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Abstract

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells1,2,3,4,5,6 or used bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.

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Change history

  • 14 August 2018

    In the HTML and PDF versions of this article initially published, μ's appeared as m's throughout the scale bar lengths in the figure legends, as well as in the Methods section in the phrases "added to give a final volume of 150 μl per well," "an average hPSC cell size of 15 μm" and "sectioned on a vibratome to collect 300-μm sections." In the HTML version, μ's appeared as m's in all instances throughout the Methods section except "35 μg/ml of pure laminin" and "35 μl laminin/entactin + 25 μl collagen." The errors have been corrected in the HTML and PDF versions of the article.

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References

  1. 1.

    , , & Development and application of human adult stem or progenitor cell organoids. Nat. Rev. Nephrol. 11, 546–554 (2015).

  2. 2.

    , & Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).

  3. 3.

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

  4. 4.

    , , & Organoid models of human gastrointestinal development and disease. Gastroenterology 150, 1098–1112 (2016).

  5. 5.

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

  6. 6.

    & Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125–1247125 (2014).

  7. 7.

    & Advances in tissue engineering. J. Pediatr. Surg. 51, 8–12 (2016).

  8. 8.

    , & Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248–260 (2015).

  9. 9.

    , & Organoid models and applications in biomedical research. Nephron 130, 191–199 (2015).

  10. 10.

    & Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine. Stem Cells 31, 417–422 (2013).

  11. 11.

    & Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).

  12. 12.

    et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532 (2008).

  13. 13.

    et al. Modeling human cortical development in vitro using induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 109, 12770–12775 (2012).

  14. 14.

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

  15. 15.

    et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

  16. 16.

    et al. Defined three-dimensional microenvironments boost induction of pluripotency. Nat. Mater. 15, 344–352 (2016).

  17. 17.

    , , , & A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat. Methods 11, 847–854 (2014).

  18. 18.

    et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. (Amst.) 2, 219–230 (2009).

  19. 19.

    , , , & Regeneration of peripheral nerve through a polyglactin tube. Muscle Nerve 5, 54–57 (1982).

  20. 20.

    & Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297, 365–369 (2002).

  21. 21.

    , , & Signals from the edges: the cortical hem and antihem in telencephalic development. Semin. Cell Dev. Biol. 20, 712–718 (2009).

  22. 22.

    et al. Transcriptional landscape of the prenatal human brain. Nature 508, 199–206 (2014).

  23. 23.

    Specification of cerebral cortical areas. Science 241, 170–176 (1988).

  24. 24.

    & Radial columns in cortical architecture: it is the composition that counts. Cereb. Cortex 20, 2261–2264 (2010).

  25. 25.

    & Cortical evolution: judge the brain by its cover. Neuron 80, 633–647 (2013).

  26. 26.

    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).

  27. 27.

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

  28. 28.

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

  29. 29.

    , , & Meningeal cells organize the superficial glia limitans of the cerebellum and produce components of both the interstitial matrix and the basement membrane. J. Neurocytol. 23, 135–149 (1994).

  30. 30.

    , , , & A critical function of the pial basement membrane in cortical histogenesis. J. Neurosci. 22, 6029–6040 (2002).

  31. 31.

    Evolution of the neocortex: a perspective from developmental biology. Nat. Rev. Neurosci. 10, 724–735 (2009).

  32. 32.

    , , & A new subtype of progenitor cell in the mouse embryonic neocortex. Nat. Neurosci. 14, 555–561 (2011).

  33. 33.

    , , & Guiding neuronal cell migrations. Cold Spring Harb. Perspect. Biol. 2, a001834 (2010).

  34. 34.

    , , , & 2D and 3D stem cell models of primate cortical development identify species-specific differences in progenitor behavior contributing to brain size. Cell Stem Cell 18, 467–480 (2016).

  35. 35.

    & Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. USA 101, 16–22 (2004).

  36. 36.

    , & Gateway compatible vectors for analysis of gene function in the zebrafish. Dev. Dyn. 236, 3077–3087 (2007).

  37. 37.

    et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761 (2009).

  38. 38.

    , & Organotypic slice culture of embryonic brain tissue. CSH Protoc. 2007, t4914 (2007).

  39. 39.

    & Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  40. 40.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

  41. 41.

    et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

  42. 42.

    , , , & Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol. 12, R22 (2011).

  43. 43.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

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Acknowledgements

We thank members of the Lancaster laboratory for helpful discussion and technical support, especially S. Giandomenico and M. Sutcliffe, as well as A.G. Gianni for sea sponge sample. We also thank members of the Knoblich laboratory for insight and technical help, particularly A. Peer. T.O. was supported by the Cambridge Wellcome Trust PhD program in developmental biology, and F.J.L. is a Wellcome Trust Investigator. M.A.L. was funded by a Marie Curie Postdoctoral fellowship, and work in M.A.L.'s laboratory is supported by the Medical Research Council (MC_UP_1201/9). N.S.C. was funded by an EMBO long-term fellowship and a Deutsche Forschungsgemeinschaft research fellowship (DFG CO 1324/1-1). Work in J.A.K.'s laboratory is supported by the Austrian Academy of Sciences, the Austrian Science Fund (grants I_1281-B19 and Z_153_B09), and an advanced grant from the European Research Council. pCAGEN and pCAG-GFP were gifts from Connie Cepko, Harvard Medical School. pT2/HB was a gift from Perry Hackett, University of Minnesota. pENTR-EGFP2 was a gift from Nathan Lawson, University of Massachusetts Medical School. pCMV(CAT)T7-SB100 was a gift from Zsuzsanna Izsvak, Max Delbruck Center for Molecular Medicine.

Author information

Affiliations

  1. IMBA—Institute of Molecular Biotechnology of the Austrian Academy of Science, Vienna, Austria.

    • Madeline A Lancaster
    • , Nina S Corsini
    • , Simone Wolfinger
    • , E Hilary Gustafson
    • , Thomas R Burkard
    •  & Juergen A Knoblich
  2. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.

    • Madeline A Lancaster
    •  & Alex W Phillips
  3. IMP—Institute of Molecular Pathology, Vienna, Austria.

    • Thomas R Burkard
  4. Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, UK.

    • Tomoki Otani
    •  & Frederick J Livesey

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Contributions

M.A.L. conceived the project, planned and performed experiments, and wrote the manuscript. N.S.C. performed experiments and analyzed data. S.W. and E.H.G. prepared samples and performed various treatments. A.W.P. performed cloning and prepared samples. T.R.B. performed bioinformatics analysis of RNA-seq data. T.O. prepared samples and performed experiments under the supervision of F.J.L. J.A.K. supervised the project, planned and interpreted experiments, and wrote the manuscript.

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Competing interests

M.A.L. and J.A.K. have filed a patent application for use of this technology in future disease modeling and toxicology testing.

Corresponding authors

Correspondence to Madeline A Lancaster or Juergen A Knoblich.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

Excel files

  1. 1.

    Supplementary Table 1

    Upregulated and downregulated genes in enCORs at 20 days and 60 days, and the associated GO term analysis

  2. 2.

    Supplementary Table 2

    Hierarchical clusters of up and down regulated genes and the associated GO term analysis

Videos

  1. 1.

    Supplementary Video 1

    Live imaging of GFP electroporated slice culture of H1 1009 enCOR electroporated with GFP on day 63, followed by vibratome sectioning four 1010 days later and live imaging 24-hours later

  2. 2.

    Supplementary Video 2

    Live image of GFP electroporated H9 organoid displaying several labeled radial glial processes and their endfeet (asterisks) as well as several migrating neurons (arrows).

  3. 3.

    Supplementary Video 3

    Higher magnification of the multipolar neuron marked by blue arrow in Supplementary Video 2.

  4. 4.

    Supplementary Video 4

    Live imaging of farnesyl-GFP electroporated cells in slice culture taken 13 days after electroporation

  5. 5.

    Supplementary Video 5

    Higher magnification of live imaging of farnesyl-GFP electroporated slice culture shown in Supplementary Video 4

  6. 6.

    Supplementary Video 6

    Higher magnification of the neuron marked by the orange arrow in Supplementary Video 2

  7. 7.

    Supplementary Video 7

    Live imaging of spontaneous calcium surges using the calcium dye Fluo-4 in a slice culture taken 13 days after electroporation

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DOI

https://doi.org/10.1038/nbt.3906