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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Oncogene Open Access 12 July 2023
Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types
Cell Communication and Signaling Open Access 14 June 2023
Nature Communications Open Access 12 January 2023
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Mertens, J., Marchetto, M.C., Bardy, C. & Gage, F.H. Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nat. Rev. Neurosci. 17, 424–437 (2016).
Shanks, N., Greek, R. & Greek, J. Are animal models predictive for humans? Philos. Ethics Humanit. Med. 4, 2 (2009).
Kelava, I. & Lancaster, M.A. Dishing out mini-brains: current progress and future prospects in brain organoid research. Dev. Biol. 420, 199–209 (2016).
Eiraku, M. 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).
Sasai, Y., Eiraku, M. & Suga, H. In vitro organogenesis in three dimensions: self-organising stem cells. Development 139, 4111–4121 (2012).
Mariani, J. et al. Modeling human cortical development in vitro using induced pluripotent stem cells. Proc. Natl. Acad. Sci. USA 109, 12770–12775 (2012).
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).
Lancaster, M.A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Paşca, A.M. et al. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12, 671–678 (2015).
Qian, X. et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165, 1238–1254 (2016).
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).
Renner, M. et al. Self-organized developmental patterning and differentiation in cerebral organoids. EMBO J. 36, 1316–1329 (2017).
Quadrato, G. et al. Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545, 48–53 (2017).
Mariani, J. et al. FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015).
Bershteyn, M. et al. Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell 20, 435–449.e4 (2017).
Iefremova, V. et al. An organoid-based model of cortical development identifies non-cell-autonomous defects in Wnt signaling contributing to Miller-Dieker syndrome. Cell Rep. 19, 50–59 (2017).
Shen, Q. et al. Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells. Science 304, 1338–1340 (2004).
Yin, X. et al. Engineering stem cell organoids. Cell Stem Cell 18, 25–38 (2016).
Giandomenico, S.L. & Lancaster, M.A. Probing human brain evolution and development in organoids. Curr. Opin. Cell Biol. 44, 36–43 (2017).
Schwartz, M.P. et al. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proc. Natl. Acad. Sci. USA 112, 12516–12521 (2015).
Stenevi, U., Björklund, A. & Svendgaard, N.A. Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival. Brain Res. 114, 1–20 (1976).
Gage, F.H. & Björklund, A. Intracerebral grafting of neuronal cell suspensions into the adult brain. Cent. Nerv. Syst. Trauma 1, 47–56 (1984).
Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat. Med. 18, 618–623 (2012).
Watson, C.L. et al. An in vivo model of human small intestine using pluripotent stem cells. Nat. Med. 20, 1310–1314 (2014).
Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).
Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).
Kretzschmar, K. & Clevers, H. Organoids: modeling development and the stem cell niche in a dish. Dev. Cell 38, 590–600 (2016).
O'Rourke, K.P. et al. Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer. Nat. Biotechnol. 35, 577–582 (2017).
Roper, J. et al. In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis. Nat. Biotechnol. 35, 569–576 (2017).
Dye, B.R. et al. A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids. eLife 5, e19732 (2016).
Gage, F.H. & Fisher, L.J. Intracerebral grafting: a tool for the neurobiologist. Neuron 6, 1–12 (1991).
Gage, F.H., Björklund, A. & Stenevi, U. Denervation releases a neuronal survival factor in adult rat hippocampus. Nature 308, 637–639 (1984).
Lancaster, M.A. & Knoblich, J.A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).
Camp, J.G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl. Acad. Sci. USA 112, 15672–15677 (2015).
Cahoy, J.D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).
Jin, X. & Costa, R.M. Start/stop signals emerge in nigrostriatal circuits during sequence learning. Nature 466, 457–462 (2010).
Falkner, S. et al. Transplanted embryonic neurons integrate into adult neocortical circuits. Nature 539, 248–253 (2016).
Golshani, P. et al. Internally mediated developmental desynchronization of neocortical network activity. J. Neurosci. 29, 10890–10899 (2009).
Espuny-Camacho, I. et al. Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Neuron 77, 440–456 (2013).
Grealish, S. et al. Monosynaptic tracing using modified rabies virus reveals early and extensive circuit integration of human embryonic stem cell-derived neurons. Stem Cell Rep. 4, 975–983 (2015).
Cunningham, M. et al. hPSC-derived maturing GABAergic interneurons ameliorate seizures and abnormal behavior in epileptic mice. Cell Stem Cell 15, 559–573 (2014).
Qi, Y. et al. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat. Biotechnol. 35, 154–163 (2017).
Thompson, L.H. & Björklund, A. Reconstruction of brain circuitry by neural transplants generated from pluripotent stem cells. Neurobiol. Dis. 79, 28–40 (2015).
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).
Bachoud-Lévi, A.C. & Perrier, A.L. Regenerative medicine in Huntington's disease: current status on fetal grafts and prospects for the use of pluripotent stem cell. Rev. Neurol. (Paris) 170, 749–762 (2014).
Barker, R.A., Drouin-Ouellet, J. & Parmar, M. Cell-based therapies for Parkinson disease—past insights and future potential. Nat. Rev. Neurol. 11, 492–503 (2015).
Lindvall, O. Clinical translation of stem cell transplantation in Parkinson's disease. J. Intern. Med. 279, 30–40 (2016).
Steinbeck, J.A. & Studer, L. Moving stem cells to the clinic: potential and limitations for brain repair. Neuron 86, 187–206 (2015).
Kikuchi, T. et al. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model. Nature 548, 592–596 (2017).
Di Lullo, E. & Kriegstein, A.R. The use of brain organoids to investigate neural development and disease. Nat. Rev. Neurosci. 18, 573–584 (2017).
Perrin, S. Preclinical research: make mouse studies work. Nature 507, 423–425 (2014).
Mak, I.W., Evaniew, N. & Ghert, M. Lost in translation: animal models and clinical trials in cancer treatment. Am. J. Transl. Res. 6, 114–118 (2014).
Assawachananont, J. et al. Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice. Stem Cell Rep. 2, 662–674 (2014).
Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).
Huch, M., Boj, S.F. & Clevers, H. Lgr5(+) liver stem cells, hepatic organoids and regenerative medicine. Regen. Med. 8, 385–387 (2013).
Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Mansour, A.A., Khazanov-Zisman, S., Netser, Y., Klar, A. & Ben-Arie, N. Nato3 plays an integral role in dorsoventral patterning of the spinal cord by segregating floor plate/p3 fates via Nkx2.2 suppression and Foxa2 maintenance. Development 141, 574–584 (2014).
Gonçalves, J.T., Anstey, J.E., Golshani, P. & Portera-Cailliau, C. Circuit level defects in the developing neocortex of Fragile X mice. Nat. Neurosci. 16, 903–909 (2013).
Gonçalves, J.T. et al. In vivo imaging of dendritic pruning in dentate granule cells. Nat. Neurosci. 19, 788–791 (2016).
Heiney, S.A., Wohl, M.P., Chettih, S.N., Ruffolo, L.I. & Medina, J.F. Cerebellar-dependent expression of motor learning during eyeblink conditioning in head-fixed mice. J. Neurosci. 34, 14845–14853 (2014).
Pologruto, T.A., Sabatini, B.L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).
Jin, X., Tecuapetla, F. & Costa, R.M. Basal ganglia subcircuits distinctively encode the parsing and concatenation of action sequences. Nat. Neurosci. 17, 423–430 (2014).
Perkel, D.H., Gerstein, G.L. & Moore, G.P. Neuronal spike trains and stochastic point processes. II. Simultaneous spike trains. Biophys. J. 7, 419–440 (1967).
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).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Microscopic image shows a single colony of H9-GFP+ hESCs (Left), and a representative example of 46-days old GFP+ brain organoids used for the transplantation experiments. (b) Immunofluorescence staining for GFP of GFP-expressing cerebral organoids at day 60. Note, unlike the perimeter areas, the center of the organoid lacks GFP expression. Solid line indicates the border of the low-GFP expressing region. (c) Cerebral organoids immunostained for PAX6 and CTIP2 at 38 days. (d) 38-days old cerebral organoids immunostained for SOX2 and NeuN. Right panel is magnification of the boxed area in the left panel. Scale bar is 1 mm in a, 200 μm in b, 20 μm in c, d (right), and 200 μm in d (left).
(a) Immunofluorescence staining for GFP and human mitochondria (hMito), which specifically labels human cells, of the grafted brain organoid at 14 dpi. Insets show staining form the same section of neurites projection into the host brain. (b) Lower power slide scanner image of a coronal brain section stained with anti-GFP antibody showing robust integration of GFP+ human brain organoids at 90 dpi. Unlike organoids grown in culture before grafting (Supplementary Fig. 1b), there are no detectable signs of regions that lack GFP expression in the center of the organoid 3 months post-implantation. (c) Left, confocal stitched tile scan of 90dpi-organoid graft stained with GFP. Right, Histogram of GFP intensity at various positions of the graft across the red line. Nuclei were counterstained with DAPI. Scale bar is, 100 μm in a, 1 mm in b, 200 μm in c.
(a) Immunofluorescence staining for the mature astrocyte marker GFAP (left) and microglial marker Iba1 (right) on a whole organoid cross section at 46 days in culture. (b) Immunofluorescence staining of the mature neuronal dendrite marker MAP2 (left panel), astrocyte marker S100B (middle panel), and triple staining of GFP, hGFAP and SOX2 (right panel) inside the organoid graft at 50 dpi. Note the cellular colocalization between hGFAP and SOX2; co-association of SOX2 and hGFAP suggests that SOX2+ cells are glia and not NPCs.
(c) Immunofluorescence staining of GFP, Sox2, and NeuN within the grafted brain organoid at 90 dpi.(d) Immunofluorescence staining of GFP and MAP2 within the grafted brain organoid at 90 dpi. (e) Immunofluorescence staining of GFP, hNuclei, and SOX2 within the grafted brain organoid at 233 dpi. (f) Immunofluorescence staining of GFP and NeuN, within the grafted organoid at 233 dpi. (g) Immunofluorescence staining of GFP, Olig2, and Iba1 within the grafted brain organoid at 233 dpi. (h) Example image of coronal section obtained from grafted mouse brain at 90 dpi. Nuclei were counterstained with DAPI. Scale bar is, 500 μm in a, 50 μm in b-g, 1 mm in h.
Supplementary Figure 4 Brain organoids send long-distance axonal projections with synaptic connectivity after implantation in mouse brain.
(a) Slide scanner images of coronal brain sections stained for GFP and obtained from 90-dpi grafted mouse brain show robust integration of GFP+ organoids and very large numbers of axons extending into the grafting region and into more rostral section of the host brain. Dashed boxes indicate the sampled region from which higher magnification views in (b) were obtained. Note that images were over-saturated for the signal in the graft region to show the GFP signal in the axons trajectories. Note that the high background on the edge of the section is a nonspecific signal. Scale bars:1 mm in a and 200 μm in b.
(a) Double immunofluorescence staining of GFP and the endothelial marker Endoglin in the indicated post-implantation stage showing the intensive growth of blood vessels inside the organoid graft. (b) Double immunofluorescence staining for human-specific CD31 and CD31 (recognizing both mouse and human) of organoid graft harvested at 90 dpi. Note that the organoid graft is negative for human CD31, suggesting a host origin of the infiltrated blood vessels at the examined time point. (c) Quantification of vascularization success rate in the grafted organoids. Average value is 85.4%±6.4; data represent mean±s.e.m (n=10 independent experiments, total of 55 animals). (d) Surface area change of single grafted organoids during the first 2 weeks of implantation. (e) The average change of total organoid surface area during the first 2 weeks of implantation normalized to day 0 (100%). Data represent mean ± s.e.m from at least 3 grafted animals; day 0-5 (n=9), day 6 (n=8), day 7 and 8 (n=6), day 13 (n=4), day 14 (n=3). (f) Deep in vivo two-photon imaging of GFP signal of the implanted organoids in a head-fixed awake mouse through the cranial window, demonstrating the feasibility of the imaging system. Image shows maximum projection of 300 μm from 200-500 μm inside the grafted organoid from a 30 dpi mouse. (g) Two-photon imaging of blood vessels inside the grafted organoid. Dextran was infused in engrafted animal at 120 dpi. Single z-plane acquired at 418 μm depth below the organoid surface, showing blood flow inside the vascular network (see Supplementary video 4). Scale bar is 50 μm in a,b and 100 μm in h,g.
Supplementary Figure 6 Electrophysiological recording of neuronal activity in the grafted brain organoids.
(a) Firing rate changes of single neurons obtained from 50-dpi organoid at two different levels of depth (top: −0.9 mm; bottom: −1.0 mm) from the graft surface. Each line (color-coded) indicates the firing rate of an individual neuron. Arrows on the top denote the time isoflurane was turned ON (filled arrows) and OFF (empty arrows). (b) Spike raster plots from the neurons in (a) at two different depths (top: −0.9 mm; bottom: −1.0 mm). Each vertical bar indicates a single spike. (c) Cross-correlation of neuron pairs in (a) at depths of −0.9 mm (top) and −1.0 mm (bottom). (d,e) Example of cross correlation analysis performed on one neuron pair from a 115 dpi organoid graft (Fig. 6f). Analysis was performed by testing the significance of the central peak observed in cross-correlation (d). The threshold for the test was calculated based on mean + 3*SD of the baseline, which was defined as the time window from −100 ms to −20 ms in cross-correlation (d, dashed blue line). Obviously, although the magnitude is relatively small, the central peak significantly exceeds the threshold, which is evident of correlation between neurons. Note that when shuffling the neuronal activity with a random duration, the shuffled cross-correlation between the same neurons in (d) became completely flat with no peak (e), indicating that the correlation is indeed from the correlated activity, rather than noise. (f) Spikes with magnitude exceeding the threshold were tagged and recorded as waveforms (red arrows). Then we applied PCA analysis to these waveforms to discriminate the neuronal spikes from noise. On PC plane, waveforms showing distinct shapes are classified into separate clusters.
Supplementary Figure 7 Electrophysiological recording of neuronal activity in the grafted brain organoids.
Examples of in vivo recording from the organoid graft and the host brain. (a-b) Left panels, firing rate changes of single neurons obtained from the indicated time points and the dorsal-ventral (DV) depth from the graft surface. Each line (color-coded) indicates the firing rate of an individual neuron. Arrows on the top denote the time isoflurane was turned ON (filled arrows) and OFF (empty arrows). Right panels, spike raster plots from the neurons in the left panel. Each vertical bar indicates a single spike. (c) Firing rate changes of single neurons obtained from the host cortical region.
Organoid graft was injected with AAV-hSyn-ChR2-YFP and analyzed at 155 dpi. (a) Immunostaining for GFP (labeling hSyn::ChR2-YFP) and hNuclei inside the organoid graft. (b) Immunostaining for tdTomato (labeling organoid graft) and GFP (labeling hSyn::ChR2-YFP) in the cortex of the host brain at 155 dpi. Bottom panels are magnification of the boxed area in the top panel. Note the fragmented tdTomato expression along the axons perhaps due to long term expression. Nuclei were counterstained with DAPI. Scale bar is 50 μm.
Supplementary Figure 9 ptogenetic stimulation evokes neuronal responses in the organoid-graft expressing ChR2.
(a) Diagram of optogenetic stimulation and electrophysiological recording in un-injected control organoid-graft. (b) Light-evoked responses from the same single neuron to 1s constant (left panel), 50 Hz (middle panel), or 20 Hz (right panel) light stimulation in control graft. Top panels: raster plot of neuronal responses in 30 trials. Each tick indicates a single spike. Bottom panels: PETH of the average firing rate across 30 trials. (c,d) Organoid graft was injected with AAV-hSyn-ChR2-YFP and analyzed at 155dpi. (c) Diagram of optogenetic stimulation and electrophysiological recording in organoid graft. (d) Light-evoked responses from the same single neuron to 1s constant (left panel), 50 Hz (middle panel), or 20 Hz (right panel) light stimulation. The stimulation onset is at 0. Top panels: raster plot of neuronal responses in 30 trials. Each tick indicates a single spike. Bottom panels: PETH of the average firing rate across 30 trials. The response latency to the laser onset was less than 5 milliseconds and no detectable response was observed before or after the period of light illumination. n= 2 animals. All recordings were performed while animals were under isoflurane anesthesia.
Supplementary Figure 10 Optogenetic stimulation reveals intragraft neuronal activity and functional output connectivity from the organoid graft to host brain.
(a) Diagram of optogenetic stimulation in tdTomato-expressing uninfected-control organoid and electrophysiological recording in the host brain. (b) Local field potential (LFP) recorded from a single electrode in the control host brain after optogenetic stimulation of organoid with 20 Hz light stimulation. The voltage is color coded for 30 trials (top). Averaged LFP across trials (bottom). (c) LFP changes recorded from a different electrode from the same brain region in (b). (d-h) Organoid graft was injected with AAV-hSyn-ChR2-YFP and analyzed at 155dpi. (d) Diagram of optogenetic stimulation in tdTomato-expressing, AAV-hSyn-ChR2-YFP-infected, organoid and electrophysiological recording in the host brain (fiber location: DV -1.7mm; array location: AP -2.54 mm, ML -1.5 mm, DV -2.2 mm). (e) Local field potential recorded from a single electrode in the host brain region. Optogenetic stimulation of organoid with 20 Hz drives LFP changes in the host brain region. The voltage is color coded for 30 trials (top). Averaged LFP across trials (bottom). Inset: Averaged LFP with a finer time scale. (f) Power spectral density of the averaged LFP in (e). (g) LFP changes recorded from a different electrode from the same brain region in (e). Inset: Averaged LFP with a finer time scale. (h) Power spectral density of the averaged LFP in (g). (i-k) LFP changes recorded from the same animal and brain region as in (d) but from different, more dorsal array location (AP -2.54 mm, ML -1.5 mm, DV -0.8mm). n=2 animals. All recordings were performed while animals were under isoflurane anesthesia.
Supplementary Figure 11 Behavioral examination of spatial learning abilities in organoid-grafted mice
(a) Latency to locate target during Barnes maze training of 3 trials per day for 3 days. Values for individual mice were averaged within each day for analysis. Training performance was analyzed by two-way ANOVA with group as a between subject factor and day as a within-subject factor. There was a significant effect of day (F(2,24) = 24.3, p < 0.0001), but no effect of group (F(1,12) = 0.8116, p = 0.3854) or group X day interaction (F(2,24) = 2.878, p = 0.0758). (b) Latency to locate target during Barnes maze probe trial on day 4, 24 hr after final training day. Probe trial performance was analyzed by the Mann-Whitney test. Unengrafted vs. grafted p = 0.1474. (c) Errors prior to locating target during Barnes maze training of 3 trials per day for 3 days. Values for individual mice were averaged within each day for analysis. There was a significant effect of day (F(2,24) = 7.244, p = 0.0035) and a group X day interaction (F(2,24) = 7.878, p = 0.0023; **post-hoc Sidak’s multiple comparison test p = 0.0052 for training Day 1, p = 0.7258 for Day 2, p = 0.9438 for Day 3). No main effect was observed for group (F(1,12) = 0.8008, p = 0.3884). (d) Pokes to target location during Barnes maze probe trial on day 4, 24 hr after final training day. **Unengrafted vs. grafted p = 0.0076 (e) Location of errors during probe trial for Nod-Scid mice. (f) Location of errors during probe trial for organoid implanted mice. Values in a-f represent mean ± s.e.m. for each group, n=7 mice per group.
Supplementary Figures 1–11 (PDF 2391 kb)
List of primary and secondary antibodies. (PDF 81 kb)
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. (AVI 40870 kb)
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. (AVI 4165 kb)
Two-photon time series of grafted organoid after dextran injection at 120 dpi showing active blood flow at 141 μm depth. (MP4 18013 kb)
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. (AVI 3980 kb)
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. (AVI 2567 kb)
This representative 60 s movie of calcium activity was acquired at 109 dpi. (AVI 2266 kb)
About this article
Cite this article
Mansour, A., Gonçalves, J., Bloyd, C. et al. An in vivo model of functional and vascularized human brain organoids. Nat Biotechnol 36, 432–441 (2018). https://doi.org/10.1038/nbt.4127
This article is cited by
Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types
Cell Communication and Signaling (2023)
Gene Therapy (2023)
Molecular Psychiatry (2023)
Acta Pharmacologica Sinica (2023)
Opportunities and limitations for studying neuropsychiatric disorders using patient-derived induced pluripotent stem cells
Molecular Psychiatry (2023)