Alizarin red S staining results after temporary exposure of CoCl2 on hTERT-hfMSCs. (A) hTERT-hfMSCs were exposed to 100 μM CoCl2 for 0, 24 or 48hours. After exposure, the media were replaced with osteogenic medium and hTERT-hfMSCs were cultured in control conditions for 28 days. At the end of time periods, calcified nodule was stained with Alizarin red S. (B) Results from (A) were quantified by spectrophotometer.

Discussion

Significant advances have been made in the use of human primary mesenchymal stem cells (hMSCs), which can be easily isolated from bone marrow aspirates and rapidly expanded in vitro, to replace damaged human bone [5,7, 10,38-40]. However, hMSCs isolated from bone marrow have limitations in ex vivo culture and expansion. Properties of hMSCs with short life spans led to develop immortalized cell lines using hTERT which play a key role in sustaining cell division [31,41]. This study was conducted with hTERThfMSCs which were derived from primary human fetal bone marrow and immortalized using hTERT. With the exception of the fact that hTERT-hfMSCs proliferate faster than primary hMSCs, it also shares three common characteristics with primary hMSC; among them are that their morphological phenotype is analogous to that of primary cells, both have not developed chromosomal abnormalities through karyotyping, and that they are able to differentiate 3 mesenchymal lineages [35-37].

 HIF-1α protein expression is regulated by an oxygensensitive mechanism. Although HIF-1α is rapidly degraded under normoxic condition, it becomes stabilized and its activity progressively increased under hypoxic condition. Subsequently, it stimulates the transcription of several genes that are associated with low oxygen tension [42,43] In this study. only transcriptional changes of HIF-1α were laid out after CoCl2 exposure by RT-PCR. Presentation of changes at protein levels were accepted as distinguishable method between normoxia and hypoxia because HiF-1α is detectable on the mRNA level under both normoxic and hypoxic condition [31,44], Detection of HIF-1α protein was seemed to be closely related to experimental conditions such as exposure time to CoCl2 and oxygen concentration used [34]. Instead of HIF-1α protein induction, VEGF expression, which was increased by hypoxia and oxygen deprivation [30,45,46] was observed after CoCl2 exposure. This establishes the fact that CoCl2 treatment during hMSC differentiation can mimic the hypoxic condition.

As mentioned earlier, reactivities of MSC to hypoxic condition considerably varied due to several differences of experimental procedures. Overall, reduced oxygen concentration within the physiological range increased MSC number obtained after culture [15,47-49] in spite of some negative results [50]. Meanwhile, although it is difficult to clearly state the effects of hypoxia on osteogenic differentiation, several reports documented reduced osteogenic capacity of hMSCs cultured under hypoxia or following hypoxia [51-53]. On the contrary to this views, recent study revealed that hypoxic preconditioning prior to osteogenic differentiation can improve cellular differentiation of hMSCs under low oxygen atmosphere [31]. In addition, it was proposed that activation of HIF-1α pathway accelerates bone regeneration in vivo [54]. These considerations are in line with the results in this study; preservation and enhancement of osteogenic potential of hMSC by temporary CoCl2 exposure to hMSC are feasible prior to osteogenic differentiation. 

 In the present study, transcriptional changes of osteogenic differentiation related genes were investigated to assess the effects of temporary CoCl2 exposure to hMSC osteogenic capacity. Runx-2 transcription factor which plays an essential role in controlling osteoblasts and bone mineralizationrelated genes such as ALP, OC, OP and BSP expression were generally up-regulated during osteoblast differentiation. However, the expression of type I collagen which is the main component of bone matrix was not affected. These results may suggest that temporary exposure to CoCl2 may accelerate osteoblastic differentiation, especially mineral depositions of hMSCs. Meanwhile, ALP activities were at their peaks after 2 weeks of osteogenic differentiation and afterwards those activities were reduced irrespective of CoCl2 exposure. This result is consistent with the reports denoting that ALP is an early differentiation marker for hard tissueforming cells [55]. The changes of gene expression were different from some data that were obtained from after hypoxic condition was adopted in the hypoxic chamber incubation [30,31,56]. The reason behind these differences is assumed to be the disparity in the activated signal pathways. Although both hypoxia and CoCl2 exposure could stabilize HIF-1α, they activated following some other signal pathways such as ERK1/2 and AMP-activated protein kinase. In addition, the levels of HIF-1α were not the same under low oxygen tension and CoCl2 exposure [56].

 Taken together, the present study might address that osteogenic differentiation potential of hMSC could be accelerated after temporary exposure of CoCl2. Therefore, when hMSCs will be implanted to the site with low-oxygen tension, temporary pre-treatment with CoCl2 to them might raise the probability of clinical success. In addition, like CoCl2-induced apoptosis, temporary CoCl2 exposure to hMSC can serve as a simple and convenient in vitro model to estimate hypoxic reactivities of hMSC. However, the proposition that CoCl2 can reproduce real hypoxic condition is still in debate. Therefore, further research would be needed to investigate a precise molecular mechanism of hypoxia on hMSC in vitro and revolutionary experimental condition to recreate in vivo hMSC niche area.

Acknowledgements

 This study was financially supported by National Research Foundation of Korea Grant funded by the Korean Government (KRF-2010-002598) and Chonnam National University, 2009.

Reference

1.Van der Kooy D, Weiss S. Why stem cells? Science. 2000, 287(5457):1439-41.
2.Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: entity or function? Cell. 2001, 105(7):829-41.
3.Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci. 1999, 96(19):10711-6.
4.Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284(5411):143-7.
5.Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Moseley AM, Deans R, Marshak DR, Flake AW. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med. 2000, 6(11):1282-6.
6.Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W, Patel N, Cooper DR, Sanberg PR. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000, 164(2):247-56.
7.Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. 2001, 105(3):369-77.
8.Oh SY, Park HW, Cho JS, Jung HK, Lee SP, Park KS, Chang MS. Induction of a Neuronal Phenotype from Human Bone Marrow-Derived Mesenchymal Stem Cells. Int J Oral Biol. 2009, 34(4):177-83.
9.Park EG, Cho TJ, Oh KH, Kwon SK, Lee DS, Park SB, Cho JJ. Establishment of High Throughput Screening System Using Human Umbilical Cord-derived Mesenchymal Stem Cells. Int J Oral Biol. 2012, 37(2):43-50.
10.Cakouros, D., Isenmann, S., Cooper, L., Zannettino, A., Anderson, P., Glackin, C., and Gronthos, S. Twist-1 induces Ezh2 recruitment regulating histone methylation along the Ink4A/Arf locus in mesenchymal stem cells. Mol Cell Biol . 2012, 32:1433-41.
11.Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997, 276(5309):71-4.
12.Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells. 2001, 19(3):219-25.
13.Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood. 1993, 82(7):2031-7.
14.Lennon DP, Edmison JM, Caplan AI. Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J Cell Physiol. 2001, 187(3):345-55.
15.Csete M. Oxygen in the cultivation of stem cells. Ann N Y Acad Sci. 2005, 1049:1-8.
16.Ma T, Grayson WL, Fröhlich M, Vunjak-Novakovic G. Hypoxia and stem cell-based engineering of mesenchymal tissues. Biotechnol Prog. 2009, 25(1):32-42.
17.Calvi, L. M., Bromberg, O., Rhee, Y., Weber, J. M., Smith, J. N., Basil, M. J., Frisch, B. J., and Bellido, T. Osteoblastic expansion induced by parathyroid hormone receptor signaling in murine osteocytes is not sufficient to increase hematopoietic stem cells. Blood. 2012, 119:2489-99.
18.Polykandriotis E, Arkudas A, Euler S, Beier JP, Horch RE, Kneser U. Prevascularisation strategies in tissue engineering. Handchir Mikrochir Plast Chir. 2006, 38(4):217-23.
19.Malda J, Klein TJ, Upton Z. The roles of hypoxia in the in vitro engineering of tissues. Tissue Eng. 2007, 13(9):2153-62.
20.Volkmer E, Drosse I, Otto S, Stangelmayer A, Stengele M, Kallukalam BC, Mutschler W, Schieker M. Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone. Tissue Eng Part A. 2008, 14(8):1331-40.
21.Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol. 2006, 207(2):331-9.
22.Potier E, Ferreira E, Meunier A, Sedel L, Logeart-Avramoglou D, Petite H. Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death. Tissue Eng. 2007, 13(6):1325-31.
23.Mylotte LA, Duffy AM, Murphy M, O'Brien T, Samali A, Barry F, Szegezdi E. Metabolic flexibility permits mesenchymal stem cell survival in an ischemic environment. Stem Cells. 2008, 26(5):1325-36.
24.Tuncay OC, Ho D, Barker MK. Oxygen tension regulates osteoblast function. Am J Orthod Dentofacial Orthop. 1994, 105(5):457-63.
25.Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, Gittes GK, Longaker MT. Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro. Plast Reconstr Surg. 1999, 104(3):738-47.
26.Warren SM, Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Bouletreau PJ, Longaker MT. Hypoxia regulates osteoblast gene expression. J Surg Res. 2001, 99(1):147-55.
27.Park JH, Park BH, Kim HK, Park TS, Baek HS. Hypoxia decreases Runx2/Cbfa1 expression in human osteoblastlike cells. Mol Cell Endocrinol. 2002, 192(1-2):197-203.
28.Salim A, Nacamuli RP, Morgan EF, Giaccia AJ, Longaker MT. Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts. J Biol Chem. 2004, 279(38):40007-16.
29.Pacary E, Legros H, Valable S, Duchatelle P, Lecocq M, Petit E, Nicole O, Bernaudin M. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells. J Cell Sci. 2006, 119(Pt 13):2667-78.
30.Potier E, Ferreira E, Andriamanalijaona R, Pujol JP, Oudina K, Logeart-Avramoglou D, Petite H. Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression. Bone. 2007, 40(4):1078-87.
31.Volkmer E, Kallukalam BC, Maertz J, Otto S, Drosse I, Polzer H, Bocker W, Stengele M, Docheva D, Mutschler W, Schieker M. Hypoxic preconditioning of human mesenchymal stem cells overcomes hypoxia-induced inhibition of osteogenic differentiation. Tissue Eng Part A. 2010, 16(1):153-64.
32.Jiang BH, Zheng JZ, Leung SW, Roe R, Semenza GL. Transactivation and inhibitory domains of hypoxia-inducible factor 1 alpha. Modulation of transcriptional activity by oxygen tension. J Biol Chem. 1997, 272(31):19253-60.
33.Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci. 1998, 95(20):11715-20.
34.Jung JY, Mo HC, Yang KH, Jeong YJ, Yoo HG, Choi NK, Oh WM, Oh HK, Kim SH, Lee JH, Kim HJ, Kim WJ. Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells. Life Sci. 2007, 80(15):1355-63.
35.Isenmann, S., Arthur, A., Zannettino, A. C., Turner, J. L., Shi, S., Glackin, C. A., and Gronthos, S. TWIST family of basic Helix-Loop-Helix Transcription Factors Mediate Human Mesenchymal Stromal/Stem Cell Growth and Commitment. Stem Cells. 2009, 27(10):2457-68
36.Gunn, E.J., Williams, J.T., Huynh, D.T., Iannotti, M.J., Han, C., Barrios, F.J., Kendall, S., Glackin, C.A., Colby, D.A., and Kirshner, J. The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma, Leukemia & lymphoma. 2011, 52:1085-97.
37.Samineni, S., Glackin, C., and Shively, J.E. Role of CEACAM1, ECM, and mesenchymal stem cells in an orthotopic model of human breast cancer, International journal of breast cancer. 2011, 381080.
38.Mackenzie TC, Flake AW. Human mesenchymal stem cells persist, demonstrate site-specific multipotential differentiation, and are present in sites of wound healing and tissue regeneration after transplantation into fetal sheep. Blood Cells Mol Dis. 2001, 27(3):601-4.
39.Reyes GD, Esterling LE, Corona W, Ferraren D, Rollins DY, Padigaru M, Yoshikawa T, Monje VD, Detera-Wadleigh SD. Map of candidate genes and STSs on 18p11.2, a bipolar disorder and schizophrenia susceptibility region. Mol Psychiatry. 2002, 7(4):337-9.
40.Wiedswang G, Borgen E, Karesen R, Naume B. Detection of isolated tumor cells in BM from breast-cancer patients:significance of anterior and posterior iliac crest aspirations and the number of mononuclear cells analyzed. Cytotherapy. 2003, 5(1):40-5.
41.Huang G, Zheng Q, Sun J, Guo C, Yang J, Chen R, Xu Y, Wang G, Shen D, Pan Z, Jin J, Wang J. Stabilization of cellular properties and differentiation mutilpotential of human mesenchymal stem cells transduced with hTERT gene in a long-term culture. J Cell Biochem. 2008, 103(4):1256-69.
42.Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006, 441(7092):437-43.
43.Riddle RC, Khatri R, Schipani E, Clemens TL. Role of hypoxia-inducible factor-1alpha in angiogenic-osteogenic coupling. J Mol Med. 2009, 87(6):583-90.
44.Wang Y, Wan C, Deng L, Liu X, Cao X, Gilbert SR, Bouxsein ML, Faugere MC, Guldberg RE, Gerstenfeld LC, Haase VH, Johnson RS, Schipani E, Clemens TL. The hypoxiainducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development. J Clin Invest. 2007, 117(6):1616-26.
45.Maxwell PH, Dachs GU, Gleadle JM, Nicholls LG, Harris AL, Stratford IJ, Hankinson O, Pugh CW, Ratcliffe PJ. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci. 1997, 94(15):8104-9.
46.Dachs GU, Patterson AV, Firth JD, Ratcliffe PJ, Townsend KM, Stratford IJ, Harris AL. Targeting gene expression to hypoxic tumor cells. Nat Med. 1997, 3(5):515-20.
47.Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999, 13(22):2905-27.
48.Rosová I, Dao M, Capoccia B, Link D, Nolta JA. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells. 2008, 26(8):2173-82.
49.Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006, 20(6):661-9.
50.Liu X, Saboo RR, Pizer SM, Mageras GS. Lysophosphatidic acid protects mesenchymal stem cells against ischemia induced apoptosis in vivo. Stem cells Dev. 2009, 18(7):947-54.
51.Zhu W, Chen J, Cong X, Hu S, Chen X. Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells. 2006, 24(2):416-25.
52.Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, Ladilov Y, Furlani D, Piechaczek C, Moebius JM, Lützow K, Lendlein A, Stamm C, Li RK, Steinhoff G. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells. 2007, 25(8):2118-27.
53.Lord-Dufour S, Copland IB, Levros LC Jr, Post M, Das A, Khosla C, Galipeau J, Rassart E, Annabi B. Evidence for transcriptional regulation of the glucose-6-phosphate transporter by HIF-1alpha: Targeting G6PT with mumbaistatin analogs in hypoxic mesenchymal stromal cells. Stem Cells. 2009, 27(3):489-97.
54.Wan C, Gilbert SR, Wang Y, Cao X, Shen X, Ramaswamy G, Jacobsen KA, Alaql ZS, Eberhardt AW, Gerstenfeld LC, Einhorn TA, Deng L, Clemens TL. Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration. Proc Natl Acad Sci. 2008, 105(2):686-91.
55.Yang ZH, Zhang XJ, Dang NN, Ma ZF, Xu L, Wu JJ, Sun YJ, Duan YZ, Lin Z, Jin Y. Apical tooth germ cellconditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues. J Periodontal Res. 2009, 44(2):199-210.
56.Ren H, Cao Y, Zhao Q, Li J, Zhou C, Liao L, Jia M, Zhao Q, Cai H, Han ZC, Yang R, Chen G, Zhao RC. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions. Biochem Biophys Res Commun. 2006, 347(1):12-21.