Structural and functional integration of human forebrain organoids with the injured adult rat visual system

Abstract

Brain organoids created from human pluripotent stem cells represent a promising approach for brain repair. They acquire many structural features of the brain and raise the possibility of patient-matched repair. Whether these entities can integrate with host brain networks in the context of the injured adult mammalian brain is not well established. Here, we provide structural and functional evidence that human brain organoids successfully integrate with the adult rat visual system after transplantation into large injury cavities in the visual cortex. Virus-based trans-synaptic tracing reveals a polysynaptic pathway between organoid neurons and the host retina and reciprocal connectivity between the graft and other regions of the visual system. Visual stimulation of host animals elicits responses in organoid neurons, including orientation selectivity. These results demonstrate the ability of human brain organoids to adopt sophisticated function after insertion into large injury cavities, suggesting a translational strategy to restore function after cortical damage.

Keywords: brain organoid; brain repair; functional integration; stem cells; visual cortex.

Figure 1:. Human cortical organoids exhibit robust survival after transplantation into the injured visual cortex of adult rats.

(A) Schematic illustrating the protocol used to generate cortical organoids (see Methods for additional details). NS1: necrostatin-1, Dtx: day of transplantation. (B-C) Brightfield (B) and fluorescence (C) micrographs of whole organoids at different times points in vitro demonstrate the presence and growth of multiple cortical units. (D) Immunofluorescence staining of sectioned organoids in vitro show cortical units consisting of a central lumen surrounded by layers of neural progenitors (Pax6⁺) and differentiated neurons (MAP2⁺). (E) Makers for upper- (Satb2) and lower-layer cortical neurons (CTIP2) show the formation of rudimentary laminar structure. (F) Schematic of the transplantation paradigm, including the location of the 5 mm craniotomy. The micrographs on the right show the appearance of the intact brain and brain after an organoid has been inserted into an aspiration cavity (arrow). (G) Brightfield images of the same animal 2 months after transplantation under visible (left) and ultraviolet light (right) show the integrated organoid with intact surrounding brain tissue and vasculature. The graft can be clearly identified by its GFP signal. (H), Coronal sections of animal brains 1, 2, and 3 months after transplantation demonstrate organoid growth over time. Composites of individual images are stitched together. (I). Quantification of the organoid volumes over time. The red line at the bottom of the graph represents the average organoid volume at the time of transplantation. (J-L) Brightfield (J) and fluorescence (K-L) images depict blood vessel growth (CD31⁺) within the transplanted organoid that is most likely of host origin (GFP⁻). (M) The number of distinct CD31⁺ vascular structures in the entire cross-section of an organoid is quantified. (N) Representative fluorescent images depict the temporal progression of apoptotic cells (cleaved caspase 3⁺) within organoid grafts. (O) The percentage of cleaved caspase 3⁺ cells within organoid grafts is quantified. In (I), (M), and (O), n=6 animals with long horizontal bars representing means and short horizontal bars representing 1 standard deviation. *p<0.05, **p<0.001 (Kruskal-Wallis test). Scale bars: 500 μm (B-C), 100 μm (D-E, K), 1 mm (F-H), 20 μm (L, N).

Figure 2:. Organoid grafts demonstrate progressive cellular maturation.

A broad panel of immunohistochemical markers was imaged with confocal microscopy to evaluate the cellular composition of the organoid grafts. Fluorescent micrographs in this figure are all at 2 months after transplantation. (A) This low-magnification micrograph (composite of individual images that are stitched together) depicting STEM121 staining shows the location of the high-magnification images in B. The other high-magnification micrographs in this figure are similar in location to B. (B) STEM121 confirms the human origin of the vast majority of the graft cells (GFP⁺). Neural progenitors are identified with Pax6 (C) and Sox2 (E). (D) Quantification of Pax6⁺ cells shows a decline in this population over time.The presence of differentiated neurons is shown using MAP2 (F) and NeuN (G). (H) Quantification of NeuN demonstrates an increase in the number of mature neurons in the organoid graft over time. (I) The majority of graft cells are positive for FoxG1, a telencephalic maker. (J-N), Cells in the organoid graft express markers for cortical layers V (Tbr1, CTIP2), IV (Necab1), and II/III (Cux1, Satb2). (O) Quantification of these cortical layer markers demonstrates increasing CTIP⁺, Cux1⁺, and Satb2⁺ cells and decreasing Tbr1⁺ cells over time. (P-Q) Astrocytes are identified with GFAP, which is quantified in (Q) Reconstructions of confocal z stacks show no co-localization of GFAP with GFP. (R-S) Oligodendrocytes are identified with Olig2, which is quantified in (S). In (D), (H), (O), (Q), and (S), n=6 animals with long horizontal bars representing means and short horizontal bars representing 1 standard deviation*p<0.05, **p<0.001 (Kruskal-Wallis test). Scale bars: 1 mm (A), 20 μm in all other micrograph panels.

Figure 3:. Transplanted organoids send projections widely into the host brain.

(A) This schematic illustrates the approximate location of projections from the organoid graft 2 months after transplantation. (B-C) Low- (B) and high-magnification (C) views of the host cortex adjacent to the transplanted organoid on a coronal section show a high-density of GFP⁺ projections adjacent to the graft. (D-G) More remote GFP⁺ projections are found in the retrosplenial cortex medial to the graft (D-E), auditory cortex laterally (F), and visual cortex lateral to the graft (G). The dotted line in the upper right corner of (D) defines the border between the organoid graft and host brain. (H-L) Graft projections are also identified in the ipsilateral (H) and contralateral corpus callosum (I), as well as the thalamus (J). GFP⁺ processes within the thalamus co-localize with STEM121, confirming their human origin. The schematics in the upper right corner of (E-H) and (J) show the location of the GFP⁺ processes in the brain for that panel. In (D-I), green is GFP and blue is DAPI. (K) GFP⁺ processes co-localize with NF-200 (red) but not MAP2, a somatic and dendritic marker (white). (L) They also co-localize with tau (red), an axonal marker. These processes do not appear to be myelinated (myelin basic protein, white). (M) GFP⁺ processes in the host brain also co-localize with human synapsin (red). (B) and (I) are composites of individual images that are stitched together. (N) The number of GFP⁺ projections is quantified in different regions of the brain at 1-3 mpt. (O), The number of GFP⁺ projections is quantified in different thalamic nuclei at 2 and 3 mpt. In (N-O), n=6 animals with long horizontal bars representing means and short horizontal bars representing 1 standard deviation. Scale bars: 500 μm (B, D, I), 100 μm (C, F), 50 μm (E, G, H, J-L), and 10 μm (M).

Figure 4:. Organoid grafts receive afferent inputs from multiple regions of the rodent brain.

(A) This schematic illustrates the timeline for using the modified rabies system for monosynaptic retrograde tracing to analyze organoid graft afferents. (B) Transplanted organoids were immunostained with STEM121 (red) to confirm the human origin of the graft. (C-D) Uninfected cells within the organoid express DsRed⁺ alone. Organoid cells infected by RABV-GFP express both DsRed and GFP and are thus yellow. Cells that only express GFP (arrows in the right panel of (D)) are trans-synaptic partners that are upstream of the yellow “starter” cells. All GFP⁺ cells in the organoid co-localize with STEM121 and are thus of human origin (see Figure S6). (E-R) Regions of the host brain providing monosynaptic input to the organoid graft include adjacent visual cortex (F-G, more remote somatosensory cortex (H-I), ipsilateral (J-L) and contralateral hippocampi (M-O), and the ipsilateral thalamus (P-R). (B), (C), (E), (J), (M), and (P) are composites of individual images that are stitched together. (S-T) The number of GFP⁺ cells that are monosynaptically connected with organoid grafts are quantified by brain region (S) and thalamic nuclei (T). In (S-T), n=4 animals with long horizontal bars representing means and short horizontal bars representing 1 standard deviation. *p<0.05, **p<0.001 (Kruskal-Wallis test). Scale bars: 1 mm (E, J, M, P), 500 μm (B-C) 300μm (F, H, K, N, Q), 50 μm (G, I, L, O, R), 10 μm (D).

Figure 5:. A polysynaptic pathway exists between the retina of the host animal and transplanted organoids.

(A) This schematic shows the timeline for using modified herpes simplex virus (HSV) for polysynaptic anterograde tracing. (B) A low-magnification micrograph of the right hemisphere of the brain (coronal section) shows GFP+ cells within the organoid (white arrow) and multiple regions of the host brain. The densest area of GFP+ cells in the host brain corresponded to the entorhinal cortex (asterisk). (C) A human antigen marker (STEM121, red) was used to identify the non-fluorescent graft. (D) A high-magnification view of the organoid shows a high density of GFP⁺ cells co-localizing with STEM121. (E) HSV-labeled cells (GFP⁺, green) within organoid grafts are neuronal in nature, as evidenced by co-labeling with the neuronal marker, beta-III tubulin (red). (F) In contrast, these HSV-labeled cells (GFP⁺ green) do not co-localize with GFAP (red) and are thus unlikely to be astrocytes. (G-K) Micrographs demonstrate the presence of GFP⁺ cells in expected parts of the visual pathway, including the visual cortex adjacent to the organoid (G), lateral geniculate nucleus of the thalamus (H-I), and the optic nerve contralateral, but not ipsilateral, to the organoid graft (J-K). (L-M) The presence of GFP⁺ cells in the visual cortex of naïve animals treated with retinal injections of HSV-GFP is shown. (B), (C), (H), and (L) are composites of individual images that are stitched together. (N) Quantification of the GFP⁺ cells demonstrates a significantly higher density of these cells in the organoid compared to adjacent visual cortex and visual cortex in naïve animals without organoid grafts. In (N), n=4 animals with long horizontal bars representing means and short horizontal bars representing 1 standard deviation. **p<0.01 (Kruskal-Wallis test). Scale bars: 1 mm (B), 500 μm (C, H, L), 100 μm (G, I, J, K, M), 50 μm (D-F).

Figure 6:. Transplanted organoids functionally integrate with the host visual system.

Neural activity from naïve visual cortex and an organoid graft at 2 mpt is shown on the left and right, respectively. (A) Schematic of experimental design with visual stimulation and electrophysiological recordings. (B-C) Representative single-unit waveforms recorded from the visual cortex of animal naïve-1 (B) and the organoid graft in animal 2M-1 (C) are depicted. The grey lines are single spikes, and the black line indicates the average waveform across all recorded spikes. (D-E) Raster plots of single units recorded from naïve-1 (D) and the organoid graft in 2M-1 (E) are shown across 140 and 120 trials, respectively. The yellow line corresponds to the timing of a 0.5 Hz flashing screen stimulus. (F-G) Peri-stimulus time histograms (PSTH; grey bars, left axis) and the event-related potentials detected at the channel where the unit had the greatest amplitude (ERP; black line, right axis) are plotted for representative evoked units recorded from the visual cortex in naïve-1 (E) and the organoid graft in 2M-1 (F). These are the same units as depicted in (D-E). Again, the yellow lines correspond to the 0.5 Hz flashing screen stimulus. (H-K), Rose plots show the number of times a single unit fired based on the orientation of a presented drifting grating for 2 example units recorded from naïve-1 (H-I) and the organoid in 2M-1 (J-K). These units show preferential firing for drifting gratings in a particular orientation. Black lines indicate the average number of spikes across trials and grey lines correspond to 1 standard error above and below the mean. Angle measures around the periphery of the rose plot indicate the orientation of drifting grating presentation (degrees). The radial axis indicates the firing rate of the neuron in response to that orientation of drifting grating (Hz). OSI: orientation selectivity index.