Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation of human brain region–specific organoids using a miniaturized spinning bioreactor

Nature Protocols volume 13, pages 565–580 (2018)Cite this article

Subjects

Abstract

Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14–84 d to complete (depending on the type of brain region–specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Building a SpinΩ bioreactor.
Figure 2: Schematic diagram of brain region–specific organoid protocols.
Figure 3: Progression and quality control of forebrain organoid development.
Figure 4: Immunofluorescence characterization of brain region–specific organoids.

Similar content being viewed by others

References

  1. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  2. Wen, Z., Christian, K.M., Song, H. & Ming, G.L. Modeling psychiatric disorders with patient-derived iPSCs. Curr. Opin. Neurobiol. 36, 118–127 (2016).

    Article  CAS  Google Scholar 

  3. Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    Article  CAS  Google Scholar 

  4. Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).

    Article  CAS  Google Scholar 

  5. Kadoshima, T. et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex. Proc. Natl. Acad. Sci. USA 110, 20284–20289 (2013).

    Article  CAS  Google Scholar 

  6. Pasca, A.M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).

    Article  CAS  Google Scholar 

  7. Jo, J. et al. Midbrain-like organoids from human pluripotent stem cells contain functional dopaminergic and neuromelanin-producing neurons. Cell Stem Cell 19, 248–257 (2016).

    Article  CAS  Google Scholar 

  8. Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016).

    Article  CAS  Google Scholar 

  9. Nguyen, H.N.N., Song, H. & Ming, G. Engineering human pluripotent stem cell-derived 3D brain tissue for drug discovery. J. Transl. Neurosci. 1, 38–48 (2016).

    Google Scholar 

  10. Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

    Article  Google Scholar 

  11. Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell 18, 25–38 (2016).

    Article  CAS  Google Scholar 

  12. Danjo, T. et al. Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals. J. Neurosci. 31, 1919–1933 (2011).

    Article  CAS  Google Scholar 

  13. Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K. & Sasai, Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep. 10, 537–550 (2015).

    Article  CAS  Google Scholar 

  14. Sakaguchi, H. et al. Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue. Nat. Commun. 6, 8896 (2015).

    Article  CAS  Google Scholar 

  15. Sasai, Y. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).

    Article  CAS  Google Scholar 

  16. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480, 547–551 (2011).

    Article  CAS  Google Scholar 

  17. Blackshaw, S. et al. Molecular pathways controlling development of thalamus and hypothalamus: from neural specification to circuit formation. J. Neurosci. 30, 14925–14930 (2010).

    Article  CAS  Google Scholar 

  18. Byerly, M.S. & Blackshaw, S. Vertebrate retina and hypothalamus development. Wiley Interdiscip. Rev. Syst. Biol. Med. 1, 380–389 (2009).

    Article  CAS  Google Scholar 

  19. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  Google Scholar 

  20. Wen, Z. et al. Synaptic dysregulation in a human iPS cell model of mental disorders. Nature 515, 414–418 (2014).

    Article  CAS  Google Scholar 

  21. Yoon, K.J. et al. Modeling a genetic risk for schizophrenia in iPSCs and mice reveals neural stem cell deficits associated with adherens junctions and polarity. Cell Stem Cell 15, 79–91 (2014).

    Article  CAS  Google Scholar 

  22. Chiang, C.H. et al. Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Mol. Psychiatry 16, 358–360 (2011).

    Article  CAS  Google Scholar 

  23. Yoon, K.J. et al. Zika-virus-encoded NS2A disrupts mammalian cortical neurogenesis by degrading adherens junction proteins. Cell Stem Cell 21, 349–358.e6 (2017).

    Article  CAS  Google Scholar 

  24. Xu, M. et al. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat. Med. 22, 1101–1107 (2016).

    Article  CAS  Google Scholar 

  25. Yoon, K.J. et al. Temporal control of mammalian cortical neurogenesis by m6A methylation. Cell 171, 877–889.e17 (2017).

    Article  CAS  Google Scholar 

  26. Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).

    Article  CAS  Google Scholar 

  27. Bagley, J.A., Reumann, D., Bian, S., Levi-Strauss, J. & Knoblich, J.A. Fused cerebral organoids model interactions between brain regions. Nat. Methods 14, 743–751 (2017).

    Article  CAS  Google Scholar 

  28. Xiang, Y. et al. Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration. Cell Stem Cell 21, 383–398.e7 (2017).

    Article  CAS  Google Scholar 

  29. Lui, J.H., Hansen, D.V. & Kriegstein, A.R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).

    Article  CAS  Google Scholar 

  30. Qian, X., Nguyen, H.N., Jacob, F., Song, H. & Ming, G.L. Using brain organoids to understand Zika virus-induced microcephaly. Development 144, 952–957 (2017).

    Article  CAS  Google Scholar 

  31. Ming, G.L., Tang, H. & Song, H. Advances in Zika virus research: stem cell models, challenges, and opportunities. Cell Stem Cell 19, 690–702 (2016).

    Article  CAS  Google Scholar 

  32. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    Article  CAS  Google Scholar 

  33. Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568 (2015).

    Article  CAS  Google Scholar 

  34. Chen, Y.W. et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat. Cell Biol. 19, 542–549 (2017).

    Article  CAS  Google Scholar 

  35. Juopperi, T.A. et al. Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington′s disease patient cells. Mol. Brain 5, 17 (2012).

    Article  CAS  Google Scholar 

  36. Lancaster, M.A. et al. Guided self-organization and cortical plate formation in human brain organoids. Nat. Biotechnol. 35, 659–666 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K.M. Christian for feedback on the manuscript, members of Ming and Song laboratories for discussions, and L. Liu, Y. Cai and D.G. Johnson for technical assistance. The research was supported by grants from the National Institutes of Health (R37NS047344, U19MH106434, P01NS097206 and R01AG057497 to H.S.; R21NS095348, R01MH105128, R35NS097370 and U19AI131130 to G.M.), the Simons Foundation (to H.S. and G.M.) and The Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to G.M.).

Author information

Author notes

  1. Xuyu Qian and Fadi Jacob: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Xuyu Qian, Fadi Jacob, Hongjun Song & Guo-li Ming

  2. Biomedical Engineering Graduate Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Xuyu Qian

  3. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Fadi Jacob

  4. Department of Biology, Brandeis University, Waltham, Massachusetts, USA

    Mingxi Max Song

  5. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Ha Nam Nguyen

  6. The Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Hongjun Song & Guo-li Ming

Authors
  1. Xuyu Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fadi Jacob
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mingxi Max Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ha Nam Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongjun Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guo-li Ming
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.Q., F.J., M.M.S., H.N.N., H.S. and G.M. designed, built and tested the systems and wrote the manuscript.

Corresponding author

Correspondence to Guo-li Ming.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Purchased bioreactor parts. (XLSX 10 kb)

Supplementary Data 1

Bioreactor CAD zip file. (ZIP 179 kb)

Supplementary Data 2

SpinΩ 3D PDF. (ZIP 451 kb)

Supplementary Data 3

SpinΩ 3D PDF, exploded view. (ZIP 451 kb)

SpinΩ assembly. (MOV 18655 kb)

Embedding Matrigel. (MOV 27987 kb)

Breaking Matrigel. (MOV 19796 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Qian, X., Jacob, F., Song, M. et al. Generation of human brain region–specific organoids using a miniaturized spinning bioreactor. Nat Protoc 13, 565–580 (2018). https://doi.org/10.1038/nprot.2017.152

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.152

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research