Osteogenic induction of human adipose-derived stem cells

doi:10.3969/j.issn.2095-4344.2014.19.006

Abstract

Abstract:

BACKGROUND: A great amount of mesenchymal stem cells can be successfully derived from fat tissue and induced to differentiate into osteoblasts, chondrocytes, adipocytes and myocardial cells.
OBJECTIVE: To establish the method of isolating, culturing and osteogenic differentiation of adipose-derived stem cells in vitro, and to explore the potential of adipose-derived stem cells as seed cells for bone tissue engineering.
METHODS: Collagenase enzymatic digestion was used to isolate adipose-derived stem cells from human fat tissue which were then cultured in vitro. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 assay was performed to examine cell viability. Adipose-derived stem cells were induced by osteogenic induced reagent to differentiate into bone cells. In addition, we also performed BCIP/NBT method to detect alkaline phosphatase activity. Alizarin red staining was used to detect the formation of calcium node. RT-PCR was performed to examine alkaline phosphatase and osteopontin expression.
RESULTS AND CONCLUSION: We successfully obtained adipose-derived stem cells from fat aspirated liquid. Adipose-derived stem cells obtained could passage stably and proliferate highly. Flow cytometry data showed the expression of stem cell surface markers. Adipose-derived stem cells showed typical osteoblast morphology after osteogenic differentiation. Alkaline phosphatase staining was positive and alizarin red staining displayed the formation of calcium node. Furthermore, we found that alkaline phosphatase and osteopontin mRNA was expressed after differentiation 0, 3, 7, 14, 21, 28 days. These findings indicate that adipose-derived stem cells can be obtained from fat tissue through enzymatic digestion, differentiate towards bone cells, and express alkaline phosphatase and osteopontin, which can become potential seed cells for bone tissue engineering.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, mesenchymal stem cells, subcutaneous fat, osteoblasts, osteopontin

CLC Number:

R394.2

Guo Yan-ping, Tao Chang-bo, Zhang Ai-jun, Li Xue-yang, Shen Cai-qi, Li Zheng-dao, Jin Pei-sheng . Osteogenic induction of human adipose-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 2987-2992.

Figures/Tables 5

References

[1] 孙宝谦,兑振华,王增良等.颅骨修补与颅骨保存研究进展[J].中国医药导刊,2009,12(1):9-10.
[2] 张贺,张蔷,王锡亮.颅骨缺损修补的研究与进展[J].中华现代外科学杂志,2005,2(18):1679-1681.
[3] 于明琨,王永谦.自体骨移植修补颅骨缺损的材料与方法[J].中国临床康复,2006,10(41):126-129.
[4] 李景峰,郑启新.异种,异体骨材料的研究进展[J].国际生物医学工程杂志,2008,31(4):249-253.
[5] 张永光,王志强.骨移植替代材料研究进展[J].中国修复重建外科杂志,2008,22(10):1264-1268.
[6] 王呈,曾炳芳.骨移植替代材料在创伤骨科的应用[J].国际骨科学杂志,2012,33(1):37-38.
[7] De Peppo GM, Thomsen P, Karlsson C,et al, Human progenitor cells for bone engineering applications.curr mol med.2013;13(5):723-734.
[8] Yuan J,Cui L,Zhang WJ,et al.Repair of canine mandibular bone defets with marrow stromal cells and porous beta-tricalcium phosphate.Biomaterials.2007;28(6): 1005-1013.
[9] 王宇玫,张浩,李君,等.不同年龄人群骨髓间充质干细胞的生长变化[J].中华老年多器官疾病杂志,2008,9(3):223-226.
[10] Zuk PA, Zhu M, Ashjian P,et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell.2002;13(12): 4279-4295.
[11] Tapp H,Hanley EN Jr,Patt JC,et al.Adipose-derived stem cells:characterization and current application in orthopaedic tissue repair.Exp Biol Med(Maywood).2009;234(1):1-9.
[12] Zaminy A, Ragerdi Kashani I, Barbarestani M, et al. Osteogenic differentiation of rat mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells: melatonin as a differentiation factor. Iran Biomed J. 2008;12(3):133-141.
[13] Mahmoudifar N, Doran PM.Osteogenic Differentiation and Osteochondral Tissue Engineering Using Human Adipose-Derived Stem Cells.Biotechnol Prog. 2013;29(1): 176-185.
[14] Hamid AA, Idrus RB, Saim AB, Sathappan S,et al. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation.Clinics (Sao Paulo). 2012;67(2): 99-106.
[15] Moioli EK, Chen M, Yang R, et al.Hybrid adipogenic implants from adipose stem cells for soft tissue reconstruction in vivo. Tissue Eng Part A. 2010;16(11):3299-3307.
[16] Palpant NJ, Metzger JM.Aesthetic cardiology: adipose-derived stem cells for myocardial repair.Curr Stem Cell Res Ther.2010;5(2):145-152.
[17] 张钦,马真胜,刘建,等.成骨诱导自体脂肪源干细胞用于修复兔颅骨缺损[J].中国骨肿瘤病,2009,8(2):104-107.
[18] 鲍慧婧,邹俊,尹烁,等.兔脂肪间充质干细胞复合PLGA支架的生物相容性研究[J].中华实验眼科杂志,2001,29(6):511-516.
[19] 李强,陶常波,张爱君,等.EGF刺激对脂肪间充质干细胞促血管生成作用的影响[J].中华医学美学杂志,2012,18(6):440-442.
[20] Bose S, Roy M, Bandyopadhyay A.Recent advances in bone tissue engineering scaffolds.Trends Biotechnol. 2012;30(10): 546-554.
[21] Khan WS, Hardingham TE.Mesenchymal stem cells, sources of cells and differentiation potential.J Stem Cells. 2012;7(2): 75-85.
[22] Amini AR, Laurencin CT, Nukavarapu SP.Bone tissue engineering: recent advances and challenges.Crit Rev Biomed Eng. 2012;40(5):363-408.
[23] Han J, Koh YJ, Moon HR,et al.Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells.Blood. 2010 ;115(5):957-964.
[24] Nakao N, Nakayama T, Yahata T,et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo:advantages over bone marrow-derived mesenchymal stem cells. Am J Pathol.2010;177(2):547-554.
[25] 黄宏,朱方强,孙宏振,等.脂肪来源干细胞体外成骨诱导和成脂分化[J].第三军医大学学报,2008,30(13):1219-1222.
[26] Datta P, Ghosh P, Ghosh K, et al .In vitro ALP and osteocalcin gene expression analysis and in vivo biocompatibility of N-methylene phosphonic chitosan nanofibers for bone regeneration. J Biomed Nanotechnol. 2013;9(5):870-879.
[27] McKee MD, Pedraza CE, Kaartinen MT. Osteopontin and wound healing in bone.Cells Tissues Organs. 2011;194(2-4): 313-319.
[28] 柳向东,唐朝晖,白祥军,等.人骨髓间充质干细胞定向成骨诱导中碱性磷酸酶对成骨能力的影响[J].中华创伤杂志,2009,25(10): 945-948.
[29] Strem BM, Hicok KC, Zhu M,et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med. 2005;54(3): 132-141.
[30] Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN,et al. Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies.Cell Tissue Res. 2008;332(3):415-426.
[31] Arthur A, Zannettino A, Gronthos S.The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair.J Cell Physiol. 2009;218(2):237-245.
[32] Sen A, Lea-Currie YR, Sujkowska D, et al.Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J Cell Biochem.2001;81(2): 312-319.
[33] Tchkonia T, Giorgadze N, Pirtskhalava T,et al.Fat depot origin affects adipogenesis in primary cultured and cloned human preadipocytes.Am J Physiol Regul Integr Comp Physiol. 2002; 282(5):R1286-1296.
[34] Strong AL, Strong TA, Rhodes LV, et al.Obesity associated alterations in the biology of adipose stem cells mediate enhanced tumorigenesis by estrogen dependent pathways. Breast Cancer Res. 2013;15(5):R102.