REGULATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS OSTEOGENIC AND ADIPOGENIC DIFFERENTIATIONS BY Wnt10b ADENOVIRAL VECTOR IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHOUYong , ZOUChang , MINLi , SHIRui , ZHANGWenli , LUOYi , PENGJing , DUANHong , TUChongqi ,  ZHANGHui

Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

ZHANGHui, Email: caesarzh@163.com

Keywords：

Wnt10b gene; Human bone marrow mesenchymal stem cells; Osteogenesis; Adipogenesis

DOI：

10.7507/1002-1892.20140278

Video：

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Objective To investigate the regulation of human bone marrow mesenchymal stem cells (hBMSCs) osteogenic and adipogenic differentiations mediated by Wnt10b adenoviral vector in vitro. Methods The hBMSCs from ilial bone tissue in adults at passage 4 were infected by Wnt10b gene expression adenoviral vector (group A), Wnt10b-shRNA adenoviral vector (group B), and empty vector (group C), and non-transfected hBMSCs served as the blank control group. Then the cells were cultured separately in the circumstance of osteogenic induction, adipogenic induction, and non-induction. The alkaline phosphatase (ALP) staining, alizarin red staining, and oil red O staining were used to detect the osteogenic and adipogenic differentiations; real-time fluorescent quantitative PCR and Western blot were used to analyze the expressions of osteoblast and adipocyte genes and proteins. Results The results of ALP staining were positive after osteogenic induction, group A showed strong staining, and group B showed the weakest staining. The results of alizarin red staining showed that there were a lot of patchy confluent brown mineralized nodules in group A; a few punctate brown mineralized nodules were seen in group B; and many punctuate brown mineralized nodules were found in groups C and D. The results of oil red O staining showed strong staining in groups B, C, and D after adipogenic induction, especially in group B; scattered or small clustered staining was observed in group A. The expressions of osteoblast genes and proteins were significantly higher in group A than groups B, C, and D, and in groups C and D than group B by real-time fluorescent quantitative PCR and Western blot test; however, the expressions of adipocyte genes and proteins showed a contrary tendency. Conclusion The high level expression of Wnt10b can enhance osteogenic differentiation of hBMSCs, and the low level expression of Wnt10b can increase adipogenic differentiation of hBMSCs.

Citation： ZHOUYong, ZOUChang, MINLi, SHIRui, ZHANGWenli, LUOYi, PENGJing, DUANHong, TUChongqi, ZHANGHui. REGULATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS OSTEOGENIC AND ADIPOGENIC DIFFERENTIATIONS BY Wnt10b ADENOVIRAL VECTOR IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(10): 1284-1291. doi: 10.7507/1002-1892.20140278 Copy

1.	Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science, 1999, 284(5411):143-147.
2.	Gimble JM, Morgan C, Kelly K, et al.Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells.J Cell Biochem, 1995, 58(3):393-402.
3.	Hartsock RJ, Smith EB, Petty CS.Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest.A study made from 177 cases of sudden death examined by necropsy.Am J Clin Pathol, 1965, 43:326-331.
4.	Nelson-Dooley C, Della-Fera MA, Hamrick M, et al.Novel treatments for obesity and osteoporosis:targeting apoptotic pathways in adipocytes.Curr Med Chem, 2005, 12(19):2215-2225.
5.	Verma S, Rajaratnam JH, Denton J, et al.Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis.J Clin Pathol, 2002, 55(9):693-698.
6.	Sekiya I, Larson BL, Vuoristo JT, et al.Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs).J Bone Miner Res, 2004, 19(2):256-264.
7.	Gimble JM, Zvonic S, Floyd ZE, et al.Playing with bone and fat.J Cell Biochem, 2006, 98(2):251-266.
8.	Johnson ML, Kamel MA.The Wnt signaling pathway and bone metabolism.Curr Opin Rheumatol, 2007, 19(4):376-382.
9.	Baron R, Rawadi G, Roman-Roman S.Wnt signaling:a key regulator of bone mass.Curr Top Dev Biol, 2006, 76:103-127.
10.	Boudin E, Fijalkowski I, Piters E, et al.The role of extracellular modulators of canonical Wnt signaling in bone metabolism and diseases.Semin Arthritis Rheum, 2013, 43(2):220-240.
11.	Kramer I, Halleux C, Keller H, et al.Osteocyte Wnt/β-catenin signaling is required for normal bone homeostasis.Mol Cell Biol, 2010, 30(12):3071-3085.
12.	Monroe DG, McGee-Lawrence ME, Oursler MJ, et al.Update on Wnt signaling in bone cell biology and bone disease.Gene, 2012, 492(1):1-18.
13.	Longo KA, Wright WS, Kang S, et al.Wnt10b inhibits development of white and brown adipose tissues.J Biol Chem, 2004, 279(34):35503-35509.
14.	Hoeppner LH, Secreto FJ, Westendorf JJ.Wnt signaling as a therapeutic target for bone diseases.Expert Opin Ther Targets, 2009, 13(4):485-496.
15.	Wagner ER, Zhu G, Zhang BQ, et al.The therapeutic potential of the Wnt signaling pathway in bone disorders.Curr Mol Pharmacol, 2011, 4(1):14-25.
16.	Bennett CN, Longo KA, Wright WS, et al.Regulation of osteoblastogenesis and bone mass by Wnt10b.Proc Natl Acad Sci U S A, 2005, 102(9):3324-3329.
17.	Reya T, O'Riordan M, Okamura R, et al.Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.Immunity, 2000, 13(1):15-24.
18.	Bennett CN, Ouyang H, Ma YL, et al.Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation.J Bone Miner Res, 2007, 22(12):1924-1932.
19.	Stevens JR, Miranda-Carboni GA, Singer MA, et al.Wnt10b deficiency results in age-dependent loss of bone mass and progressive reduction of mesenchymal progenitor cells.J Bone Miner Res, 2010, 25(10):2138-2147.
20.	Zhang JW, Klemm DJ, Vinson C, et al.Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein β gene during adipogenesis.J Biol Chem, 2004, 279(6):4471-4478.

1. Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science, 1999, 284(5411):143-147.
2. Gimble JM, Morgan C, Kelly K, et al.Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells.J Cell Biochem, 1995, 58(3):393-402.
3. Hartsock RJ, Smith EB, Petty CS.Normal variations with aging of the amount of hematopoietic tissue in bone marrow from the anterior iliac crest.A study made from 177 cases of sudden death examined by necropsy.Am J Clin Pathol, 1965, 43:326-331.
4. Nelson-Dooley C, Della-Fera MA, Hamrick M, et al.Novel treatments for obesity and osteoporosis:targeting apoptotic pathways in adipocytes.Curr Med Chem, 2005, 12(19):2215-2225.
5. Verma S, Rajaratnam JH, Denton J, et al.Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis.J Clin Pathol, 2002, 55(9):693-698.
6. Sekiya I, Larson BL, Vuoristo JT, et al.Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs).J Bone Miner Res, 2004, 19(2):256-264.
7. Gimble JM, Zvonic S, Floyd ZE, et al.Playing with bone and fat.J Cell Biochem, 2006, 98(2):251-266.
8. Johnson ML, Kamel MA.The Wnt signaling pathway and bone metabolism.Curr Opin Rheumatol, 2007, 19(4):376-382.
9. Baron R, Rawadi G, Roman-Roman S.Wnt signaling:a key regulator of bone mass.Curr Top Dev Biol, 2006, 76:103-127.
10. Boudin E, Fijalkowski I, Piters E, et al.The role of extracellular modulators of canonical Wnt signaling in bone metabolism and diseases.Semin Arthritis Rheum, 2013, 43(2):220-240.
11. Kramer I, Halleux C, Keller H, et al.Osteocyte Wnt/β-catenin signaling is required for normal bone homeostasis.Mol Cell Biol, 2010, 30(12):3071-3085.
12. Monroe DG, McGee-Lawrence ME, Oursler MJ, et al.Update on Wnt signaling in bone cell biology and bone disease.Gene, 2012, 492(1):1-18.
13. Longo KA, Wright WS, Kang S, et al.Wnt10b inhibits development of white and brown adipose tissues.J Biol Chem, 2004, 279(34):35503-35509.
14. Hoeppner LH, Secreto FJ, Westendorf JJ.Wnt signaling as a therapeutic target for bone diseases.Expert Opin Ther Targets, 2009, 13(4):485-496.
15. Wagner ER, Zhu G, Zhang BQ, et al.The therapeutic potential of the Wnt signaling pathway in bone disorders.Curr Mol Pharmacol, 2011, 4(1):14-25.
16. Bennett CN, Longo KA, Wright WS, et al.Regulation of osteoblastogenesis and bone mass by Wnt10b.Proc Natl Acad Sci U S A, 2005, 102(9):3324-3329.
17. Reya T, O'Riordan M, Okamura R, et al.Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.Immunity, 2000, 13(1):15-24.
18. Bennett CN, Ouyang H, Ma YL, et al.Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation.J Bone Miner Res, 2007, 22(12):1924-1932.
19. Stevens JR, Miranda-Carboni GA, Singer MA, et al.Wnt10b deficiency results in age-dependent loss of bone mass and progressive reduction of mesenchymal progenitor cells.J Bone Miner Res, 2010, 25(10):2138-2147.
20. Zhang JW, Klemm DJ, Vinson C, et al.Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein β gene during adipogenesis.J Biol Chem, 2004, 279(6):4471-4478.

Chinese Journal of Reparative and Reconstructive Surgery

REGULATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS OSTEOGENIC AND ADIPOGENIC DIFFERENTIATIONS BY Wnt10b ADENOVIRAL VECTOR IN VITRO

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