Mesenchymal Stem Cell Purification from the Articular Cartilage Cell Culture

Document Type: Original Article

Authors

Stem Cell Department, Royan Institute, Tehran, Iran

Abstract

Objective
Articular cartilage as an avascular skeletal tissue possesses limited capacity to heal. On the other hand, it is believed that the regeneration capacity of each tissue is largely related to its stem cell contents. Little is known about the presence of mesenchymal stem cells in articular cartilage tissue. This subject is investigated in the present study.
Materials and Methods
Articular cartilage was obtained from rats of Wistar strain and subjected to enzymatic digestion using 1:1 ratio of 0.2% collagenase I and 0.1% pronase. The released cells were then collected and cultivated in        25 cm2 culture flask, which was resulted in the appearance of two morphologically distinct cell populations. Fibroblastic population were purified and expanded through several subcultures, using their loose adherence on culture surfaces and examined in terms of differentiation potential.
Results
The primary culture mainly contained polygonal cells with few fibroblastic cells among them. Upon the first subculture, the fibroblastic cells appeared as aggregates of few cells among the polygonal cartilage cells. It was found that the fibroblastic cell population could easily detach from the culture surface following to a brief exposure to tripsin solution. Using this property, these cells purified and expanded by several subcultures. According to the results, the isolated cells were able to easily differentiate into chondrocytic, osteocytic and adipocytic cell lineages.
Conclusion
It seems that articular cartilage contains a distinct fibroblastic cell population with mesenchymal stem cell nature. These cells can be purified and expanded by cell culture methods, thanks to their loose adherence on the culture dish surfaces.    

Keywords


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

2. Conget PA, Miguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999; 181: 67-73.

3. Caplan AI. Mesenchymal stems cells. J Orthop Res 1991; 9:641-650.

4. 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.

5. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone marrow as a potential source of hepatic oval cells.  Science 1999; 284: 1168-1170.

6. Pousom R, Alison MR, cook T, Jeffery R, Ryan E, Forbes SJ, et al.one marrow stem cells contribute to healing of the kidney. J Am Soc Nephrol 2003; 14: 48S-54S.

7. Orlic D, Kajstura J, Chimenti S, Jakoniuk J, Anderson SM, Li B, Pickel J, et al . Bone marrow cells regenerate infracted myocardium. Nature 2001;  410:701-705.

8. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000; 290: 1770-1782.

9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 248: 143-147.

10. Martin DR, Cox NR, Hathcock TL, Niemeyer GP, Baker HJ. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp Hematol 2002; 30: 879-886.

11. Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 1997; 6:125-134.

12. Ringe J, Kaps C, Schmitt B, Buscher K, Bartel J, Smolian H, et al. Porcine mesenchymal stem cells: Induction of distinct mesenchymal cell lineages. Cell Tissue Res 2002; 307: 321-327.

13. Eslaminejad MB, Nadri S, Hosseini RH. Expression of thy 1.2 surface antigen increases significantly during the murine mesenchymal stem cells cultivation period. Dev Growth Differ 2007; 49: 351-364.

14.          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.

15.          Petite H, Viateau V, Bensaid W, Meunier A, Depollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nat Biotechnol 2000; 18: 959-963.

16. Eslaminejed MB, Mirzadeh H, Mohamadi Y, Nickmahzar A. Bone differentiation of the marrow-derived mesenchymal stem cells using tricalcium phosphate/alginate/gelatine scaffolds.  J Tissue Engin Regen Med 2007; in press.

17. Noth 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. 

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

19.  Nakahara H, Bruder SP, Haynesworth SE, Holecek JJ, Baber MA, et al. Bone and cartilage formation in diffusion chambers by subcultured cells derived from the periosteum. Bone 1990; 11:181-188.

20.  Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol 2001; 189:54-63.

21.  Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: Stem cells from human exfoliated deciduous teeth.  Proc Natl Acad Sci  2003; 1013:5807-5812. 

22.   Jankowski RJ, Deasy BM, Huard J.  Muscle-derived stem cells. Gene Ther 2002; 9:642-647. 

23.  Fickert S, Fiedler J, Brenner RE. Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers. Arthrit Res Ther 2004; 6: R422-R432.

24.  Tallhheden T, Dennis JE, Lennon DP, Sjogern-Jansson DDSE, Caplan AI, Lindahl A. Phenotypic plasticity of human articular chondrocytes. J Bone Joint Surg 2003; 85: 93-100.

25. Barbero A, Ploegert S, Herberer M, Martin I. Plasticity of clonal populations of dedifferentiated adult human articular chondrocyte. Am Colleg Rheum 2003; 5: 1315-1325.

26.  Kuçi S, Wessels JT, Bühring HJ, Schilbach K, Schumm M, Seitz G, et al. Identification of a novel class of human adherent CD34- stem cells that give rise to SCID-repopulating cells. Blood 2003; 3:869-876.

27. Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.  Exp Hematol 2002; 7:783-791.

28. Gronthose S, Simmons PJ. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-derived conditions in vitro. Blood 1995; 85: 929-940.

29.   Minguell JJ, Erices A, Conget P.  Mesenchymal stem cells.  Exp Biol Med 2001; 226: 507-520.