Stem Cell Reviews and Reports

, Volume 11, Issue 2, pp 228–241 | Cite as

Synergistic Effects of Hypoxia and Morphogenetic Factors on Early Chondrogenic Commitment of Human Embryonic Stem Cells in Embryoid Body Culture

  • Supansa Yodmuang
  • Darja Marolt
  • Ivan Marcos-Campos
  • Ivana Gadjanski
  • Gordana Vunjak-NovakovicEmail author


Derivation of articular chondrocytes from human stem cells would advance our current understanding of chondrogenesis, and accelerate development of new stem cell therapies for cartilage repair. Chondrogenic differentiation of human embryonic stem cells (hESCs) has been studied using supplemental and cell-secreted morphogenetic factors. The use of bioreactors enabled insights into the effects of physical forces and controlled oxygen tension. In this study, we investigated the interactive effects of controlled variation of oxygen tension and chondrocyte-secreted morphogenetic factors on chondrogenic differentiation of hESCs in the embryoid body format (hESC-EB). Transient hypoxic culture (2 weeks at 5 % O2 followed by 1 week at 21 % O2) of hESC-EBs in medium conditioned with primary chondrocytes up-regulated the expression of SOX9 and suppressed pluripotent markers OCT4 and NANOG. Pellets derived from these cells showed significant up-regulation of chondrogenic genes (SOX9, COL2A1, ACAN) and enhanced production of cartilaginous matrix (collagen type II and proteoglycan) as compared to the pellets from hESC-EBs cultured under normoxic conditions. Gene expression profiles corresponded to those associated with native cartilage development, with early expression of N-cadherin (indicator of cell condensation) and late expression of aggrecan (ACAN, indicator of proteoglycan production). When implanted into highly vascularized subcutaneous area in immunocompromised mice for 4 weeks, pellets remained phenotypically stable and consisted of cartilaginous extracellular matrix (ECM), without evidence of dedifferentiation or teratoma formation. Based on these results, we propose that chondrogenesis in hESC can be synergistically enhanced by a control of oxygen tension and morphogenetic factors secreted by chondrocytes.


Human embryonic stem cells Hypoxia Chondrogenic differentiation Embryoid body Morphogenetic factors 



We gratefully acknowledge research funding received from the NIH (grants DE016525, EB002520 and AR061988 to GVN), New York Stem Cell Foundation (NYSCF-Helmsley Investigator award to DM; grant CU09-3055 to GVN), the Ministry of Education and Science of Serbia (grants ON174028 and III41007 to IG) and Royal Thai Graduate Fellowship (to SY). We thank Dr. Jenny Yuan and Chandhanarat Chandhanayingyong for their help with animal studies.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary material

12015_2015_9584_MOESM1_ESM.pdf (604 kb)
ESM 1 (PDF 604 kb)


  1. 1.
    Tuan, R. S., Boland, G., & Tuli, R. (2003). Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Research and Therapy, 5(1), 32–45.CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Langer, R., & Vacanti, J. P. (1993). Tissue engineering. Science, 260(5110), 920–926.CrossRefPubMedGoogle Scholar
  3. 3.
    Oldershaw, R. A. (2012). Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. International Journal of Experimental Pathology, 93(6), 389–400.PubMedCentralPubMedGoogle Scholar
  4. 4.
    Meinel, L., Hofmann, S., Karageorgiou, V., Zichner, L., Langer, R., Kaplan, D., et al. (2004). Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotechnology and Bioengineering, 88(3), 379–391.CrossRefPubMedGoogle Scholar
  5. 5.
    Steck, E., Fischer, J., Lorenz, H., Gotterbarm, T., Jung, M., & Richter, W. (2009). Mesenchymal stem cell differentiation in an experimental cartilage defect: restriction of hypertrophy to bone-close neocartilage. Stem Cells and Development, 18(7), 969–978.CrossRefPubMedGoogle Scholar
  6. 6.
    Estes, B. T., Diekman, B. O., Gimble, J. M., & Guilak, F. (2010). Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nature Protocols, 5(7), 1294–1311.CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Stolzing, A., Jones, E., McGonagle, D., & Scutt, A. (2008). Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mechanisms of Ageing and Development, 129(3), 163–173.CrossRefPubMedGoogle Scholar
  8. 8.
    Long, F., & Ornitz, D. M. (2013). Development of the endochondral skeleton. Cold Spring Harbor Perspectives in Biology, 5(1), a008334.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Pelttari, K., Winter, A., Steck, E., Goetzke, K., Hennig, T., Ochs, B. G., et al. (2006). Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis and Rheumatism, 54(10), 3254–3266.CrossRefPubMedGoogle Scholar
  10. 10.
    Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282(5391), 1145–1147.CrossRefPubMedGoogle Scholar
  11. 11.
    Sa, S., & McCloskey, K. E. (2012). Stage-specific cardiomyocyte differentiation method for h7 and h9 human embryonic stem cells. Stem Cell Reviews, 8(4), 1120–1128.CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang, J., Wilson, G. F., Soerens, A. G., Koonce, C. H., Yu, J., Palecek, S. P., et al. (2009). Functional cardiomyocytes derived from human induced pluripotent stem cells. Circulation Research, 104(4), e30–41.CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Marolt, D., Campos, I. M., Bhumiratana, S., Koren, A., Petridis, P., Zhang, G., et al. (2012). Engineering bone tissue from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 109(22), 8705–8709.CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Oldershaw, R. A., Baxter, M. A., Lowe, E. T., Bates, N., Grady, L. M., Soncin, F., et al. (2010). Directed differentiation of human embryonic stem cells toward chondrocytes. Nature Biotechnology, 28(11), 1187–1194.CrossRefPubMedGoogle Scholar
  15. 15.
    Nakagawa, T., Lee, S. Y., & Reddi, A. H. (2009). Induction of chondrogenesis from human embryonic stem cells without embryoid body formation by bone morphogenetic protein 7 and transforming growth factor beta1. Arthritis and Rheumatism, 60(12), 3686–3692.CrossRefPubMedGoogle Scholar
  16. 16.
    Toh, W. S., Lee, E. H., Guo, X. M., Chan, J. K., Yeow, C. H., Choo, A. B., et al. (2010). Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells. Biomaterials, 31(27), 6968–6980.CrossRefPubMedGoogle Scholar
  17. 17.
    Amarilio, R., Viukov, S. V., Sharir, A., Eshkar-Oren, I., Johnson, R. S., & Zelzer, E. (2007). HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis. Development, 134(21), 3917–3928.CrossRefPubMedGoogle Scholar
  18. 18.
    Ezashi, T., Das, P., & Roberts, R. M. (2005). Low O2 tensions and the prevention of differentiation of hES cells. Proceedings of the National Academy of Sciences of the United States of America, 102(13), 4783–4788.CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Koay, E. J., & Athanasiou, K. A. (2008). Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality. Osteoarthritis and Cartilage, 16(12), 1450–1456.CrossRefPubMedGoogle Scholar
  20. 20.
    Hwang, N. S., Varghese, S., & Elisseeff, J. (2008). Derivation of chondrogenically-committed cells from human embryonic cells for cartilage tissue regeneration. PLoS One, 3(6), e2498.CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Hwang, N. S., Varghese, S., Lee, H. J., Zhang, Z., Ye, Z., Bae, J., et al. (2008). In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proceedings of the National Academy of Sciences of the United States of America, 105(52), 20641–20646.CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Lima, E. G., Grace Chao, P. H., Ateshian, G. A., Bal, B. S., Cook, J. L., Vunjak-Novakovic, G., et al. (2008). The effect of devitalized trabecular bone on the formation of osteochondral tissue-engineered constructs. Biomaterials, 29(32), 4292–4299.CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Yodmuang, S., Gadjanski, I., Chao, P. H., & Vunjak-Novakovic, G. (2013). Transient hypoxia improves matrix properties in tissue engineered cartilage. Journal of Orthopaedic Research, 31(4), 544–553.CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Yang, L., Soonpaa, M. H., Adler, E. D., Roepke, T. K., Kattman, S. J., Kennedy, M., et al. (2008). Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature, 453(7194), 524–528.CrossRefPubMedGoogle Scholar
  25. 25.
    Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 3(6), 1101–1108.CrossRefPubMedGoogle Scholar
  26. 26.
    Farndale, R. W., Buttle, D. J., & Barrett, A. J. (1986). Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica et Biophysica Acta, 883(2), 173–177.CrossRefPubMedGoogle Scholar
  27. 27.
    Bruckner, P., Horler, I., Mendler, M., Houze, Y., Winterhalter, K. H., Eich-Bender, S. G., et al. (1989). Induction and prevention of chondrocyte hypertrophy in culture. Journal of Cell Biology, 109(5), 2537–2545.CrossRefPubMedGoogle Scholar
  28. 28.
    Kisiday, J., Jin, M., Kurz, B., Hung, H., Semino, C., Zhang, S., et al. (2002). Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proceedings of the National Academy of Sciences of the United States of America, 99(15), 9996–10001.CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Zhao, H., Ito, Y., Chappel, J., Andrews, N. W., Teitelbaum, S. L., & Ross, F. P. (2008). Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteblast secretion. Developmental Cell, 14(6), 914–925.CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Tavella, S., Raffo, P., Tacchetti, C., Cancedda, R., & Castagnola, P. (1994). N-CAM and N-cadherin expression during in vitro chondrogenesis. Experimental Cell Research, 215(2), 354–362.CrossRefPubMedGoogle Scholar
  31. 31.
    Meier, S., Solursh, M., & Vaerewyck, S. (1973). Modulation of extracellular matrix production by conditioned medium. American Zoologist, 13, 1051–1060.Google Scholar
  32. 32.
    Keller, G. M. (1995). In vitro differentiation of embryonic stem cells. Current Opinion in Cell Biology, 7(6), 862–869.CrossRefPubMedGoogle Scholar
  33. 33.
    Kramer, J., Hegert, C., Guan, K., Wobus, A. M., Muller, P. K., & Rohwedel, J. (2000). Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mechanisms of Development, 92(2), 193–205.CrossRefPubMedGoogle Scholar
  34. 34.
    Vats, A., Bielby, R. C., Tolley, N., Dickinson, S. C., Boccaccini, A. R., Hollander, A. P., et al. (2006). Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Engineering, 12(6), 1687–1697.CrossRefPubMedGoogle Scholar
  35. 35.
    Benjamin, S., Sheyn, D., Ben-David, S., Oh, A., Kallai, I., Li, N., et al. (2013). Oxygenated environment enhances both stem cell survival and osteogenic differentiation. Tissue Engineering Part A, 19(5–6), 748–758.CrossRefPubMedGoogle Scholar
  36. 36.
    Muller, J., Benz, K., Ahlers, M., Gaissmaier, C., & Mollenhauer, J. (2011). Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures. Cell Transplantation, 20(10), 1589–1602.CrossRefPubMedGoogle Scholar
  37. 37.
    Egli, R. J., Bastian, J. D., Ganz, R., Hofstetter, W., & Leunig, M. (2008). Hypoxic expansion promotes the chondrogenic potential of articular chondrocytes. Journal of Orthopaedic Research, 26(7), 977–985.CrossRefPubMedGoogle Scholar
  38. 38.
    Meretoja, V. V., Dahlin, R. L., Wright, S., Kasper, F. K., & Mikos, A. G. (2013). The effect of hypoxia on the chondrogenic differentiation of co-cultured articular chondrocytes and mesenchymal stem cells in scaffolds. Biomaterials, 34(17), 4266–4273.CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Mackie, E. J., Ahmed, Y. A., Tatarczuch, L., Chen, K. S., & Mirams, M. (2008). Endochondral ossification: how cartilage is converted into bone in the developing skeleton. The International Journal of Biochemistry & Cell Biology, 40, 46–62.CrossRefGoogle Scholar
  40. 40.
    Gadjanski, I., Spiller, K., & Vunjak-Novakovic, G. (2012). Time-dependent processes in stem cell-based tissue engineering of articular cartilage. Stem Cell Reviews, 8(3), 863–881.CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Brighton C.T. (1985). Epiphyseal bone formation. In: Textbook of Small Animal Orthopaedics. Newton CD and Nunamaker DM, eds. Lippincott Williams & Wilkins, pp 50–55.Google Scholar
  42. 42.
    Zhu, L. L., Wu, L. Y., Yew, D. T., & Fan, M. (2005). Effects of hypoxia on the proliferation and differentiation of NSCs. Molecular Neurobiology, 31(1–3), 231–242.CrossRefPubMedGoogle Scholar
  43. 43.
    Van Winkle, A. P., Gates, I. D., & Kallos, M. S. (2012). Mass transfer limitations in embryoid bodies during human embryonic stem cell differentiation. Cells, Tissues, Organs, 196(1), 34–47.CrossRefPubMedGoogle Scholar
  44. 44.
    Wilson, J. L., Suri, S., Singh, A., Rivet, C. A., Lu, H., & McDevitt, T. C. (2014). Single-cell analysis of embryoid body heterogeneity using microfluidic trapping array. Biomedical Microdevices, 16(1), 79–90.CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Stachelscheid, H., Wulf-Goldenberg, A., Eckert, K., Jensen, J., Edsbagge, J., Bjorquist, P., et al. (2013). Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors. Journal of Tissue Engineering and Regenerative Medicine, 7(9), 729–741.CrossRefPubMedGoogle Scholar
  46. 46.
    Yamashita, A., Krawetz, R., & Rancourt, D. E. (2009). Loss of discordant cells during micro-mass differentiation of embryonic stem cells into the chondrocyte lineage. Cell Death and Differentiation, 16(2), 278–286.CrossRefPubMedGoogle Scholar
  47. 47.
    Schipani, E. (2010). Posttranslational modifications of collagens as targets of hypoxia and Hif-1alpha in endochondral bone development. Annals of the New York Academy of Sciences, 1192, 317–321.CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    Gilkes, D. M., Bajpai, S., Chaturvedi, P., Wirtz, D., & Semenza, G. L. (2013). Hypoxia-inducible factor 1 (HIF-1) promotes extracellular matrix remodeling under hypoxic conditions by inducing P4HA1, P4HA2, and PLOD2 expression in fibroblasts. Journal of Biological Chemistry, 288(15), 10819–10829.CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Eisinger-Mathason, T. S., Zhang, M., Qiu, Q., Skuli, N., Nakazawa, M. S., Karakasheva, T., et al. (2013). Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis. Cancer Discov, 3(10), 1190–1205.CrossRefPubMedGoogle Scholar
  50. 50.
    Oberlender, S. A., & Tuan, R. S. (1994). Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development, 120(1), 177–187.PubMedGoogle Scholar
  51. 51.
    Ornitz, D. M., & Marie, P. J. (2002). FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes and Development, 16(12), 1446–1465.CrossRefPubMedGoogle Scholar
  52. 52.
    Butler, W. T. (1989). The nature and significance of osteopontin. Connective Tissue Research, 23(2–3), 123–136.CrossRefPubMedGoogle Scholar
  53. 53.
    Goldring, M. B., Tsuchimochi, K., & Ijiri, K. (2006). The control of chondrogenesis. Journal of Cellular Biochemistry, 97(1), 33–44.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Supansa Yodmuang
    • 1
  • Darja Marolt
    • 2
  • Ivan Marcos-Campos
    • 1
  • Ivana Gadjanski
    • 3
  • Gordana Vunjak-Novakovic
    • 1
    Email author
  1. 1.Department of Biomedical EngineeringColumbia UniversityNew YorkUSA
  2. 2.The New York Stem Cell Foundation Research InstituteNew YorkUSA
  3. 3.Center for Bioengineering-BioIRCBelgrade Metropolitan UniversityKragujevacSerbia

Personalised recommendations