Abstract
Human neural organoids represent promising models for studying neural function; however, organoids grown in vitro lack certain microenvironments and sensory inputs that are thought to be essential for maturation. The transplantation of patient-derived neural organoids into animal hosts helps overcome some of these limitations and offers an approach for neural organoid maturation and circuit integration. Here, we describe a method for transplanting human stem cell–derived cortical organoids (hCOs) into the somatosensory cortex of newborn rats. The differentiation of human induced pluripotent stem cells into hCOs occurs over 30–60 days, and the transplantation procedure itself requires ~0.5–1 hours per animal. The use of neonatal hosts provides a developmentally appropriate stage for circuit integration and allows the generation and experimental manipulation of a unit of human neural tissue within the cortex of a living animal host. After transplantation, animals can be maintained for hundreds of days, and transplanted hCO growth can be monitored by using brain magnetic resonance imaging. We describe the assessment of human neural circuit function in vivo by monitoring genetically encoded calcium responses and extracellular activity. To demonstrate human neuron–host functional integration, we also describe a procedure for engaging host neural circuits and for modulating animal behavior by using an optogenetic behavioral training paradigm. The transplanted human neurons can then undergo ex vivo characterization across modalities including dendritic morphology reconstruction, single-nucleus transcriptomics, optogenetic manipulation and electrophysiology. This approach may enable the discovery of cellular phenotypes from patient-derived cells and uncover mechanisms that contribute to human brain evolution from previously inaccessible developmental stages.
Key points
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The protocol involves surgical implantation of human cortical organoids in the cerebral cortex of rat pups. Organoid growth is monitored by using MRI, whereas their functional integration in the host neural circuitry is carried out by using behavioral, electrophysiological and optogenetic approaches.
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Transplanted organoids enable multimodal genomic measurements from millions of cells, facilitating characterization of human–human and human–rodent cellular interactions, including neural circuit activity patterns and relationships between glia and neurons.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper18. Single-cell gene expression raw data are available under the Gene Expression Omnibus accession number GSE190815. Additional raw datasets are available for research purposes from the corresponding author upon request.
Code availability
Code used for data processing and analysis are available on request from the corresponding author. For additional details on processing calcium imaging data, see our recently published STAR protocol45. The code used to analyze snRNA-seq data is available for download from https://github.com/kkelley85/Transplant_organoid_snRNAseq.
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Acknowledgements
We thank members of the Pașca laboratory at Stanford University for insightful discussions and technical support. This work was supported by the Stanford Big Idea Project on Brain Organogenesis (Wu Tsai Neuroscience Institute) (to S.P.P. and K.D.), the National Institute of Mental Health (R01 MH115012; to S.P.P.), the Kwan Funds (to S.P.P.), the Senkut Funds (to S.P.P.), the Coates Foundation (to S.P.P.), the Ludwig Family Foundation (to S.P.P.), the Alfred E. Mann Foundation (to S.P.P.), the Stanford Maternal & Child Health Research Institute (MCHRI) Postdoctoral Fellowship (to F.G. and O.R.), the Walter V. and Idun Berry Postdoctoral Fellowship (to F.G.), the NARSAD Young Investigator Award (to F.G.) and an NIH NIDA K99/R00 (K99 DA050662) (to F.G.). S.P.P. is a New York Stem Cell Foundation (NYSCF) Robertson Stem Cell Investigator, a CZI Ben Barres Investigator and a CZ BioHub Investigator. We thank the Stanford Center for Innovation in In vivo Imaging (SCi 3)—Small Animal Imaging Center, which is supported by the NIH S10 Shared Instrumentation grant (S10RR026917-01), and the Stanford Behavioral and Functional Neuroscience Laboratory, which is supported by an NIH S10 Shared Instrumentation for Animal Research grant (1S10OD030452-01).
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All authors contributed to the development of the methods described in this protocol. K.W.K. and S.P.P. wrote the manuscript with input and corrections from all authors.
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Stanford University holds patents for the generation of cortical organoids/spheroids (listing S.P.P. as an inventor) and a provisional patent application for transplantation of organoids (listing S.P.P., O.R., F.G., K.D. and K.W.K. as inventors).
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Nature Protocols thanks Yanhong Shi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Revah, O. et al. Nature 610, 319–326 (2022): https://doi.org/10.1038/s41586-022-05277-w
Miura, Y. et al. Nat. Biotechnol. 38, 1421–1430 (2020): https://doi.org/10.1038/s41587-020-00763-w
Yoon, S.-J. et al. Nat. Methods 16, 75–78 (2019): https://doi.org/10.1038/s41592-018-0255-0
Sloan, S. A. et al. Nat. Protoc. 13, 2062–2085 (2018): https://doi.org/10.1038/s41596-018-0032-7
Chen, X. et al. Nature 628, 818–825 (2024): https://doi.org/10.1038/s41586-024-07310-6
Extended data
Extended Data Fig. 1 Lentiviral expression in an hCO.
Representative example images of hChR2-EYFP expression 6 d after infection in an hCO. Confocal images were overexposed to enable visualization of neurons, including those with low expression.
Extended Data Fig. 2 Example whisker images of FOXN1 transgenic pups.
a, Representative image of heterozygous FOXN1+/− whisker growth in rat pups. b, Representative image of characteristic immature whisker growth of homozygous FOXN1−/− rat pups.
Extended Data Fig. 3
Images of small-animal stereotactic instrument (left) with animal placed in the stereotactic frame (middle and right).
Extended Data Fig. 4 Vasculature in neonatal rat to target S1.
Representative image of neonatal rat brain vasculature landmarks for targeting S1.
Extended Data Fig. 5 Example images during transplantation of an hCO.
a, Representative surgical landmarks for targeting S1. b, Representative images after craniotomy and after puncturing the dura. c, Representative images of a needle at the dura surface (left) and within S1 (right). d, Representative image after organoid transplantation.
Extended Data Fig. 6 Images of nuclei after t-hCO dissociation.
Representative images of nuclei stained with trypan blue after dissociation from three separate examples.
Supplementary information
Supplementary Video 1
Surgical preparation, incision and exposure of the dorsal surface of the skull
Supplementary Video 2
Identifying surgical landmarks, craniotomy and transplantation of an hCO
Supplementary Video 3
Example craniotomy and puncturing of the dura
Supplementary Video 4
Retraction of the syringe and closing of the surgical site
Supplementary Video 5
Example failure: bubbles in media
Supplementary Video 6
Example failure: significant hCO backflow from the syringe
Supplementary Video 7
Minor hCO backflow from the syringe
Supplementary Video 8
Aspiration of hCO with the syringe
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Kelley, K.W., Revah, O., Gore, F. et al. Host circuit engagement of human cortical organoids transplanted in rodents. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01029-4
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DOI: https://doi.org/10.1038/s41596-024-01029-4
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