HYPOXIA INDUCIBLE FACTOR 1α/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GONGMing ¹ , HUANGSheng ¹ , LUOJiaquan ¹ , HUANGBaoding ² , ZHOUZhiyu ¹ , DAIXuejun ³ , GAOManman ¹ , LILiangping ¹ ,  ZOUXuenong ¹

1. Department of Spinal Surgery, Orthopaedic Research Institute, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, 510080, P. R. China;
2. Department of Orthopedics, the Sixth Affiliated Hospital of Sun Yat-sen University Guangzhou Guangdong, 510080, P. R. China;
3. Department of Spinal Surgery, Yan'an Hospital of Kunming City;

Corresponding?author：

ZOUXuenong, Email: zxnong@hotmail.com

Keywords：

Hypoxia inducible factor; Human bone marrow mesenchymal stem cells; Chondrogenic differentiation

DOI：

10.7507/1002-1892.20150186

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Objective To observe the genes expression of hypoxia inducible factor 1α (HIF-1α) and HIF-2α by inducing chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) so as to provide a fundamental basis for HIF involving in the mechanism of chondrogenesis. Methods High density pellet of hBMSCs was obtained by centrifugation and cultured with H-DMEM medium containing 2% fetal bovine serum (control group) and with chondrogenic medium (chondrogenic induction group) under hypoxia (2%O₂) for 3 weeks. Immunohistochemistry staining was utilized to identify extracellular proteoglycan and collagen type Ⅱ at 3 weeks after culture. Western blot was applied for measuring HIF-1α and HIF-2α protein levels at 1 week after culture. Real-time quantitative PCR was performed to detect the genes expressions of HIF-1α, HIF-2α, Sox-9, collagen type Ⅱ, collagen type X, and Aggrecan at 1, 2, and 3 weeks after culture. Results Toluidine blue staining showed sparse nucleus in the control group, and dense nucleus in the chondrogenic induction group;extracellular matrix staining was deeper in the chondrogenic induction group than the control group. Immunohistochemical staining for collagen type Ⅱ was positive in cytoplasm;when compared with the chondrogenic induction group, the control group showed sparse and light-coloured nucleus. At 1 week after culture, the protein expression levels of HIF-1α and HIF-2α in the chondrogenic induction group were significantly lower than those in the control group (t=8.345, P=0.001;t=7.683, P=0.002). When compared with control group, the HIF-1α mRNA expression was significantly down-regulated at 1 week and significantly up-regulated at 2 weeks in chondrogenic induction group (P<0.05), but no significant difference was found at 3 weeks between the 2 groups (P>0.05). And the mRNA expression of HIF-2α was significantly down-regulated and mRNA expression of Sox-9 was significantly up-regulated after chondrogenic differentiation when compared with the control group (P<0.01). The mRNA expressions of collagen type Ⅱ and collagen type X were significantly up-regulated at 2 and 3 weeks after chondrogenic differentiation when compared with the control group (P<0.05). And the mRNA expression of Aggrecan was significantly up-regulated at each time point after chondrogenic differentiation (P<0.05). Conclusion HIF-1α may involve the hBMSCs chondrogenic differentiation under hypoxia, while HIF-2α expression is depressed throughout the period and may have negative effect on differentiation.

Citation： GONGMing, HUANGSheng, LUOJiaquan, HUANGBaoding, ZHOUZhiyu, DAIXuejun, GAOManman, LILiangping, ZOUXuenong. HYPOXIA INDUCIBLE FACTOR 1α/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(7): 857-862. doi: 10.7507/1002-1892.20150186 Copy

1.	Studer D,Millan C,?ztürk E,et al.Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.Eur Cell Mater,2012,24:118-135.
2.	Araldi E,Schipani E.Hypoxia,HIFs and bone development.Bone,2010,47(2):190-196.
3.	Loboda A,Jozkowicz A,Dulak J.HIF-1 versus HIF-2-is one more important than the other? Vascul Pharmacol,2012,56(5-6):245-251.
4.	Tew SR,Li Y,Pothacharoen P,et al.Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes.Osteoarthritis Cartilage,2005,13(1):80-89.
5.	Adesida AB,Mulet-Sierra A,Jomha NM.Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells.Stem Cell Res Ther,2012,3(2):9.
6.	Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-147.
7.	Zell S,Schmitt R,Witting S,et al.Hypoxia induces mesenchymal gene expression in renal tubular epithelial cells:an in vitro model of kidney transplant fibrosis.Nephron Extra,2013,3(1):50-58.
8.	Johnstone B,Hering TM,Caplan AI,et al.In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells.Exp Cell Res,1998,238(1):265-272.
9.	Majmundar AJ,Wong WJ,Simon MC.Hypoxia-inducible factors and the response to hypoxic stress.Mol Cell,2010,40(2):294-309.
10.	Pelttari K,Steck E,Richter W.The use of mesenchymal stem cells for chondrogenesis.Injury,2008,39 Suppl 1:S58-65.
11.	Rankin EB,Giaccia AJ,Schipani E.A central role for hypoxic signaling in cartilage,bone,and hematopoiesis.Curr Osteoporos Rep,2011,9(2):46-52.
12.	Seghatoleslami MR,Tuan RS.Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells.J Cell Biochem,2002,84(2):237-248.
13.	Markway BD,Tan GK,Brooke G,et al.Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures.Cell Transplant,2010,19(1):29-42.
14.	Schipani E,Ryan HE,Didrickson S,et al.Hypoxia in cartilage:HIF-1alpha is essential for chondrocyte growth arrest and survival.Genes Dev,2001,15(21):2865-2876.
15.	Patel SA,Simon MC.Biology of hypoxia-inducible factor-2alpha in development and disease.Cell Death Differ,2008,15(4):628-634.
16.	Lee JH,Kemp DM.Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions.Biochem Biophys Res Commun,2006,341(3):882-888.
17.	Utting JC,Robins SP,Brandao-Burch A,et al.Hypoxia inhibits the growth,differentiation and bone-forming capacity of rat osteoblasts.Exp Cell Res,2006,312(10):1693-1702.
18.	D'Ippolito G,Diabira S,Howard GA,et al.Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.Bone,2006,39(3):513-522.
19.	Wiesener MS,Jürgensen JS,Rosenberger C,et al.Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.FASEB J,2003,17(2):271-273.
20.	Malladi P,Xu Y,Chiou M,et al.Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells.Am J Physiol Cell Physiol,2006,290(4):C1139-1146.

1. Studer D,Millan C,?ztürk E,et al.Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells.Eur Cell Mater,2012,24:118-135.
2. Araldi E,Schipani E.Hypoxia,HIFs and bone development.Bone,2010,47(2):190-196.
3. Loboda A,Jozkowicz A,Dulak J.HIF-1 versus HIF-2-is one more important than the other? Vascul Pharmacol,2012,56(5-6):245-251.
4. Tew SR,Li Y,Pothacharoen P,et al.Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes.Osteoarthritis Cartilage,2005,13(1):80-89.
5. Adesida AB,Mulet-Sierra A,Jomha NM.Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells.Stem Cell Res Ther,2012,3(2):9.
6. Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-147.
7. Zell S,Schmitt R,Witting S,et al.Hypoxia induces mesenchymal gene expression in renal tubular epithelial cells:an in vitro model of kidney transplant fibrosis.Nephron Extra,2013,3(1):50-58.
8. Johnstone B,Hering TM,Caplan AI,et al.In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells.Exp Cell Res,1998,238(1):265-272.
9. Majmundar AJ,Wong WJ,Simon MC.Hypoxia-inducible factors and the response to hypoxic stress.Mol Cell,2010,40(2):294-309.
10. Pelttari K,Steck E,Richter W.The use of mesenchymal stem cells for chondrogenesis.Injury,2008,39 Suppl 1:S58-65.
11. Rankin EB,Giaccia AJ,Schipani E.A central role for hypoxic signaling in cartilage,bone,and hematopoiesis.Curr Osteoporos Rep,2011,9(2):46-52.
12. Seghatoleslami MR,Tuan RS.Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells.J Cell Biochem,2002,84(2):237-248.
13. Markway BD,Tan GK,Brooke G,et al.Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures.Cell Transplant,2010,19(1):29-42.
14. Schipani E,Ryan HE,Didrickson S,et al.Hypoxia in cartilage:HIF-1alpha is essential for chondrocyte growth arrest and survival.Genes Dev,2001,15(21):2865-2876.
15. Patel SA,Simon MC.Biology of hypoxia-inducible factor-2alpha in development and disease.Cell Death Differ,2008,15(4):628-634.
16. Lee JH,Kemp DM.Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions.Biochem Biophys Res Commun,2006,341(3):882-888.
17. Utting JC,Robins SP,Brandao-Burch A,et al.Hypoxia inhibits the growth,differentiation and bone-forming capacity of rat osteoblasts.Exp Cell Res,2006,312(10):1693-1702.
18. D'Ippolito G,Diabira S,Howard GA,et al.Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells.Bone,2006,39(3):513-522.
19. Wiesener MS,Jürgensen JS,Rosenberger C,et al.Widespread hypoxia-inducible expression of HIF-2alpha in distinct cell populations of different organs.FASEB J,2003,17(2):271-273.
20. Malladi P,Xu Y,Chiou M,et al.Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells.Am J Physiol Cell Physiol,2006,290(4):C1139-1146.

Chinese Journal of Reparative and Reconstructive Surgery

HYPOXIA INDUCIBLE FACTOR 1α/2α GENES EXPRESSION IN CHONDROGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

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