Maturation and circuit integration of transplanted human cortical organoids methods
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Main
To study human-derived cortical neurons within in vivo circuits, we stereotactically transplanted intact, 3D hCO into the S1 of early postnatal athymic rats (postnatal days 3-7) (Fig. and Extended Data Fig. ). At this time point, thalamocortical and corticocortical axonal projections have not yet completed their innervation of the S1 (ref. ). This approach therefore aims to maximize integration of t-hCO while minimally compromising endogenous circuitry. To visualize the location of t-hCO in living animals, we performed T2-weighted MRI reconstructions of the rat brain at 2-3 months post-transplantation (Fig. and Extended Data Fig. ). t-hCO were readily observed and volume measurements of t-hCO were similar to those calculated from fixed slices (Extended Data Fig.; P > 0.05). We identified t-hCO in 81% of engrafted animals at approximately 2 months post-transpl...
Main
We next asked to what extent t-hCO cells functionally integrate into the rat S1. The S1 in rodents receives robust synaptic input from the ipsilateral ventrobasal nucleus and the posterior nucleus of the thalamus, as well as from the ipsilateral motor and secondary somatosensory cortices and contralateral S1 (Fig. ). To reconstruct innervation patterns, we infected hCO with rabies-dG-GFP/AAV-G and, after 3 days, hCO were transplanted into the rat S1. At 7-14 days post-transplantation, we observed dense GFP expression in neurons in the ipsilateral S1 and ventrobasal nucleus (Fig. ). In addition, antibody staining for the thalamic marker netrin G1, revealed the presence of thalamic terminals in t-hCO (Fig. ). To assess whether these afferent projections were capable of evoking synaptic responses in t-hCO cells, we performed whole-cell recordings from human cells in acute thalamoco...
Main
We next asked whether t-hCO could be activated by sensory stimuli within an in vivo context. We transplanted hCO expressing the genetically encoded calcium indicator GCaMP6s into the rat S1. After 150 days, we conducted fibre photometry or two-photon calcium imaging (Fig. and Extended Data Fig. ). We found that t-hCO cells exhibited synchronous, rhythmic activity (Fig., Extended Data Fig. and Supplementary Video ). To characterize the spiking activity of t-hCO, we performed extracellular electrophysiological recordings in anaesthetized, transplanted rats (Extended Data Fig. ). We generated stereotactic coordinates based on images acquired from MRI; these recorded units thus represent putative human neurons, although electrophysiology alone does not permit species-of-origin identification. We observed synchronous bursts of activity (Extended Data Fig. ). Spiking bursts were appr...
Main
We next investigated whether t-hCO can engage rat circuits to drive behaviour. We first examined whether t-hCO neurons send axonal projections into surrounding rat tissue. We infected hCO with a lentivirus encoding hChR2 fused to EYFP (hChR2-EYFP). We observed EYFP expression in ipsilateral cortical regions including auditory, motor and somatosensory cortices, as well as in subcortical regions including the striatum, hippocampus and thalamus, 110 days later (Fig. ). To assess whether these efferent projections were capable of evoking synaptic responses in rat cells, we optically activated hChR2-EYFP-expressing t-hCO cells while recording from cortical rat cells in acute brain slices. Activation of t-hCO axons with blue light evoked short-latency EPSCs in rat pyramidal cortical neurons, which were blocked by NBQX (Fig. ). Moreover, these responses could be blocked by a...
Main
We finally asked whether t-hCO were capable of modulating rat behaviour. To test this, we transplanted hCO expressing hChR2-EYFP into the S1, and 90 days later, we implanted an optical fibre into t-hCO for light delivery. We then trained rats on a modified operant conditioning paradigm (Fig. ). We placed animals into a behavioural testing chamber and applied randomly interleaved presentations of 5-s-long blue (473 nm) and red (635 nm) laser stimulations. Animals received a water reward if they licked during the blue light stimulation, but not if they licked during the red light stimulation. On the first day of training, animals showed no difference in their licking behaviour during either blue or red light stimulation. However, on day 15, animals transplanted with hCO expressing hChR2-EYFP showed increased licking during blue light stimulation compared with red...
Methods
We generated hCO from hiPS cells as previously described,. To initiate the generation of hCO from hiPS cells cultured on feeders, intact hiPS cell colonies were lifted from the plates using dispase (0.35 mg ml -1 ) and transferred to ultra-low attachment plastic dishes (Corning) in hiPS cell medium supplemented with the two SMAD inhibitors dorsomorphin (5 µM; P5499, Sigma-Aldrich) and SB-431542 (10 µM; 1614, Tocris) and the ROCK inhibitor Y-27632 (10 µM; S1049, Selleckchem). For the first 5 days, the hiPS cell medium was changed every day and supplemented with dorsomorphin and SB-431542. On the sixth day in suspension, neural spheroids were transferred to neural medium containing neurobasal-A (10888, Life Technologies), B-27 supplement without vitamin A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin and...
MRI of transplanted rats
All animal procedures followed animal care guidelines approved by Stanford University's APLAC. Rats (more than 60 days post-transplantation) were anaesthetized with 5% isoflurane for induction and 1-3% isoflurane during imaging. For imaging, an actively shielded Bruker 7 Tesla horizontal bore scanner (Bruker Corp.) with International Electric Company (IECO) gradient drivers, a 120-mm inner diameter shielded gradient insert (600 mT/m, 1,000 T/m/s), AVANCE III electronics, eight-channel multi-coil radiofrequency and multinuclear capabilities, and the supporting Paravision 6.0.1 platform were used. Acquisitions were performed with an 86 mm inner diameter actively de-couplable volume radiofrequency coil with a four-channel cryo-cooled receive-only radiofrequency coil. Axial 2D Turbo-RARE (repetition time = 2,500 ms, echo time = 33 ms, 2 averages) 16 slic...
Lentivirus labelling and G-deleted rabies infections
To stably express GCaMP6s in hCO for two-photon calcium imaging, hiPS cells were infected with pLV[Exp]-EF1a::GcaMP6s-WPRE-Puro followed by antibiotic selection. In brief, the cells were dissociated with EDTA and suspended at a density of approximately 300,000 cells in 1 ml in Essential 8 medium in the presence of polybrene (5 µg ml -1 ) and 15 µl of virus. Cells were then incubated in suspension for 60 min and plated at a density of 50,000 cells per well. Once confluent, cells were treated with 5-10 µg ml -1 puromycin for 5-10 days or until stable colonies appeared. Acute infections of hCO were performed as previously described with a few modifications. In brief, day 30-45 hCO were transferred to 1.5-ml microcentrifuge Eppendorf tubes containing 100 µl neural medium. Next, approximately 9...
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To study human-derived cortical neurons within in vivo circuits, we stereotactically transplanted intact, 3D hCO into the S1 of early postnatal athymic rats (postnatal days 3-7) (Fig. and Extended Data Fig. ). At this time point, thalamocortical and corticocortical axonal projections have not yet completed their innervation of the S1 (ref. ). This approach therefore aims to maximize integration of t-hCO while minimally compromising endogenous circuitry. To visualize the location of t-hCO in living animals, we performed T2-weighted MRI reconstructions of the rat brain at 2-3 months post-transplantation (Fig. and Extended Data Fig. ). t-hCO were readily observed and volume measurements of t-hCO were similar to those calculated from fixed slices (Extended Data Fig.; P > 0.05). We identified t-hCO in 81% of engrafted animals at approximately 2 months post-transplantation ( n = 72 animals; hCO from 10 hiPS cell lines; hiPS cell lines are listed in Supplementary Table ). Of these, 87% were located in the cerebral cortex (Fig. ). By performing consecutive MRI scans at multiple time points in the same transplanted rats, we found that t-hCO increase...
We next asked to what extent t-hCO cells functionally integrate into the rat S1. The S1 in rodents receives robust synaptic input from the ipsilateral ventrobasal nucleus and the posterior nucleus of the thalamus, as well as from the ipsilateral motor and secondary somatosensory cortices and contralateral S1 (Fig. ). To reconstruct innervation patterns, we infected hCO with rabies-dG-GFP/AAV-G and, after 3 days, hCO were transplanted into the rat S1. At 7-14 days post-transplantation, we observed dense GFP expression in neurons in the ipsilateral S1 and ventrobasal nucleus (Fig. ). In addition, antibody staining for the thalamic marker netrin G1, revealed the presence of thalamic terminals in t-hCO (Fig. ). To assess whether these afferent projections were capable of evoking synaptic responses in t-hCO cells, we performed whole-cell recordings from human cells in acute thalamocortical slices. Electrical stimulation of the nearby fibres in the rat S1, internal capsule, white matter, t-hCO or optogenetic activation of opsin-expressing thalamic terminals in t-hCO evoked short-latency EPSCs in t-hCO neurons, which were blocked by application of the AMPA receptor antagonist NB...
We next asked whether t-hCO could be activated by sensory stimuli within an in vivo context. We transplanted hCO expressing the genetically encoded calcium indicator GCaMP6s into the rat S1. After 150 days, we conducted fibre photometry or two-photon calcium imaging (Fig. and Extended Data Fig. ). We found that t-hCO cells exhibited synchronous, rhythmic activity (Fig., Extended Data Fig. and Supplementary Video ). To characterize the spiking activity of t-hCO, we performed extracellular electrophysiological recordings in anaesthetized, transplanted rats (Extended Data Fig. ). We generated stereotactic coordinates based on images acquired from MRI; these recorded units thus represent putative human neurons, although electrophysiology alone does not permit species-of-origin identification. We observed synchronous bursts of activity (Extended Data Fig. ). Spiking bursts were approximately 460 ms in length and were separated by approximately 2-s-long silent periods (Extended Data Fig. ). Individual units fired on average approximately three spikes per burst, and each burst recruited approximately 73% of recorded units. The activity of individual units was highly correl...
We next investigated whether t-hCO can engage rat circuits to drive behaviour. We first examined whether t-hCO neurons send axonal projections into surrounding rat tissue. We infected hCO with a lentivirus encoding hChR2 fused to EYFP (hChR2-EYFP). We observed EYFP expression in ipsilateral cortical regions including auditory, motor and somatosensory cortices, as well as in subcortical regions including the striatum, hippocampus and thalamus, 110 days later (Fig. ). To assess whether these efferent projections were capable of evoking synaptic responses in rat cells, we optically activated hChR2-EYFP-expressing t-hCO cells while recording from cortical rat cells in acute brain slices. Activation of t-hCO axons with blue light evoked short-latency EPSCs in rat pyramidal cortical neurons, which were blocked by NBQX (Fig. ). Moreover, these responses could be blocked by application of tetrodotoxin (TTX) and recovered by 4-amino-pyridine (4-AP), indicating that they were evoked by monosynaptic connections (Fig. ). Fig. 5 Transplanted hCO make functional connections onto rat neurons and modulate behaviour. a, Schematic of axon tracing (left). t-hCO EYFP expression (r...
We finally asked whether t-hCO were capable of modulating rat behaviour. To test this, we transplanted hCO expressing hChR2-EYFP into the S1, and 90 days later, we implanted an optical fibre into t-hCO for light delivery. We then trained rats on a modified operant conditioning paradigm (Fig. ). We placed animals into a behavioural testing chamber and applied randomly interleaved presentations of 5-s-long blue (473 nm) and red (635 nm) laser stimulations. Animals received a water reward if they licked during the blue light stimulation, but not if they licked during the red light stimulation. On the first day of training, animals showed no difference in their licking behaviour during either blue or red light stimulation. However, on day 15, animals transplanted with hCO expressing hChR2-EYFP showed increased licking during blue light stimulation compared with red light stimulation. These changes in licking behaviour were not observed in control animals transplanted with hCO expressing a control fluorophore (learning success rate: hChR2 89%, EYFP 0%, Fig. and Supplementary Video ). These data suggest that t-hCO cells can activate rat neurons to drive r...
We generated hCO from hiPS cells as previously described,. To initiate the generation of hCO from hiPS cells cultured on feeders, intact hiPS cell colonies were lifted from the plates using dispase (0.35 mg ml -1 ) and transferred to ultra-low attachment plastic dishes (Corning) in hiPS cell medium supplemented with the two SMAD inhibitors dorsomorphin (5 µM; P5499, Sigma-Aldrich) and SB-431542 (10 µM; 1614, Tocris) and the ROCK inhibitor Y-27632 (10 µM; S1049, Selleckchem). For the first 5 days, the hiPS cell medium was changed every day and supplemented with dorsomorphin and SB-431542. On the sixth day in suspension, neural spheroids were transferred to neural medium containing neurobasal-A (10888, Life Technologies), B-27 supplement without vitamin A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin and streptomycin (1:100, Life Technologies) and supplemented with the epidermal growth factor (EGF; 20 ng ml -1; R&D Systems) and fibroblast growth factor 2 (FGF2; 20 ng ml -1; R&D Systems) until day 24. From day 25 to day 42, the medium was supplemented with brain-...
All animal procedures followed animal care guidelines approved by Stanford University's APLAC. Rats (more than 60 days post-transplantation) were anaesthetized with 5% isoflurane for induction and 1-3% isoflurane during imaging. For imaging, an actively shielded Bruker 7 Tesla horizontal bore scanner (Bruker Corp.) with International Electric Company (IECO) gradient drivers, a 120-mm inner diameter shielded gradient insert (600 mT/m, 1,000 T/m/s), AVANCE III electronics, eight-channel multi-coil radiofrequency and multinuclear capabilities, and the supporting Paravision 6.0.1 platform were used. Acquisitions were performed with an 86 mm inner diameter actively de-couplable volume radiofrequency coil with a four-channel cryo-cooled receive-only radiofrequency coil. Axial 2D Turbo-RARE (repetition time = 2,500 ms, echo time = 33 ms, 2 averages) 16 slice acquisitions were performed with 0.6-0.8-mm slice thickness, with 256 × 256 samples. Signal was received with a 2-cm inner diameter quadrature transmit-receive volume radiofrequency coil (Rapid MR International, LLC). Finally, 3D volume rendering and analysis were performed using...
To stably express GCaMP6s in hCO for two-photon calcium imaging, hiPS cells were infected with pLV[Exp]-EF1a::GcaMP6s-WPRE-Puro followed by antibiotic selection. In brief, the cells were dissociated with EDTA and suspended at a density of approximately 300,000 cells in 1 ml in Essential 8 medium in the presence of polybrene (5 µg ml -1 ) and 15 µl of virus. Cells were then incubated in suspension for 60 min and plated at a density of 50,000 cells per well. Once confluent, cells were treated with 5-10 µg ml -1 puromycin for 5-10 days or until stable colonies appeared. Acute infections of hCO were performed as previously described with a few modifications. In brief, day 30-45 hCO were transferred to 1.5-ml microcentrifuge Eppendorf tubes containing 100 µl neural medium. Next, approximately 90 µl of medium was removed and 3-6 µl of high-titre lentivirus (0.5 × 10 8 to 1.2 × 10 9 ) was added to the tube, and the hCO were moved to the incubator for 30 min. Next, 90-100 µl medium was added to each tube and the tubes were returned...
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