Induction of Chondrogenic Differentiation of Human Adipose-Derived Stem Cells with TGF-β3 in Pellet Culture System

Document Type: Original Article


1 Department of Anatomical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Medical Physics and Biomedical Engineering, Isfahan University of Medical Sciences, Isfahan, Iran

3 Pathology Lab of Azzahra Hospital, Isfahan University of Medical Sciences, Isfahan, Iran


Adult stem cells which are derived from different tissues, with their unique abilities to self-renew and differentiate into various phenotypes have the potential for cell therapy and tissue engineering. Human adipose tissue is an appropriate source of mesenchymal stem cells with wide differentiation potential for tissue engineering research. In this study isolated stem cells from human subcutaneous adipose tissue were investigated for chondrogenic potential of adipose-derived stem cells (ADSCs) in pellet culture system treated withtransforming growth factor- β3 (TGF-β3).
Materials and Methods
Human ADSCs were isolated from subcutaneous adipose tissue and digested with collagenase type I. Immunocytochemical method for cell surface antigens was done in order to characterize the cells. The isolated cells were treated with chondrogenic medium, supplemented with TGF-β3 in pellet culture system and harvested after 21 days. Histological staining was used to evaluate the presence of proteoglycan, with alcian blue. Immunohistochemical method performed for the assessment of cartilage–specific type II collagen and aggrecan. Also, in order to confirm our results, we managed RT-PCR technique.
Chondrogenesis of ADSCs in pellet culture, induced by TGF-β3 growth factor. Histological and immunohistochemical methods showed deposition of typical cartilage extracellular matrix components in pellets. RT-PCR analysis of cartilage matrix genes, such as type II collagen and aggrecan, also, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with TGF-β3. 
TGF-β3 promoted chondrogenesis of ADSC in pellet culture system. We suggest that human subcutaneous adipose stem cells could be excellent candidates for the cartilage tissue engineering.


1.   Anonymous. Prevalence and impact of arthritis by race and ethnicity-US, 1989-1991. Morb Mortal Wkly Rep 1996; 10:373-378.   

2.   Wang Y, Kim UJ, Blasioli DJ, Kim HJ, Kaplan DL. In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffold and mesenchymal stem cells. Biomaterial 2005; 26:7082-7094.

3.  Gillogly SD, Voight M, Blackburn T. Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation. J Orthop Sports Phys Ther 1998; 28:241-251.

4.   Furukawa T, Eyre DR, Koide S, Glimcher MJ. Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee. J Bone Joint Surg Am 1980; 62:79-89.

5.   Mitchell N.  Healing of articular cartilage in intra-articular fractures in rabbits. Clin Orthop Relat Res 2004; 62:628-634.

6.   Tew SR, Kwan AP, Hann A, Thomson BM, Archer CW. The reactions of articular cartilage to experimental wounding: role of apoptosis. Arthrit Rheum 2000; 431:215-225.

7.   Minas T, Nehrer S. Current concepts in the treatment of articular cartilage defects. Orthop 1997; 20:525-538.

8.  Lee JD, Hwang O, Kim SW, Han S. Primary cultured chondrocytes of different origins respond differently to         b-FGF and TGF-ß. J Life Sci 1997; 61:293-299.

9.  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.  Sekiya I, Larson BL, Vuoristo JT, Cui JG, Prockop DJ. Adipogenic differentiation of human adult stem cells from bone marrow stroma (MSCs). J Bone Miner Res  2004; 19:256-264.

11.  Wakitani S, Imoto K, Saito M, Yamamoto T, Kawabata HA. Case report: reconstruction of a damaged knee following treatment of giant cell tumor of the proximal tibia with cryosurgery and cementation. Osteoarthritis Cartilage 2002; 10:402-407.

12.  Horwitz EM, Hanlon AL, Pinover WH, Hanks GE. There a role for short-term hormone use in the treatment of nonmetastatic prostate cancer? Radiat Oncol Investig 1999; 7:249-259.

13.  Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 4, 276:71-74.

14.  Fukumoto T, Sperling JW, Sanyal A, Fitzsimmons JS, Reinholz GG, Conover CA, et al. Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro. Osteoarthritis Cartilage 2003; 11:55-64.

15.  De Bari C, Dell'Accio F, Tylzanowski P, Luyten FP. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 2001; 44:1928-1942.

16.  Cao B,  Zheng B, Jankowski RJ, Kimura S, Ikezawa M, Deasy B, et al. Cummins J, Epperly M, Qu-Petersen Z, Huard J. Muscle stem cells differentiate into hematopoietic lineages but retain myogenic potential. Nat Cell Biol 2003; 5:640-646.

17.  Zuk  PA, Zhu  M, Ashjian P, De Ugarte  DA, Huang  JI, Mizuno H, et al. A  Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002; 13:4279-4295.

18. Young  HE, Steele  TA, Bray  RA, Hudson  J, Floyd  JA, Hawkins  K,  et al. Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 2001; 264:51-62.

19.  Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN. Mesenchymal precursor cells in the blood of normal individuals. Arthrit Res  2000; 2:477-488.

20.   Nöth U, Osyczka AM, Tuli R, Hickok NJ, Danielson KG, Tuan RS. Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J Orthop Res  2002; 20:1060-1069.

21.   Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, Goldberg VM. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 1994; 76:579-592.

22.  Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, Helder MN, Klein-Nulend J, Schouten TE, Ritt MJ, van Milligen FJ. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006; 8:166-177.

23.   Awad HA, Wickham MQ, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials 2004; 25:3211-3222.

24.  Holtzer H, Abbott J, Lash J, Holtzer S. The loss of phenotypic traits by differentiated cells in vitro dedifferentiation of cartilage cells. Proc Natl Acad Sci USA 1960; 46:1533-1542.

25.    Fell HB. Confocal images of marrow stromal (Westen-Bainton) cells. J Morphol Physiol 1925; 40:417-459.       

26.  Majumdar MK, Wang E, Morris EA. BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-1. J Cell Physiol 2001; 189:275-284.

27.   Johnston  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.

28.   Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow.Tissue Eng 1998; 4:415-428.

29.   Minguell JJ, Conget P, Erices A. Biology and clinical utilization of mesenchymal progenitor cells. Braz J Med Biol Res  2000; 33:881-887.

30.  Indrawattana N,Chen G, Tadokoro M, Shann LH, Ohgushi H, Tateishi T, et al.  A growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun  2004; 320:914-919.

31.  Jin XB, Sun YS, Zhang K, Wang J, Ju XD, Lou SQ. Neocartilage formation from predifferentiated human adipose derived stem cells in vivo. Acta Pharmacol Sin 2007; 28:663-671.

32.  Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis  Rheum 2006;54:1222-1232.

33.  Bruder SP, Horowitz MC, Mosca JD, Haynesworth SE. Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone. 1997; 21:225-235.

34.  Pittenger  MF, Mackay  AM, Beck  SC, Jaiswal  RK, Douglas  R, Mosca  JD, et al.  Multilineage potential of adult human mesenchymal stem cells. Science 1999 2; 284:143-147.

35.  Sun S, Guo Z, Xiao X, Liu B, Liu X, Tang PH. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 2003; 21:527-535.

36.  Eslaminejad MB, Nikmahzar A, Taghiyar L, Nadri S, Massumi M. Murine  mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ 2006; 48:361-370.

37.  Sekiya I, Colter DC, Prockop DJ. BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem Biophys Res Commun 2001; 284:411-418.

38.  Sekiya I, Larson BL, Vuoristo JT, Reger RL, Prockop DJ. Comparison of effect of  BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res 2005; 320:269-276.

39.  Lee  RH, Kim  BC, Choi  IS, Kim  H, Choi  HS, Suh  KT. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose. Tissue Cell Physiol Biochem 2004; 14:311-324.

40.  Erickson GR, Gimble JM, Franklin DM, Rice HE,Awad  H, Guilak  F. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 2002; 290:763-769.