High-Throughput Screening for Modulators of Mesenchymal Stem Cell Chondrogenesis

  • Alice H. Huang
  • Nuzhat A. Motlekar
  • Ashley Stein
  • Scott L. Diamond
  • Eileen M. Shore
  • Robert L. Mauck


Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and the study of skeletal development. Despite considerable interest in MSC chondrogenesis, the signal transduction and molecular mechanisms underlying this process remain largely undefined. To explore the signaling topology regulating chondrogenic differentiation, as well as to discover novel modulators, we developed and validated a high-throughput screening (HTS) assay for MSC chondrogenesis. Adapting standard assay procedures to enable HTS, we successfully minimized cell number, handling, and culture duration. Using our optimized methodology with automation, we evaluated a comprehensive screen using four growth factors, TGF-β3, BMP-2, IGF-1, and FGF-2, to demonstrate the feasibility of large combinatorial screens. We examined the chondrogenic effects of these growth factors in different combinations and doses (81 combinations total with 16 replicates per group) and found variable effects on GAG content with different combinations. In general, TGF-β3 had a pro-chondrogenic effect while FGF-2 had a proliferative effect. BMP-2 was both proliferative and pro-chondrogenic while the effect of IGF-1 in our system was variable. We also carried out an HTS campaign of the National Institute of Neurological Disorders and Stroke (NINDS) chemical library of small molecules (1040 compounds) and identified 5 potential inducers and 24 potential inhibitors of chondrogenesis. Of these compounds, several were identified from the hypnotic, anti-neoplastic, or anti-protein synthesis classes of molecules. These studies demonstrate our ability to carry out high-throughput screening assays for modulators of chondrogenesis.


Cartilage Chondrogenesis High-throughput screening Mesenchymal stem cells Tissue engineering 

Supplementary material

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  1. 1.
    Adamson L. F., C. S. Anast 1966 Amino acid, potassium, and sulfate transport and incorporation by embryonic chick cartilage: the mechanism of the stimulatory effects of serum. Biochim. Biophys. Acta 121(1):10–20PubMedGoogle Scholar
  2. 2.
    Angele P., R. Kujat, M. Nerlich, J. Yoo, V. Goldberg, B. Johnstone 1999 Engineering of osteochondral tissue with bone marrow mesenchymal progenitor cells in a derivatized hyaluronan-gelatin composite sponge. Tissue Eng. 5(6):545–554 doi:10.1089/ten.1999.5.545 PubMedCrossRefGoogle Scholar
  3. 3.
    Antoci V. Jr., C. S. Adams, N. J. Hickok, I. M. Shapiro, J. Parvizi 2007 Antibiotics for local delivery systems cause skeletal cell toxicity in vitro. Clin. Orthop. Relat. Res. 462, 200–206 doi:10.1097/BLO.0b013e31811ff866 PubMedCrossRefGoogle Scholar
  4. 4.
    Audhya T. K., K. D. Gibson 1976 Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine. Biochim. Biophys. Acta 437(2):364–376PubMedGoogle Scholar
  5. 5.
    Awad H. A., Y. D. Halvorsen, J. M. Gimble, F. Guilak 2003 Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells. Tissue Eng. 9(6):1301–1312 doi:10.1089/10763270360728215 PubMedCrossRefGoogle Scholar
  6. 6.
    Barry F., R. E. Boynton, B. Liu, J. M. Murphy 2001 Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp. Cell Res. 268(2):189–200. doi:10.1006/excr.2001.5278 PubMedCrossRefGoogle Scholar
  7. 7.
    Brown P. D., P. D. Benya 1988 Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin b-induced reexpression. J. Cell Biol. 106(1):171–179. doi:10.1083/jcb.106.1.171 PubMedCrossRefGoogle Scholar
  8. 8.
    Caplan, A. I. 1991 Mesenchymal stem cells. J. Orthop. Res. 9(5):641–650 doi:10.1002/jor.1100090504 PubMedCrossRefGoogle Scholar
  9. 9.
    Derfoul A., A. D. Miyoshi, D. E. Freeman, R. S. Tuan 2007 Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits mmp-13 expression and matrix degradation. Osteoarthr. Cartil. 15(6):646–655 doi:10.1016/j.joca.2007.01.014 PubMedCrossRefGoogle Scholar
  10. 10.
    Estes B. T., A. W. Wu, F. Guilak 2006 Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis Rheum. 54(4):1222–1232 doi:10.1002/art.21779 PubMedCrossRefGoogle Scholar
  11. 11.
    Farndale R. W., D. J. Buttle, A. J. Barrett 1986 Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 883(2):173–177PubMedGoogle Scholar
  12. 12.
    Huang C. Y., K. L. Hagar, L. E. Frost, Y. Sun, H. S. Cheung 2004 Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem Cells 22(3):313–323. doi:10.1634/stemcells.22–3-313 PubMedCrossRefGoogle Scholar
  13. 13.
    Huang A. H., M. Yeger-McKeever, A. Stein, R. L. Mauck 2008 Tensile properties of engineered cartilage formed from chondrocyte- and msc-laden hydrogels. Osteoarthr. Cartil. 16:1074–1082PubMedCrossRefGoogle Scholar
  14. 14.
    Huddleston P. M., J. M. Steckelberg, A. D. Hanssen, M. S. Rouse, M. E. Bolander, R. Patel 2000 Ciprofloxacin inhibition of experimental fracture healing. J. Bone Joint Surg. Am. 82(2):161–173PubMedGoogle Scholar
  15. 15.
    Im G. I., N. H. Jung, S. K. Tae 2006 Chondrogenic differentiation of mesenchymal stem cells isolated from patients in late adulthood: the optimal conditions of growth factors. Tissue Eng. 12(3):527–536. doi:10.1089/ten.2006.12.527 PubMedCrossRefGoogle Scholar
  16. 16.
    Indrawattana N., G. Chen, M. Tadokoro, L. H. Shann, H. Ohgushi, T. Tateishi, J. Tanaka, A. Bunyaratvej 2004 Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem. Biophys. Res. Commun. 320(3):914–919. doi:10.1016/j.bbrc.2004.06.029 PubMedCrossRefGoogle Scholar
  17. 17.
    Kafienah W., S. Mistry, M. J. Perry, G. Politopoulou, A. P. Hollander 2007 Pharmacological regulation of adult stem cells: chondrogenesis can be induced using a synthetic inhibitor of the retinoic acid receptor. Stem Cells 25(10):2460–2468 doi:10.1634/stemcells.2007-0059 PubMedCrossRefGoogle Scholar
  18. 18.
    Li W. J., R. Tuli, X. Huang, P. Laquerriere, R. S. Tuan 2005 Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 26(25):5158–5166 doi:10.1016/j.biomaterials.2005.01.002 PubMedCrossRefGoogle Scholar
  19. 19.
    Longobardi L., L. O’Rear, S. Aakula, B. Johnstone, K. Shimer, A. Chytil, W. A. Horton, H. L. Moses, A. Spagnoli 2006 Effect of igf-i in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of tgf-beta signaling. J. Bone Miner. Res. 21(4):626–636. doi:10.1359/jbmr.051213 PubMedCrossRefGoogle Scholar
  20. 20.
    Majumdar M. K., E. Wang, E. A. Morris 2001 Bmp-2 and bmp-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of il-1. J. Cell Physiol. 189(3):275–284. doi:10.1002/jcp.10025 PubMedCrossRefGoogle Scholar
  21. 21.
    Mauck R. L., B. A. Byers, X. Yuan, R. S. Tuan 2007 Regulation of cartilaginous ecm gene transcription by chondrocytes and mscs in 3d culture in response to dynamic loading. Biomech. Model Mechanobiol. 6(1–2):113–125. doi:10.1007/s10237-006-0042-1 PubMedCrossRefGoogle Scholar
  22. 22.
    Mauck R. L., X. Yuan, R. S. Tuan 2006 Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture. Osteoarthr. Cartil. 14(2):179–189. doi:10.1016/j.joca.2005.09.002 PubMedCrossRefGoogle Scholar
  23. 23.
    McMahon L. A., P. J. Prendergast, V. A. Campbell 2008 A comparison of the involvement of p38, erk1/2 and pi3k in growth factor-induced chondrogenic differentiation of mesenchymal stem cells. Biochem. Biophys. Res. Commun. 368(4):990–995PubMedGoogle Scholar
  24. 24.
    Mueller M. B., R. S. Tuan 2008 Functional characterization of hypertrophy in chondrogenesis of human mesenchymal stem cells. Arthritis Rheum. 58(5):1377–1388. doi:10.1002/art.23370 PubMedCrossRefGoogle Scholar
  25. 25.
    Murphy J. M., K. Dixon, S. Beck, D. Fabian, A. Feldman, F. Barry 2002 Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum. 46(3):704–713. doi:10.1002/art.10118 PubMedCrossRefGoogle Scholar
  26. 26.
    Palmer G. D., A. Steinert, A. Pascher, E. Gouze, J. N. Gouze, O. Betz, B. Johnstone, C. H. Evans, S. C. Ghivizzani 2005 Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro. Mol. Ther. 12(2):219–228. doi:10.1016/j.ymthe.2005.03.024 PubMedCrossRefGoogle Scholar
  27. 27.
    Penick K. J., L. A. Solchaga, J. F. Welter 2005 High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells. Biotechniques 39(5):687–691PubMedGoogle Scholar
  28. 28.
    Pittenger M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, D. R. Marshak 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147. doi:10.1126/science.284.5411.143 PubMedCrossRefGoogle Scholar
  29. 29.
    Prockop D. J. 1997 Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276(5309):71–74. doi:10.1126/science.276.5309.71 PubMedCrossRefGoogle Scholar
  30. 30.
    Reginster J. Y., R. Deroisy, L. C. Rovati, R. L. Lee, E. Lejeune, O. Bruyere, G. Giacovelli, Y. Henrotin, J. E. Dacre, C. Gossett 2001 Long-term effects of glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial. Lancet 357(9252):251–256. doi:10.1016/S0140-6736(00)03610-2 PubMedCrossRefGoogle Scholar
  31. 31.
    Schmitt B., J. Ringe, T. Haupl, M. Notter, R. Manz, G. R. Burmester, M. Sittinger, C. Kaps 2003 Bmp2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. Differentiation 71(9–10):567–577. doi:10.1111/j.1432-0436.2003.07109003.x PubMedCrossRefGoogle Scholar
  32. 32.
    Solchaga L. A., K. Penick, J. D. Porter, V. M. Goldberg, A. I. Caplan, J. F. Welter 2005 Fgf-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J. Cell Physiol. 203(2):398–409. doi:10.1002/jcp.20238 PubMedCrossRefGoogle Scholar
  33. 33.
    Song L., R. S. Tuan 2004 Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J. 18(9):980–982PubMedGoogle Scholar
  34. 34.
    Stewart A. A., C. R. Byron, H. Pondenis, M. C. Stewart 2007 Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am. J. Vet. Res. 68(9):941–945. doi:10.2460/ajvr.68.9.941 PubMedCrossRefGoogle Scholar
  35. 35.
    Toh W. S., H. Liu, B. C. Heng, A. J. Rufaihah, C. P. Ye, T. Cao 2005 Combined effects of tgfbeta1 and bmp2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation. Growth Factors 23(4):313–321. doi:10.1080/08977190500252763 PubMedCrossRefGoogle Scholar
  36. 36.
    Tuli R., M. R. Seghatoleslami, S. Tuli, M. S. Howard, K. G. Danielson, R. S. Tuan 2002 P38 map kinase regulation of ap-2 binding in tgf-beta1-stimulated chondrogenesis of human trabecular bone-derived cells. Ann. N. Y. Acad. Sci. 96, 172–177CrossRefGoogle Scholar
  37. 37.
    Tuli R., S. Tuli, S. Nandi, X. Huang, P. A. Manner, W. J. Hozack, K. G. Danielson, D. J. Hall, R. S. Tuan 2003 Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves n-cadherin and mitogen-activated protein kinase and wnt signaling cross-talk. J. Biol. Chem. 278(42):41227–41236. doi:10.1074/jbc.M305312200 PubMedCrossRefGoogle Scholar
  38. 38.
    Verbruggen G. 2006 Chondroprotective drugs in degenerative joint diseases. Rheumatology (Oxford) 45(2):129–138. doi:10.1093/rheumatology/kei171 CrossRefGoogle Scholar
  39. 39.
    Walters W. P., M. Namchuk 2003 Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2(4):259–266. doi:10.1038/nrd1063 PubMedCrossRefGoogle Scholar
  40. 40.
    Welter, J. F., L. A. Solchaga, and K. J. Penick. Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay. Biotechniques 42(6):732, 734–737, 2007Google Scholar
  41. 41.
    Woods A., F. Beier 2006 Rhoa/rock signaling regulates chondrogenesis in a context-dependent manner. J. Biol. Chem. 281(19):13134–13140. doi:10.1074/jbc.M509433200 PubMedCrossRefGoogle Scholar
  42. 42.
    Woods A., G. Wang, F. Beier 2005 Rhoa/rock signaling regulates sox9 expression and actin organization during chondrogenesis. J. Biol. Chem. 280(12):11626–11634. doi:10.1074/jbc.M409158200 PubMedCrossRefGoogle Scholar
  43. 43.
    Woods A., G. Wang, H. Dupuis, Z. Shao, F. Beier 2007 Rac1 signaling stimulates n-cadherin expression, mesenchymal condensation, and chondrogenesis. J. Biol. Chem. 282(32):23500–23508. doi:10.1074/jbc.M700680200 PubMedCrossRefGoogle Scholar
  44. 44.
    Wu X., S. Ding, Q. Ding, N. S. Gray, P. G. Schultz 2002 A small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells. J. Am. Chem. Soc. 124(49):14520–14521. doi:10.1021/ja0283908 PubMedCrossRefGoogle Scholar
  45. 45.
    Yu P. B., C. C. Hong, C. Sachidanandan, J. L. Babitt, D. Y. Deng, S. A. Hoyng, H. Y. Lin, K. D. Bloch, R. T. Peterson 2008 Dorsomorphin inhibits bmp signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4(1):33–41. doi:10.1038/nchembio.2007.54 PubMedCrossRefGoogle Scholar
  46. 46.
    Zhang J. H., T. D. Chung, K. R. Oldenburg 1999 A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4(2):67–73. doi:10.1177/108705719900400206 PubMedCrossRefGoogle Scholar
  47. 47.
    Zhang Z., J. Messana, N. S. Hwang, J. H. Elisseeff 2006 Reorganization of actin filaments enhances chondrogenic differentiation of cells derived from murine embryonic stem cells. Biochem. Biophys. Res. Commun. 348(2):421–427. doi:10.1016/j.bbrc.2006.07.073 PubMedCrossRefGoogle Scholar
  48. 48.
    Zhao Y., S. Ding 2007 A high-throughput sirna library screen identifies osteogenic suppressors in human mesenchymal stem cells. Proc. Natl. Acad. Sci. USA 104(23):9673–9678. doi:10.1073/pnas.0703407104 PubMedCrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2008

Authors and Affiliations

  • Alice H. Huang
    • 1
    • 2
  • Nuzhat A. Motlekar
    • 3
  • Ashley Stein
    • 1
  • Scott L. Diamond
    • 2
    • 3
  • Eileen M. Shore
    • 1
    • 4
  • Robert L. Mauck
    • 1
    • 2
  1. 1.McKay Orthopaedic Research Laboratory, Department of Orthopaedic SurgeryUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of BioengineeringUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Penn Center for Molecular DiscoveryUniversity of PennsylvaniaPhiladelphiaUSA
  4. 4.Department of GeneticsUniversity of PennsylvaniaPhiladelphiaUSA

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