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, 36 (5), 432-441

An in Vivo Model of Functional and Vascularized Human Brain Organoids

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An in Vivo Model of Functional and Vascularized Human Brain Organoids

Abed AlFatah Mansour et al. Nat Biotechnol.

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.

Conflict of interest statement

COMPETING INTERESTS

The author declares no competing interests.

Figures

Figure 1
Figure 1
Intracerebral grafting of brain organoids into mouse brain. (a) Illustration of the experimental procedure for generation of GFP+ organoids from hESCs and intracerebral implantation into immunodeficient mouse brain. (b) Whole-mount dorsal view image of mouse brain grafted with a GFP+ cerebral organoid and harvested at 50 dpi; the graft is outlined in white. Right, magnified graft displaying neurite outgrowth from the organoid toward the host brain (arrowheads). (c) Kaplan–Meier survival curve for overall survival of mice after engraftment with brain organoids. (91.8% survival beyond 180 dpi, n = 61 mice from ten experiments). (d) GFP+ organoids were grafted into mouse brain and harvested at the indicated dpi. Coronal sections were analyzed using immunofluorescence and confocal microscopy. Immunofluorescence staining for GFP and human nuclear antigen (hNuclei), demonstrates that the implant survived well and distributed throughout the lesion cavity at 14 dpi. Left image shows confocal stitched tile scan; image was vertically inverted. White solid lines indicate apical/ventricular surface. Dotted white line indicates the radial glia VZ-L regions. Yellow lines indicate the graft-host border. (e) Graft immunostained for GFP, dorsal telencephalic progenitor marker PAX6 and the deep-layer subcortical neuron marker CTIP2. Radially organized cells (arrowhead, left panel) represent the PAX6+ VZ-L region (dotted white lines). n = 4 animals in b, and n = 3 animals in d,e. Nuclei were counterstained with DAPI. Scale bars: 1 μm in b, 100 μm in d, and 20 μm in e.
Figure 2
Figure 2
Survival and differentiation of grafted organoids. Organoids were grafted and harvested at the indicated dpi, coronal sections were analyzed using immunofluorescence and confocal microscopy. (a) Double immunofluorescence staining for SOX2 and NeuN (left), and GFP and SMI312 (right). At 14 dpi, the graft expresses both the NPCs marker SOX2 and the mature neuronal marker NeuN but shows low numbers of SMI312-positive processes. At 50 dpi, the graft retains a lower expression domain of SOX2, NeuN+ cells, and the SMI312-positive area increase. White solid lines indicate the apical/ventricular surface. Dotted white lines indicate the radial glia VZ-L regions. Yellow lines indicate the graft-host border. (b) Quantification of the percentage (mean ± s.e.m.) of SOX2+/DAPI+ and of NeuN+/DAPI+ cells in the graft at the indicated grafting time point. P values were calculated using one-way ANOVA with post hoc Tukey’s test between group comparisons (F(2, 6) = 18.81; P = 0.0026 for NeuN, F(2, 6) = 24.3; P < 0.0001 for SOX2), n = 3 independent animals per group. Asterisks indicate pair-wise comparisons with 14 dpi. (c) Comparative quantification of the percentage of SOX2+/DAPI+ and of NeuN+/DAPI+ cells in the graft compared with stage-matched organoids in culture at the indicated grafting or culture time point. Data are presented as mean ± s.e.m., unpaired two-tailed t-test. For SOX2, 53 day vs. 14 dpi (t = 3.059, df = 8, P = 0.0156) and 102 d vs. 50 dpi (t = 1.617, df = 9, P = 0.1369, not significant). For NeuN, 53 d vs. 14 dpi (t = 1.208, df = 8, P < 0.2617, not significant) and 102 d vs. 50 dpi (t = 12.15, df = 9, P < 0.0001). Day 53 (n = 7 organoids), day 102 (n = 8 organoids) from three independent patches. n = 3 animals for 14 dpi and 50 dpi. (d) Immunofluorescence staining for GFP and human-specific GFAP (hGFAP) at the indicated time points showing astrocyte differentiation in the graft with increased abundance over time. Right panel is a higher magnification of the boxed area. (e) Immunostaining for oligodendrocyte marker Olig2 in the graft at 50 and 90 dpi. (f) Human graft contains Iba1+ microglia that do not co-localize with GFP. (g) Double immunofluorescence staining for presynaptic marker Synapsin (Syn) and the postsynaptic marker PSD95 at 50 dpi, showing a co-association between pre- and post-synaptic compartments and the formation of synaptic connections in the graft. The box indicates the region of magnification from the left panel; arrowheads indicate direct contact between a pre- and post-synapses puncta. Image shows a single plane confocal-Z-section. Nuclei were counterstained with DAPI. Scale bars: 100 μm in a, 50 μm in d–f, 5 μm in f (right panel) and g. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant.
Figure 3
Figure 3
Organoid graft sends axonal projections with synaptic connectivity. (a,b) Confocal images of brain sections stained for GFP and obtained from ipsilateral (a) and contralateral cortex (b) of 90-dpi grafted mouse brain. Images show robust integration of GFP+ organoids and very large numbers of axons extending into the cortical regions and corpus callosum (CC) of the host brain. (c) Single focal plane confocal image of immunostaining for GFP, human-specific Synaptophysin (hSyn), and PSD95 in the host cortex at 90 dpi. Colocalization (arrowheads) and close association of hSyn and PSD95 indicate synaptic connectivity between the organoid axons and the host brain. Right panels show magnification of the boxed region in the left panel. (d) GFP signal with Z-stack 3D reconstruction of hSyn and colocalized only PSD95 puncta. (e) Quantification of the number of hSyn, PSD95, and hSyn/PSD95 colocalized puncta normalized to area and obtained from ipsilateral host cortical regions of 90-dpi grafted mouse brains. Data are presented as mean ± s.e.m. n = 3 grafted animals. Nuclei were counterstained with DAPI. Scale bars: 50 μm in a,b and 5 μm in c,d.
Figure 4
Figure 4
Functional vasculature and decreased apoptosis in grafts. (a) Illustration of the imaging approach for live two-photon microscopy imaging of the organoid graft. (b) Serial macroscopic tracking of grafts showing dynamics of blood vessel perfusion by the recipient vascular system. Dotted area indicates the graft. (c) Grafts co-immunostained for the endothelial markers CD31 and hNuclei at the indicated time points. (d) Organoids have an elevated degree of cell death that is rescued after grafting. Top, TUNEL staining of grafted organoids and stage-matched cultured organoids at the indicated stage. Left panels show staining obtained from two different organoids and organoid grafts at day 31 and 5 dpi, respectively. Bottom, quantification of TUNEL+/DAPI+ cells in grafted organoids and nearly stage-matched cultured organoids of indicated ages. Values are represented as mean ± s.e.m., (n = 3, except for 102-d organoid, n = 4, and 233 dpi n = 2); unpaired two-tailed t-test was used to compare mean difference between each group. Day 31 vs. 5 dpi (t = 0.03656, df = 4, P = 0.9726, not significant), day 53 vs. 14 dpi (t = 14.67, df = 4, P = 0.0001), day 102 vs. 50 dpi (t = 5.943, df = 5, P = 0.0019), and day 279 vs. 233 dpi (t = 6.267, df = 3, P = 0.0082). (e–g) In vivo two-photon imaging of blood vessels via dextran infusion as viewed through the cranial window. Organoids were implanted and TexasRed-dextran was injected at different time points of post-implantation (30 dpi in e, 120 dpi in f). (e) maximum projection of a 300-μm stack taken in a 30-dpi graft (Supplementary Video 2). (f) top view of a three-dimensional reconstruction of a 500-μm Z section in the organoids from a 120-dpi grafted animal (Supplementary Video 3). (g) Single z-plane obtained from 120-dpi graft and acquired at 141-μm depth below the organoid surface, showing blood flow in the vasculature network (Supplementary Video 4). Nuclei were counterstained with DAPI. Scale bars: 1 mm in b, 50 μm in c,d, and 100 μm in e–g. **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Two-photon imaging reveals neuronal activity in the graft. (a) illustration of our imaging system for Ca2+ imaging to monitor neuronal activity of the organoid graft using a two-photon microscope. (b) Representative images (from three frames) of two-photon Ca2+ imaging shown in grayscale (upper) and pseudo-color (lower). The images illustrate epochs of low (left and right panels) and high (middle) Ca2+ concentration in the graft during a Ca2+ transient at 78 dpi. The fluorescence intensity of the calcium sensor is markedly higher during periods of neuronal activity. Graph is a plot of full-frame average intensity during a Ca2+ transient, and red lines indicate time points corresponding to the frames in the left panels. (c) Typical field of view of neurons expressing jRGECO1a in the graft at 168 dpi. (d) Representative ΔF/F calcium traces of cells from the field of view depicted in c. Note the rhythmic and perfectly synchronized activity. (e) Representative ΔF/F traces from the same mouse at different time points after implantation. (f) The same cells in the graft can be imaged over different imaging sessions. n = 3 grafted animals in b–e, n = 1 grafted animal in f. Scale bar, 10 μm in b,c.
Figure 6
Figure 6
In vivo electrophysiological recording in the graft. (a) Illustration of in vivo multielectrode array recording of the organoid graft. (b) Waveforms of action potentials (red) from neuron 3 shown in d,e, third row (DV −1.8 mm). (c) Principal component analysis (PCA) of waveforms from the same neuron in b. (d) Firing rate changes of single neurons (color-coded) obtained from a 115-dpi grafted organoid at four different DV depths (from top to bottom: −0.6 mm, −1.5 mm, −1.8 mm, and −2.0 mm) from the organoid 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). (e) Spike raster plots from the neuronsin d at four different depths (from top to bottom: −0.6 mm, −1.5 mm, −1.8 mm, and −2.0 mm). Each vertical bar indicates a single spike. (f) Cross-correlation of neuron pairs shown in d. At depths of −1.5 mm and −2.0 mm, neuron pairs show strong correlated activity. n = 3 grafted animals.

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