Article

An in vivo model of functional and vascularized human brain organoids

  • Nature Biotechnology volume 36, pages 432441 (2018)
  • doi:10.1038/nbt.4127
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Abstract

Differentiation of human pluripotent stem cells to small brain-like structures known as brain organoids offers an unprecedented opportunity to model human brain development and disease. To provide a vascularized and functional in vivo model of brain organoids, we established a method for transplanting human brain organoids into the adult mouse brain. Organoid grafts showed progressive neuronal differentiation and maturation, gliogenesis, integration of microglia, and growth of axons to multiple regions of the host brain. In vivo two-photon imaging demonstrated functional neuronal networks and blood vessels in the grafts. Finally, in vivo extracellular recording combined with optogenetics revealed intragraft neuronal activity and suggested graft-to-host functional synaptic connectivity. This combination of human neural organoids and an in vivo physiological environment in the animal brain may facilitate disease modeling under physiological conditions.

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Acknowledgements

We thank members of the Gage laboratory for helpful discussions; S. Schafer for the pCSC-CAG-GFP lentivirus and I. Verma for the pBOB-CAG-Td-Tomato construct. We also thank M. Shtrahman for assistance and two-photon imaging expertise, L. Moore, S. Baktvar, S. Kim, B. Miller, C. Lim, and I. Guimont for their technical assistance, M.L. Gage for editorial comments, V. Mertens for illustrations, I. Farhy-Tselnicker and J. Klug for technical advice, and M. Shtrahman, and T. Toda for critical reading and comments on the manuscripts. We thank U. Manor and the Waitt Advanced Biophotonics Core, K. Diffenderfer and the Salk Stem Cell Core, C. O'Connor and C. Fitzpatrick and the FACS Core, and the Salk Institute for generously providing critical infrastructural and financial support. We apologize to those whose work was not cited owing to space limitations. This work was supported by the NIH (U19 MH106434, U01 MH106882), The Paul G, Allen Family Foundation, Bob and Mary Jane Engman, The Leona M, and Harry B, Helmsley Charitable Trust Grant (2012-PG-MED), Annette C, Merle-Smith, The G, Harold and Leila Y, Mathers Foundation, JPB Foundation, Dolby Family Ventures for F.H.G. and NIH grants (R01NS083815, R01AG047669) for X.J. S.F. was funded by CIRM Bridges to Stem Cell Research Internship Program. A.A.M. received funding from the EMBO Postdoctoral Long-term Fellowship (ALTF 1214-2014, EMBO fellowship is co-funded also by the European Commission FP7-Marie Curie Actions, LTFCOFUND2013, GA-2013-609409), and is currently supported by the Human Frontiers Science Program (HFSP Long-Term Fellowship- LT001074/2015).

Author information

Author notes

    • J Tiago Gonçalves

    Present address: Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA.

Affiliations

  1. Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, USA.

    • Abed AlFatah Mansour
    • , J Tiago Gonçalves
    • , Cooper W Bloyd
    • , Sarah Fernandes
    • , Daphne Quang
    • , Stephen Johnston
    • , Sarah L Parylak
    •  & Fred H Gage
  2. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA.

    • Hao Li
    •  & Xin Jin
  3. Department of Biology, San Diego State University, San Diego, California, USA.

    • Sarah Fernandes

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Contributions

A.A.M. and F.H.G. conceived the idea for the project and wrote the manuscript. A.A.M. designed and performed the experiments and analyzed the data. A.A.M. generated hESC lines and organoids culture, performed cellular, molecular and histological assays and analyzed the data. S.F. and D.Q. performed cell culture and histological experiments under the supervision of A.A.M. A.A.M., C.W.B., F.H.G., J.T.G., and S.J. performed surgeries. T.G. and C.W.B. performed two-photon imaging and analyzed the data. S.L.P. performed behavioral experiments. H.L. performed electrophysiological microelectrode and optogenetic experiments under the supervision of X.J. F.H.G. supervised the project and provided funding.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Fred H Gage.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Table 1

    List of primary and secondary antibodies.

Videos

  1. 1.

    Two-photon in vivo imaging of organoid graft in awake, head-fixed mouse.

    GFP+ organoid (green) was grafted into mouse brain and was imaged under a two-photon microscope at 59 dpi. Video shows serial images acquired from 1 to 500 μm below the organoid surface. Note that higher laser power (compared to other experiments) was used to achieve significant signal upon penetration to 500 μm.

  2. 2.

    Two-photon in vivo imaging of blood vessels inside the organoids.

    GFP+ organoid (green) was grafted into mouse brain and the mouse was injected with Dextran (red) at 30 dpi, and imaged directly. Video shows serial images acquired from 200 to 500 μm below the organoid surface.

  3. 3.

    Active blood flow within the grafted organoid.

    Two-photon time series of grafted organoid after dextran injection at 120 dpi showing active blood flow at 141 μm depth.

  4. 4.

    In vivo two-photon imaging of organoid tissue labeled with jRGECO1a, a genetically encoded calcium sensor.

    This representative 60 s movie of calcium activity was acquired at 78 dpi. The mouse was head-fixed to the microscope but unanesthetized and allowed to run on a cylindrical treadmill.

  5. 5.

    In vivo two-photon imaging of organoid tissue labeled with jRGECO1a.

    This representative 60 s movie of calcium activity was acquired at 108 dpi. The mouse was head-fixed to the microscope but unanesthetized and allowed to run on a cylindrical treadmill.

  6. 6.

    Additional example of in vivo two-photon imaging of organoid tissue labeled with jRGECO1a.

    This representative 60 s movie of calcium activity was acquired at 109 dpi.