Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells
© Adesida et al.; licensee BioMed Central Ltd. 2012
Received: 26 September 2011
Accepted: 2 March 2012
Published: 2 March 2012
The capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has drawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow stromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on how BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic development pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.
Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.
After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.
Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.
KeywordsChondrogenesis chondrocytes hypoxia bone marrow stem cells tissue engineering cartilage repair
Articular cartilage covers the end of long bones in articulating joints where it provides near frictionless movement. Unfortunately, articular cartilage has a very limited capacity to repair after injury. If left untreated, cartilage defects progressively lead to more extensive lesions and, ultimately, require joint arthroplasty. Cell-based strategies using culture expanded autologous chondrocytes from non-loading regions of articular cartilage are currently used to treat focal cartilage defects . However, there is some evidence of progressive degenerative changes in the joint using this technique . Furthermore, there is evidence that the matrix-forming capacity of expanded chondrocytes is compromised due to de-differentiation processes [3, 4]. Thus, there is interest in other cell sources for cartilage repair.
Adherent bone marrow stromal cells or bone marrow mesenchymal stromal cells (BMSCs) have received much interest for cartilage repair because of their multipotent capacity to differentiate into different cell types including chondrocytes [5–10]. While there has been much study related to the potential of BMSCs to form cartilaginous tissue, there has been a limited number of reports of the implantation of human BMSCs for cartilage repair . The reason for this is unclear but may be related to hypertrophic differentiation or the lack of a consensus on how human BMSCs are to be cultured for reproducible and optimal chondrogenic differentiation. Human BMSCs have been estimated to account for a mere 0.001% to 0.01% of the total bone marrow mononuclear cells (MNCs) in the stromal compartment of bone marrow [5, 12, 13]. Thus, in vitro culture expansion is a requisite for increasing cell numbers for research and clinical applications. Since the first published report of Friedenstein and co-workers , describing the isolation and expansion of an adherent and spindle-shaped population of cells from whole human bone marrow aspirates, little has changed in the methodology of isolation and expansion of BMSCs. While it is practiced within the art that human BMSCs are isolated after initial cell adherence to tissue culture plastic ware and subsequent cell expansion under normal mammalian conditions of air containing 21% oxygen tension (normoxia), there is increasing evidence that BMSCs are adapted to limiting metabolic conditions . In agreement with this observation, hypoxic (3% O2) conditions have been reported to favor the multi-potentiality of a subpopulation of human bone marrow stromal cells over osteogenic differentiation . Accordingly, human BMSCs showed enhanced proliferative activity under hypoxic (1.5% to 3% O2) conditions relative to normoxia [16–18]. While these studies demonstrated the effect of hypoxia on human BMSC expansion in vitro, the downstream effect of hypoxic conditions during isolation and expansion on human BMSCs' chondrogenic differentiation capacity was unexplored. D'Ippolito et al.  and Grayson et al.  investigated osteogenic differentiation of BMSCs after hypoxia mediated expansion, while the study of Martin-Rendon et al.  investigated chondrogenesis on commercially acquired human BMSCs that lacked initial isolation and cell expansion culture history prior to further cell propagation under normoxia and subsequent chondrogenic differentiation under normoxia and hypoxia for their studies; hypoxia enhanced BMSC chondrogenic differentiation potential.
Ovine BMSCs have been isolated and propagated under hypoxia (5% O2) and shown to increase in proliferation rate relative to cells expanded under normoxia. Moreover, it was demonstrated that ovine BMSCs isolated and expanded under hypoxic conditions and subsequent chondrogenic differentiation under normoxia displayed an enhanced chondrogenic phenotype compared to their counterparts after normoxia mediated isolation and expansion . However, it is not clear whether hypoxia isolated and propagated ovine BMSCs would have similarly displayed improved chondrogenesis had they been differentiated under hypoxic conditions.
While it has been shown that normoxia isolated and expanded non-human and human BMSCs undergo enhanced chondrogenic differentiation under hypoxic conditions [9, 17, 20], it is unknown whether human BMSCs isolated and expanded under hypoxia are predisposed towards improved subsequent chondrogenesis regardless of the oxygen tension. The study of Mueller et al. , showed that hypoxic conditions during expansion culture of human BMSCs resulted in improved chondrogenesis even under normoxic conditions. However, the study used BMSCs that had been initially isolated and expanded under normoxia prior to further culture expansion under hypoxia and subsequent chondrogenic differentiation culture under hypoxia and normoxia. Perhaps the improved chondrogenesis noted by Mueller et al. was not surprising because, during embryonic development, cartilage formation occurs when mesenchymal stem cells condense and differentiate into chondrocytes during an avascular period in a low oxygen tension microenvironment . Thus, oxygen tension is known to play a significant role in the fate of mesenchymal stem cells.
In this study, we investigated the effect of oxygen tension from the onset of human BMSCs isolation and expansion on subsequent in vitro chondrogenesis under normoxic and hypoxic conditions. We hypothesized that persistent hypoxic culture during human BMSC isolation and expansion enhances the chondrogenic differentiation capacity under normoxia and hypoxia by altering the expression of genes that facilitate chondrogenesis.
Typically, in vitro chondrogenesis of BMSCs is performed in 3D cell pellet culture, in order to mimic mesenchymal cell condensation during chondrogenesis in embryonic development, in the presence of serum free chondrogenic factors comprising TGF-β1 or -β3, dexamethasone, ascorbate and ITS+1 under normoxic conditions [6, 9, 10, 19, 22–25]. In this study, we adopted the pellet culture method to investigate in vitro chondrogenesis of both normoxia and hypoxia isolated and expanded human BMSCs. In vitro chondrogenesis was implemented under normoxic conditions only, in order to isolate the downstream effect of cell isolation and expansion under hypoxic conditions on BMSC chondrogenesis; several studies have reported that hypoxic culture conditions enhance chondrogenic differentiation [9, 20, 26–32].
Materials and methods
Collection of bone marrow specimens and culture bone marrow stem cells
Donor information of bone marrow aspirates from the iliac crest
Colony forming unit fibroblastic (cfu-f) assay
The proportion of adherent cell population within each bone marrow aspirate specimen was assessed by Cfu-f assay. Cfu-f was performed by plating 2.5 × 105 nucleated cells in 100 mm sterile petri dishes in triplicate (Becton Dickinson, Mississauga, Ontario, Canada). The cells were cultured as described before using αMEM supplemented with 10% heat inactivated fetal bovine serum, penicillin-streptomycin, HEPES, sodium pyruvate and 5 ng/ml FGF-2 for two weeks under normoxia or hypoxia. After the first week, the non-adherent cell population was removed by aspiration and culture media were replenished every three days until two weeks of culture. During feeds, hypoxia cultivated cells experienced a short period (< 5 minutes) of exposure to normal oxygen tension. After two weeks the developed cell colonies were visualized after fixing with 4% phosphate buffered formalin, washing by PBS and staining using 0.25% crystal violet solution (Sigma-Aldrich). The number of cell colonies developed was recorded as well as their respective diameters. Data were compared in a Student t-test and a significant difference between the two culture conditions was considered when P < 0.05.
Flow cytometry analysis
Antibodies used to characterize normoxia and hypoxia expanded BMSC
Santa Cruz Biotechnology
Santa Cruz Biotechnology
CD44 (Pgp-1, H-CAM, Ly 24)
CD 105 (endoglin)
Santa Cruz Biotechnology
Not specified (Isotype control)
Santa Cruz Biotechnology
Not specified (Isotype control)
Santa Cruz Biotechnology
Not specified (Isotype control)
Santa Cruz Biotechnology
Mouse pooled immunoglobulin (Isotype control)
TNP-KHL (Isotype control)
In vitro chondrogenic differentiation
BMSCs at passage 2 (P2) were re-suspended in chondrogenic culture medium consisting of high glucose (D)MEM containing 4.5 mg/ml D-Glucose, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine (all from Invitrogen) supplemented with 0.1 mM ascorbic acid 2-phosphate, 10-5 M dexamethasone, 1x ITS+1 premix (Sigma-Aldrich), and 10 ng/ml TGFβ1 (Humanzyme-Medicorp Inc.). For chondrogenesis in pellet culture, a total of 2.5 × 105 cells were spun in 1.5 ml sterile conical polypropylene microfuge tubes (Enzymax LLC, Kentucky, USA) at 1500 rpm for three minutes to form spherical cell pellets. The final volume of chondrogenic media was 250 μl per pellet. Medium change was performed three times a week. Hypoxia cultivated pellets experienced a short period (< 5 minutes) of exposure to normal oxygen tension during media changes. The pellets were cultured for three weeks under normal or low oxygen tension to allow appreciable matrix accumulation. Thereafter, the pellets were processed biochemically for glycosaminoglycan (GAG) and DNA contents, histologically, immunohistochemically and gene expression analysis via quantitative reverse transcription polymerase chain reaction (qRT-PCR) for cartilage specific matrix gene and proteins expression.
In vitro cultivated pellets were rinsed in PBS (Invitrogen) and were digested in proteinase K (1 mg/ml in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 mg/ml pepstatin A; all from Sigma-Aldrich) for 16 hours at 56°C. The sulfated GAG content was measured by 1,9-dimethymethylene blue binding (Sigma-Aldrich) using chondroitin sulfate (Sigma-Aldrich) as standard. The DNA content was determined using the CyQuant cell proliferation assay kit (Invitrogen) with supplied bacteriophage λ DNA as standard. Statistical differences between test groups were evaluated by one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-tests. Statistical analyses were performed using SPSS (version 18). A significant difference was considered when P < 0.05.
Histology and immunohistochemistry
Tissues generated from the pellet cultures were fixed in 4% phosphate buffered formalin, processed into paraffin wax, sectioned at 5 μm and stained with 0.1% safranin O and counterstained with 1% fast green to reveal sulfated proteoglycan (GAG) matrix depositions. Other sections were probed with antibodies raised against collagen types I and II. Sections were treated with trypsin and then incubated with antibodies against collagen I (MAB3391, Millipore, Temecula, California, USA) or collagen II (II-II6B3 from Developmental Studies Hybridoma Bank at University of Iowa, USA). Immuno-localized antigens were visualized with goat anti-mouse IgG biotinylated secondary antibody (Dako Canada Inc, Mississauga, Ontario, Canada) and a streptavidin-horseradish peroxidase labeling kit with 3,3'-diaminobenzidine (Dako). Images were captured using an Omano OM159T biological trinocular microscope (Microscope Store, Virginia, USA) fitted with an Optixcam summit series 5MP digital camera and Optixcam software and assembled in Adobe Photoshop (Adobe Systems Inc. San Jose, USA).
Gene expression analysis
Primer sequences used in quantitative real-time PCR (all primers were purchased from Invitrogen, Mississauga, Ontario, Canada)
All studies were implemented with stem cells propagated from bone marrow aspirates taken from surgical discards of patients undergoing routine orthopedic procedures after approval and a waiver of informed consent of the local ethical committee of the University of Alberta (Edmonton, Canada).
Effect of oxygen tension on the colony forming characteristics
Effect of oxygen tension on the expression of cell surface markers
Downstream effect of oxygen tension on extracellular matrix formation
Downstream effect of oxygen tension on chondrogenic gene and protein expression, and TGFβ receptor expression
Immunohistochemical staining (brown coloration) of pellets from the four donors confirmed that the pellets formulated from normoxia- and hypoxia-expanded BMSCs expressed types I and II collagens. Representative photomicrographs of pellets (from donor BM74) labeled with antibodies to collagen I and II are shown in Figure 6a-6h. The pellets derived from hypoxia-expanded BMSCs were strongly and uniformly labelled with anti-collagen type II (Figures 6f (under normoxia) and 6h (under hypoxia)). In contrast, the pellets formulated from normoxia-expanded BMSCs labeled only strongly with anti-collagen type II at the pellet periphery and weakly in the central core (Figures 6e (under normoxia) and 6 g (under hypoxia)). Collagen type I was uniformly distributed in all pellets regardless of the oxygen tension during pellet culture or whether the pellets were formed from normoxia or hypoxia-expanded BMSCs (Figure 6a-6d). Primary antibody and non-specific IgG controls showed no false positive staining (data not included).
Differential expression of TGFβ receptor proteins
TGFβ-RI and TGFβ-RII have been implicated in the reduced chondrogenic potential of adipose derived mesenchymal stem cells under standard chondrogenic culture conditions using TGF-β1 or TGF-β3 . We therefore investigated the expression of these receptor proteins in our pellets. The mRNA expression of TGFβ-RI and TGFβ-RII were statistically similar between the pellets derived from normoxia- and hypoxia-expanded BMSCs under normoxic conditions. However, under hypoxic conditions the expression of TGFβ-RI in pellets derived from hypoxia-expanded BMSCs were significantly up-regulated by 3.5-fold relative to its expression in pellets formed from normoxia-expanded BMSCs. Similarly, the mRNA expression of TGFβ-RII was increased under hypoxic condition in pellets derived from hypoxia-expanded BMSCs; however, the increment was not statistically significant relative to its expression in pellets formed from normoxia-expanded BMSCs (Figure 6i).
The cellular response to low oxygen tension in many mammalian cells is regulated by the transcriptional activity of HIF-1, a heterodimer of HIF-1α and HIF-1β . HIF-2, a heterodimer of HIF-2α and HIF-1β, has also been identified as a regulator of the response of mammalian cells to low oxygen tension [41, 42]. In addition, both HIF-1 and HIF-2 have been implicated in hypoxia-mediated enhancement of chondrogenesis of mesenchymal cells [29, 30, 41–43]. Thus, we investigated the gene expression of HIF-1α and HIF-2α in our study pellets after cultivation under normoxic and hypoxic conditions. The mRNA expression level of HIF-1α was similar in pellets regardless of whether the pellets were formed from normoxia- or hypoxia-expanded BMSC or cultivation of pellets under normoxic or hypoxic conditions (Figure 6i). The expression of HIF-2α was the same in pellets derived from normoxia- and hypoxia-expanded BMSCs under normal oxygen tension. However, under low oxygen tension the expression of HIF-2α increased regardless of whether the pellets were derived from normoxia- or hypoxia-expanded BMSCs. The expression of HIF-2α increased significantly by 3.2-fold in pellets derived from hypoxia-expanded BMSCs when compared to its expression in pellets under normoxic conditions. In contrast, the increment determined (1.6-fold) in pellets derived from normoxia-expanded BMSCs under hypoxic conditions relative to its expression in the same pellets under normoxic conditions was not statistically significant (Figure 6i).
In this study we have compared the chondrogenic potential of human BMSCs obtained under low (3%) and normal (21%) oxygen tension. The BMSCs were isolated via plastic adherence and cell culture mediated propagation. We have used flow cytometry to characterize the cell surface protein expression of the BMSCs and used an in vitro pellet model of chondrogenesis with TGF-β1 to investigate their capacity to undergo chondrogenic differentiation.
Our data showed that the BMSCs obtained after plating of bone marrow nucleated cells and subsequent cell culture propagation under low oxygen tension expressed a panel of conventionally used mesenchymal stem cell surface markers but with a consistently reduced concentration (that is, reduced mean fluorescence intensity) of CD90. The BMSCs obtained via propagation under low oxygen tension underwent a more robust chondrogenesis than their counterparts under normal oxygen tension albeit with dependence on the donor. The reduced CD90 expression in the BMSCs harvested under hypoxia may be associated with improved chondrogenesis. A link between CD90 expression and chondrogenic potential of BMSCs has not been reported in the literature. However, the lack of CD90 expression in in vitro expanded human articular chondrocytes has been observed with chondrocytes with higher chondrogenic potential . Thus, there is reason to speculate on a potential link between its expression and the chondrogenic capacity of BMSCs. Furthermore, in vitro expanded human articular chondrocytes have also been reported to display plasticity features that are similar to those of mesenchymal stem cells . Regardless of the oxygen tension during pellet culture, the BMSCs isolated and propagated under hypoxic conditions displayed a superior chondrogenic capacity than their counterparts from normal oxygen tension. The superior chondrogenic capacity was characterized by higher GAG per DNA content, enhanced transcript expression of a panel of chondrogenic genes (aggrecan, Col2a1 and Sox9) as well as the intense and uniform distribution of collagen II and safranin O staining for sulfated proteoglycans. Our data mirrors the findings of Tew et al. , that demonstrated that pellets derived from de-differentiated and Sox9 transduced articular chondrocytes displayed a phenotype that was consistent with a more robust chondrogenic response (increased safranin O staining and Col2a1 expression) compared to control pellets formed from non-transduced and de-differentiated articular chondrocytes. Furthermore, our data of increased mRNA expression of Sox9 and aggrecan supports previously reported evidence that transcriptional activity of Sox9 enhances the gene promoter activities of aggrecan in chondrocytes [46–49]. Transcriptional factors Sox9, L-Sox5 and Sox6 have been reported to be essential for regulating the expression of Col2a1 as well as other genes involved in chondrogenesis [48, 50, 51].
The chondrogenic capacity of all pellets in our study improved further under low oxygen tension. The improvement was characterized by a marginal (that is, non-statistically significant) increase in transcript expression of aggrecan, Col2a1 and COMP. Furthermore, and more significantly, the improvement in chondrogenic potential under hypoxia was accompanied by a concomitant suppression in Col10a1 expression, a marker of hypertrophic chondrogenesis and terminally differentiated chondrocytes. This finding is in accordance with the observation of Hirao et al. that hypoxia (that is, 5% O2) supports commitment of C3HT10T1/2 (a pluripotent mesenchymal cell line) to chondrogenic differentiation rather than osteogenesis through mechanisms involving a concomitant down-regulation of Col10a1 and Runx2 activity via Smad suppression and histone deactylase 4 activation (HDAC4) .
Our results indicate that the response of BMSCs to hypoxic conditions involves up-regulation of the transcriptional expression of HIF-2α rather than HIF-1α, which remained unperturbed. This finding was surprising since hypoxia has been reported to induce chondrocyte-specific gene expression (aggrecan, Col2a and Sox9) in mesenchymal cells (mouse ST2 stromal cells and C3HT10T1/2) through transcriptional activity of HIF-1α . Furthermore, HIF-1α, has been implicated in hypoxia-mediated inhibition of senescence and maintenance of human mesenchymal stem cell properties . Our finding, therefore suggests that the response of the BMSCs to hypoxia was mediated by the transcriptional activity of HIF-2α. The involvement of HIF-2α here is in accordance with a report that hypoxia (that is, 5% O2) enhances the expression of several chondrogenic genes including Col2a1, aggrecan and Sox9 during chondrogenesis of adipose derived mesenchymal stem cells . However, in contrast to the hypoxia-mediated down-regulation of Col10a1 noted in our present study, hypoxia (5% O2) enhanced the expression of Col10a1 during the chondrogenic differentiation of adipose derived mesenchymal stem cells . A plausible explanation for the differential outcome may depend on the difference in oxygen tension or cell type. Nonetheless, our data opens the perspective of a possible mechanistic link between the transcriptional activity of HIF-2α and collagen X expression.
Differential expression of TGFβ-receptors
TGFβ-RI and TGFβ-RII have been implicated in the reduced chondrogenic potential of adipose derived mesenchymal stem cells relative to the chondrogenic potential of BMSCs. Thus, we investigated the expression of these receptor proteins in our BMSCs pellets. Our data showed that the expression of these receptors was generally higher in pellets derived from hypoxia-expanded BMSCs after culture in hypoxic conditions. However, it was surprising that the expression of TGFβ-RI was statistically higher in pellets formulated from hypoxia-expanded BMSCs during pellet culture under hypoxic conditions.
We have shown that the isolation and expansion of BMSCs under hypoxic conditions of 3% oxygen tension increases the propensity of the BMSCs to undergo a more robust chondrogenesis under normoxic and hypoxic culture conditions relative to BMSCs isolated and propagated under normoxic conditions. Our results also show that the response of these cells to low oxygen tension is mediated by HIF-2α. Taken together, our finding highlights the need to isolate and propagate BMSCs under hypoxic conditions for improved in vitro chondrogenesis and for in vivo cartilage repair and/or regeneration. The outcome of this study suggests that oxygen tension is an important factor in the determination of the chondrogenic differentiation fate of adherent populations of BMSCs.
We would like to thank Dr Thomas Churchill and Jacek Studzinski (Department of Surgery, University of Alberta, Canada) for histological assistance. Financial support was provided in part by: Edmonton Orthopaedic Research Committee, University of Alberta Hospital Foundation to AA, Edmonton Civic Employee's Charitable Fund to AA and new investigator startup fund by Department of Surgery, University of Alberta to AA.
bone marrow mesenchymal stromal cells
complementary deoxyribonucleic acid
type I collagen α2 chain
type II collagen α1 chain
cartilage oligomeric matrix protein
basic fibroblast growth factor
hypoxia inducible factor-1 alpha
hypoxia inducible factor-2 alpha
messenger ribonucleic acid
mesenchymal stem cells
quantitative real-time polymerase chain reaction
Sry-related HMG box-9
- TGF-β1 or β3:
transforming growth factor -β1 or β3
transforming growth factor β receptor I
transforming growth factor β receptor II.
- Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994, 331: 889-895. 10.1056/NEJM199410063311401.View ArticlePubMedGoogle Scholar
- Lee CR, Grodzinsky AJ, Hsu HP, Martin SD, Spector M: Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J Orthop Res. 2000, 18: 790-799. 10.1002/jor.1100180517.View ArticlePubMedGoogle Scholar
- von der Mark K, Gauss V, von der Mark H, Muller P: Relationship between cell shape and type of collagen synthesized as chondrocytes lose their cartilage phenotype in culture. Nature. 1977, 267: 531-532. 10.1038/267531a0.View ArticlePubMedGoogle Scholar
- Watt FM: Effect of seeding density on stability of the differentiated phenotype of pig articular chondrocytes in culture. J Cell Sci. 1988, 89: 373-378.PubMedGoogle Scholar
- 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: 143-147. 10.1126/science.284.5411.143.View ArticlePubMedGoogle Scholar
- Mackay A, Beck S, Murphy J, Barry F, Chichester C, Pittenger M: Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 1998, 4: 415-428. 10.1089/ten.1998.4.415.View ArticlePubMedGoogle Scholar
- Lee JW, Kim YH, Kim SH, Han SH, Hahn SB: Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. Yonsei Med J. 2004, 45 (Suppl): 41-47.View ArticlePubMedGoogle Scholar
- Sekiya I, Vuoristo JT, Larson BL, Prockop DJ: In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA. 2002, 99: 4397-4402. 10.1073/pnas.052716199.PubMed CentralView ArticlePubMedGoogle Scholar
- Khan WS, Adesida AB, Tew SR, Lowe ET, Hardingham TE: Bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions. J Orthop Res. 2010, 28: 834-840.PubMedGoogle Scholar
- Acharya C, Adesida A, Zajac P, Mumme M, Riesle J, Martin I, Barbero A: Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J Cell Physiol. 2012, 227: 88-97. 10.1002/jcp.22706.View ArticlePubMedGoogle Scholar
- Kuroda R, Ishida K, Matsumoto T, Akisue T, Fujioka H, Mizuno K, Ohgushi H, Wakitani S, Kurosaka M: Treatment of a full-thickness articular cartilage defect in the femoral condyle of an athlete with autologous bone-marrow stromal cells. Osteoarthritis Cartilage. 2007, 15: 226-231. 10.1016/j.joca.2006.08.008.View ArticlePubMedGoogle Scholar
- Krebsbach PH, Kuznetsov SA, Bianco P, Gehron Robey P: Bone marrow stromal cells: characterization and clinical application. Crit Rev Oral Biol Med. 1999, 10 (2): 165-181. 10.1177/10454411990100020401.View ArticlePubMedGoogle Scholar
- Friedenstein AJ, Chailakhjan RK, Lalykina KS: The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970, 3: 393-403.PubMedGoogle Scholar
- Friedenstein A, Gorskaja J, Kulagina N: Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976, 4: 267-274.PubMedGoogle Scholar
- Cross M, Alt R, Niederwieser D: The case for a metabolic stem cell niche. Cells Tissues Organs. 2008, 188: 150-159. 10.1159/000114206.View ArticlePubMedGoogle Scholar
- 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: 513-522. 10.1016/j.bone.2006.02.061.View ArticlePubMedGoogle Scholar
- Martin-Rendon E, Hale SJM, Ryan D, Baban D, Forde SP, Roubelakis M, Sweeney D, Moukayed M, Harris AL, Davies K, Watt SM: Transcriptional profiling of human cord blood CD133+ and cultured bone marrow mesenchymal stem cells in response to hypoxia. Stem Cells. 2007, 25: 1003-1012. 10.1634/stemcells.2006-0398.View ArticlePubMedGoogle Scholar
- Grayson WL, Zhao F, Bunnell B, Ma T: Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Resh Commun. 2007, 358: 948-953. 10.1016/j.bbrc.2007.05.054.View ArticleGoogle Scholar
- Krinner A, Zscharnack M, Bader A, Drasdo D, Galle J: Impact of oxygen environment on mesenchymal stem cell expansion and chondrogenic differentiation. Cell Prolif. 2009, 42: 471-484. 10.1111/j.1365-2184.2009.00621.x.View ArticlePubMedGoogle Scholar
- Mueller J, Benz K, Ahlers M, Gaissmaier C, Mollenhauer J: Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures. Cell Transplant.
- Gerber HP, Ferrara N: Angiogenesis and bone growth. Trends Cardiovasc Med. 2000, 10: 223-228. 10.1016/S1050-1738(00)00074-8.View ArticlePubMedGoogle Scholar
- Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU: In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. 1998, 238: 265-272. 10.1006/excr.1997.3858.View ArticlePubMedGoogle Scholar
- Murdoch AD, Grady LM, Ablett MP, Katopodi T, Meadows RS, Hardingham TE: Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage. Stem Cells. 2007, 25: 2786-2796. 10.1634/stemcells.2007-0374.View ArticlePubMedGoogle Scholar
- Oldershaw RA, Tew SR, Russell AM, Meade K, Hawkins R, McKay TR, Brennan KR, Hardingham TE: Notch signaling through jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells but must be switched off to complete chondrogenesis. Stem Cells. 2008, 26: 666-674. 10.1634/stemcells.2007-0806.View ArticlePubMedGoogle Scholar
- Pittenger M, Mackay A, Beck S, Jaiswal R, Douglas R, Mosca J, Moorman M, Simonetti D, Craig S, Marshak D: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147. 10.1126/science.284.5411.143.View ArticlePubMedGoogle Scholar
- Adesida A, Tweats L, Millward-Sadler J, Salter D, Hardingham T: Cultured human meniscus cells are chondrogenic in pellet culture: this is enhanced by hypoxia and involves upregulation of prolyl 4-hydroxylase type I. Trans Annu Meet Orthop Res Soc. 2005, 1721-Google Scholar
- Domm C, Schunke M, Christesen K, Kurz B: Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthritis Cartilage. 2002, 10: 13-22. 10.1053/joca.2001.0477.View ArticlePubMedGoogle Scholar
- Lafont JE, Talma S, Hopfgarten C, Murphy CL: Hypoxia promotes the differentiated human articular chondrocyte phenotype through SOX9-dependent and -independent pathways. J Biol Chem. 2008, 283: 4778-4786.View ArticlePubMedGoogle Scholar
- Lafont JE, Talma S, Murphy CL: Hypoxia-inducible factor 2alpha is essential for hypoxic induction of the human articular chondrocyte phenotype. Arthritis Rheum. 2007, 56: 3297-3306. 10.1002/art.22878.View ArticlePubMedGoogle Scholar
- Khan WS, Adesida AB, Hardingham TE: Hypoxic conditions increase hypoxia-inducible transcription factor 2alpha and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients. Arthritis Res Ther. 2007, 9: R55-10.1186/ar2211.PubMed CentralView ArticlePubMedGoogle Scholar
- Mizuno S, Glowacki J: Low oxygen tension enhances chondroinduction by demineralized bone matrix in human dermal fibroblasts in vitro. Cells Tissues Organs. 2005, 180: 151-158. 10.1159/000088243.View ArticlePubMedGoogle Scholar
- Grimshaw MJ, Mason RM: Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension. Osteoarthritis Cartilage. 2001, 9: 357-364. 10.1053/joca.2000.0396.View ArticlePubMedGoogle Scholar
- Martin I, Muraglia A, Campanile G, Cancedda R, Quarto R: Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology. 1997, 138: 4456-4462. 10.1210/en.138.10.4456.PubMedGoogle Scholar
- Adesida AB, Grady LM, Khan WS, Hardingham TE: The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia. Arthritis Res Ther. 2006, 8: R61-10.1186/ar1929.PubMed CentralView ArticlePubMedGoogle Scholar
- Adesida AB, Grady LM, Khan WS, Millward-Sadler SJ, Salter DM, Hardingham TE: Human meniscus cells express hypoxia inducible factor-1alpha and increased SOX9 in response to low oxygen tension in cell aggregate culture. Arthritis Res Ther. 2007, 9: R69-10.1186/ar2267.PubMed CentralView ArticlePubMedGoogle Scholar
- Foldager CB, Munir S, Ulrik-Vinther M, Soballe K, Bunger C, Lind M: Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes. BMC Mol Biol. 2009, 10: 94-10.1186/1471-2199-10-94.PubMed CentralView ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-[delta][delta]CT method. Methods. 2001, 25 (4): 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Diaz-Romero J, Nesic D, Grogan SP, Heini P, Mainil-Varlet P: Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells. J Cell Physiol. 2008, 214: 75-83. 10.1002/jcp.21161.View ArticlePubMedGoogle Scholar
- Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F, Richter W: Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol. 2007, 211: 682-691. 10.1002/jcp.20977.View ArticlePubMedGoogle Scholar
- Semenza GL: HIF-1 and mechanisms of hypoxia sensing. Curr Opin Cell Biol. 2001, 13: 167-171. 10.1016/S0955-0674(00)00194-0.View ArticlePubMedGoogle Scholar
- Hu CJ, Iyer S, Sataur A, Covello KL, Chodosh LA, Simon MC: Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells. Mol Cell Biol. 2006, 26: 3514-3526. 10.1128/MCB.26.9.3514-3526.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC: Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol. 2003, 23: 9361-9374. 10.1128/MCB.23.24.9361-9374.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsai CC, Chen YJ, Yew TL, Chen LL, Wang JY, Chiu CH, Hung SC: Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST. Blood. 2011, 117: 459-469. 10.1182/blood-2010-05-287508.View ArticlePubMedGoogle Scholar
- Giovannini S, Diaz-Romero J, Aigner T, Mainil-Varlet P, Nesic D: Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes. J Cell Physiol. 2010, 222: 411-420. 10.1002/jcp.21965.View ArticlePubMedGoogle Scholar
- Barbero A, Ploegert S, Heberer M, Martin I: Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes. Arthritis Rheum. 2003, 48: 1315-1325. 10.1002/art.10950.View ArticlePubMedGoogle Scholar
- Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE: Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage. 2005, 13: 80-89. 10.1016/j.joca.2004.10.011.View ArticlePubMedGoogle Scholar
- Sekiya I, Tsuji K, Koopman P, Watanabe H, Yamada Y, Shinomiya K, Nifuji A, Noda M: SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6. J Biol Chem. 2000, 275: 10738-10744. 10.1074/jbc.275.15.10738.View ArticlePubMedGoogle Scholar
- Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B: SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. Mol Cell Biol. 1997, 17: 2336-2346.PubMed CentralView ArticlePubMedGoogle Scholar
- Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B: Sox9 is required for cartilage formation. Nat Genet. 1999, 22: 85-89. 10.1038/8792.View ArticlePubMedGoogle Scholar
- Lefebvre V, Behringer RR, de Crombrugghe B: L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway. Osteoarthritis Cartilage. 2001, 9 (Suppl A): S69-75.View ArticlePubMedGoogle Scholar
- de Crombrugghe B, Lefebvre V, Behringer RR, Bi W, Murakami S, Huang W: Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol. 2000, 19: 389-394. 10.1016/S0945-053X(00)00094-9.View ArticlePubMedGoogle Scholar
- Hirao M, Tamai N, Tsumaki N, Yoshikawa H, Myoui A: Oxygen tension regulates chondrocyte differentiation and function during endochondral ossification. J Biol Chem. 2006, 281: 31079-31092. 10.1074/jbc.M602296200.View ArticlePubMedGoogle Scholar
- Robins JC, Akeno N, Mukherjee A, Dalal RR, Aronow BJ, Koopman P, Clemens TL: Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9. Bone. 2005, 37: 313-322. 10.1016/j.bone.2005.04.040.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.