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Generation of Pluripotent Stem Cells and Their Differentiation to the Chondrocytic Phenotype

Protocol
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 100)

Abstract

It is well documented that adult cartilage has minimal self-repair ability. Current methods for treatment of cartilage injury focus on the relief of pain and inflammation and have met with limited long-term success. In the forefront of new therapeutic approaches, autologous chondrocyte transplantation is still only applied to a very small percentage of the patient population.

Our laboratory has focused on cartilage repair using progenitor cells and studied their differentiation into cartilage. Adult mesenchymal stem cells are an attractive candidate as progenitor cells for cartilage repair because of their documented osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture; furthermore, their use will obviate the need for harvesting precious healthy cartilage from a patient to obtain autologous chondrocytes for transplantation. However, the need to induce chondrogenic differentiation in the mesenchymal stem cells is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes.

This chapter focuses on the methods involved in the isolation of human mesenchymal stem cells and their differentiation along the chondrogenic lineage. Although we have the technology to accomplish chondrogenic differentiation of stem cells, much is still to be learned regarding the regulatory mechanisms controlling the lineage transitions and maturation of the cartilaginous tissue.

Key Words

Mesenchymal stem cell chondrogenesis differentiation tissue culture transforming growth factor-β cartilage chondrocyte aggregate culture 

References

  1. 1.
    Langer, R. and Vacanti, J. P. (1993) Tissue engineering. Science 260, 920–926.CrossRefPubMedGoogle Scholar
  2. 2.
    Vacanti, C. A. and Vacanti, J. P. (2000) The science of tissue engineering. Orthop. Clin. North Am. 31, 351–356.CrossRefPubMedGoogle Scholar
  3. 3.
    Vacanti, J. and Langer, R. (1999) Tissue engineering: the design and fabrication of living replacement devices for surgical re-construction and transplantation. Lancet 354(Suppl. 1), SI32–SI34.PubMedGoogle Scholar
  4. 4.
    Bonassar, L. J. and Vacanti, C. A. (1998) Tissue engineering: the first decade and beyond. J. Cell Biochem. Suppl. 30-31, 297–303.CrossRefPubMedGoogle Scholar
  5. 5.
    Pearson, R. G., Bhandari, R., Quirk, R. A., and Shakesheff, K. M. (2002) Recent advances in tissue engineering: an invited review. J. Long Term Eff. Med. Implants 12, 1–33.PubMedGoogle Scholar
  6. 6.
    Temenoff, J. S. and Mikos, A. G. (2000) Review: tissue engineering for regeneration of articular cartilage. Biomaterials 21, 431–440.CrossRefPubMedGoogle Scholar
  7. 7.
    O’Driscoll, S. W. (2001) Preclinical cartilage repair: current status and future perspectives. Clin. Orthop. 391(Suppl.), S397–S401.PubMedGoogle Scholar
  8. 8.
    Luyten, F. P., Dell’Accio, F., and De Bari, C. (2001) Skeletal tissue engineering: opportunities and challenges. Best Pract. Res. Clin. Rheumatol. 15, 759–769.CrossRefPubMedGoogle Scholar
  9. 9.
    Musgrave, D. S., Fu, F. H., and Huard, J. (2002) Gene therapy and tissue engineering in orthopaedic surgery. J. Am. Acad. Orthop. Surg. 10, 6–15.PubMedGoogle Scholar
  10. 10.
    Risbud, M. (2001) Tissue engineering: implications in the treatment of organ and tissue defects. Biogerontology 2, 117–125.CrossRefPubMedGoogle Scholar
  11. 11.
    Guilak, F., Butler, D. L., and Goldstein, S. A. (2001) Functional tissue engineering: the role of biomechanics in articular cartilage repair. Clin. Orthop. 391(Suppl.), S295–S305.PubMedGoogle Scholar
  12. 12.
    Risbud, M. V. and Sittinger, M. (2002) Tissue engineering: advances in in vitro cartilage generation. Trends Biotechnol. 20, 351–356.CrossRefPubMedGoogle Scholar
  13. 13.
    Caplan, A. I. and Goldberg, V. M. (1999) Principles of tissue engineered regeneration of skeletal tissues. Clin. Orthop. 367(Suppl.), S12–S16.PubMedGoogle Scholar
  14. 14.
    Caplan, A. I., Elyaderani, M., Mochizuki, Y., Wakitani, S., and Goldberg, V. M. (1997) Principles of cartilage repair and regeneration. Clin. Orthop. Rel. Res. 342, 254–269.CrossRefGoogle Scholar
  15. 15.
    Buckwalter, J. (1998) Articular cartilage: injuries and potential for healing. J. Orthop. Sports Phys. Ther. 28, 192–202.PubMedGoogle Scholar
  16. 16.
    Buckwalter, J. and Mankin, H. (1998) Articular cartilage repair and transplantation. Arthritis Rheum. 41, 1331–1342.CrossRefPubMedGoogle Scholar
  17. 17.
    Buckwalter, J. A. and Mankin, H. J. (1998) Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr. Course Lect. 47, 487–504.PubMedGoogle Scholar
  18. 18.
    Mankin, H. J. (1974) The reaction of articular cartilage to injury and osteoarthritis (first of two parts) N. Engl. J. Med. 291, 1285–1292.CrossRefPubMedGoogle Scholar
  19. 19.
    Mankin, H. J. (1974) The reaction of articular cartilage to injury and osteoarthritis (second of two parts) N. Engl. J. Med. 291, 1335–1340.CrossRefPubMedGoogle Scholar
  20. 20.
    Mankin, H. J. and Buckwalter, J. A. (1996) Restoration of the osteoarthrotic joint [editorial]. J. Bone Joint Surg. Am. 78, 1–2.PubMedGoogle Scholar
  21. 21.
    Chesterman, P. J. and Smith, A. U. (1968) Homotransplantation of articular cartilage and isolated chondrocytes. An experimental study in rabbits. J. Bone Joint Surg. Br. 50, 184–197.PubMedGoogle Scholar
  22. 22.
    Bentley, G. and Greer, R. B. d. (1971) Homotransplantation of isolated epiphyseal and articular cartilage chondrocytes into joint surfaces of rabbits. Nature 230, 385–388.CrossRefPubMedGoogle Scholar
  23. 23.
    Wakitani, S., Kimura, T., Hirooka, A., et al. (1989) Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel. J. Bone Joint Surg. Br. 71, 74–80.PubMedGoogle Scholar
  24. 24.
    Vacanti, C. A., Kim, W., Schloo, B., Upton, J., and Vacanti, J. P. (1994) Joint resurfacing with cartilage grown in situ from cell-polymer structures. Am. J. Sports Med. 22, 485–488.CrossRefPubMedGoogle Scholar
  25. 25.
    Freed, L. E., Grande, D. A., Lingbin, Z., Emmanual, J., Marquis, J. C., and Langer, R. (1994) Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J. Biomed. Mater. Res. 28, 891–899.CrossRefPubMedGoogle Scholar
  26. 26.
    Frenkel, S. R., Toolan, B., Menche, D., Pitman, M. I., and Pachence, J. M. (1997) Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair. J. Bone Joint Surg. Br. 79, 831–836.CrossRefPubMedGoogle Scholar
  27. 27.
    Nehrer, S., Breinan, H. A., Ramappa, A., et al. (1997) Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro. [erratum appears in (1997) J. Biomed. Mater. Res. 38(4), 288]. J. Biomed. Mater. Res. 38, 95–104.CrossRefPubMedGoogle Scholar
  28. 28.
    Breinan, H. A., Minas, T., Hsu, H. P., Nehrer, S., Sledge, C. B., and Spector, M. (1997) Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model. J. Bone Joint Surg. Am. 79, 1439–1451.PubMedGoogle Scholar
  29. 29.
    Minas, T. (1998) Chondrocyte implantation in the repair of chondral lesions of the knee: economics and quality of life. Am. J. Orthop. 27, 739–744.PubMedGoogle Scholar
  30. 30.
    Giannini, S., Buda, R., Grigolo, B., and Vannini, F. (2001) Autologous chondrocyte transplantation in osteochondral lesions of the ankle joint. Foot Ankle Int. 2, 513–517.Google Scholar
  31. 31.
    Breinan, H. A., Minas, T., Hsu, H. P., Nehrer, S., Shortkroff, S., and Spector, M. (2001) Autologous chondrocyte implantation in a canine model: change in composition of reparative tissue with time. J. Orthop. Res. 19, 482–492.CrossRefPubMedGoogle Scholar
  32. 32.
    Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O., and Peterson, L. (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N. Engl. J. Med. 331, 889–895.CrossRefPubMedGoogle Scholar
  33. 33.
    Amiel, D., Coutts, R. D., Abel, M., Stewart, W., Harwood, F., and Akeson, W. H. (1985) Rib perichondrial grafts for the repair of full-thickness articular-cartilage defects. A morphological and biochemical study in rabbits. J. Bone Joint Surg. Am. 67, 911–920.PubMedGoogle Scholar
  34. 34.
    O’Driscoll, S. W., Keeley, F. W., and Salter, R. B. (1986) The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit. J. Bone Joint Surg. Am. 68, 1017–1035.PubMedGoogle Scholar
  35. 35.
    Amiel, D., Coutts, R. D., Harwood, F. L., Ishizue, K. K., and Kleiner, J. B. (1988) The chondrogenesis of rib perichondrial grafts for repair of full thickness articular cartilage defects in a rabbit model: a one year postoperative assessment. Connect. Tissue Res. 18, 27–39.CrossRefPubMedGoogle Scholar
  36. 36.
    Shahgaldi, B. F., Amis, A. A., Heatley, F. W., McDowell, J., and Bentley, G. (1991) Repair of cartilage lesions using biological implants. A comparative histological and biomechanical study in goats. J. Bone Joint Surg. Br. 73, 57–64.PubMedGoogle Scholar
  37. 37.
    Wakitani, S., Goto, T., Pineda, S. J., et al. (1994) Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 76, 579–592.PubMedGoogle Scholar
  38. 38.
    Wakitani, S., Goto, T., Pined, S. J., et al. (1994) Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 76, 579–592.PubMedGoogle Scholar
  39. 39.
    Chu, C. R., Coutts, R. D., Yoshioka, M., Harwood, F. L., Monosov, A. Z., and Amiel, D. (1995) Articular cartilage repair using allogeneic perichondrocyteseeded biodegradable porous polylactic acid (PLA): a tissue-engineering study. J. Biomed. Mater. Res. 29, 1147–1154.CrossRefPubMedGoogle Scholar
  40. 40.
    Butnariu-Ephrat, M., Robinson, D., Mendes, D. G., Halperin, N., and Nevo, Z. (1996) Resurfacing of goat articular cartilage by chondrocytes derived from bone marrow. Clin. Orthop. 330, 234–243.CrossRefPubMedGoogle Scholar
  41. 41.
    Hunziker, E. B. and Rosenberg, L. C. (1996) Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. J. Bone Joint Surg. Am. 78, 721–733.PubMedGoogle Scholar
  42. 42.
    Chu, C. R., Dounchis, J. S., Yoshioka, M., Sah, R. L., Coutts, R. D., and Amiel, D. (1997) Osteochondral repair using perichondrial cells. A 1-year study in rabbits. Clin. Orthop. 340, 220–229.CrossRefPubMedGoogle Scholar
  43. 43.
    Goldberg, V. M., Solchaga, L. A., Lundberg, M., et al. (1999) Mesenchymal stem cell repair of osteochondral defects of articular cartilage. Semin. Arthroplasty 10, 30–36.Google Scholar
  44. 44.
    Johnstone, B. and Yoo, J. U. (1999) Autologous mesenchymal progenitor cells in articular cartilage repair. Clin. Orthop. 367(Suppl.), S156–S162.PubMedGoogle Scholar
  45. 45.
    Dounchis, J. S., Bae, W. C., Chen, A. C., Sah, R. L., Coutts, R. D., and Amiel, D. (2000) Cartilage repair with autogenic perichondrium cell and polylactic acid grafts. Clin. Orthop. 377, 248–264.CrossRefPubMedGoogle Scholar
  46. 46.
    Solchaga, L. A., Gao, J., Dennis, J. E., et al. (2002) Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle. Tissue Eng. 8, 333–347.CrossRefPubMedGoogle Scholar
  47. 47.
    Friedenstein, A. J. (1976) Precursor cells of mechanocytes. Int. Rev. Cytol. 47, 327–359.CrossRefPubMedGoogle Scholar
  48. 48.
    Caplan, A. I. (1991) Mesenchymal stem cells. J. Orthop. Res. 9, 641–650.CrossRefPubMedGoogle Scholar
  49. 49.
    Haynesworth, S. E., Goshima, J., Goldberg, V. M., and Caplan, A. I. (1992) Characterization of cells with osteogenic potential from human bone marrow. Bone 13, 81–88.CrossRefPubMedGoogle Scholar
  50. 50.
    Ashton, B. A., Allen, T. D., Howlett, C. R., Eaglesom, C. C., Hattori, A., and Owen, M. (1980) Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin. Orthop. 151, 294–307.PubMedGoogle Scholar
  51. 51.
    Johnstone, B., Hering, T. M., Caplan, A. I., Goldberg, V. M., and Yoo, J. U. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272.CrossRefPubMedGoogle Scholar
  52. 52.
    Yoo, J. U., Barthel, T. S., Nishimura, K., et al. (1998) The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J. Bone Joint Surg. Am. 80, 1745–1757.PubMedGoogle Scholar
  53. 53.
    Angele, P., Yoo, J. U. Smith, C., et al. (2000) Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro. J. Orthop. Res. 21, 451–457.CrossRefGoogle Scholar
  54. 54.
    Hanada, K., Solchaga, L. A., Caplan, A. I., et al. (2001) BMP-2 induction and TGF-β1 modulation of rat periosteal cell chondrogenesis. J. Cell Biochem. 81, 284–294.CrossRefPubMedGoogle Scholar
  55. 55.
    Lennon, D. P., Haynesworth, S. E., Bruder, S. P., Jaiswal, N., and Caplan, A. I. (1996) Human and animal mesenchymal progenitor cells from bone marrow: identification of serum for optimal selection and proliferation. In Vitro Cell. Dev. Biol. 32, 602–611.CrossRefGoogle Scholar
  56. 56.
    Jaiswal, N., Haynesworth, S. E., Caplan, A. I., and Bruder, S. P. (1997) Osteogenic differentiation of purified culture-expanded human mesenchymal stem cells in vitro. J. Cell. Biochem. 64, 295–312.CrossRefPubMedGoogle Scholar
  57. 57.
    Ballock, R. T. and Reddi, A. H. (1994) Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J. Cell Biol. 126, 1311–1318.CrossRefPubMedGoogle Scholar
  58. 58.
    Pittenger, M. F., Mackay, A. M., Beck, S. C., et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  1. 1.Department of OrthopaedicsCase Western Reserve University and University Hospitals of ClevelandCleveland
  2. 2.Skeletal Research Center, Department of BiologyCase Western Reserve UniversityCleveland

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