Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Maturation and circuit integration of transplanted human cortical organoids.

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1-5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

Imaging neuronal migration and network activity in human forebrain assembloids.

Assembloids generated from human pluripotent stem cells are self-organizing, multicellular in vitro models that recapitulate aspects of cell-cell interactions and circuit assembly during neural development. Here, we present protocols to functionally monitor, in forebrain assembloids, the migration of GABAergic interneurons from the ventral to the dorsal forebrain and the activity in early cortical networks. Specifically, we describe high-resolution imaging and analysis of neuronal migration as well as calcium imaging of network activity in forebrain assembloids. For complete details on the use and execution of this protocol, please refer to Birey et al. (2022).

Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome.

Defects in interneuron migration can disrupt the assembly of cortical circuits and lead to neuropsychiatric disease. Using forebrain assembloids derived by integration of cortical and ventral forebrain organoids, we have previously discovered a cortical interneuron migration defect in Timothy syndrome (TS), a severe neurodevelopmental disease caused by a mutation in the L-type calcium channel (LTCC) Cav1.2. Here, we find that acute pharmacological modulation of Cav1.2 can regulate the saltation length, but not the frequency, of interneuron migration in TS. Interestingly, the defect in saltation length is related to aberrant actomyosin and myosin light chain (MLC) phosphorylation, while the defect in saltation frequency is driven by enhanced γ-aminobutyric acid (GABA) sensitivity and can be restored by GABA-A receptor antagonism. Finally, we describe hypersynchronous hCS network activity in TS that is exacerbated by interneuron migration. Taken together, these studies reveal a complex role of LTCC function in human cortical interneuron migration and strategies to restore deficits in the context of disease.

Primate cell fusion disentangles gene regulatory divergence in neurodevelopment.

Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.

Generation of human striatal organoids and cortico-striatal assembloids from human pluripotent stem cells.

Cortico-striatal projections are critical components of forebrain circuitry that regulate motivated behaviors. To enable the study of the human cortico-striatal pathway and how its dysfunction leads to neuropsychiatric disease, we developed a method to convert human pluripotent stem cells into region-specific brain organoids that resemble the developing human striatum and include electrically active medium spiny neurons. We then assembled these organoids with cerebral cortical organoids in three-dimensional cultures to form cortico-striatal assembloids. Using viral tracing and functional assays in intact or sliced assembloids, we show that cortical neurons send axonal projections into striatal organoids and form synaptic connections. Medium spiny neurons mature electrophysiologically following assembly and display calcium activity after optogenetic stimulation of cortical neurons. Moreover, we derive cortico-striatal assembloids from patients with a neurodevelopmental disorder caused by a deletion on chromosome 22q13.3 and capture disease-associated defects in calcium activity, showing that this approach will allow investigation of the development and functional assembly of cortico-striatal connectivity using patient-derived cells.

Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals.

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

Generation and assembly of human brain region-specific three-dimensional cultures.

The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region-specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain-specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region-specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.

Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells.

There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

Assembly of functionally integrated human forebrain spheroids.

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring.

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

Genetic and Stress-Induced Loss of NG2 Glia Triggers Emergence of Depressive-like Behaviors through Reduced Secretion of FGF2.

NG2-expressing glia (NG2 glia) are a uniformly distributed and mitotically active pool of cells in the central nervous system (CNS). In addition to serving as progenitors of myelinating oligodendrocytes, NG2 glia might also fulfill physiological roles in CNS homeostasis, although the mechanistic nature of such roles remains unclear. Here, we report that ablation of NG2 glia in the prefrontal cortex (PFC) of the adult brain causes deficits in excitatory glutamatergic neurotransmission and astrocytic extracellular glutamate uptake and induces depressive-like behaviors in mice. We show in parallel that chronic social stress causes NG2 glia density to decrease in areas critical to Major Depressive Disorder (MDD) pathophysiology at the time of symptom emergence in stress-susceptible mice. Finally, we demonstrate that loss of NG2 glial secretion of fibroblast growth factor 2 (FGF2) suffices to induce the same behavioral deficits. Our findings outline a pathway and role for NG2 glia in CNS homeostasis and mood disorders.

Age-Dependent Netrin-1 Signaling Regulates NG2+ Glial Cell Spatial Homeostasis in Normal Adult Gray Matter.

Neuron-glial antigen 2-positive (NG2(+)) glial cells are the most proliferative glia type in the adult CNS, and their tile-like arrangement in adult gray matter is under tight regulation. However, little is known about the cues that govern this unique distribution. To this end, using a NG2(+) glial cell ablation model in mice, we examined the repopulation dynamics of NG2(+) glial cells in the mature and aged mice gray matter. We found that some resident NG2(+) glial cells that escaped depletion rapidly enter the cell cycle to repopulate the cortex with altered spatial distribution. We reveal that netrin-1 signaling is involved in the NG2(+) glial cell early proliferative, late repopulation, and distribution response after ablation in the gray matter. However, ablation of NG2(+) glial cell in older animals failed to stimulate a similar repopulation response, possibly because of a decrease in the sensitivity to netrin-1. Our findings indicate that endogenous netrin-1 plays a role in NG2(+) glial cell homeostasis that is distinct from its role in myelination.