Functional neuromodulatory assembloids

Abstract

Human raphe nucleus organoids or spheroids (hRNS) can be generated in vitro, at least in part, from human pluripotent stem (hPS) cells. Such spheroids model the human raphe nucleus, contain specific cell sets associated with the human raphe nucleus, such as serotonergic neurons, and can be assembled with cortical spheroids (hCS) to generate functional human neuromodulatory circuits.

Description

[Background technology]

[0001] The mammalian neuromodulatory system consists of discrete neuronal populations projecting from the brainstem, pontine nuclei, or basal forebrain, which fine-tune brain function and are involved in various neuropsychiatric disorders. Neuromodulators such as serotonin, norepinephrine, acetylcholine, and dopamine can act on these cells on multiple timescales, ranging from short-term adjustments of neuronal and synaptic function to long-term circuit adaptations.

[0002] During early brain development, neuromodulators actively participate in the assembly of the nervous system by regulating cell division, differentiation, migration, synaptogenesis, synaptic transmission, and dendritic pruning. One of the earliest neuromodulatory innervations to the developing cerebral cortex is via serotonergic neurons originating from the raphe nuclei located in the midline of the brainstem.

[0003] The serotonin (5HT) system innervates nearly every region of the central nervous system, despite consisting of only about 500,000 neurons in the human brain. Serotonin acts through at least 14 different G protein-coupled receptors, which have been shown to vary considerably in the human cerebral cortex compared to other mammals and primates. Depending on the subtype, these receptors can exert either inhibitory or excitatory regulatory neuronal activity.

[0004] In the human central nervous system, serotonergic neurons are generated as early as the fifth week of gestation, and by the 15th week of gestation, the raphe nuclei already contain a typical arrangement of serotonin neurons. Importantly, dysfunction of serotonergic neurotransmission has long been associated with major depressive disorder (MDD), and many of the pharmacological agents used in MDD, such as selective serotonin reuptake inhibitors (SSRIs), function by modulating this pathway. SSRIs are the first-line treatment for MDD. However, a significant proportion of patients remain SSRI-resistant, and it is unclear whether and how alterations in serotonergic neurons contribute to SSRI resistance in these patients. Furthermore, serotonin signaling is involved in multiple other neuropsychiatric disorders, such as schizophrenia, affective disorders, anxiety, and autism spectrum disorder.

[0005] To date, region-specific brain organoids or spheroids do not contain neuromodulatory systems, and there is no human platform to study, manipulate, or interrogate neuromodulatory pathways using cells derived from human patients. Summary of the Invention

[0006] Provided are compositions and methods for the in vitro generation of human raphe nucleus organoid or spheroid (hRNS), which can be at least partially generated from human pluripotent stem (hPS) cells.Such spheroid models human raphe nucleus and comprises the specific cell set associated with human raphe nucleus, such as serotonergic neuron, GABAergic neuron, etc.

[0007] hRNS can be functionally integrated with human cerebral cortical spheroids (hCS), which contain human cortical neurons, e.g., glutamatergic neurons, to form a cortico-raphe nuclei assembloid (hCS-hRNS). Human serotonergic neurons form bidirectional projections between hRNS and hCS neurons, generating a neuromodulatory assembloid. This assembloid consists of functionally integrated cells, including neurons that interact in a physiologically relevant manner, e.g., by forming synapses between neuronal classes to provide a physiologically relevant, functional neural circuit. Using a combination of viral tracing and live imaging, evidence for the formation of in vitro generated human cortico-raphe neural circuits is provided herein, providing a useful model for the development and dysfunction of the cortico-raphe pathway.

[0008] In some embodiments, assembloids are provided in which one or more of the cells are genetically modified to provide additional functionality for screening. For example, one or both of the cortical neurons and serotonergic neurons can be genetically modified to express fluorescent calcium indicators known and used in the art. One or both of the cortical neurons and serotonergic neurons can be genetically modified to express light-activated opsins. In some embodiments, the assembloids comprise opsin-expressing serotonergic neurons and cortical neurons (e.g., glutamatergic neurons) that express fluorescent calcium indicators, where the functional relationship between the neurons is demonstrated by activating the serotonergic neurons using light and observing the calcium indicator response from the cortical neurons. 5HT lineage-specific viral tools, such as AAV-based FEV minipromoter-driven reporters, are available and have been shown to work well to specifically explore and study 5HT lineage cells within hRNS and hCS-hRNS.

[0009] hRNS spheroids and hCS-hRNS assembloids offer unique opportunities for analyzing the development and function of serotonergic neural circuits between the raphe nuclei and the cortex (and vice versa), as well as serotonergic regulation of cortical neural circuits. Furthermore, these spheroids or brain region-specific organoids and assembloids provide models for studying the effects of neurological or psychiatric disorders on the neural circuits of these brain regions. Of particular relevance are neurological or psychiatric disorders associated with serotonin dysfunction, such as myeloma, schizophrenia, and other psychoses, affective disorders (e.g., depression, bipolar disorder, or anxiety disorder), and autism spectrum disorder (ASD).

[0010] Furthermore, these spheroids and assembloids can be used to establish screening platforms for SSRI function, for example, to model various pharmacological modulations of the serotonergic system by SSRIs and atypical antipsychotics, to analyze serotonergic-related disorders such as serotonin syndrome, and to analyze the in utero effects of SSRIs on cortical development. For example, in neuromodulatory assembloid systems in which cortical neuron activity is monitored with calcium indicators (gCamp6 or Fluo-4) or voltage indicators and / or serotonergic neuron activity is modulated with electrodes, optogenetics, etc., this system can be utilized in high-throughput assays for candidate drug libraries, such as modulators of 5-HT receptors (e.g., antipsychotics) or 5-HT transporters (SSRIs), to test their relative physiological effects (i.e., calcium amplitude, calcium spike frequency, neuronal membrane voltage changes, etc.) compared with known active drugs. The model provided herein also allows for testing the effects of genetic background, for example, using multiple assemblies from different human participants, optionally including genetic variants that affect the function of the receptor or transporter of interest. The composition of 5HT receptors expressed by postsynaptic neurons varies with neuronal subtype and developmental age, determining the effects of stimulation and pharmacological applications in this system, resulting in altered neuromodulatory responses, such as long-term inhibition or excitation, tonicity, adaptation, or burst modes of firing. Comparison with one or more known control agents can be used to determine the desired response.

[0011] This system also offers the advantage of providing the opportunity to use patient-derived hiPSCs. This could enable screening approaches to clarify mechanisms of SSRI resistance in hRNS-hCS assembloids derived from patients suffering from neurological or psychiatric disorders (e.g., major depressive disorder (MDD)), with or without SSRI resistance. Furthermore, assembloids can be generated by combining control and patient cells (e.g., control-hCS and patient-hRNS) to dissect cell-autonomous contributions. This platform can also be used to study genetic forms of autism spectrum disorders associated with impairments of the 5-HT system, such as microdeletions or duplications of 16p11.2 in the FEV1 gene and rare disease-causing mutations.

[0012] Also provided is a method for determining the activity of a candidate agent neural circuit within an assembloid, the method comprising contacting the assembloid with a candidate agent. Cells present in the assembloid optionally contain at least one allele encoding a mutation associated with or potentially associated with a neurological or psychiatric disorder, and determining the effect of the agent on morphological, genetic, or functional parameters, including, but not limited to, neuron number, neuronal function, gene expression profiling, cell death, single-cell gene expression (RNA-seq), calcium imaging using pharmacological screens, patch clamp recording, modulation of synaptogenesis, etc.

[0013] These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the subject methods and compositions as more fully described below. [Brief explanation of the drawings]

[0014] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0015] [Figure 1A] FIG. 1 is a schematic diagram showing hRNS-hCS assembly. [Figure 1B] FIG. 1 is a schematic diagram showing the recipe for inducing human raphe nucleus-like spheroids (hRNS). [Figure 1C] RT-qPCR profiling of genes expressed in the developing human hindbrain in human cortical spheroids (hCS) versus hRNS, with different hiPSC lines represented in different colors. [Figure 1D] Representative images of immunocytochemistry (ICC) showing hindbrain precursors are shown. [Figure 1E] 5-HT neuronal lineage cells are shown. [Figure 1F] 5-HT neurotransmitter levels measured by HPLC from hRNS across different in vitro stages and hiPSC lines are shown. [Figure 1G] RT-qPCR profiling of different 5-HT receptors (HTRs) in hCSs at day 100 of in vitro differentiation, with HTR subtypes represented by color. [Figure 2A] Shown are UMAP projections of single-cell transcriptomics data from 13,708 hRNS cells harvested from nine spheroids derived from three hiPSC lines at days 79–82 of in vitro differentiation. [Figure 2B] Cluster marker expression representing different neuronal populations in the hRNS is shown. [Figure 2C] Shown are FEV+ subclusterings representing subclusters separated from caudal and rostral identities (top), and a heatmap of the top 10 differentially expressed genes in each cluster (bottom). [Figure 3A] Assembly of hCS and AAV-Syn1::mCherry-labeled hRNS (left) and 3D reconstruction of the hCS-hRNS assembler with mCherry-labeled hRNS cells (right) are shown. [Figure 3B]Representative ICC images showing NKX6-1+ hindbrain precursors and TPH2+5-HT expressing cells labeled in hRNS-hCS assembloids are shown. [Figure 3C] Assembling AAV-Syn1::mCherry-labeled hCS and AAV-Syn1::eYFP-labeled hRNS (left) and live imaging of the co-infected hRNS-hCS (right), demonstrating bidirectional projection. The inset shows the axonal morphology with fusiform varicosities often seen in forebrain-projecting 5-HT+ neuronal processes. [Figure 4A] Schematic diagram showing the characterization of the 5HT lineage-specific FEV reporter Ple67 on dissociated hRNS cells (left), along with representative immunocytochemistry images (center) and quantification of colocalization (right). [Figure 4B] Assembly of hCS labeled with AAV-SYN1::mCherry-labeled hCS with AAV-Ple67iCRE and hRNS labeled with AAV-EF1α-DIO-eYFP (left), and live imaging of infected hRNS-hCS showing the projection of 5HT lineage cells to hCS (right). [Figure 4C] Assembly of hCS labeled with AAV-Syn1::GCaMP7s-labeled hCS with hRNS labeled with AAV-Ple67iCRE and AAV-EF1α-DIO-ChRmine-Kv2.1-mScarlet (left), and exemplary traces of optically evoked calcium transients in hCS neurons induced by 625 nm light stimulation of 5-HT lineage FEV+ cells in hRNS (right) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawing figures.

[0017] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compositions and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0018] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Any method and material similar or equivalent to the method and material described herein can be used to implement or test this invention, but here we will describe some potential and preferred methods and materials.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material in connection with which the publication is cited.In the event of any discrepancy, it should be understood that the present disclosure shall prevail over any disclosure of the incorporated publication.

[0020] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "reprogramming factor polypeptide" includes a plurality of such polypeptides, reference to an "induced pluripotent stem cell" includes a reference to one or more induced pluripotent stem cells and equivalents thereof known to those skilled in the art, and so forth.

[0021] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0022] definition The terms "pluripotency" and "pluripotent stem cells" refer to cells that have the ability to differentiate into all cell types within an organism. The term "induced pluripotent stem cells" encompasses pluripotent cells, which, like embryonic stem (ES) cells, can be cultured for long periods while maintaining the ability to differentiate into all cell types within an organism. However, unlike ES cells, pluripotent cells are derived from differentiated somatic cells, i.e., cells with a narrower, more defined potential and cannot give rise to all cell types within an organism in the absence of experimental manipulation. hiPS cells have a human ES-like morphology and grow as flat colonies with a large nucleus-to-cytoplasm ratio, clear borders, and prominent nuclei. In addition, hiPS cells express pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. In addition, hiPS cells are capable of forming teratomas, and they are capable of forming or giving rise to ectodermal, mesodermal, or endodermal tissues in vivo.

[0023] As used herein, "reprogramming factor" refers to one or more biologically active factors, i.e., a cocktail of biologically active factors, that act on cells to alter transcription, thereby reprogramming the cells to pluripotency or pluripotency. Reprogramming factors can be provided to cells, e.g., fibroblasts, adipocytes, and other cells derived from an individual with a family history or genetic makeup of interest for cardiac disease, individually or as a single composition, i.e., a premixed composition of reprogramming factors. Factors may be provided in the same or different molar ratios. Factors may be provided one or more times during the course of culturing the cells of the subject invention. In some embodiments, the reprogramming factors are transcription factors, including, but not limited to, Oct3 / 4, Sox2, Klf4, c-Myc, Nanog, and Lin-28.

[0024] Somatic cells are contacted with reprogramming factors as defined above in sufficient combination and amount to reprogram cells to pluripotency. Reprogramming factors can be provided to somatic cells individually or as a single composition, i.e., a premixed composition of reprogramming factors. In some embodiments, reprogramming factors are provided as multiple coding sequences on a vector. Somatic cells can be fibroblasts, adipocytes, stromal cells, etc., as known in the art. Somatic cells or hiPS cells can be obtained from cell banks, normal donors, individuals with the target neurological or psychiatric disease, etc.

[0025] Following induction of pluripotency, hiPS cells are cultured according to any convenient method, for example, on irradiated feeder cells and commercially available medium.hiPS cells can be dissociated from feeder cells by digesting with protease, for example, dispase, preferably at a concentration and for a period sufficient to separate intact colonies of pluripotent stem cells from the feeder layer.Spheroids can also be generated from hiPS cells grown under feeder-free conditions by dissociating and aggregating into single cell suspensions using various approaches, including centrifugation in plates.

[0026] Genes can be introduced into somatic cells or into hiPS cells derived therefrom for various purposes, such as to replace genes with loss-of-function mutations or to provide marker genes. Alternatively, vectors expressing antisense mRNA, siRNA, ribozymes, etc., can be introduced to block the expression of undesirable genes. Another method of gene therapy is the introduction of drug resistance genes to allow normal progenitor cells to have an advantage and be exposed to selection pressure, for example, multiple drug resistance genes (MDR) or anti-apoptotic genes such as BCL-2. As discussed above, nucleic acids can be introduced into target cells using various techniques known in the art, such as electroporation, calcium-precipitated DNA, fusion, transfection, lipofection, infection, etc. The particular method by which DNA is introduced is not critical to the practice of the present invention.

[0027] Disease-associated or disease-causing genotypes can be generated in healthy hiPS cells through targeted genetic manipulation (e.g., CRISPR / Cas9), or hiPS cells can be derived from individual patients with disease-associated genotypes or diagnosed with the disease. Furthermore, neurological and neuromuscular diseases with poorly defined or no genetic component can be studied within the model system. A particular advantage of this method is the fact that edited hiPS cell lines share the same genetic background as the corresponding non-edited hiPS cell line. This reduces the variability associated with strain-to-strain differences in genetic background. Neurodevelopmental and neuropsychiatric disorders and neurological disease states that have a strong genetic component or are directly caused by genetic or genomic alterations can be modeled in the system of the present invention.

[0028] The methods and compositions described herein relate to brain region-specific spheroids. Brain region-specific spheroids are three-dimensional (3D) aggregates of cells that resemble specific regions of the human brain and contain functional neurons typically associated with that region of the brain. These spheroids can be maintained in suspension culture for extended periods, such as 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or longer, without adhering to a surface, such as a culture dish. Functional neurons are intended to mean that the neurons can form functional synapses with other neurons, either in the same spheroid or in a different spheroid. The formation of functional synapses can be demonstrated using calcium imaging, as described in more detail in the Examples. For example, the human raphe nucleus spheroids described herein contain raphe nucleus neurons, such as serotonergic neurons.

[0029] The methods and compositions described herein also relate to assembloids containing two or more (e.g., two or more) of these brain region-specific spheroids. The assembloids described herein resemble multiple brain regions and contain functional neural circuits between neurons of one spheroid (representing one region) and another spheroid (representing another region). For example, cortical-raphe nucleus assembloids resemble the cortex and raphe nuclei of the human brain and contain neurons (e.g., serotonergic neurons) with projections between the raphe nucleus spheroid and the cortical spheroid. These neurons can functionally synapse with human cortical neurons (e.g., glutamatergic neurons) in the cortical spheroid and regulate the activity of the neural circuits in the cortical spheroid. Like spheroids, these assembloids can also be maintained for long periods of time without adhering to a surface.

[0030] Raphe nuclei. The human raphe nuclei are clusters of neurons located within the brainstem. The majority of neurons originating from the raphe nuclei are serotonergic neurons that project to multiple locations in the human central nervous system, including the forebrain cortex, ventral striatum, hippocampus, and amygdala. The raphe nuclei also receive connections from the cerebral cortex and other brain regions. Through interactions with these regions and other neuromodulatory systems, serotonin influences a wide range of functions, including reward valuation, impulsivity, harm aversion, and anxiety states. Disruptions to these systems are associated with a variety of neuropsychiatric disorders, some of which are described in more detail below.

[0031] Serotonergic neurons are neurons that produce the neurotransmitter serotonin, also known as 5-hydroxytryptamine or 5-HT. Their presence can be detected by the expression of enzymes involved in the serotonin pathway, such as tryptophan 5-hydroxylase 2 (TPH2), and / or markers of mature serotonin neurons, such as vesicular monoamine transporter 2 (VMAT2) and serotonin reuptake transporter (SERT). Serotonin acts through at least 14 different G protein-coupled receptors, which can exert either excitatory or inhibitory neuronal activity, depending on their subtype.

[0032] The present disclosure provides an in vitro spheroid structure (also known as region-specific organoid) containing serotonergic neurons and the assembloid derived therefrom.The presence of serotonergic neurons can be verified by determining the presence of neurons that express the above-mentioned markers and by determining the presence of the serotonin produced by these neurons.hRNS can contain at least 1% serotonergic neurons, at least 5%, at least 10%, at least 15%, at least 20%, at least 25% or more serotonergic neurons as defined by these markers as a proportion of the total cell population.

[0033] When hCSs and hRNSs are fused to generate an assembler, this structure provides a model for serotonergic (5-HT) regulation of cortical circuits. Functional integration of serotonergic neurons and cortical neurons can be verified microscopically by the presence of bidirectional axonal projections, in which axons of hCS-derived neurons project to hRNSs and axons of hRNS-derived neurons project to hCSs. Bidirectional axonal projections in hRNS-hCSs can be visualized by labeling hRNSs and hCSs with neuron-specific viral reporters (e.g., AAV-DJ-hSyn1::mCherry for hCSs and AAV-DJ-hSyn1:::eYFP for hRNSs) before assembly and monitoring the emergence of projections with long-term confocal imaging. A subset of these projections is expected to make synaptic connections. Notably, in addition to synaptic connections, some serotonergic neurons release 5-HT diffusively in the absence of closely associated postsynaptic sites, such that cells distal to the release site can bind the released 5-HT (termed "diffusive transmission"). Thus, a true measure of serotonergic connectivity to hCS can also involve non-junctional neurotransmitter transmission.

[0034] Functional assays for circuit integration may involve, for example, determining signaling between classes of neurons or determining the effects of neuromodulatory systems on regulating cell division, differentiation, migration, synaptogenesis, and dendritic pruning. For example, in optogenetic systems, light stimulation of serotonergic neurons can reveal patterns of responses in functionally integrated cortical neurons, such as an increase in calcium activity in response to light stimulation or a decrease in calcium activity in response to light stimulation, either transiently or throughout the post-stimulation period. (As discussed above, there are various responses that can indicate functional connectivity and SSRI responsiveness in hRNS-hCS.) For example, calcium responses after stimulation can be determined, where the number of activated neurons per assembly can be 10 or more, 100 or more, or even 10 or more. The use of two-photon (2P) systems with optimized imaging equipment can be used to capture a greater number of events.

[0035] Cerebral Cortex. The adult cerebral cortex contains two major classes of neurons: glutamatergic cortical neurons (also known as pyramidal cells) and GABAergic interneurons.

[0036] Glutamatergic Neurons. The mature cerebral cortex harbors a heterogeneous population of glutamatergic neurons organized into a highly complex histological architecture. So-called excitatory neurons are typically classified according to the lamina in which their soma is located, their specific combination of gene expression, dendritic morphology, and electrophysiological properties. GABAergic interneurons are inhibitory neurons of the nervous system that play a key role in neural circuits and activity. They are so named because of their release of the neurotransmitter gamma-aminobutyric acid (GABA). Interneurons are a specialized type of neuron whose primary role is to regulate the activity of other neurons within a neural network. Cortical interneurons are so named because of their localization in the cerebral cortex.

[0037] Disease Relevance: Dysfunction in serotonergic neural circuits has been associated with a variety of neurological and psychiatric disorders, including schizophrenia, affective disorders, and autism spectrum disorder (ASD). The system described herein offers a unique opportunity to study the role of these circuits in these disorders and enable the screening of potential therapeutic agents.

[0038] Affective disorders, also known as mood disorders, are a group of mental disorders, including depression, bipolar disorder, and anxiety disorders. Altered serotonin activity has been associated with various mood disorders, and selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat affective disorders such as major depressive disorder (MDD). The underlying role of serotonin in affective disorders has not been fully elucidated, and the system described herein therefore provides an opportunity to further study the role of serotonin in these disorders, screen for potential new SSRIs, and model interactions between SSRIs and serotonergic neurons. Extremely high levels of serotonin can cause a condition known as serotonin syndrome, which has toxic and potentially fatal effects, and this can also be further investigated using the system described herein.

[0039] Schizophrenia. Schizophrenia is a chronic and severe mental disorder that affects an individual's behavior. Although the underlying cause of schizophrenia is still unclear, the disorder has been linked to abnormal serotonin and dopamine signaling in the central nervous system. The system described herein provides an opportunity to further study the role of serotonin in schizophrenia and develop potential therapeutic treatments.

[0040] Autism spectrum disorder (ASD) is a developmental disorder associated with cognitive impairments that affect communication and behavior. Elevated whole-blood serotonin levels were the first biomarker identified in ASD and are present in over 25% of affected children. However, the contribution of the serotonin system to ASD pathophysiology remains incompletely understood (Muller et al., "The serotonin system in autism spectrum disorder: from biomarkers to animal models," Neuroscience 321:24-41 (2016)). The system described here provides an opportunity to study the role of serotonin in ASD.

[0041] Calcium Sensor. Neuronal activity causes rapid changes in intracellular free calcium, which can be used to track the activity of neuronal populations. Art-recognized sensors for this purpose include fluorescent proteins that fluoresce in the presence of changes in calcium concentration. These proteins can be introduced into cells, e.g., hiPS cells, by including the coding sequence in a suitable expression vector, e.g., a viral vector, to genetically modify neurons generated by the methods described herein. GCaMP is a widely used protein calcium sensor composed of a fluorescent protein, e.g., GFP, the calcium-binding protein calmodulin (CaM), and the CaM-interacting M13 peptide, although a variety of other sensors are also available. Many different proteins are available, including those described, for example, in Zhao et al. (2011) Science 333:1888-1891, Mank et al. (2008) Nat. Methods 5(9):805-11, Akerboom et al. (2012) J. Neurosci. 32(40):13819-40, Chen et al. (2013) Nature 499(7458):295-300, and in U.S. Patent Nos. 8,629,256, 9,518,980, 9,488,642, and 9,945,844.

[0042] Optogenetics integrates optics and genetic engineering to measure and manipulate neurons. Actuators are genetically encoded tools for light-activated control of proteins, such as opsins and photoswitches. Opsins are light-activated ion channels or pumps that absorb light at specific wavelengths. Opsins can be targeted and expressed in specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning a light source on and off. Channelrhodopsins typically enable rapid depolarization of neurons upon exposure through direct stimulation of ion channels. Chlamydomonas reinhardtii channelrhodopsin-1 (ChR1) is excited by blue light and allows nonspecific cation influx into cells upon stimulation. Examples of ChRs from other species include CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama), and SdChR (from Scherffelia dubia). Synthetic mutants, such as ChR2(H134R), C1V1(t / t), ChIEF;ChETA, VChR1, Chrimson, ChrimsonR, Chronos, PsChR2, CoChR, CsChR, and CheRiff, have been created. Alternatively, neuron-inhibiting ChR mutants, such as GtACR1 and GtACR2 (derived from the cryptophyte Guillardia theta), as well as mutants such as iChloC, SwiChRca, Phobos, and Aurora, have been created and identified. Halorhodopsin, known as NpHR (derived from Natronomonas pharaoni), causes cell hyperpolarization when triggered by yellow light, and mutants include Halo, eNpHR, eNpHR2.0, eNpHR3.0, and Jaws. Archaerhodopsin-3 (Arch), derived from Halorubrum sodomense, is also used to inhibit neurons.

[0043] The terms "treatment," "treating," "treating," and the like are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely stabilizes or cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in mammals, particularly humans, and includes (a) preventing a disease or condition from occurring in a subject who is susceptible to the disease or condition but has not yet been diagnosed with the disease, (b) inhibiting disease symptoms, i.e., preventing their occurrence, or (c) alleviating the disease, i.e., causing regression of the disease or condition.

[0044] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0045] Methods for Producing Spheroids and Assembloids Methods are provided for obtaining and using in vitro cell cultures of spheroids (also known as brain region-specific organoids) and assembloids, wherein the spheroids and assembloids are produced from human pluripotent stem cells. The generation of human raphe nucleus spheroids (hRNS) utilizes a multi-step process. Various differentiated spheroid structures, such as hRNS and hCS, are differentiated from spheroids of neural progenitor cells. In some embodiments, the human pluripotent stem cells are induced human pluripotent stem (hiPS) cells. In some embodiments, the hiPS cells are derived from somatic cells obtained from an unaffected individual. In other embodiments, the hiPS cells are derived from somatic cells obtained from an individual containing at least one allele encoding a mutation associated with a disease, including, but not limited to, the above-mentioned neurological or psychiatric disorders.

[0046] Human neural precursor spheroids. Neural precursor spheroids can be differentiated from pluripotent stem cells, including, but not limited to, human induced pluripotent stem cells (hiPS cells). Initially, hiPS cells can be obtained from any convenient source or generated from somatic cells using art-recognized methods. hiPS cells are dissociated from feeders into single cells, preferably as intact colonies, and then grown in suspension culture. In certain embodiments, the culture does not contain a feeder layer, for example, when cultured in a vitronectin-coated vessel. The culture may further lack non-human components, i.e., be xeno-free. hiPS cells may be cultured in any medium suitable for the growth and expansion of hiPS cells. For example, the medium may be Essential 8 Medium. Suspension growth optionally includes in the culture medium an effective dose of a selective Rho-associated kinase (ROCK) inhibitor for the initial period of culture, up to about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, or about 48 hours (see, e.g., Watanabe et al. (2007) Nature Biotechnology 25:681-686). Inhibitors useful for such purposes include, but are not limited to, Y-27632; thiazovivin (Cell Res, 2013, 23(10):1187-200; fasudil (HA-1077) HCl (J Clin Invest, 2014, 124(9):3757-66); GSK429286A (Proc Natl Acad Sci USA, 2014, 111(12):E1140-8); RKI-1447, AT13148, and the like. In certain embodiments, the ROCK inhibitor Y-27632 is used.

[0047] The suspension culture of hiPS cells is then induced to neural fate. This culture may be feeder-free. For neural induction, an effective dose of an inhibitor of the BMP and TGFβ pathway is added to the culture medium (e.g., Essential 8 medium) for a period of at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, and up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, or up to about 5 days. Such inhibitors are also referred to as inhibitors of the SMAD pathway. For example, dorsomorphin (DM) can be added at an effective dose of at least about 0.1 μM, at least about 1 μM, at least about 5 μM, at least about 10 μM, at least about 50 μM, or up to about 100 μM, which inhibits bone morphogenetic protein (BMP) type I receptors (ALK2, ALK3, and ALK6). Other useful BMP inhibitors include, but are not limited to, A83-01, DMH-1, K02288, ML347, SB505124, etc. SB-431542 is a TGFβ inhibitor and can be added at an effective dose of at least about 0.1 μM, at least about 1 μM, at least about 5 μM, at least about 10 μM, at least about 50 μM, up to about 100 μM, which inhibits TGFβ signaling but does not affect BMP signaling.Other useful inhibitors of TGFβ include LDN-193189 (J Clin Invest, 2015, 125(2):796-808), galunisertib (LY2157299) (Cancer Res, 2014, 74(21):5963-77), LY2109761 (Toxicology, 2014, 326C-:9-17), SB525334 (Cell Signal, 2014, 26(12):3027-35), SD-208, EW-7197, kartogenin, DMH1, LDN-212854, ML347, and LDN-193189 HCl (Proc Natl Acad Sci USA, 2013, 110(52):E5039-48), SB505124, pirfenidone (Histochem Cell Biol, 2014, 10.1007 / s00418-014-1223-0), RepSox, K02288, hesperetin, GW788388, LY364947, etc. The medium containing TGFβ and BMP inhibitors may be changed daily.

[0048] An effective dose of a GSK-3 inhibitor can be included in the culture medium. For example, CHIR99021 can be added at an effective dose of about 0.5 μM to about 50 μM, about 1 μM to about 25 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, or about 1 μM to about 3 μM, or about 1.5 μM. Other useful GSK-3 inhibitors include, but are not limited to, CT98014, CT98023, CT99021, TWS119, SB-216763, SB-41528, AR-A014418, AZD-1080 6-BIO, dibromocantharelline, hymenialdesine, indirubin, meridianin, alsterpaullone, cadspaullone, and kenpaullone. The GSK-3 inhibitor may be added to the culture medium simultaneously with the BMP inhibitor and the TGFβ inhibitor, or may be added to the culture medium about 1, 2, or 3 days after the addition of the BMP inhibitor and the TGFβ inhibitor. For example, the culture medium may be supplemented with the GSK-3 inhibitor together with the BMP inhibitor and the TGFβ inhibitor after 1 to 2 days of culture, e.g., after 1 day of culture.

[0049] The method may include culturing in a medium containing a BMP inhibitor, a TGFβ inhibitor, and a GSK-3 inhibitor for at least about 2 days, at least about 3 days, at least about 4 days, or at least about 5 days, and up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, or up to about 5 days. For example, the neural induction step may include culturing in a medium containing a BMP inhibitor and a transforming growth factor β (TGFβ) inhibitor for a period of 1 to 2 days, e.g., 1 day, supplementing the medium with a GSK-3 inhibitor, and culturing in a medium containing a BMP inhibitor, a TGFβ inhibitor, and a GSK-3 inhibitor for 4 to 10 days, e.g., 7 days. The medium containing the TGFβ, BMP, and GSK-3 inhibitors may be changed daily.

[0050] The concentration of the BMP inhibitor may be reduced during culture in the medium. For example, culturing in a medium containing a BMP inhibitor, a TGFβ inhibitor, and a GSK-3 inhibitor can include (1) culturing for 2 to 5 days in a medium containing a BMP inhibitor, a TGFβ inhibitor, and a GSK-3 inhibitor, where the TGFβ inhibitor is present at a concentration of about 5 μM to about 20 μM, about 5 μM to about 15 μM, about 8 μM to about 12 μM, or about 10 μM, followed by (2) culturing for 2 to 5 days in a medium containing a BMP inhibitor, a TGFβ inhibitor, and a GSK-3 inhibitor, where the TGFβ inhibitor is present at a concentration of about 1 μM to about 5 μM, about 2 μM to about 4 μM, or about 2.5 μM.

[0051] Human raphe nucleus spheroids. After about 5, 6, 7, 8, 9, or 10 days in suspension culture, the floating neural precursor spheroids are transferred to neural medium to differentiate into neural precursors. An exemplary neural medium is neurobasal medium, B-27 supplement minus vitamin A, and GlutaMAX supplement. The neural medium is supplemented with a GSK-3 inhibitor, a sonic hedgehog pathway agonist, and FGF4.

[0052] The GSK-3 inhibitor can be as described above. In certain embodiments, the neuronal culture medium is supplemented with CHIR99021 at a concentration that can be, for example, about 0.5 μM to about 50 μM, about 1 μM to about 25 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM, about 1 μM to about 3 μM, or about 1.5 μM.

[0053] Suitable Sonic Hedgehog pathway agonists include Smoothened agonist, SAG, CAS364590-63-6, which modulates the binding of Smo to its downstream effectors by interacting with the Smo heptahelical domain (K D SAG can be provided in the neuronal medium at a concentration that can be about 10 nM to about 1 μM, about 50 nM to about 0.5 μM, about 75 nM to about 0.25 μM, or about 100 nM.

[0054] In certain embodiments, the neural medium is supplemented with FGF4, e.g., at a concentration of about 1 ng / ml to about 100 ng / ml, about 5 ng / ml to about 50 ng / ml, about 5 ng / ml to about 15 ng / ml, or about 10 ng / ml. FGF4 may be added to the neural medium simultaneously with the GSK-3 inhibitor and the Sonic Hedgehog pathway agonist, or may be added to the neural medium at least about 2 days, at least about 3 days, and up to about 10 days, up to about 7 days, up to about 6 days, or up to about 5 days after the addition of the BMP inhibitor and the TGFβ inhibitor. For example, the neural medium may be supplemented with FGF4 after 1 to 5 days, e.g., after 3 days, of culture in the neural medium with the GSK-3 inhibitor and the Sonic Hedgehog pathway agonist.

[0055] The process of differentiating neural spheroids into hRNS may include culturing them in neural medium containing a GSK-3 inhibitor and a Sonic Hedgehog pathway agonist for at least about 2 days, at least about 3 days, and up to about 10 days, up to about 7 days, up to about 6 days, or up to about 5 days. For example, the process of differentiating neural spheroids into hRNS may include culturing them in neural medium containing a GSK-3 inhibitor and a Sonic Hedgehog pathway agonist for 2-5 days, e.g., 3 days, supplementing the neural medium with FGF4, and culturing them in neural medium containing a GSK-3 inhibitor, a Sonic Hedgehog agonist, and FGF4 for at least 1 week, at least 2 weeks, at least 3 weeks, up to about 5 weeks, up to about 4 weeks, or 1-3 weeks. The medium containing a GSK-3 inhibitor, a Sonic Hedgehog agonist, and FGF4 may be changed daily.

[0056] As demonstrated in the Examples, the combined use of a GSK-3 inhibitor, a Sonic Hedgehog pathway agonist, and FGF4 results in the formation of hRNS with high levels of markers indicative of human raphe nuclei, for example, at least two weeks after suspension cultures of hiPS cells are induced to adopt a neural fate. For example, hRNS may have high levels of transcription factors that drive caudal midbrain / hindbrain development, such as NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, and EN1, but low levels of forebrain markers, such as FOXG1. Methods for determining transcription factor expression levels include RT-qPCR, as further described in the Examples. In some embodiments, the methods disclosed herein further comprise determining whether hRNS express transcription factors that drive caudal midbrain / hindbrain development. hRNS with high or low levels of transcription factors may have significantly higher or lower levels of gene expression compared to gene expression in non-raphe spheroids, e.g., cortical spheroids (hCS), as calculated using standard statistical tests.

[0057] To promote the differentiation of neural precursors into neurons, about 1 week, 2 weeks, 3 weeks, or 4 weeks after transferring the neural spheroids to the neural medium, the neural medium is replaced with an effective dose of BDNF and NT3 to replace the GSK-3 inhibitor and the Sonic Hedgehog pathway agonist. The growth factors can be provided at concentrations of at least about 0.5 ng / ml, at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, up to about 500 ng / ml, up to about 250 ng / ml, up to about 100 ng / ml, up to about 20 ng / ml, or about 10 ng / ml.

[0058] The neuronal culture medium at this stage may optionally be supplemented with one or more of the following agents that generally promote neuronal activity: a gamma-secretase inhibitor, e.g., DAPT, at a concentration of about 1-25 mM, about 2-10 mM, and about 2.5 mM; L-ascorbic acid, at a concentration of about 10-500 nM, about 50-250 nM, and about 200 nM; cAMP, at a concentration of about 10-500 nM, about 50-150 nM, and about 100 nM; and docosahexaenoic acid (DHA), at a concentration of about 1 μM-100 μM, about 5 μM to about 50 μM, 5 μM-25 μM, or about 10 μM. In some embodiments, the neuronal culture medium comprises effective doses of BDNF, NT3, a gamma-secretase inhibitor, L-ascorbic acid, cAMP, and DHA.

[0059] To promote differentiation of neural precursors into neurons, neural spheroids can be cultured in a neural medium containing the above-mentioned factors for at least about 1 week, at least about 2 weeks, at least about 3 weeks, up to about 6 weeks, up to about 5 weeks, up to about 4 weeks, about 1 to about 3 weeks, or about 2 weeks. The neural medium may further contain FGF4 for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, up to about 10 days, up to about 8 days, up to about 6 days, about 2 to about 10 days, or about 5 days.

[0060] For example, the step of promoting differentiation of neural precursors into neurons may include (4) culturing neural spheroids in suspension culture for 2 to 10 days in a neural medium containing FGF4 and at least one compound selected from the group consisting of brain-derived neurotrophic factor (BDNF), NT-3, L-ascorbic acid 2-phosphate trisodium salt (AA), N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (cAMP), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), and DAPT, and (5) culturing neural spheroids in suspension culture for at least 1 week in a neural medium containing at least one compound in the absence of FGF4.

[0061] After about 1 week, 2 weeks, 4 weeks, about 5 weeks, about 6 weeks, or about 7 weeks after transferring the neural spheroids to the neural medium, the spheroids can be maintained in the neural medium for an extended period of time, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer. In some embodiments, the spheroids are maintained for 3 months or longer. The spheroids can be maintained in the neural medium in the absence of growth factors.

[0062] The human RNS contains functional serotonergic neurons. As mentioned above, the majority of neurons derived from the human raphe nuclei are serotonergic neurons that project to multiple locations in the human central nervous system, including regions of the human cortex. The presence of serotonergic neurons can be detected by using methods such as immunohistochemistry to determine the expression of serotonin, enzymes involved in the serotonin pathway, such as tryptophan 5-hydroxylase 2 (TPH2), and / or markers of mature serotonin neurons, such as vesicular monoamine transporter 2 (VMAT2) and serotonin reuptake transporter (SERT). Neuronal functionality can be assessed by, for example, measuring Ca 2+ This can be determined by monitoring neuronal activity by imaging activity.

[0063] Human cortical spheroids. hCS may be generated, for example, by the methods previously described in Pasca et al. (2015) Nat. Methods 12(7):671-678, entitled "Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture," which is specifically incorporated herein by reference.

[0064] For example, as described above, a suspension culture of hiPS cells is cultured to produce neural precursor spheroids. After about 5, 6, 7, 8, 9, or 10 days in suspension culture, the suspension neural precursor spheroids are transferred to a neural medium to differentiate into neural precursors. The medium is supplemented with effective doses of FGF2 and EGF. The growth factors can be provided at concentrations of at least about 0.5 ng / ml, at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 20 ng / ml, up to about 500 ng / ml, up to about 250 ng / ml, or up to about 100 ng / ml.

[0065] After about one week, two weeks, three weeks, or four weeks of FGF2 / EGF exposure, the neural medium is changed to replace FGF2 and EGF with effective doses of BDNF and NT3 to promote differentiation of neural precursors into hCS containing glutamatergic neurons. The growth factors can be provided at concentrations of at least about 0.5 ng / ml, at least about 1 ng / ml, at least about 5 ng / ml, at least about 10 ng / ml, at least about 20 ng / ml, up to about 500 ng / ml, up to about 250 ng / ml, or up to about 100 ng / ml. The cortical spheroids contain functional glutamatergic neurons.

[0066] Assembloids. hRNS can be functionally integrated with separately cultured human cortical spheroids (hCS) to form cortico-raphe nuclei assembloids (hCS-hRNS) containing glutamatergic and serotonergic neurons. The resulting hCS-hRNS contains neural circuits between the cortex and raphe nuclei, providing functional integration of these circuits. For example, functionally integrated cells interact in physiologically relevant ways, such as forming synapses, transmitting signals, and forming multicellular structures.

[0067] The cortical spheroids are co-cultured with human raphe nucleus spheroids in neural medium under conditions that allow cell fusion, which may include culturing the hRNS and hCS in close proximity, e.g., in direct contact with each other.

[0068] Assembly may be performed using spheroids after about 30, 60, or 90 days of culture for hRNS, and after about 30, 60, or 90 days of culture for hCS. hRNS and hCS spheroids may be co-cultured for 2, 3, 5, 8, 10, 14, 18, 21, or more days. Assembly may be performed in neuronal medium. The resulting cortical-raphe nucleus assembloids were demonstrated to contain functional neural circuits, where the assembloids contained bidirectional projections between the cortical spheroids and the raphe nucleus spheroids, and serotonergic neurons in the raphe nucleus spheroids were able to regulate the activity of the cortical neural circuits. Methods for confirming neuronal functionality are known in the art and include optogenetic methods and imaging of calcium activity in neurons, such as those described in the Examples. In some embodiments, the method may include confirming the functionality of neurons within the cortical-raphe nucleus assembloids.

[0069] Screening assays Also disclosed herein is a screening assay that involves determining the effect of a candidate agent on spheroids, such as hRNS, or assembloids, such as hCS-hRNS, or cells derived therefrom. The candidate agent may be a small molecule or a genetic factor. The screening assay can involve contacting a candidate agent with spheroids, assembloids, or cells derived therefrom, and determining the effect of the candidate agent on parameters of the spheroids, assembloids, or cells, including morphological, genetic, or functional changes.

[0070] For example, screening assays can involve determining the effect of a candidate drug (e.g., an SSRI inhibitor) on the function of neural circuits within spheroids or assembloids. As described herein, hCS-hRNS assembloids were demonstrated to contain neurons with bidirectional projections between hCS and hRNS, and serotonergic neurons in hRNS were able to regulate the function of cortical neural circuits, as revealed by a combination of viral labeling and calcium imaging with photostimulation. Therefore, screening assays can involve determining whether a candidate drug can alter the ability of serotonergic neurons to regulate the function of cortical neural circuits in hCS.

[0071] As also described herein, various diseases and disorders are associated with serotonergic dysfunction.Therefore, the assay described herein can be particularly useful when spheroid or assembloid comprises at least one allele associated with neurological or psychiatric disorders, schizophrenia, affective disorders (e.g., MDD, bipolar disorder or anxiety disorder) and autism spectrum disorder (ASD).For example, candidate drugs that can restore the function of neural circuits (e.g., cortical neural circuits) in spheroids or assembloids that comprise these disorder-related alleles can have therapeutic utility in treating the disorder.

[0072] Furthermore, the assembloids described herein can be used to dissect cell-autonomous contributions to these disorders. For example, assembloids can be generated such that one spheroid (e.g., hRNS or hCS) is derived from a patient suffering from a disorder described herein, and the other spheroid is derived from an unaffected individual, i.e., a subject not suffering from the same disorder. For example, in a method for generating assembloids from first and second human pluripotent stem cells, either the first or second human pluripotent stem cells can contain at least one allele associated with a neurological or psychiatric disorder.

[0073] Neuronal activity induces rapid changes in intracellular free calcium. Therefore, calcium imaging assays can be used to determine the function of neuronal circuits. This can involve modifying neurons to contain genetically encoded calcium indicator proteins, such as the fluorophore sensor GCaMP, and imaging those cells. GCaMP contains circularly permuted green fluorescent protein, the calcium-binding protein calmodulin (CaM), and the CaM-interacting M13 peptide, which increases the brightness of GFP upon calcium binding. Further details regarding calcium imaging assays are described in Chen et al. (2013) Nature 499(7458):295-300. Other calcium imaging assays include Fura-2 calcium imaging, Fluo-4 calcium imaging, and Cal-590 calcium imaging.

[0074] For example, neurons can be modified to express GCamP6f. This can be combined with methods to activate specific neurons in response to external stimuli, such as optogenetic methods to activate neurons in response to light. For example, to test functionality between two types of neurons involved in a neural circuit, a "first" neuron can be modified to express an optogenetic actuator (e.g., ChrimsonR) and a "second" neuron can be modified to express a calcium indicator (e.g., GCamP6f), and calcium release can be monitored using imaging. If the first neuron is functionally connected (synaptically connected) to the second neuron, optogenetic activation of the first neuron affects intracellular calcium levels and a visible readout within the second neuron.

[0075] As described above, serotonin can produce a variety of different responses, which may depend on the receptor at which serotonin acts. Therefore, a method for determining the ability of serotonergic neurons to regulate cortical neural circuits can involve labeling hRNS cells with an optogenetic actuator (e.g., Chrimson®), labeling hCS cells with a calcium indicator, stimulating hRNS cells in an hRNS-hCS assembloid, and determining whether there is an increase or decrease in calcium activity in the hCS cells in the assembloid. Such an increase or decrease can be transient or occur throughout the entire post-stimulation period. As described in the Examples herein, such a method was used to confirm serotonergic regulation of cortical neural circuits within hRNS-hCS assembloids. To determine the effect of a candidate drug on this serotonergic regulation, this optogenetic method can be performed in the presence and absence of the candidate drug, and the results in the two conditions can be compared.

[0076] Analytical methods at the single-cell level are also of interest, such as live imaging (including confocal or light-sheet microscopy), single-cell gene expression or single-cell RNA sequencing, calcium imaging, immunocytochemistry, patch clamping, flow cytometry, etc., as described above. Various parameters can be measured to determine the effects of drugs or treatments on spheroids, assembloids, or cells derived therefrom. For example, single-cell RNA sequencing of cells constituting spheroids or assembloids can be used to characterize the identity of these cells and can be utilized in assays aimed at determining whether a candidate drug affects cell fate.

[0077] A parameter is a quantifiable component of a cell, preferably one that can be accurately measured in a high-throughput system. A parameter may also be any cellular component or cellular product, including cell surface determinants, receptors, proteins or their conformations or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids such as mRNA, DNA, etc., or a portion or combination thereof derived from such cellular components. Most parameters provide quantitative readouts, but in some cases, semi-quantitative or qualitative results are acceptable. Readouts may include a single determined value or may include a mean, median, variance, etc. Variability is expected, and the range of values ​​for each set of test parameters is obtained using standard statistical methods, with common statistical methods used to provide a single value.

[0078] Parameters of interest include the detection of cytoplasmic, cell surface, or secreted biomolecules, biopolymers, such as polypeptides, polysaccharides, polynucleotides, and lipids. Cell surface and secreted molecules are preferred parameter types and are more easily assayed because they mediate intracellular signaling and cellular effector responses. In one embodiment, the parameter comprises a specific epitope. Epitopes are frequently identified using specific monoclonal antibodies or receptor probes. In some cases, the molecular entity comprising the epitope is derived from two or more substances and comprises a defined structure, such as the combinatorially determined epitope associated with heterodimeric integrins. The parameter can be the detection of a specifically modified protein or oligosaccharide. The parameter can be defined by a specific monoclonal antibody or a ligand or receptor binding determinant.

[0079] Candidate agents of interest are biologically active agents encompassing numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, and the like. An important aspect of the present invention is the evaluation of candidate drugs, selected therapeutic antibodies, and protein-based therapeutics with desirable biological response functions. Candidate agents contain functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, typically including at least an amine, carbonyl, hydroxyl, or carboxyl group, and often including at least two of the functional chemical groups. Candidate agents often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0080] Also included are pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modulators, and neuroactive agents. Exemplary pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, New York, (1996), 9th Edition, in the following sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins, Dermatology; and Toxicology, all of which are incorporated herein by reference.

[0081] An important class of candidate drugs for use in the compositions and methods described herein are selective serotonin reuptake inhibitors (SSRIs). SSRIs are a class of drugs typically used as antidepressants in the treatment of major depressive disorder (MDD) and anxiety disorders. SSRIs typically function by increasing the extracellular level of serotonin by limiting its reabsorption. Examples of known SSRIs include citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, and dapoxetine. In addition to investigating the role of these known SSRIs, the system described herein can also be used as part of a screening assay to discover new SSRIs.

[0082] Test compounds include all classes of molecules mentioned above, and may also include samples with unknown content. Targets are complex mixtures of natural compounds derived from natural sources, such as plants. Many samples contain compounds in solution, but solid samples that can be dissolved in a suitable solvent can also be assayed. Target samples include environmental samples, such as groundwater, seawater, and mining waste; biological samples, such as lysates prepared from crops, tissue samples, and the like; manufacturing samples, such as time-lapse samples during pharmaceutical preparation; and libraries of compounds prepared for analysis. Target samples include compounds to be evaluated for potential therapeutic value, i.e., drug candidates.

[0083] The term "sample" also includes the above-mentioned fluids to which additional components have been added, such as components that affect ionic strength, pH, total protein concentration, etc. Additionally, samples may be processed to achieve at least partial fractionation or concentration. Biological samples may be stored, for example, under nitrogen, frozen, or a combination thereof, provided care is taken to reduce compound degradation. The volume of sample used is sufficient to allow measurable detection; typically, approximately 0.1-1 mL of biological sample is sufficient.

[0084] Compounds, including candidate drugs, can be obtained from a variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for the random and directed synthesis of a wide variety of organic compounds, including biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified through conventional chemical, physical, and biochemical means and used to generate combinatorial libraries. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, and amidation, to generate structural analogs.

[0085] As used herein, the term "genetic factor" refers to polynucleotides and their analogs, and these agents are tested in the screening assays of the present invention by adding the genetic factor to cells. The introduction of the genetic factor results in a change in the overall genetic composition of the cell. Genetic factors such as DNA can be experimentally introduced into the genome of a cell, typically through the integration of the sequence into a chromosome, using, for example, CRISPR-mediated genome engineering (see, e.g., Shmakov et al. (2017) Nature Reviews Microbiology 15:169). Genetic changes can also be transient, such as when the exogenous sequence is not integrated but is maintained as an episomal agent. Genetic factors such as antisense oligonucleotides can also affect protein expression without changing the genotype of the cell by interfering with the transcription or translation of mRNA. The effect of the genetic factor is to increase or decrease the expression of one or more gene products in the cell.

[0086] The introduction of an expression vector encoding a polypeptide can be used to express the encoded product in cells that lack the sequence or to overexpress the product.A variety of promoters can be used, either constitutive or subject to external regulation, where in the latter case, gene transcription can be turned on or off.These coding sequences can include full-length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine naturally occurring sequences with the functional or structural domains of other coding sequences.Alternatively, the introduced sequence can code for antisense sequences, antisense oligonucleotides, RNAi, dominant negative mutations, or dominant or constitutively active mutations of natural sequences, modified regulatory sequences, etc.The expression vector can be a viral vector, such as adeno-associated virus, adenovirus, herpes simplex virus, retrovirus, lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus and picornavirus vector.

[0087] Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified from the natural phosphodiester structure to increase intracellular stability and binding affinity. Several such modifications, which change the chemical properties of the backbone, sugar, or heterocyclic base, have been described in the literature. Useful modifications to backbone chemistry include phosphorothioates, phosphorodithioates in which both non-bridging oxygens are replaced with sulfur, phosphoramidites, alkylphosphotriesters, and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioates, 3'-S-5'-O-phosphorothioates, 3'-CH2-5'-O-phosphonates, and 3'-NH-5'-O-phosphoramidates. Peptide nucleic acids replace the entire ribose phosphodiester backbone with peptide bonds. Sugar modifications are also used to enhance the stability and affinity of, for example, morpholino oligonucleotide analogs.

[0088] Multiple assays can be performed in parallel using different drug concentrations to obtain the response difference for various concentrations.As known in the art, determining the effective concentration of a drug typically uses a concentration range resulting from 1:10 or other logarithmic dilutions.Concentration can be further refined by a second series of dilutions as needed.Typically, one of these concentrations serves as a negative control, i.e., at zero concentration, or below the detection level of the drug, or below the concentration of the drug that does not cause detectable changes in phenotype.

[0089] In addition to the functional parameters mentioned above, various methods are available for quantifying the presence of selected parameters. To measure the amount of a molecule present, a convenient method is to label the molecule with a detectable moiety, which can be fluorescent, luminescent, radioactive, enzymatic, etc. In particular, molecules specific for binding to parameters with high-affinity fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only specific proteins, but also specific conformations, cleavage products, or site modifications such as phosphorylation. Individual peptides and proteins can be engineered to fluoresce, for example, by expressing them as green fluorescent protein chimeras in cells (for a review, see Jones et al. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can be genetically modified to provide fluorescent dyes as part of their structure.

[0090] Depending on the label selected, parameters may be measured using immunoassay techniques other than fluorescent labels, such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules, which are particularly useful due to their high specificity for attaching to a single molecular target. U.S. Patent No. 4,568,649 describes a ligand detection system using scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cellular readout of proteins and other cellular determinants can be obtained using fluorescently or otherwise tagged reporter molecules. Cell-based ELISA or related non-enzymatic or fluorescence-based methods allow for the measurement of cell surface and secreted parameters. Capture ELISA and related non-enzymatic methods typically use two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry is useful for analyzing cell surface and intracellular parameters, as well as for measuring shape changes and granularity, and for analyzing beads used as antibody or probe binding reagents. The readout from such an assay may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the mean fluorescence intensity, median fluorescence intensity, variance of fluorescence intensity, or some relationship between these.

[0091] In the art, both single-cell and multi-cell multi-parameter multiplex assays in which input cells are typed and parameters are read by quantitative imaging, using fluorescence and confocal microscopy, are described in Confocal Microscopy Methods and Protocols (Methods in Molecular Biology Vol. 122) Paddock, Ed., Humana Press, 1998. These methods are described in U.S. Patent No. 5,989,833, issued November 23, 1999.

[0092] The results of the assay can be input into a data processor to provide a data set. Algorithms are used to compare and analyze data obtained under different conditions. The effects of factors and drugs are read out by determining changes in multiple parameters. The data includes results from the assay combination with the drug, and may also include one or more of results from control conditions, simulated conditions, and other assay combinations using other drugs or performed under other conditions. For quick and easy comparison, the results may be presented visually in a graph, and may include numbers, graphs, color representations, etc.

[0093] Datasets are generated from values ​​obtained by measuring parameters in the presence and absence of different cells, e.g., genetically modified cells, i.e., cells cultured in the presence of a specific factor or agent that affects neuronal function, and comparing the presence of the agent of interest with at least one other control condition, which may typically include no agent or a different agent. Parameters include functional states such as synaptogenesis and calcium ions in response to stimulation, whose levels change in the presence of the agent. Desirably, results are normalized to a standard, usually a "control value or condition," to provide a normalized dataset. Values ​​obtained from test conditions can be normalized by subtracting the unstimulated control value from the test value and dividing the corrected test value by the corrected stimulated control value. Other normalization methods can also be used, such as the logarithm or other derivative of the measurement, or the ratio of the test value to the stimulated or other control value. While data are normalized to control data for the same cell type under control conditions, datasets can include normalized data from one, two, or multiple cell types and assay conditions.

[0094] The dataset can include values ​​for the levels of a set of parameters obtained under different assay combinations. A compilation is developed that provides values ​​for a sufficient number of alternative assay combinations to allow for comparison of the values.

[0095] The database can be compiled from a set of experiments, for example, the database can include data obtained from a panel of assay combinations involving multiple different environmental variables, each variable can be a set of related compounds, or compounds representing a different class of molecules.

[0096] Mathematical systems can be used to compare datasets and provide quantitative measures of similarities and differences between them. For example, datasets can be analyzed by pattern recognition algorithms or clustering methods (e.g., hierarchical or k-means) that use statistical analysis (e.g., correlation coefficients) to quantify associations. These methods can be modified (e.g., by weighting, employing classification strategies, etc.) to optimize the dataset's ability to discriminate between different functional effects. For example, individual parameters can be weighted more or less when analyzing a dataset to enhance the discriminatory power of the analysis. The effect of altering the weights assigned to each parameter is evaluated, and an iterative process is used to optimize the identification of pathways or cellular functions.

[0097] Comparison of the data set obtained from the test compound with the reference data set is achieved by using appropriate deduction protocols, AI systems, statistical comparisons, etc. Preferably, the data set is compared with a database of reference data. Similarity with the reference data with known pathway stimuli or inhibitors can provide an early indication of the cellular pathways targeted or altered by the test stimuli or agents.

[0098] Reference databases can be compiled.These databases can include the reference data from panels that contain known drugs or drug combinations that target specific pathways, as well as the reference data from the analysis of cells that are treated under single or multiple environmental conditions, or environmental conditions that remove or specifically change parameters.Reference data can also be generated from panels that contain cells that have gene constructs that selectively target or regulate specific cellular pathways.In this way, a database can be developed that can reveal the contribution of individual pathways to complex responses.

[0099] The effectiveness of pattern search algorithms in classification can involve optimizing the number of parameters and assay combinations. The disclosed techniques for parameter selection provide computational requirements that result in physiologically relevant outputs. Furthermore, these techniques for pre-filtering data sets (or potential data sets) using cellular activity and disease-related biological information improve the likelihood that the output returned from the database search will be relevant to predicting drug mechanisms and in vivo drug effects.

[0100] The following procedure is used to develop an expert system for the selection and classification of biologically active drug compounds or other interventions. For all reference and test patterns, a data matrix is ​​typically generated, where each point in the data matrix represents a readout from a parameter, and the data for each parameter can be obtained, for example, from multiple replicate measurements of individual cells of the same type. As mentioned above, the data points can be quantitative, semi-quantitative, or qualitative, depending on the nature of the parameter.

[0101] The readout may be the mean, average, median, or variance, or other statistically or mathematically derived value associated with the measured value. Parameter readouts may be further refined by direct comparison with the corresponding reference readout. Absolute values ​​obtained for each parameter under identical conditions reflect the inherent variability of the biological system and also reflect the variability of individual cells and individuals.

[0102] Classification rules are constructed from a set of training data (i.e., a data matrix) obtained from multiple repeated experiments. Classification rules are selected to correctly identify repeated reference patterns and successfully distinguish between different reference patterns. Classification rule learning algorithms can include decision tree methods, statistical methods, Naive Bayes algorithms, etc.

[0103] The knowledge database must be sufficiently complex to allow for effective identification and classification of novel test data. Some approach to generating a sufficiently comprehensive set of classification patterns, and sufficiently powerful mathematical / statistical methods to distinguish between them, can achieve this.

[0104] Data from cells treated with specific drugs known to interact with particular markers or pathways provide a more detailed and differentiated readout set. Data generated from cells that have been genetically modified using overexpression and antisense techniques allows for the examination of the effects of individual genes on phenotype.

[0105] A preferred knowledge database includes reference data from an optimized panel of cells, environments, and parameters. For complex environments, data reflecting small variations in the environment may also be included in the knowledge database, such as environments in which one or more factors or cell types of interest are excluded, included, or quantitatively altered, for example, in the concentration or time of exposure.

[0106] For further details of the general techniques useful in carrying out the present invention, practitioners can refer to standard textbooks and reviews in cell biology, tissue culture, embryology, and neurobiology. Regarding tissue culture and embryonic stem cells, readers can refer to "Teratocarcinomas and embryonic stem cells: A practical approach" (EJ Robertson, ed., IRL Press Ltd. 1987), "Guide to Techniques in Mouse Development" (P.M. Wasserman et al. eds., Academic Press 1993), "Embryonic Stem Cell Differentiation in Vitro" (M.V. Wiles, Meth. Enzymol. 225:900, 1993), and "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

[0107] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999), Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999), Viral Vectors (Kaplift & Loewy eds., Academic Press 1995), Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Genetic engineering reagents, cloning vectors, and kits referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0108] Each publication cited herein is hereby incorporated by reference in its entirety for all purposes.

[0109] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. [Example]

[0110] Described herein is a novel approach to studying functional neuromodulatory systems using human pluripotent stem cells (hPSCs) to generate three-dimensional (3D) assembloids containing forebrain organoids combined with organoids modeling the raphe nuclei and capable of transmitting serotonergic projections. We first generated hPSC-derived raphe nuclei spheroids (hRNSs) containing functional serotonergic neurons along with other resident cell types of the raphe nuclei. Assembly of human cerebral cortex spheroids (hCSs) containing pyramidal glutamatergic neurons from various cortical layers along with hRNSs resulted in hRNS-hCS assembloids in which bidirectional projections exist between the cortex and the raphe nuclei. Using a combination of single-cell RNA sequencing, viral labeling, and photostimulated calcium imaging, we demonstrate that an in vitro human cell model of serotonergic regulation of cortical circuits can be used as a platform to understand the assembly of these circuits and to model disorders such as MDD, autism spectrum disorder, and schizophrenia, as well as to develop drug screens to identify better drugs that target this neuromodulatory pathway.

[0111] Example 1 method Generation of human raphe nucleus spheroids (hRNS). Human pluripotent stem cells (hPSCs) were maintained on 6-well plates coated with recombinant human vitronectin (VTN-N, Life Technologies, A14700) in Essential 8 medium (Life Technologies, A1517001) supplemented with penicillin and streptomycin (1:100, Gibco, 15140122). To generate hRNS, hiPSCs were incubated with Accutase (Innovate Cell Technologies, AT-104) at 37°C for 7 minutes to dissociate into single cells. Approximately 3 million single cells per well were added to AggreWell 800 (STEMCELL Technologies, 34815) in Essential 8 medium supplemented with the ROCK inhibitor Y-27632 (10 μM, Selleckchem, S1049), centrifuged at 100 g for 3 minutes, and the cells were trapped within the microwells and incubated at 37°C with 5% CO. After 24 hours, spheroids were harvested from each microwell by carefully pipetting the medium up and down within the well (using the cut end of a P1000 tip) and transferring to an ultra-low attachment plastic dish (Corning, 3262) in Essential 6 medium (Life Technologies, A1516401) supplemented with dorsomorphin (2.5 μM, Sigma-Aldrich, P5499) and SB-431542 (10 μM, Tocris, 1614). From days 2 to 8, the medium was changed daily and supplemented with dorsomorphin, SB-431542 (10 μM from days 2 to 5, 2.5 μM from days 5 to 8), and the GSK-3 inhibitor CHIR 99021 (1.5 μM).

[0112] On day 6, neural spheroids were transferred to neural medium containing Neurobasal A (Life Technologies, 10888), vitamin A-free B-27 supplement (Life Technologies, 12587), GlutaMax (1:100, Life Technologies, 35050), and penicillin and streptomycin. From days 5 to 15, neural medium was changed daily and supplemented with CHIR 99021 (1.5 μM) and the smoothened agent SAG (100 nM). From days 8 to 20, neural medium was also supplemented with fibroblast growth factor-4 (FGF4, 10 ng / mL).

[0113] To promote differentiation, from days 16 to 30, neural medium was changed every other day and supplemented with BDNF (10 ng / mL), NT-3 (10 ng / mL), IGF-1 (10 ng / mL), cAMP (100 nM), L-ascorbic acid (200 µM), and docosahexaenoic acid (DHA, 10 µM). A schematic diagram illustrating the different recipes is presented in Figure 1A. To characterize cellular diversity in hRNS, single-cell transcriptomics was performed on dissociated hRNS at day 42, based on the supplier's recommendations (10x Genomics, 120262). To quantify serotonin release in hRNS, two to three intact hRNS per time point per hiPS cell line were flash-frozen at various time points and processed for high-performance liquid chromatography (HPLC). For serotonin uncaging experiments, NPEC-caged serotonin (Tocris, 3991) was used at a final concentration of 50 μM. Glutamate was uncaged using UV light (405 nm) using the FRAP module of a Leica SP8 confocal microscope.

[0114] Generation of cortical-raphe nucleus assembloids (hCS-hRNS). (Figure 1A) To generate cortical-raphe nucleus assembloids (hCS-hRNS), hCS and hRNS were generated separately and then assembled by placing them in close proximity to each other in 1.5 mL microcentrifuge tubes in an incubator for 3 days. The neuronal medium used for assembly contained Neurobasal-A, vitamin A-free B-27 supplement, GlutaMax (1:100), penicillin, and streptomycin (1:100). The medium was carefully changed on day 2, and on day 3, the assembloids were placed into 24-well ultra-low attachment plates in the neuronal medium described above using a cut P1000 pipette tip. After this, the medium was changed every 3–4 days, and hCS were generated by previously described methods. Assembly was performed over a period of 45–60 days. In some experiments, hCSs or hRNSs were virally labeled with AAV-DJ1-hSyn1::YFP 7–10 days prior to assembly. For optogenetic photostimulation experiments, hRNSs were virally labeled with AAV1-hSyn1::ChrimsonR-tdTomato virus and assembled with EF1a-GCaMP6s-expressing hCSs as described above.

[0115] Example 2 Generation of functional hRNS To identify spheroids (organoids) resembling the raphe nuclei, we first patterned aggregated hPSCs in microwells with dual SMAD inhibition of the neuroectoderm and subsequently exposed them to CHIR, the SHH agonist SAG, and FGF4 (Figure 1B). Gene and protein expression analysis by RT-qPCR and immunocytochemistry at day 15 of patterning revealed upregulation of transcription factors driving caudal midbrain / hindbrain development (NKX6-1, NKX2-2, OLIG2, GATA2, GATA3, LMX1B, FOXA2, EN1; Figures 1C and 1D) and downregulation of the forebrain marker FOXG1 (Figure 1C).

[0116] Immunocytochemistry at day 52 revealed the presence of serotonergic neurons, characterized by the presence of 5-hydroxytryptamine (serotonin, 5-HT) and tryptophan 5-hydroxylase 2 (TPH2), one of the key enzymes in the serotonin synthesis pathway. The core molecular phenotype of mature serotonergic neurons includes vesicular monoamine transporter 2 (VMAT2), which packages 5-HT into synaptic vesicles and the serotonin reuptake transporter SERT, which recycles extracellular 5-HT. Immunocytochemistry at day 52 revealed cells positive for both SERT and VMAT2 within the hRNS (Figure 1E). Next, we measured 5-HT release in the hRNS using HPLC. Across three different time points and strains, we consistently measured 5-HT in hRNS ranging from 20 to 175 ng / mL / mg of protein, whereas there was no detectable 5-HT at any time point in hCS, demonstrating the specificity of hRNS patterning to the raphe nuclei (Figure 1F). Next, we investigated the gene expression profile for 11 5HT metabotropic receptors (HTRs) in hCS. We observed that a combination of excitatory Go / Gs / G11-coupled (pink, HTR2a, HTR6, HTR2c) and inhibitory Gi / Go-coupled (green, HTR1b, 1d) genes was expressed in hCS at day 100 (Figure 1G).

[0117] To investigate the cell type diversity in the hRNS, we performed single-cell transcriptomics of the hRNS at days 79–82. Unsupervised clustering of hRNS cells revealed a large population of hindbrain lineage neurons expressing classical markers for 5HT lineage neurons ("5HT neurons") as well as other neuronal subtypes ("GABAergic neurons," "glutamatergic neurons") (Figure 2A–B). Closer examination of the 5HT cluster revealed caudal and rostral subpopulations (Figure 2C).

[0118] Example 3 Assembly of hCS-hRNS To model the development and function of the cortico-raphe circuit, hRNSs were virally labeled using AAV-DJ1-hSYN1::mCherry between days 45 and 60 and then assembled with hCSs 7–8 days later, resulting in hRNS-hCS assemblies. Live imaging of intact hRNS-hCSs 16 days after assembly (days post-fusion; daf) revealed extensive hRNS-derived mCherry projections to the hCSs. + Immunocytochemistry showed that TPH2 cells project to hCS (Figure 3A). + The cells were further shown (Figure 3B). To evaluate the directionality of projections in hRNS-hCS, we virally labeled hRNS and hCS with AAV-DJ1-hSYN1::eYFP and AAV-DJ1-hSYN1::mCherry, respectively, and then assembled them. Live imaging of intact hRNS-hCS 50 days after assembly revealed bidirectional projections between hRNS and hCS. Forebrain-projecting serotonergic cells in the raphe nucleus exhibit distinct axonal morphology with large and oval varicosities along thin axons [4]. Similar structures were observed in the eYFP-derived hRNS in hCS. + were observed along the axons of the projections and not in the mCherry+ projections derived from hCSs (Fig. 3C).

[0119] Example 4 Functional investigation of neuromodulatory connections in hCS-hRNS To label 5HT-lineage neurons in the hRNS for functional studies, we used a viral reporter driving expression of emGFP under the FEV minipromoter Ple67 (AAV-Ple67::emGFP). We characterized its specificity using immunostaining for 5-HT and TPH2 on dissociated hRNS cells infected with Ple67::emGFP (Figure 4A). This experiment demonstrated that all emGFP neurons were immunostained. + 80-90% of cases are highly specific 5HT + or TPH2 +Next, we used an iCRE-dependent version of this reporter (AAV-DJ-Ple67iCRE) and co-infected hRNS with AAV-EF1α-DIO-eYFP to induce recombination and drive eYFP expression in 5-HT lineage cells. We then co-infected hRNS with hCS infected with AAV-hSYN1::mCherry. The resulting hCS-hRNS gene expressed eYFP in a wide range of 5-HT lineage cells, from hRNS to hCS. + To functionally investigate the serotonergic input to hCS in the hCS-hRNS, we used the same iCRE-dependent Ple67 reporter to transfect 5HT lineage cells of the hRNS (AAV-Ple67iCRE and AAV-EF1α-DIO-ChRmine-K). V We expressed the soma-targeted red-shifted opsin ChRmine-Kv2.1 in 2.1 and assembled them with hCSs labeled with a genetically encoded calcium indicator (AAV-hSYN1-GCamP7s). Light stimulation of 5-HT lineage cells using high-frequency light stimulation at a wavelength of 625 nm reliably evoked responses in hCS neurons, as demonstrated by stimulus-locked calcium responses (Figure 4C).

[0120] References Nadim,F.&Bucher,D.Neuromodulation of Neurons and Synapses.Curr.Opin.Neurobiol.0,48-56(2014).Bucher,D.&Marder,E.SnapShot:Neuromodulation.Cell 155,482-482.e1(2013).Vitalis,T.&Parnavelas,J.G.The Role of Serotonin in Early Cortical Development.Dev.Neurosci.25,245-256(2003).Hornung,J.-P.The human raphe nuclei and the serotonergic system.J.Chem.Neuroanat.26,331-343(2003).Jacobs,B.L.&Azmitia,E.C.Structure and function of the brain serotonin system.Physiol.Rev.72,165-229(1992).Hodge,R.D.et al.Conserved cell types with divergent features between human and mouse cortex.bioRxiv 384826(2018).doi:10.1101 / 384826 Sundstrom,E.et al.Neurochemical differentiation of human bulbospinal monoaminergic neurons during the first trimester.Dev.Brain Res.75,1-12(1993).Takahash et al.Distribution of serotonin-containing cell bodies in the brainstem of the human fetus determined with immunohistochemistry using antiserotonin serum.Brain Dev.8,355-365(1986).Adell,A.Revisiting the role of raphe and serotonin in neuropsychiatric disorders.J.Gen.Physiol.145,257-259(2015).Sodhi,M.S.K.&Sanders-Bush,E.Serotonin and brain development.in International Review of Neurobiology 59,111-174(Academic Press,2004).Bonnin,A.et al.A transient placental source of serotonin for the fetal forebrain.Nature 472,347-350(2011).Whitaker-Azmitia,P.M.Serotonin and brain development:role in human developmental diseases.Brain Res.Bull.56,479-485(2001).Pasca,A.M.et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat.Methods 12,671-678(2015).Birey,F.et al.Assembly of functionally integrated human forebrain spheroids.Nature 545,54-59(2017).Okaty,B.W.,Commons,K.G.&Dymecki,S.M.Embracing diversity in the 5-HT neuronal system.Nat.Rev.Neurosci.1(2019).doi:10.1038 / s41583-019-0151-3.Celada,P.,Puig,M.V.&Artigas,F.Serotonin modulation of cortical neurons and networks.Front.Integr.Neurosci.7,(2013).Frank,C.Recognition and treatment of serotonin syndrome.Can.Fam.Physician 54,988-992(2008).Walsh,JJet al.5-HT release in nucleus accumbens rescues social deficits in mouse autism model.Nature 560,589(2018).Doan,RNet al.Recessive gene disruptions in autism spectrum disorder.Nat.Genet.1(2019).doi:10.1038 / s41588-019-0433-8.

[0121] cross reference This application claims priority to U.S. Provisional Patent Application No. 62 / 898,430, filed September 10, 2019, which is incorporated herein in its entirety for all purposes.

Claims

1. 1. A method for producing human raphe nucleus / brainstem spheroids or organoids (hRNS) in vitro, comprising: (a) inducing human pluripotent stem cells in 3D suspension culture to a neural fate to generate neural progenitor spheroids by culturing in a medium comprising an inhibitor of bone morphogenetic protein (BMP) and an inhibitor of transforming growth factor beta (TGFβ), wherein the medium is supplemented with an inhibitor of GSK-3, and the culture in the medium comprising the inhibitor of BMP, the inhibitor of TGFβ, and the inhibitor of GSK-3 is for a period of 4 to 8 days; (b) differentiating the neural progenitor spheroids into hRNS by culturing the neural progenitor spheroids in suspension culture in a neural medium comprising an inhibitor of GSK-3 and a sonic hedgehog (SHH) pathway agonist for a period of 1 to 5 days, supplementing the neural medium with FGF4, and culturing the neural progenitor spheroids in suspension culture in a neural medium comprising an inhibitor of GSK-3, a SHH pathway agonist, and FGF4 for a period of 1 to 3 weeks; (c) maintaining the hRNS in neuronal medium for at least one week so that the hRNS contains serotonergic neurons; A method comprising:

2. 1. A method for producing cortico-raphe / brainstem assembloids (hCS-hRNS) in vitro, comprising: (i) generating human raphe nucleus spheroids (hRNS) from a first human pluripotent stem cell by the method of claim 1; (ii) (a) inducing second human pluripotent stem cells in a second suspension culture to a neural fate by culturing the neural progenitor spheroids in suspension culture in a neural medium comprising an inhibitor of bone morphogenetic protein (BMP) and an inhibitor of transforming growth factor beta (TGFβ) to derive second neural progenitor spheroids, and supplementing the medium with an inhibitor of GSK-3 for a period of 2 to 10 days; (b) differentiating the second neural progenitor spheroids into human cortical spheroids (hCS); (iii) culturing the hRNS and hCS in a neuronal medium under conditions that allow cell fusion, so that the cortico-raphe nucleus assembloid contains human serotonergic neurons with projections between the hRNS and hCS, and neurons from the hCS that project to the hRNS; Including, Human neurons form bidirectional projections between the hRNS and hCS. method.

3. 3. The method of claim 1 or 2, wherein the BMP inhibitor is selected from the group consisting of dorsomorphin, LDN-193189, and LY364947, the TGFβ inhibitor is SB-431542, and the GSK-3 inhibitor is CHIR99021, the medium is replenished with the GSK-3 inhibitor after a period of 1 to 2 days so that the medium contains the BMP inhibitor, the TGFβ inhibitor, and the GSK-3 inhibitor, and the culturing in the medium containing the BMP inhibitor, the TGFβ inhibitor, and the GSK-3 inhibitor is for a period of 4 to 8 days.

4. The step (a) of culturing in the medium containing the BMP inhibitor, the TGFβ inhibitor, and the GSK-3 inhibitor, (1) culturing the cells in a medium containing an inhibitor of BMP, an inhibitor of TGFβ, and an inhibitor of GSK-3 for a period of 2 to 5 days, wherein the inhibitor of TGFβ is present at a concentration of 5 μM to 20 μM; and (2) culturing the cells for a period of 2 to 5 days in a medium containing an inhibitor of BMP, an inhibitor of TGFβ, and an inhibitor of GSK-3, wherein the inhibitor of TGFβ is present at a concentration of 1 μM to 5 μM; The method according to any one of claims 1 to 3, comprising:

5. 5. The method according to claim 1, wherein in step (b), the neural progenitor spheroids are cultured in a neural medium containing the inhibitor of GSK-3 and the SHH pathway agonist for a period of 1 to 3 weeks, and the neural medium is supplemented with FGF4 after a period of 2 to 10 days.

6. step (b) further comprises supplementing the neuronal culture medium with at least one compound selected from the group consisting of brain-derived neurotrophic factor (BDNF), NT-3, L-ascorbic acid 2-phosphate trisodium salt (AA), N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (cAMP), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), and DAPT; The method according to any one of claims 1 to 5, wherein said neuronal medium is replenished with said at least one compound after a period of 1 to 3 weeks.

7. After step (b), (d) culturing the neural progenitor spheroids in suspension culture for a period of 2 to 10 days in a neural medium containing FGF4 and at least one compound selected from the group consisting of brain-derived neurotrophic factor (BDNF), NT3, L-ascorbic acid 2-phosphate trisodium salt (AA), N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (cAMP), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), and DAPT; (e) culturing the neural progenitor spheroids in suspension culture in a neural medium containing the at least one compound in the absence of FGF4 for at least one week; The method of claim 1 further comprising:

8. 8. The method of any one of claims 1 to 7, wherein the cells of the hRNS comprise at least one allele associated with a neurological or psychiatric disorder, wherein the neurological or psychiatric disorder is selected from the group consisting of schizophrenia, an affective disorder, and an autism spectrum disorder (ASD), and wherein the affective disorder is major depressive disorder, bipolar disorder, or an anxiety disorder.

9. 3. The method of claim 2, wherein step (iii) comprises culturing the hRNS and hCS for at least 3 days under conditions permissive for cell fusion and in direct physical contact.

10. The method according to any one of claims 1 to 9, wherein the one or more suspension cultures are cultured in a feeder layer-free condition.

11. 11. The method of any one of claims 1 to 10, wherein the cells of the human raphe nucleus-like spheroid (hRNS) or organoid (hRNS), or cortical-raphe nucleus assemblenoid, comprise at least one allele or genetic event associated with a neurological or psychiatric disorder, wherein the neurological or psychiatric disorder is selected from the group consisting of schizophrenia, an affective disorder, and an autism spectrum disorder (ASD), and wherein the affective disorder is major depressive disorder, bipolar disorder, or an anxiety disorder.

12. A human raphe nucleus spheroid (hRNS) obtained by the method of any one of claims 1 or 3 to 11.

13. A cortical-raphe / brainstem assembloid (hCS-hRNS) obtainable by the method according to any one of claims 2 to 6 and 8 to 11, comprising a raphe nucleus spheroid (hRNS) fused to a cortical spheroid (hCS), wherein the hCS-hRNS comprises human serotonergic neurons that project between the hRNS and hCS and form a human neuromodulatory circuit, and the hCS-hRNS can be maintained in suspension culture without attachment to a surface for at least 4 weeks.

14. 1. A method for determining the effect of a candidate agent on serotonergic modulation in a cortical neural circuit, comprising: contacting the candidate agent with the cortico-raphe / brainstem assembloid (hCS-hRNS) of claim 13; and determining the effect of the candidate agent on the ability of serotonergic neurons to modulate the function of cortical neural circuits; wherein the cells of the hCS-hRNS comprise at least one allele or genetic event associated with a neurological or psychiatric disorder, and the neurological or psychiatric disorder is selected from the group consisting of schizophrenia, affective disorders, and autism spectrum disorders (ASD).

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