Cultivation of Human Bone-Like Tissue from Pluripotent Stem Cell-Derived Osteogenic Progenitors in Perfusion Bioreactors

  • Giuseppe Maria de Peppo
  • Gordana Vunjak-Novakovic
  • Darja Marolt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1202)

Abstract

Human pluripotent stem cells represent an unlimited source of skeletal tissue progenitors for studies of bone biology, pathogenesis, and the development of new approaches for bone reconstruction and therapies. In order to construct in vitro models of bone tissue development and to grow functional, clinical-size bone substitutes for transplantation, cell cultivation in three-dimensional environments composed of porous osteoconductive scaffolds and dynamic culture systems—bioreactors—has been studied. Here, we describe a stepwise procedure for the induction of human embryonic and induced pluripotent stem cells (collectively termed PSCs) into mesenchymal-like progenitors, and their subsequent cultivation on decellularized bovine bone scaffolds in perfusion bioreactors, to support the development of viable, stable bone-like tissue in defined geometries.

Keywords

Human embryonic stem cells Human induced pluripotent stem cells Mesenchymal-like progenitors Osteogenic differentiation Osteogenic medium Bone scaffolds Cell seeding Perfusion bioreactor Medium flow rate Bone tissue development 

Notes

Acknowledgements

This work was supported by the New York Stem Cell Foundation-Helmsley Investigator Award (to D.M.); the Leona M. and Harry B. Helmsley Charitable Trust; Robin Chemers Neustein; Goldman Sachs Gives, at the recommendation of Alan and Deborah Cohen; New York State Stem Cell Science Shared Facility, Grant C024179; National Institutes of Health Grants DE016525 and EB002520, (to G.V.-N.); and the New York Stem Cell Foundation.

References

  1. 1.
    de Peppo GM, Marolt D (2013) Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cell-derived mesenchymal progenitors. Stem Cell Res Ther 5:106Google Scholar
  2. 2.
    Tandon N, Marolt D, Cimetta E, Vunjak-Novakovic G (2013) Bioreactor engineering of stem cell environments. Biotechnol Adv. 7:1020–1031Google Scholar
  3. 3.
    Marolt D, Knezevic M, Vunjak-Novakovic G (2010) Bone tissue engineering with human stem cells. Stem Cell Res Ther 2:10CrossRefGoogle Scholar
  4. 4.
    Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedgesm R, Ma H, Kang E, Fulati A, Lee HS, Sritanaudomchai H, Masterson K, Larson J, Eaton D, Sadler-Fredd K, Battaglia D, Lee D, Wu D, Jensen J, Patton P, Gokhale S, Stouffer RL, Wolf D, Mitalipov S (2013) Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 6:1228–1238CrossRefGoogle Scholar
  5. 5.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 5:861–872CrossRefGoogle Scholar
  6. 6.
    Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 4:661–680CrossRefGoogle Scholar
  7. 7.
    Kuznetsov SA, Cherman N, Robey PG (2011) In vivo bone formation by progeny of human embryonic stem cells. Stem Cells Dev 2:269–287CrossRefGoogle Scholar
  8. 8.
    Levi B, Hyun JS, Montoro DT, Lo DD, Chan CK, Hu S, Sun N, Lee M, Grova M, Connolly AJ, Wu JC, Gurtner GC, Weissman IL, Wan DC, Longaker MT (2012) In vivo directed differentiation of pluripotent stem cells for skeletal regeneration. Proc Natl Acad Sci U S A 50:20379–20384CrossRefGoogle Scholar
  9. 9.
    Fröhlich M, Grayson WL, Wan LQ, Marolt D, Drobnic M, Vunjak-Novakovic G (2008) Tissue engineered bone grafts: biological requirements, tissue culture and clinical relevance. Curr Stem Cell Res Ther 4:254–264CrossRefGoogle Scholar
  10. 10.
    Grayson WL, Fröhlich M, Yeager K, Bhumiratana S, Chan ME, Cannizzaro C, Wan LQ, Liu XS, Guo XE, Vunjak-Novakovic G (2010) Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci U S A 8:3299–3304CrossRefGoogle Scholar
  11. 11.
    Grayson WL, Bhumiratana S, Cannizzaro C, Chao PH, Lennon DP, Caplan AI, Vunjak-Novakovic G (2008) Effects of initial seeding density and fluid perfusion rate on formation of tissue-engineered bone. Tissue Eng Part A 11:1809–1820CrossRefGoogle Scholar
  12. 12.
    Grayson WL, Marolt D, Bhumiratana S, Fröhlich M, Guo XE, Vunjak-Novakovic G (2011) Optimizing the medium perfusion rate in bone tissue engineering bioreactors. Biotechnol Bioeng 5:1159–1170CrossRefGoogle Scholar
  13. 13.
    Marcos-Campos I, Marolt D, Petridis P, Bhumiratana S, Schmidt D, Vunjak-Novakovic G (2012) Bone scaffold architecture modulates the development of mineralized bone matrix by human embryonic stem cells. Biomaterials 33:8329–8342PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    de Peppo GM, Marcos-Campos I, Kahler DJ, Alsalman D, Shang L, Vunjak-Novakovic G, Marolt D (2013) Engineering bone tissue substitutes from human induced pluripotent stem cells. Proc Natl Acad Sci U S A 21:8680–8685CrossRefGoogle Scholar
  15. 15.
    Marolt D, Campos IM, Bhumiratana S, Koren A, Petridis P, Zhang G, Spitalnik PF, Grayson WL, Vunjak-Novakovic G (2012) Engineering bone tissue from human embryonic stem cells. Proc Natl Acad Sci U S A 22:8705–8709CrossRefGoogle Scholar
  16. 16.
    de Peppo GM, Sjovall P, Lennerås M, Strehl R, Hyllner J, Thomsen P, Karlsson C (2010) Osteogenic potential of human mesenchymal stem cells and human embryonic stem cell-derived mesodermal progenitors: a tissue engineering perspective. Tissue Eng Part A 11:3413–3426CrossRefGoogle Scholar
  17. 17.
    de Peppo GM, Svensson S, Lennerås M, Synnergren J, Stenberg J, Strehl R, Hyllner J, Thomsen P, Karlsson C (2010) Human embryonic mesodermal progenitors highly resemble human mesenchymal stem cells and display high potential for tissue engineering applications. Tissue Eng Part A 7:2161–2182CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Giuseppe Maria de Peppo
    • 2
  • Gordana Vunjak-Novakovic
    • 3
  • Darja Marolt
    • 1
  1. 1.The New York Stem Cell Foundation Research InstituteNew YorkUSA
  2. 2.The New York Stem Cell Foundation Research InstituteNew YorkUSA
  3. 3.Department of Biomedical Engineering and Department of MedicineColumbia UniversityNew YorkUSA

Personalised recommendations