Human sensory ascending pathway in assembloids derived from human pluripotent stem cells

Abstract

The present disclosure provides a method of producing human sensory organoids in vitro. The present disclosure provides a method of producing human dorsal hindbrain / cervical spinal cord organoids in vitro. The present disclosure provides a method of producing human ascending somatosensory assembloids in vitro. The present disclosure also provides a method of determining the effectiveness of a candidate agent on a human ascending somatosensory pathway assembloid. Also provided are human sensory organoids, human dorsal hindbrain / cervical spinal cord organoids, and human ascending somatosensory assembloids.

Description

HUMAN SENSORY ASCENDING PATHWAY IN ASSEMBLOIDS DERIVED FROM HUMANPLURIPOTENT STEM CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 626,424 filed January 29, 2024, the disclosure of which application is herein incorporated by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (STAN-2174WO_SEQ-LIST.xml; Size: 5,069 bytes; and Date of Creation: January 21 , 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0002] The sensory ascending pathway is responsible for transmitting sensory information from the peripheral nervous system to the brain, including pain and itch information. Dysfunctions in this pathway have been associated with human disorders, such as peripheral neuropathy or autism spectrum disorder. However, the mechanisms underlying these conditions are not fully understood, particularly due to the lack of reliable models to functionally probe and manipulate the human sensory ascending pathway in tractable experimental models. Therefore, there is a critical need to develop human multi-cellular models that model the circuit and integrate the components of the human sensory ascending pathway in vitro to model disorders of sensory processing and to develop effective therapies.SUMMARY

[0003] Provided herein are methods for the production of human sensory organoids, human dorsal hindbrain / cervical spinal cord organoids, and human ascending somatosensory assembloids (hASA). Also provided are methods for determining the effect of a candidate agent on the human functional cells and neural circuits in the intact 3D organoids / assembloids preparations.

[0004] The present disclosure provides a method of producing human sensory organoids in vitro, the method containing: (a) inducing a human pluripotent stem cell in suspension culture to a neural fate to generate a neural organoid; (b) differentiating the neural organoid into a human sensory organoid (hSeO); and (c) maintaining the hSeO for an extended period of time in neural medium such that the hSeO comprises human somatosensory neurons, neural crest cells, and Schwann cells.

[0005] In some cases, the present disclosure provides a method of producing human dorsal hindbrain / cervical spinal cord organoids in vitro, the method containing: (a) inducing a human pluripotent stem cell in suspension culture to a neural fate to generate a neural organoid; (b) differentiating the neural organoid into a human dorsal hindbrain / cervical spinal cord organoid (hdSpO); and (c) maintaining the hdSpO for an extended period of time in neural medium such that the hdSpO comprises spino-thalamic and other projection neurons.

[0006] In some cases, the present disclosure provides a method of producing a human ascending somatosensory assembloid in vitro, the method containing: (i) inducing in a human pluripotent stem cell suspension culture a neural fate to provide a neural organoid; (ii) differentiating the neural organoid into two or more of: human cortical organoids (hCO), human diencephalon organoids (hDiO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO) and human sensory organoid (hSeO); and (iii) culturing the two or more of hCO, hDiO, hdSpO and hSeO under conditions permissive for organoid fusion into a linear assembloid while maintaining for an extended period of time in neural medium; wherein an integrated structure is differentiated comprising interacting neurons that form a linear circuit.

[0007] In some cases, the present disclosure provides a method for determining the effect of a candidate agent on a human ascending somatosensory, the method containing: contacting the candidate agent with one or a panel of functionally integrated hDiO-hdSpO-hSeO or hCO- hDiO-hdSpO-hSeO assembled organoids differentiated from induced human pluripotent stem (hiPS) cells according to the methods, or a population of cells isolated therefrom; and determining the effect of the agent on morphologic, genetic or functional parameters.

[0008] Human sensory organoids, human dorsal hindbrain / cervical spinal cord organoids, and human ascending somatosensory assembloids are also provided.

[0009] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the subject methods and compositions as more fully described below.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention is 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. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0011] FIGs. 1A-1 B. (A) Schematics illustrating human sensory ascending pathway. (B) Key components of the ascending sensory circuitry and of a human ascending pathway assembloid consists of four regionalized neural organoids derived from pluripotent stem cells.

[0012] FIGs. 2A-2H. (A) Schematic describing the generation of hSeO from hiPS cells and scRNA-seq analysis. (B) UMAP visualization of single cell gene expression of hSeO at day 68-72. (C) UMAP plots showing gene expression. (D) Dot plots showing gene expression. (E) RT-qPCR of POU4F1 and SIX1. (F) Immunostaining of VGLUT2 and NeuN in hSeO. (G,H) Dot plots showing gene expression.

[0013] FIGs. 3A-3I. (A) Schematic describing the generation of hdSpO from hiPS cells and scRNA-seq analysis. (B) UMAP visualization of single cell gene expression of hdSpO at day 68-72. (C) UMAP plots showing gene expression. (D) UMAP plot of hdSpO showing dorsoventral neuron clusters. (E) Label transfer of the human spinal cord datasets38 onto the hdSpO dataset. UMAP plots were colored by the prediction score for d 15. (F) Dot plots showing gene expression. (G) RT-qPCR of TACR1 and LBX1 . (H) Dot plots showing gene expression. (I) Representative image showing distribution of PAX2+ and PHOX2A+ neurons in hdSpO.

[0014] FIGs. 4A-4H. (A,B) hSeO calcium imaging paradigm to record (C) the response to the noxious stimulants a & -MeATP and capsaicin. (D) Quantification showing hSeO have increased calcium activity after stimulation. (E,F) Mouse DRG calcium imaging paradigm to record (G) the response to the noxious stimulants a|3-MeATP and capsaicin. (H) Quantification showing mouse DRG have increased calcium activity after stimulation.

[0015] FIGs. 5A-5E. (A) Schematic of live calcium imaging testing various concentrations of TNP-ATP combined with a|3-MeATP exposure in ex vivo mouse DRG or hSeO. (B) Representative images of a Calbryte-incubated mouse DRG (left) and GCaMP6s expression in hSeO (right). (C) Example AF / Fbase traces demonstrating decrease of a0-MeATP (30 pM) evoked responses following incubation with TNP-ATP in mouse DRG (left) but not in hSeO (right). (D,E) Quantification of mean AF / Fbase values for 60 s after exposure to a -MeATP (30 pM) in mouse DRG and hSeO at various concentrations of TNP-ATP.

[0016] FIGs. 6A-6I. (A) Sensory-dorsal spinal assembloids demonstrate (B,C) axonal projections from sensory to dorsal spinal neurons. (D) Retrograde synaptic transmission of rabies-Cre from dorsal spinal to sensory neurons demonstrates (E) projections from hSeO to hdSpO within assembloids. (F,G) Most projections are from BRNA3+ sensory neurons. (H) Schematic illustrating the patch-clamp recordings combined with optogenetic stimulation of hSeO neurons. (I) Representative traces showing oEPSCs from 3 trials in the same neuron.

[0017] FIGs. 7A-7G. (A) Dorsal spinal-thalamic assembloids. (B,C) Spinal neurons project into thalamic regions within assembloids. (D) Retrograde synaptic transmission of rabies-Cre from thalamic to dorsal spinal neurons demonstrates (E) projections from hdSpO to hDiO within assembloids. (F,G) Projections are from NK1 R+ dorsal spinal neurons.

[0018] FIGs. 8A-8D. (A) Schematic illustrating strategy for live calcium imaging of spinothalamic neurons combined with retrograde viral tracing in hSeO-hdSpO-hDiOassembloids. (B) Representative image of co-expression of GCaMP6f and dsRedExpress on the hdSpO side of hSeO-hdSpO-hDiO assembloids. (C) Example AF / Fbase traces demonstrating ap-MeATP (30 pM)-evoked responses (left) and their blockade by exposure to NBQX and APV (50 pM, each) (right). (D) Quantification of mean AF / Fbase values for baseline and for 60 s after exposure to ap-MeATP in the absence (left) or presence (right) of NBQX and APV.

[0019] FIGs. 9A-9C. (A,B) Assembloids incorporating 4 components of the ascending sensory pathway, (C) each uniquely labeled with a fluorescent reporter.

[0020] FIGs. 10A-10C. (A) Schematics of calcium imaging in single organoids and sensory ascending pathway assembloids. (B) Representative traces demonstrate that calcium raises in hdSpO, hDiO, and hCO by ap-MeATP have been emerged by assembly (a-hdSpO, a-hDiO, and a-hCO). (C) Neurons in hCO, hDiO, hdSpO, and hSeO showed significant response by ap-MeATP treatment in 4-part assembloids while only neurons in hSeO of individual organoids, but not the other organoids, showed response.

[0021] FIGs. 11A-11 C. (A) Schematic of calcium imaging in sensory ascending pathway assembloids with stimulation by glutamate uncaging. (B,C) Representative traces and quantification of calcium rise in hSeO, hdSpO, hDiO, and hCO following glutamate uncaging of hSeO.

[0022] FIGs. 12A-12H. (A) Schematic illustrating spontaneous live calcium imaging of individual organoids and four-part assembloids. (B,C) Individual organoids and four-part assembloid expressing ]GCaMP8s (left), example AF / Fbase traces of cells (middle), and representative SCA correlograms between cells from hSeO and hCO with percentage of peak time-shifts (right). (D) Correlation values averaged per cell, for combinations of the four regions of human ascending somatosensory assembloid (hASA). (E) Schematic (upper) and representative picture (bottom) of simultaneous four-part extracellular recordings in an hASA. (F) Representative traces of spontaneous activity. Asterisks denote synchronous activity across all four regions. (G) Representative cross-correlograms. Activity counts around peak area (P) and activity counts in base area (B) were used to calculate co-activation index. (H) Co-activation index values for hSeO-hdSpO, hSeO-hDiO, and hSeO-hCO pairs. Coactivation index values aligned to hSeO activities were significantly higher than values from randomly selected time points

[0023] FIGs. 13A-13I. (A) Schematics of calcium imaging in single organoids. (B,C) Representative heatmap and traces of calcium activities by various mechanical stimulation. (D,E,F) The calcium activity intensities gradually increased, accompanied by a higher proportion of neurons becoming active by stronger mechanical stimulation. (G) Schematic of calcium imaging in assembloids. (H) Representative traces of calcium activities inassembloids. (I) Calcium activities were induced by mechanical stimulation and blocked by GsMTx4.

[0024] FIGs. 14A-14I. (A) The gene SCN9A encoding Na1.7 is highly expressed in hSeO checked via qPCR. (B) CRISPR-mediated editing of SCN9A led to significantly reduced (C) mRNA and (D) protein level in hSeO. (E) SCN9A deletion led to significantly reduced spontaneous calcium activity in hSeO. (F) The sensory ascending pathway assembloids were made from control and SCN9A knock-out hi PS cells, and (G,H) underwent live calcium imaging. (I) The peak correlation of spontaneous calcium activity significantly reduced in SCN9A knock-out assembloids.

[0025] FIGs. 15A-15G. (A) CRISPR-mediated editing of SCN9A T1464I has been confirmed by (B) Sanger sequencing, which led to significantly increased (C) spontaneous calcium activity in hSeO. (D) The sensory ascending pathway assembloids were made from control and SCN9A T1464I hiPS cells, and (E,F) underwent live calcium. (G) The peak correlation of spontaneous calcium activity significantly increased in SCN9A T1464I assembloids. FIG. 15B from top to bottom SEQ ID NO:01 to SEQ ID NO: 04.

[0026] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings.DEFINITIONS

[0027] By “pluripotency” and pluripotent stem cells it is meant that such cells have the ability to differentiate into all types of cells in an organism. The term “induced pluripotent stem cell” encompasses pluripotent cells, that, like embryonic stem (ES) cells, can be cultured over a long period of time while maintaining the ability to differentiate into all types of cells in an organism, but that, unlike ES cells, are derived from differentiated somatic cells, that is, cells that had a narrower, more defined potential and that in the absence of experimental manipulation could not give rise to all types of cells in the organism. hiPS cells have a human ES-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, hiPS cells express several pluripotency markers known by one of ordinary skill in the art, including but not limited to alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42. In addition, the hiPS cells are capable of forming teratomas. In addition, they are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.

[0028] As used herein, “reprogramming factors” refers to one or more, i.e. a cocktail, of biologically active factors that act on a cell to alter transcription, thereby reprogramming a cell to multipotency or to pluripotency. Reprogramming factors may be provided to the cells individually or as a single composition, that is, as a premixed composition, of reprogrammingfactors. The factors may be provided at the same molar ratio or at different molar ratios. The factors may be provided once or multiple times in the course of culturing the cells of the subject invention. In some embodiments the reprogramming factor is a transcription factor, including without limitation, Oct3 / 4; Sox2; Klf4; c-Myc; Nanog; and Lin-28.

[0029] Somatic cells are contacted with reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency. Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors. In some embodiments the reprogramming factors are provided as a plurality of coding sequences on a vector. The somatic cells may be fibroblasts, adipocytes, stromal cells, and the like, as known in the art. Somatic cells or hiPS cells can be obtained from cell banks, from normal donors, from individuals having a neurologic or psychiatric disease of interest, etc.

[0030] Following induction of pluripotency, hiPS cells are cultured according to any convenient method, e.g. on irradiated feeder cells and commercially available medium. The hiPS cells can be dissociated from feeders by digesting with protease, e.g., dispase, preferably at a concentration and for a period of time sufficient to detach intact colonies of pluripotent stem cells from the layer of feeders. The organoids can also be generated from hiPS cells grown in feeder-free conditions, by dissociation into a single cell suspension and aggregation using various approaches, including centrifugation in plates, etc.

[0031] Genes may be introduced into the somatic cells or the hiPS cells derived therefrom for a variety of purposes, e.g. to replace genes having a loss of function mutation, provide marker genes, etc. Alternatively, vectors are introduced that express antisense mRNA, siRNA, ribozymes, etc. thereby blocking expression of an undesired gene. Other methods of gene therapy are the introduction of drug resistance genes to enable normal progenitor cells to have an advantage and be subject to selective pressure, for example the multiple drug resistance gene (MDR), or anti-apoptosis genes, such as BCL-2. Various techniques known in the art may be used to introduce nucleic acids into the target cells, e.g. electroporation, calcium precipitated DNA, fusion, transfection, lipofection, infection and the like, as discussed above. The particular manner in which the DNA is introduced is not critical to the practice of the invention.

[0032] Disorder-associated or disorder-causing genotypes can be generated in healthy hiPS cells through targeted genetic manipulation (CRISPR / CAS9, etc.) or hiPS cells can be derived from individuals that carry a disorder-related genotype or are diagnosed with a disorder. Moreover, sensory disorders with less defined or without genetic components 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 their corresponding, non-edited hiPS cell lines. This reduces variability associated with line-line differences in genetic background.Conditions of neuropsychiatric and sensory disorders that have strong genetic components or are directly caused by genetic or genomic alterations can be modeled with the systems of the invention.

[0033] The methods described herein are associated with brain-region specific organoids. Brain-region specific organoids are three-dimensional (3D) aggregates of cells that resemble particular regions of the human brain or sensory system and contain functional neurons that are normally associated with that region of the brain. These organoids are capable of being maintained in suspension culture for long periods of time, e.g. 2 week, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more, without adhering to a surface, e.g. a surface of a culture dish. By functional neurons, it is intended to mean that the neurons are capable of forming functional synapses with other neurons, either in the same organoid, in another organoid, or with host neurons. The formation of functional synapses can be revealed using calcium imaging, as described in more details in the Examples.

[0034] Pyramidal cortical neurons are neurons that project from the cerebral cortex to other parts of the nervous system, including the striatum. These neurons are excitatory, glutamatergic neurons.

[0035] Dopaminergic neurons are collections of neurons that produce the neurotransmitter dopamine. The neurons mainly originate in two nuclei in the human midbrain - the substantia nigra and the ventral tegmental area.

[0036] GABAergic interneurons are inhibitory neurons of the nervous system that play a vital role in neural circuitry and activity. They are so named due to their release of the neurotransmitter gamma-aminobutyric acid (GABA). An interneuron is a specialized type of neuron whose primary role is to modulate the activity of other neurons in a neural network. Cortical interneurons are so named for their localization in the cerebral cortex.

[0037] There are interneuron subtypes categorized based on the surface markers they express, including parvalbumin (PV)-expressing interneurons, somatostatin (SST)-expressing interneurons, VIP-expressing, serotonin receptor 5HT3a (5HT3aR)-expressing interneurons, etc. Although these interneurons are localized in their respective layers of the cerebral cortex, they are generated in various subpallial locations.

[0038] Morphologically speaking, cortical interneurons may be described with regard to their soma, dendrites, axons, and the synaptic connections they make. Molecular features include transcription factors, neuropeptides, calcium-binding proteins, and receptors these interneurons express, among many others. Physiological characteristics include firing pattern, action potential measurements, passive or subthreshold parameters, and postsynaptic responses, to name a few.

[0039] The PV interneuron group represents approximately 40% of the GABAergic cortical interneuron population. This population of interneurons possesses a fast-spiking pattern, andfire sustained high-frequency trains of brief action potentials. Additionally, these interneurons possess the lowest input resistance and the fastest membrane time constant of all interneurons. Two types of PV-interneurons make up the PV interneuron group: basket cells, which make synapses at the soma and proximal dendrite of target neurons, and usually have multipolar morphology and chandelier cells, which target the axon initial segment of pyramidal neurons.

[0040] The SST-expressing interneuron group is the second-largest interneuron group. SST- positive interneurons are known as Martinotti cells, and possess ascending axons that arborize layer I and establish synapses onto the dendritic tufts of pyramidal neurons. Martinotti cells are found throughout cortical layers 11- VI, but are most abundant in layer V. These interneurons function by exhibiting a regular adapting firing pattern but also may initially fire bursts of two or more spikes on slow depolarizing humps when depolarized from hyperpolarized potentials. In contrast to PV-positive interneurons, excitatory inputs onto Martinotti cells are strongly facilitating.

[0041] The third group of GABAergic cortical interneurons is designated as the 5HT3aR interneuron group. VIP-expressing interneurons are localized in cortical layers II and III. VIP interneurons generally make synapses onto dendrites, and some have been observed to target other interneurons. Relative to all cortical interneurons, VIP interneurons possess a very high input resistance. In general they possess a bipolar, bitufted and multipolar morphology. Irregular spiking interneurons possess a vertically oriented, descending axon that extends to deeper cortical layers, and have an irregular firing pattern that is characterized by action potentials occurring irregularly during depolarizations near threshold, and express the calcium-binding protein calretinin (OR). Other subtypes include rapid-adapting, fast-adapting neurons IS2, as well as a minor population of VIP-positive basket cells with regular, bursting, or irregular-spiking firing patterns. Of the VIP-negative 5HT3aR group, nearly 80% express the interneuron marker Reelin. Neurogliaform cells are a type of cortical interneuron that belongs to this category: they are also known as spiderweb cells and express neuropeptide Y (NPY), with multiple dendrites radiating from a round soma.

[0042] A transcriptional network plays a role in regulating proper development and specification of GABAergic cortical interneurons, including DLX homeobox genes, LHX6, SOX6 and NKX2-1 , LHX8, GSX1 , GSX2. The DLX family of homeobox genes, specifically DLX1 , DLX2, DLX5, and DLX6, also play a role in the specification of interneuron progenitors, and are expressed in most subpallial neural progenitor cells.

[0043] Glutamatergic neurons. The mature cerebral cortex harbors a heterogeneous population of glutamatergic neurons, organized into a highly intricate histological architecture. So-called excitatory neurons are usually classified according to the lamina where their somais located, specific combinations of gene expression, by dendritic morphologies, electrophysiological properties, etc.

[0044] The terms “astrocytic cell,” “astrocyte,” etc. encompass cells of the astrocyte lineage, i.e. glial progenitor cells, astrocyte precursor cells, and mature astrocytes, which for the purposes of the present invention arise from a non-astrocytic cells (i.e., glial progenitors). Astrocytes can be identified by markers specific for cells of the astrocyte lineage, e.g. GFAP, ALDH1 L1 , AQP4, EAAT1 and EAAT2, etc. Markers of reactive astrocytes include S100, VIM, LCN2, FGFR3 and the like. Astrocytes may have characteristics of functional astrocytes, that is, they may have the capacity of promoting synaptogenesis in primary neuronal cultures; of accumulating glycogen granules in processes; of phagocytosing synapses; and the like. A "astrocyte precursor" is defined as a cell that is capable of giving rise to progeny that include astrocytes.

[0045] Astrocytes are the most numerous and diverse neuroglial cells in the CNS. An archetypal morphological feature of astrocytes is their expression of intermediate filaments, which form the cytoskeleton. The main types of astroglial intermediate filament proteins are glial fibrillary acidic protein (GFAP) and vimentin; expression of GFAP, ALDH1 L1 and / or AQP4P are commonly used as a specific marker for the identification of astrocytes.

[0046] The terms “oligodendrocyte,” “oligodendrocyte progenitor cell,” etc. can encompass cells of the oligodendrocyte lineage, i.e. neural progenitor cells that ultimately give rise to oligodendrocytes, oligodendrocyte precursor cells, and mature and myelinating oligodendrocytes, which for the purposes of the present invention arise from a nonoligodendrocyte cell by experimental manipulation. Oligodendrocytes may have functional characteristics, that is, they may have the capacity of myelinating neurons; and the like. An "oligodendrocyte precursor" or “oligodendrocyte progenitor cell” is defined as a cell that is capable of giving rise to progeny that include oligodendrocytes. Oligodendrocytes may be present in the assembloids.

[0047] Oligodendrocytes are the myelin-forming cells of the central nervous system. An oligodendrocyte extends many processes which contact and repeatedly envelope stretches of axons. Subsequent condensation of these wrapped layers of oligodendrocyte membrane form the myelin sheath. One axon may contain myelin segments from many different oligodendrocytes.

[0048] Somatosensory neurons are collections of neurons that sense and transmit somatosensory signals, such as pain and temperature, to the spinal cord.

[0049] Spinothalamic projection neurons are excitatory neurons of the spinal cord that play a vital role in ascending somatosensory circuitry. These neurons are a specialized type of neuron that receives synaptic input from sensory neurons and project their axons into the thalamus.

[0050] Calcium sensors. Neural 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 concentrations. These proteins can be introduced into cells, e.g. hiPS cells, by including the coding sequence on a suitable expression vector, e.g. a viral vector, to genetically modify neurons generated by the methods described herein. GCaMPs are widely used protein calcium sensors, which are comprised of a fluorescent protein, e.g. GFP, the calcium-binding protein calmodulin (CaM), and CaM-interacting M13 peptide, although a variety of other sensors are also available. Many different proteins are available, including, for example, those described 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; etc.; and as described in US Patent nos. 8,629,256, 9,518,980 and 9,488,642 and 9,945,844.

[0051] Optogenetics integrates optics and genetic engineering to measure and manipulate neurons. Actuators are genetically-encoded tools for light-activated control of proteins; e.g., opsins and optical switches. Opsins are light-gated 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 on and off the light source. Channel rhodopsins typically allow the fast depolarization of neurons upon exposure to light through direct stimulation of ion channels. Chlamydomonas reinhardtii Channelrhodopsin-1 (ChR1 ) is excited by blue light and permits nonspecific cation influx into the cell when stimulated. Examples of ChRs from other species include: CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama), and SdChR (from Scherffelia dubia). Synthetic variants have been created, for example ChR2(H134R), C1 V1 (t / t), ChlEF; ChETA, VChR1 , Chrimson, ChrimsonR, Chronos, PsChR2, CoChR, CsChR, CheRiff, and the like. Alternatively, ChR variants that inhibit neurons have been created and identified, for example GtACRI and GtACR2 (from the cryptophyte Guillardia theta), and variants such as iChloC, SwiChRca, Phobos, Aurora. Halorhodopsin, known as NpHR (from Natronomonas pharaoni), causes hyperpolarization of the cell when triggered with yellow light, variants include Halo, eNpHR, eNpHR2.0, eNpHR3.0, Jaws. Archaerhodopsin-3 (Arch) from Halorubrum sodomense is also used to inhibit neurons.DETAILED DESCRIPTION

[0052] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for thepurpose 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.

[0053] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0055] It must 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, and reference to "the induced pluripotent stem cells" includes reference to one or more induced pluripotent stem cells and equivalents thereof known to those skilled in the art, and so forth.

[0056] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to 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.METHODS FOR PRODUCING ORGANOIDS AND ASSEMBLOIDS

[0057] As summarized above, methods are provided for producing human sensory organoids in vitro. Methods are also provided for producing human dorsal hindbrain / cervical spinal cord organoids. Methods are also provided for producing human ascending somatosensory pathway assembloids.

[0058] The methods provided for producing human sensory organoids in vitro contain inducing a human pluripotent stem cell in suspension culture to a neural fate to provide a neural organoid; differentiating the neural organoid into a human sensory organoid (hSeO); and maintaining the hSeO for an extended period of time in neural medium such that the hSeO comprises human sensory neurons.

[0059] The methods provided for producing human dorsal hindbrain / cervical spinal cord organoids in vitro contain inducing a human pluripotent stem cell in suspension culture to a neural fate to provide a neural organoid; differentiating the neural organoid into a human dorsal hindbrain / cervical spinal cord organoid (hdSpO); and maintaining the hdSpO for an extended period of time in neural medium such that the hdSpO comprises dorsal spinal cord neurons.

[0060] The methods provided for producing a human ascending somatosensory assembloid in vitro contain inducing in a human pluripotent stem cell suspension 3D culture a neural fate to provide a neural organoid; differentiating the neural organoid into two or more of: human cortical organoids (hCO), human diencephalon organoids (hDiO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO) and human sensory organoid (hSeO); and culturing the two or more of hCO, hDiO, hdSpO and hSeO under conditions permissive for organoid fusion into a linear assembloid while maintaining for an extended period of time in neural medium; wherein an integrated structure is differentiated comprising interacting neurons that form a linear circuit.

[0061] Human induced pluripotent stem cells. Initially, hiPS cells can be obtained from any convenient source, or can be generated from somatic cells using art-recognized methods. The hiPS cells are dissociated from feeders into single cells and grown in suspension culture, preferably when dissociated as intact colonies. In certain embodiments, the culture is feeder layer free, e.g. when grown on vitronectin coated culture dishes. The culture may further be free on non-human components, i.e. xeno-free. The 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, for up to about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, (see, for example, Watanabe et al. (2007) Nature Biotechnology 25:681 686). Inhibitors useful for such purpose include, without limitation, Y-27632; Thiazovivin (Cell Res, 2013, 23(10):1 187-200; Fasudil (HA-1077) HCI (J Clin Invest, 2014, 124(9):3757-66); GSK429286A (Proc Natl Acad Sci U S A, 2014, 1 11 (12) :E1 140-8); RKI-1447; AT13148; etc. In particular embodiments the ROCK inhibitor Y-27632 is used. Optionally a WNT pathway inhibitor such as XAV-939 is added.

[0062] Human dorsal hindbrain / cervical spinal organoids (hdSpO). To generate hdSpO, organoids are maintained in a medium containing two or more SMAD inhibitors. In some embodiments, the medium is Essential 6 medium. The organoids may be maintained in the medium for a period of time. For instance, the period of time may be about 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the period is 6-8 days. The two or more SMAD inhibitors may be any SMAD inhibitors deemed useful. SMAD inhibitors include inhibitors of the BMP pathway and the TGFfS pathway. For instance SMAD inhibitors include, without limitation, dorsomorphin, A 83-01 ; DMH-1 ; K 02288; ML 347; SB 505124, SB-431542, 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; LDN- 193189 HCI (Proc Natl Acad Sci U S A, 2013, 1 10(52) :E5039-48); SB505124; Pirfenidone (Histochem Cell Biol, 2014, 10.1007 / s00418-014-1223-0); RepSox; K02288; Hesperetin; GW788388; LY364947, etc. In some embodiments, one of the two or more SMAD inhibitors is dorsomorphin. In some embodiments, one of the two or more SMAD inhibitors is dorsomorphin. In some embodiments, one of the two or more SMAD inhibitors is LDN-193189. In some embodiments, one of the two or more SMAD inhibitors is SB-431542. In some embodiments, the two or more SMAD inhibitors are dorsomorphin and SB-431542. When one of the two or more SMAD inhibitors is dorsomorphin, dorsomorphin can be added at an effective dose of at least about 0.1 y.M, at least about 1 iM, at least about 5 ) M, at least about 10 |iM, at least about 50 y.M, up to about 100 LIM concentration. When one of the two or more SMAD inhibitors is SB-431542, it can be added at an effective dose of at least about 0.1 LIM, at least about 1 g.M, at least about 5 g.M, at least about 10 |j.M, at least about 50 y.M, up to about 100 LIM concentration.

[0063] After about 4 days, about 5 days, about 6 days, about 7 days or about 8 days, a GSK inhibitor is added to the medium for a period of 1 day, 2 days, 3 days, or 4 days. In some embodiments, the GSK inhibitor is added after day 5 for a period of 2 days. In some embodiments, the GSK inhibitor is added on day 5 to day 6. The GSK inhibitor may be any GSK inhibitor that inhibits the activity of GSK-3. For instance, the GSK inhibitor may be TWS119, CHIR98014, SB216763, CHIR99021 , GSK inhibitor IX (BIO), etc. The inhibitor of GSK-3 may be CHIR 99021 added for example at a concentration of from about 0.5 ,u.M to about 50 piM, about 1 LIM to about 25 y.M, about 1 ,u.M to about 10 .M, about 1 uM to about 5 piM , or may be about 3 ^M.

[0064] After day 6, day 7, day 8, or day 9 organoids may be transferred to a medium containing EGF, retinoic acid, and a GSK inhibitor. In some embodiments, the organoids are transferred after 7 days. The EGF may be provided at a concentration for each of at leastabout 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. The retinoic acid may be at concentration of from about 10 nM to about 1 pM, from about 50 nM to about 0.5 pM, from about 75 nM to about 0.25 pM, or may be about 100 nM. An exemplary medium may contain Neurobasal™-A Medium, B-27™ Supplement minus vitamin A, and GlutaMAX™ Supplement or an equivalent medium.

[0065] On about day 19, day 20, day 21 , day 22, day 23, day 24 or day 25, the medium may be supplemented with one or more of BDNF, N6, 2’-0-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt, L-Ascorbic Acid 2-phosphate Trisodium Salt, and / or IGF-1. In some embodiments, the medium is supplemented on day 21. The BDNF can be provided at a concentration for each 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, up to about 100 ng / ml, or about 20 ng / ml. The cAMP can be provided at a concentration of from about 10 to 500 nM, from about 20 to 100 nM, and may be about 50 nM. The L-ascorbic acid may be at a concentration of from about 10 to 500 nM, from about 50 to 250 nM, and may be about 200 nM. The IGF-1 can be provided at a concentration for each 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, up to about 100 ng / ml, or about 10 ng / ml. In some embodiments, the medium is supplemented with an effective amount of BDNF, N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt, L-Ascorbic Acid 2- phosphate Trisodium Salt, and IGF-1. In some embodiments, the medium is further supplemented with a gamma secretase inhibitor. The gamma secretase inhibitor may be a range of different gamma secretase inhibitors including, without limitation, dibenzazepine, LY411575, DAPT, etc. In some embodiments, the gamma secretase inhibitor is DAPT. The DAPT may be at a concentration of from about 1 to 25 y.M, about 2 to 10 pM, and may be around about 2.5 .M. In some embodiments, the gamma secretase inhibitor is only present for 4-8 days.

[0066] Human dorsal hindbrain / cervical spinal organoids (hdSpO) comprise functional human spinothalamic projection neurons. The dorsal spinal cord neurons express one or more of the group consisting of LBX1 , TACR1 , SOX2, SLC17A6, STMN2, GAD1 , and any combination thereof. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressing LBX1. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressing TACR1. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressing SOX2. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressing SLC17A6. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressingSTMN2. In some embodiments, the hdSpO comprise dorsal spinal cord neurons expressing GAD1 .

[0067] Human sensory organoids (hSeO). To generate hSeO, that contain sensory root ganglia neurons as well Schwann cells and neural crest cells, organoids are maintained in a medium containing one or more SMAD inhibitors. In some embodiments, the medium is Essential 6 medium. The neural organoids may be maintained in the medium for a period of time. For instance, the period of time may be about 6 days, 7 days, 8 days, 9 days, or about 10 days. In some embodiments, the period is 6 days. The one or more SMAD inhibitors may be any SMAD inhibitors described above. In some embodiments, the one or more SMAD inhibitors is SB-431542. When the one or more SMAD inhibitors is SB-431542, it can be added at an effective dose of at least about 0.1 pM, at least about 1 ,uM, at least about 5 y.M, at least about 10 (iM, at least about 50 |iM, up to about 100 jiM concentration.

[0068] From about day 1 , day 2, day 3, or about day 4, the medium may be supplemented with BMP4 for a period of about 1 day, 2 days, 3 days, 4 days, 5 days, or about 6 days. In some embodiments, the medium is supplemented with BMP4 from about day 1 to about day 4. In some embodiments, the medium is supplemented with BMP4 for a period of about 2 to 6 days. The BMP4 can be provided at a concentration for 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, up to about 100 ng / ml, about 10 ng / ml, or about 5ng / mL. From about day 1 , day 2, day 3, day 4, or about day 5, the medium may be supplemented with a GSK inhibitor for a period of about 1 day, 2 days, 3 days, 4 days, 5 days, or about 6 days. The GSK inhibitor may be any of the GSK inhibitors above. In some embodiments, the medium is supplemented with the GSK inhibitor from about day 3 to about day 5. In some embodiments, the medium is supplemented with the GSK inhibitors for a period of about 2 to 6 days. The GSK inhibitor can be provided at a concentration of from about 10 to 1000 nM, from about 100 to 800 nM, and may be about 600 nM.

[0069] After about day 6, day 7, day 8, or about day 9 organoids may be transferred to a medium containing one or more SMAD inhibitors. In some embodiments, the organoids are transferred to a medium after about day 7. When the one or more SMAD inhibitors is SB- 431542, it can be added at an effective dose of at least about 0.1 y.M, at least about 1 pM, at least about 5 jiM, at least about 10 JIM, at least about 50 JIM, up to about 100 |iM concentration. In some embodiments, the organoids are transferred after 7 days. An exemplary medium may contain NeurobasalTM-A Medium, B-27™ Supplement minus vitamin A, and GlutaMAX™ Supplement or an equivalent medium.

[0070] On about day 8, day 9, day 10, day 1 1 , day 12, day 13 or about day 14, the medium may be supplemented with one or more of BDNF, GDNF, and / or NGF. The BDNF can beprovided at a concentration for each 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, up to about 100 ng / ml, or about 20 ng / ml. The GDNF and NGF can be provided at a concentration for each 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, up to about 100 ng / ml, or about 25 ng / ml. In some embodiments, the medium is supplemented on day 9. In some embodiments, the medium is supplemented with an effective amount of BDNF, GDNF, and NGF. In some embodiments, the BDNF, GDNF, and NGF are supplemented in the medium following about day 7 to about day 9. In some embodiments, the medium is further supplemented with one or more SMAD inhibitors. The one or more SMAD inhibitors may be added for a period of about 3 days, 4 days, 5 days, 6 days, 7 days, or about 8 days. In some embodiments, the one or more SMAD inhibitor is added from about day 9 to about day 14. In some embodiments, the medium is further supplemented with a gamma secretase inhibitor. The gamma secretase inhibitor may be a range of different gamma secretase inhibitors including, without limitation, dibenzazepine, LY41 1575, DAPT, etc. In some embodiments, the gamma secretase inhibitor is DAPT. The DAPT may be at a concentration of from about 1 to 25 p.M, about 2 to 10 |1M, and may be around about 2.5 p.M. In some embodiments, the gamma secretase inhibitor is only present for 5-10 days.

[0071] Human sensory organoids (hSeO) comprise functional human somatosensory neurons, Schwann cells, and neural crest cells. The cells of the human sensory organoids express one or more of the group consisting of BRN3A, SIX1 , SOX2, SOX10, STMN2, PRPH, P2RX3, SCN9A, and any combination thereof. In some embodiments, the hSeO comprise sensory neurons expressing BRN3A. In some embodiments, the hSeO comprise sensory neurons expressing SIX1. In some embodiments, the hSeO comprise sensory neurons expressing SOX2. In some embodiments, the hSeO comprise sensory neurons expressing SOX10. In some embodiments, the hSeO comprise sensory neurons expressing STMN2. In some embodiments, the hSeO comprise sensory neurons expressing PRPH. In some embodiments, the hSeO comprise sensory neurons expressing P2RX3. In some embodiments, the hSeO comprise sensory neurons expressing SCN9A. The cells in the hSeO are capable of response to a range of stimuli including, without limitation, noxious stimuli, mechanical stimuli, etc. In some embodiments, the hSeO is capable of being stimulated by noxious stimuli. The noxious stimuli may be any noxious stimuli associated with sensory responses including, without limitation, capsaicin, ap-MeATP, Zymosan, Bradykinin, MSP-3, menthol, AITC, etc. In some embodiments, the hSeO is capable of being stimulated by mechanical stimuli.

[0072] Human cortical organoids. hCO may be generated by the methods previously described, for example in Pasca et al. (2015) Nat. Methods 12(7) :671 -678, entitled “Functionalcortical neurons and astrocytes from human pluripotent stem cells in 3D culture” and in U.S. Patent No. 10,494,602, each herein specifically incorporated by reference.

[0073] For example, a suspension culture of hi PS cells is cultured to provide a neural organoid, as described above. After about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days in suspension culture, the floating neural organoids are moved to neural media to differentiate the neural organoids. The media is supplemented with an effective dose of FGF2 and EGF. The growth factors can be provided at a concentration for each 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, up to about 100 ng / ml.

[0074] To promote differentiation of neural organoids into hCO, comprising glutamatergic neurons, after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks after FGF2 / EGF exposure the neural medium is changed to replace the FGF2 and EGF with an effective dose of BDNF and NT3. The growth factors can be provided at a concentration for each 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, up to about 100 ng / ml. The cortical spheroids comprise functional glutamatergic neurons.

[0075] Human diencephalic organoids (hDiO). hDiO may be generated by the methods previously described, for example in U.S. Patent Application No. 18 / 561 ,236, which is specifically incorporated by reference herein.

[0076] To generate hDiO, on day 3 in suspension the organoids were supplemented with 1 pM CHIR (Selleckchem, S1263), in addition with two SMAD pathway inhibitors. For the first 5 days, medium was changed every day. On day 6 in suspension the organoids were transferred to neural medium containing Neurobasal™-A Medium (Thermo Fisher Scientific, 10888022), B-27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1 :100, Thermo Fisher Scientific, 35050079), Penicillin-Streptomycin (1 :100, Thermo Fisher Scientific, 15070063), and supplemented with 1 pM CHIR (Selleckchem, S1263). On day 9 of differentiation, organoids were supplemented with 100 nM SAG (Millipore Sigma, 566660-1 MG), in addition to the compounds described above. Furthermore, on day 12 of differentiation, organoids were supplemented with 30 ng / mL BMP7 (PeproTech, 120- 03P), in addition to the compounds described above. From day 19, to promote differentiation of the neural progenitors into neurons of hDiS, the neural medium was supplemented with brain-derived neurotrophic factor (BDNF; 20ng ml1, PeproTech, 450-02), NT3 (20 ng ml1, PeproTech, 450-03), L-Ascorbic Acid 2-phosphate Trisodium Salt (AA; 200 pM, Wako, 323- 44822), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP; 50 pM, Millipore Sigma, D0627), cis-4, 7, 10, 13, 16, 19-Docosahexaenoic acid (DHA; 10 pM, MilliporeSigma, D2534), and 2.5 LIM DAPT (only from day 19-25, STEMCELL Technologies, 72082). From day 43, only neural medium containing B-27™ Plus Supplement (Thermo Fisher Scientific, A3582801 ) was used for medium changes every 4 days.

[0077] In some embodiments, the hDiO comprise glutamatergic neurons expressing TCF7L2. In some embodiments, the hDiO comprise glutamatergic neurons expressing SLC17A6. In some embodiments, the hDiO comprise glutamatergic neurons expressing the thalamus- related gene TCF7L2 and SLC17A6.

[0078] Assembloids. The individual organoids can be functionally integrated with separately cultured organoids, to form a functional human ascending somatosensory assembloid containing human cortical organoids (hCO), human diencephalon organoids (hDiO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO), and human sensory organoids (hSeO). Assembloids may also be formed from hdSpO and hSeO, hDiO and hdSpO, hDiO and hdSpO and hSeO, and hCO and hDiO and hdSpO. Each of hCO, hDiO, hdSpO, and hSeO are separately differentiated, and then integrated in a desired combination (two-parts, three-parts, and four-parts) that allow formation of reciprocal connections and enable ascending pathway properties. Ascending pathway properties include, without limitation, spontaneous and synchronous neural activity, hSeO induced action potential transmission to one or more organoids contained within the sensory pathway assembloid (e.g., hCO, hDiO, and / or hdSpO), sensory-dorsal spinal projections, dorsal spinal-thalamic projections, etc.

[0079] The hCOs are co-cultured with the hDiO, hdSpO, and hSeO in neural medium under conditions permissive for cell fusion. Condition permissive for cell fusion may include culturing the hCO, hDiO, hdSpO, and hSeO in close proximity, e.g. in direct contact with one another. In some embodiments, the conditions for cell fusion include culturing the hCO, hDiO, hdSpO, and hSeO at a tilt.

[0080] Assembly may be performed with organoids after around about 30 days, about 60 days, about 90 days of culture for hCO; after around about 30 days, about 60 days, about 90 days of culture for hDiO, after around about 30 days, about 60 days, about 90 days of culture for hdSpO, and after around about 30 days, about 60 days, about 90 days of culture for hSeO. The hCO, hDiO, hdSpO, and hSeO organoids may be co-cultured for a period of 2 days, 3 days, 5 days, 7 days, 8 days, 10 days, 14 days, 18 days, 21 days or more. The resulting ascending somatosensory assembloids are demonstrated to contain functional neural circuits, where the assembloids contain neurons projecting from the hSeO to the hdSpO, from the hdSpO to hDiO, and from the hDiO to the hCO. Methods for confirming the functionality of the neurons are known in the art and include optogenetic methods and imaging of calcium activity in neurons, such as those methods described in the examples. In some embodiments, themethods may comprise confirming the functionality of the neurons in the ascending somatosensory pathway assembloids.

[0081] In some embodiments, the sensory organoids of the present disclosure are generated from cells isolated from an individual who is predicted to have or has been diagnosed with a sensory disorder. In some embodiments, the human dorsal hindbrain / cervical spinal cord organoids of the present disclosure are generated from cells isolated from an individual who is predicted to have or has been diagnosed with a sensory disorder. In some embodiments, the sensory organoids of the present disclosure contain sensory neurons containing at least one allele associated with a sensory disorder. In some embodiments, the human dorsal hindbrain / cervical spinal cord organoids of the present disclosure contain dorsal spinal cord neurons containing at least one allele associated with a sensory disorder. The sensory disorder may be any sensory disorder that is deemed suitable for organoid culture. Sensory disorders generally involve neuropathic pain and / or autonomic dysfunction. Sensory disorders that find use in the present disclosure include, with limitation, small fiber neuropathy, sodium- channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, autism spectrum disorder, etc. Alleles and mutations associated with sensory disorders are known in the art and have been described in, for example, Naureen et al. (Acta Biomed. 2020 Nov 9;91 (13-S):e2020010) and James (Br J Pain. 2013 Nov;7(4):171 -8) which are specifically incorporated by reference herein.METHODS FOR DETERMINING THE EFFECTIVENESS OF A DRUG ON A SENSORY DISORDER

[0082] As summarized above, methods are provided for determining the effect of a candidate agent on a human ascending somatosensory pathway, the method containing contacting the candidate agent with one or a panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembled organoids differentiated from induced human pluripotent stem (hiPS) cells according to the methods above, or a population of cells isolated therefrom; and determining the effect of the agent on morphologic, genetic or functional parameters. Functional parameters can include a range of different parameters including, without limitation, intrinsic electrophysiological properties, action potential propagation in response to a stimuli, e.g., a noxious stimuli or a mechanical stimuli, or , spontaneous action potential propagation, synaptic integration, calcium signaling, etc.

[0083] The methods for determining the effect of a candidate agent on the human ascending somatosensory pathway find particular use for the screening of drugs that modulate the human ascending somatosensory pathway. The modulation may be the suppression or enhancementof the human ascending somatosensory pathway such that the pathway becomes more or less sensitive or responsive to noxious or mechanical stimuli. For instance, the methods may be used to screen for agents that reduce the response to noxious or mechanical stimuli or it may be used to screen for agents that increase the response to noxious or mechanical stimuli. The methods may also be used to screen for agents that reduce the response to noxious or mechanical stimuli in presence of inflammatory mediators or in an inflammatory state. The reduction or increase to the response to noxious or mechanical stimuli may be in the form of a reduction or increase in the spiking rate or action potential propagation in response to the stimuli, a reduction or increase in the concentration or amount of the stimuli necessary to produce a response to the stimuli when compared to an absence of the agent, or a reduction or increase in intracellular free calcium with the responsive neurons. In some embodiments, the agents are being screened for their effectiveness in altering the response to pain.

[0084] Determining the effect of the agent on morphologic, genetic, or functional parameters may include contacting the panel of functionally integrated human hDiO-hdSpO-hSeO or hCO- hDiO-hdSpO-hSeO assembloid with a noxious or mechanical stimuli at the same time, before, or after contacting the panel with the candidate agent. The assembloid may also be exposed to agents that induce an inflammatory state followed by exposure to a noxious stimuli. The assembloid may be contacted with the candidate agent prior to, at the same time, or after the contacting with the inflammatory agent and the candidate agent may also be applied prior to, at the same time, or after the noxious stimuli. Agents that induce an inflammatory state may include inflammatory mediators. Inflammatory mediators include without limitation, extracellular protons, arachidonic acid, serotonin, nerve growth factor (NGF), prostaglandins (PG), bradykinin (BK), adenosine triphosphate (ATP), histamine, etc. Inflammatory meditators are known to reversibly reduce axonal density and increase the responsiveness of axonal endings to noxious stimuli, as discussed in Giorgi et al. (Mol Pain. 2023 Jan- Dec:19:17448069231 197102) which is specifically incorporated by reference herein.

[0085] The panel may otherwise be contacted with other stimuli such as electrical stimulation including alterations in ionicity, contact with other cells including without limitation neurons and neural progenitors, contact with infectious agents, e.g. rabies virus, polio virus, Zika virus, and the like, and where cells may vary in genotype, in prior exposure to an environment of interest, in the dose of agent that is provided, etc. Usually at least one control is included, for example a negative control and a positive control. Culture of cells is typically performed in a sterile environment, for example, at 37°C in an incubator containing a humidified 92-95% air / 5-8% CO2atmosphere. Cell culture may be carried out in nutrient mixtures containing undefined biological fluids such as fetal calf serum, or media which is fully defined and serum free. The effect of the altering of the environment is assessed by monitoring multiple output parameters, including morphological, functional, and genetic changes.

[0086] As also described herein, various disorders are associated with sensory disorders. Accordingly, the assays described herein may find particular utility where the pluripotent stem cells or cells that become pluripotent stem cells (i.e., induced pluripotent stem cells) are derived from an individual having a sensory disorder, such as small fibre neuropathy, sodium- channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, autism spectrum disorder, etc. Candidate agents that are able to restore the functionality of defects or abnormalities, e.g., hyposensitivity or hypersensitivity to noxious or mechanical stimuli, identified in the assembloid comprising the sensory disorder may have therapeutic utility in the treatment of said disorder.

[0087] Examples of analytic methods comprise, for example, assessing the integration of neurons. Synaptic integration of neurons to neurons can be assessed by using array tomography to detect pre- and post- synaptic proteins in hCO, hDiO, hdSpO, and / or hSeO before and after fusion, such as the presence of PSDH95 or GPHN, which are postsynaptic proteins. To further examine these synaptic puncta ‘synaptograms’ consisting of a series of high-resolution sections through a single synapse may be obtained. Whole-cell voltage clamp recordings of synaptic responses can be performed on slices on the functional assembled spheroids, and to distinguish between excitatory postsynaptic currents (EPSCs, downward deflecting) and IPSCs (upward deflecting), a low CP solution may be used in the patch pipette with cells held at -40 mV.

[0088] Synaptic integration may also be assessed using axon tracing. The axons may be traced in any way deemed useful. The axon tracing may be retrograde tracing or anterograde tracing. The axon tracing may be performed with the use of a viruses, protein or small molecule. Non-limiting examples of viruses that facilitate anterograde axon tracing include, without limitation, herpes simplex virus 1 (HSV-1 ), HSV-1 strain H129, rhabdoviruses, etc. Non-limiting examples of viruses that facilitate retrograde axon tracing include, without limitation, rabies, pseudorabies, glycoprotein, deleted rabies, etc. Non-limiting examples of proteins and small molecules that facilitate anterograde axon tracing include, without limitation, Phaseolus vulgaris-leucoagglutinin, wheat germ agglutin, dextran amines, etc. Nonlimiting examples of proteins and small molecules that facilitate retrograde axon tracing include, without limitation, horse radish peroxidase (HRP), wheat germ agglutin, cholera toxin subunit B, hydroxystilbamidine, Fast Blue, Diamidino Yellow, True Blue, the carbocyanines Dil and DiO, fluorescent lax microspheres, etc. Non-limiting examples of proteins and small molecules that facilitate retrograde axon tracing include, without limitation, horse radish peroxidase (HRP), wheat germ agglutin, cholera toxin subunit B, hydroxystilbamidine, FastBlue, Diamidino Yellow, True Blue, the carbocyanines Dil and DiO, fluorescent lax microspheres, etc. Other viruses, proteins and small molecules that facilitate axon tracing have been described in the art, for example, in Xu, X. et al. (Neuron. 2020 Sep 23;107(6):1029-1047) and in Saleeba, C. et al. (Front Neurosci. 2019 Aug 27;13:897), each of which herein specifically incorporated by reference.

[0089] Live imaging of cells may be performed, and cells modified to express a detectable marker. Calcium sensitive dyes can be used, e.g. Fura-2 calcium imaging; Fluo-4 calcium imaging, Cal-590 calcium imaging, GCaMP6 calcium imaging, voltage imaging using voltage indicators such as voltage-sensitive dyes (e.g. di-4-ANEPPS, di-8-ANEPPS, and RH237) and / or genetically-encoded voltage indicators (e.g. ASAP1 , Archer) can be used on the intact spheroids, assembled spheroids, or on cells isolated therefrom.

[0090] Calcium imaging assays can be used to determine the functional of neuronal circuits by exploiting the fact that neural activity causes rapid changes in intracellular free calcium. This may involve modifying neurons to contain genetically-encoded calcium indicator proteins or using calcium sensitive dyes such as those described above, such proteins include the fluorophore sensor GCaMP and imaging those cells. GCaMP comprises a circularly permuted green fluorescent protein, a calcium-binding protein calmodulin (CaM) and CaM-interacting M13 peptide, where brightness of the GFP increases upon calcium binding. Further details about 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.

[0091] For example, the neurons may be modified to express GCaMP6f. This can be combined with methods that activate certain neurons in response to external stimuli, for example optogenetic methods that activate neurons in response to light, application of mechanical pressure, or application of noxious stimulus such as capsaicin, a|3-MeATP, Zymosan, Bradykinin, MSP-3, menthol, AITC, etc. 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 modified to express a calcium indicator (e.g. GCaMP6f) and imaging used to monitor calcium release. If the first neuron is functionally connected (synapses with) the second neuron then optogenetic activation of the first neuron will elicit calcium release and a visible readout in the second neuron. As set out in U.S. Patent Number 17 / 773,429, such a method was used to confirm functionality of the cortico-striatal circuits.

[0092] Other optogenetic actuators may be used besides ChrimsonR. Optogenetic actuators that find use in the present disclosure include, without limitation, Channelrhodopsin-1 (ChR1 ), CsChR, CoChR, SdChR, ChR2(H134R), C1 V1 (t / t), ChlEF; ChETA, VChR1 , Chrimson, Chronos, PsChR2, CoChR, CsChR, CheRiff, etc.

[0093] Methods of analysis at the single cell level are also of interest, e.g. as described above: live imaging (including confocal or light-sheet microscopy), single cell gene expression or single cell RNA sequencing, calcium imaging, immunocytochemistry, patch-clamping, flow cytometry and the like. Various parameters can be measured to determine the effect of a drug or treatment on the functional assembled spheroids or cells derived therefrom.

[0094] Imaging of neurons can also be used to assess the effect of candidate agents on aspects of neuronal morphology. Aspects of neuronal morphology that are of interest in the present disclosure include, without limitation, soma diameter, dendrite number, dendrite length, dendrite density, dendritic spine number, dendritic spine length, dendritic spine density, axon length, etc. Imaging of neurons may be used to measure the aspects of neuronal morphology. The neuronal morphology may be measured in a variety of ways. For example, the measuring includes, without limitation, histologically staining the first human neural tissue, antibody staining the first human neural tissue, expressing a detectably labeled protein in the first human neural tissue, etc.

[0095] Histological stains that find use in the present disclosure include, without limitation, H&E staining, Nissl staining, Luxol-fast blue staining, Kluver-Barrera staining, Bodian silver staining, Holzer staining, Gallyas-Braak staining, thionine staining, Weil-Myelin staining, Solochrome staining, Peris staining, Fluoro-Jade staining, Congo Red staining, thioflavin S staining, amino cupric silver staining, Neutral Red Counter staining, cupric silver staining, Campbell-Switzer Alzheimer staining, autometallography staining, etc. Antibody stains that find use in the present disclosure include, without limitation, 4G8, 6E10, Ab1 -40, Ab1 -42, alpha synuclein, Asyn-pSer129, AT8, Brdll + hematoxylin, calbindin, caspase-3, caspase-9, cathepsin-D, CD68, c-fos, ChAT + Nissl, doublecortin, endoglin, ferritin, GAD-67, GFAP, GFP, HuIgG, Iba1 , Ki-67, LAMP1 , luciferase, MAP-2, MBP, mDectin, NeuN, Nestin, Oligo2, Orexin A, parvalbumin, p-c-jun, P.U.1 , RGMa, S830, SMI-71 , SMI-99, somatostatin, STEM-101 , TDP- 43, TH, TMEM1 19, TPH, etc.

[0096] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based and small molecule-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also foundamong biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0097] Included are pharmacologically active drugs such as opioid and non-opioid pain related drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable for this invention are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, New York, (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Cardiovascular Drugs; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference.

[0098] Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.

[0099] The term samples also includes the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 to 1 ml of a biological sample is sufficient.

[0100] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including 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. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0101] As used herein, the term “genetic agent” refers to polynucleotides and analogs thereof, which agents are tested in the screening assays of the invention by addition of the genetic agent to a cell. The introduction of the genetic agent results in an alteration of the total genetic composition of the cell. Genetic agents such as DNA can result in an experimentally introduced change in the genome of a cell, generally through the integration of the sequence into a chromosome, for example using CRISPR mediated genomic engineering (see for example Shmakov et al. (2017) Nature Reviews Microbiology 15:169). Genetic changes can also be transient, where the exogenous sequence is not integrated but is maintained as an episomal agents. Genetic agents, such as antisense oligonucleotides, can also affect the expression of proteins without changing the cell’s genotype, by interfering with the transcription or translation of mRNA. The effect of a genetic agent is to increase or decrease expression of one or more gene products in the cell. The one or more gene products may be related

[0102] Introduction of an expression vector encoding a polypeptide can be used to express the encoded product in cells lacking the sequence, or to over-express the product. Various promoters can be used that are constitutive or subject to external regulation, where in the latter situation, one can turn on or off the transcription of a gene. These coding sequences may include full-length cDNA or genomic clones, fragments derived therefrom, or chimeras that combine a naturally occurring sequence with functional or structural domains of other coding sequences. Alternatively, the introduced sequence may encode an anti-sense sequence; be an anti-sense oligonucleotide; RNAi, encode a dominant negative mutation, or dominant or constitutively active mutations of native sequences; altered regulatory sequences, etc.

[0103] Antisense and RNAi oligonucleotides can be chemically synthesized by methods known in the art. Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3’-O’-5’-S-phosphorothioate, 3'-S- 5’-O-phosphorothioate, 3’-CH2-5’-O-phosphonate and 3’-NH-5’-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity, e.g. morpholino oligonucleotide analogs.

[0104] Agents are screened for biological activity by adding the agent to at least one and usually a plurality of cells, in one or in a plurality of environmental conditions, e.g. followingstimulation with an agonist, following electric or mechanical stimulation, etc. The change in parameter readout in response to the agent is measured, desirably normalized, and the resulting screening results may then be evaluated by comparison to reference screening results, e.g. with cells having other mutations of interest, normal astrocytes, astrocytes derived from other family members, and the like. The reference screening results may include readouts in the presence and absence of different environmental changes, screening results obtained with other agents, which may or may not include known drugs, etc.

[0105] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0106] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0107] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0108] Various methods can be utilized for quantifying the presence of selected parameters, in addition to the functional parameters described above. For measuring the amount of a molecule that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the parameter 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 to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteinscan be engineered to fluoresce, e.g. by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12) :477-81 ). Thus, antibodies can be genetically modified to provide a fluorescent dye as part of their structure.

[0109] Depending upon the label chosen, parameters may be measured using other than fluorescent labels, using such immunoassay techniques as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (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 degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0110] Both single cell multiparameter and multicell multiparameter multiplex assays, where input cell types are identified and parameters are read by quantitative imaging and fluorescence and confocal microscopy are used in the art, see 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 Nov. 23, 1999.[001 11 ] Neuronal activity parameters. Of interest for the functional assembled spheroids screening system are parameters related to the electrical properties of the neurons and therefore directly informative about function and activity. Methods to measure activity may sense the occurrence of action potentials (spikes), and contractions, or twitches. The characteristics of the occurrence of a single spike or multiple spikes either in timely clustered groups (bursts) or distributed over longer time (spike train) of a single neuron or a group of neurons indicate neuronal activation patterns and thus reflect functional neuronal properties, which can be described my multiple parameters. Such parameters can be used to quantify and describe changes in neuronal activity in the systems of the invention.[001 12] Neuronal activity parameters include, without limitation, total number of spikes (per recording period); mean firing rate (of spikes); inter-spike interval (distance between sequential spikes); total number of bursts (per recording period); burst frequency; number of spikes per burst; burst duration (in milliseconds); inter-burst interval (distance between sequential bursts); burst percentage (the portion of spikes occurring within a burst); total number of network bursts (spontaneous synchronized network activity); network burst frequency; number of spikes per network burst; network burst duration; inter-network-burst interval; inter-spike interval within network bursts; network burst percentage (the portion of bursts occurring within a network burst).[001 13] Quantitative readouts of neuronal activity parameters may include baseline measurements in the absence of agents or a pre-defined genetic control condition and test measurements in the presence of a single or multiple agents or a genetic test condition. Furthermore, quantitative readouts of neuronal activity parameters may include long-term recordings and may therefore be used as a function of time (change of parameter value). Readouts may be acquired either spontaneously or in response to or presence of stimulation or perturbation of the complete neuronal network or selected components of the network. The quantitative readouts of neuronal activity parameters may further include a single determined value, the mean or median values of parallel, subsequent or replicate measurements, the variance of the measurements, various normalizations, the cross-correlation between parallel measurements, etc. and every statistic used to a calculate a meaningful and informative factor.

[0114] Comprehensive measurements of neuronal activity using electrical or optical recordings of the parameters described herein may include spontaneous activity and activity in response to targeted electrical or optical stimulation, including, for example, ChR2 delivered through lentiviruses, AAVs or pseudo rabies viruses, Neurotransmitter uncaging such glutamate uncaging, GABA uncaging, nicotine uncaging, etc.) of all neuronal cells or a subpopulation of neuronal cells within the integrated spheroids. Furthermore, spontaneous or induced neuronal activity can be measured in the self-assembled functional environment and circuitry of the neural culture or under conditions of selective perturbation or excitation of specific subpopulations of neuronal cells as discussed above.[001 15] In the provided assays, comprehensive measurements of neuronal activity can be conducted at different time points along neuronal maturation and usually include a baseline measurement directly before contacting the neural culture with the agents of interest and a subsequent measurement under agent exposure. Moreover, long-term effects of agents on neural maturation and development can be assessed by contacting the immature neural culture at an early time point with agents of interest and acquiring measurements of the samecultures after further maturation at a later time point compared to control cultures without prior agent exposure.[001 16] In some embodiments, standard recordings of neuronal activity of mature neural cultures are conducted after about 2 weeks, after about 3 weeks, after about 4 weeks, after about 6 weeks, after about 8 weeks following fusion (i.e. after mixing the different subdomain components of the culture). Recordings of neuronal activity may encompass the measurement of additive, synergistic or opposing effects of agents that are successively applied to the cultures, therefore the duration recording periods can be adjusted according to the specific requirements of the assay. In some embodiments the measurement of neuronal activity is performed for a predetermined concentration of an agent of interest, whereas in other embodiments measurements of neuronal activity can be applied for a range of concentrations of an agent of interest.[001 17] In some embodiments the provided assays are used to assess maturation of the neural culture or single components including sensory neurons, dorsal spinal neurons, etc. Maturation of neuronal cells can be measured based on morphology, by optically assessing parameters such as neuromuscular junctions, dendritic arborization, axon elongation, total area of neuronal cell bodies, number of primary processes per neuron, total length of processes per neuron, number of branching points per primary process as well as density and size of synaptic puncta stained by synaptic markers such as synapsin-1 , synaptophysin, bassoon, PSD95, anti-BTX antibodies (for neuromuscular junctions) and Homer.[001 18] The results of an assay can be entered into a data processor to provide a dataset. Algorithms are used for the comparison and analysis of data obtained under different conditions. The effect of factors and agents is read out by determining changes in multiple parameters. The data will include the results from assay combinations with the agent(s), and may also include one or more of the control state, the simulated state, and the results from other assay combinations using other agents or performed under other conditions. For rapid and easy comparisons, the results may be presented visually in a graph, and can include numbers, graphs, color representations, etc.[001 19] The dataset is prepared from values obtained by measuring parameters in the presence and absence of different cells, e.g. genetically modified cells, cells cultured in the presence of specific factors or agents that affect neuronal function, as well as comparing the presence of the agent of interest and at least one other state, usually the control state, which may include the state without agent or with a different agent. The parameters include functional states such as synapse formation and calcium ions in response to stimulation, whose levels vary in the presence of the factors. Desirably, the results are normalized against a standard, usually a "control value or state," to provide a normalized data set. Values obtainedfrom test conditions can be normalized by subtracting the unstimulated control values from the test values, and dividing the corrected test value by the corrected stimulated control value. Other methods of normalization can also be used; and the logarithm or other derivative of measured values or ratio of test to stimulated or other control values may be used. Data is normalized to control data on the same cell type under control conditions, but a dataset may comprise normalized data from one, two or multiple cell types and assay conditions.

[0120] The dataset can comprise values of the levels of sets of parameters obtained under different assay combinations. Compilations are developed that provide the values for a sufficient number of alternative assay combinations to allow comparison of values.

[0121] A database can be compiled from sets of experiments, for example, a database can contain data obtained from a panel of assay combinations, with multiple different environmental changes, where each change can be a series of related compounds, or compounds representing different classes of molecules.

[0122] Mathematical systems can be used to compare datasets, and to provide quantitative measures of similarities and differences between them. For example, the datasets can be analyzed by pattern recognition algorithms or clustering methods (e.g. hierarchical or k-means clustering, etc.) that use statistical analysis (correlation coefficients, etc.) to quantify relatedness. These methods can be modified (by weighting, employing classification strategies, etc.) to optimize the ability of a dataset to discriminate different functional effects. For example, individual parameters can be given more or less weight when analyzing the dataset, in order to enhance the discriminatory ability of the analysis. The effect of altering the weights assigned each parameter is assessed, and an iterative process is used to optimize pathway or cellular function discrimination.

[0123] For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, embryology, and neurobiology. With respect to tissue culture and embryonic stem cells, the reader may wish to refer to Teratocarcinomas and embryonic stem cells: A practical approach (E. J. 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); 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).

[0124] General methods in molecular and cellular biochemistry can be found in such standard textbooks as 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); NonviralVectors 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 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0125] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0126] The following examples are put forth so as 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 experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.EXPERIMENTALExample 1Introduction

[0127] Human sensory ascending pathway is responsible for transmitting sensory information from the peripheral nervous system to the brain, including pain and itch information. Dysfunctions in this pathway have been associated with human disorders, such as peripheral neuropathy or autism spectrum disorder. However, the mechanisms underlying these conditions are not fully understood, particularly due to the lack of reliable models to functionally probe and manipulate the human sensory ascending pathway in tractable experimental models. Therefore, there is a critical need to develop human multi-cellular models that model the circuit and integrate the components of the human sensory ascending pathway in vitro to model disorders of sensory processing and to develop effective therapies.Results

[0128] The first four-part model of the human ascending somatosensory using assembloids was developed. The method involves differentiating human induced pluripotent stem (hiPS) cells into regionalized neural organoids resembling the components of the human sensory ascending pathway: human sensory organoids (hSeO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO), human diencephalon organoids (hDiO) and human corticalorganoids (hCO). The hSeO and hdSpO developed here recapitulate transcriptomic and functional characteristics of dorsal root ganglia and dorsal spinal cord.

[0129] To determine if the sensory neurons in hSeO were functionally analogous to in vivo neurons, the genetically encoded calcium indicator GCaMP6s was transfected into hSeo and found that hSeO had significantly increased calcium signal in response to known noxious stimulants ap-MeATP and capsaicin.

[0130] Before connecting all four components, two-part assembloids were implemented to ensure proper connectivity between each component in vitro. In addition to the thalamocortical projection in thalamo-cortical assembloids, sensory-dorsal spinal projection in sensory-dorsal spinal assembloids and dorsal spinal-thalamic projection in dorsal spinal- thalamic assembloids were validated by time-lapse axon projection imaging (FIG. 5). It was found that axon projections were increased over time in both assembloids. Additionally, using trans-synaptic retrograde tracer technique, the cell-type specificity on the connectivity was exhibited in both assembloids.

[0131] Next, the 4 organoids were combined via physical proximity to generate a human sensory ascending pathway assembloid that was assembled by 7 days after fusion. This was achieved by linear assembly. Each organoid was labeled with a distinct neuron-specific promoter-driven reporter prior to fusion to be able to visually confirm regional identity.

[0132] To explore functional synaptic connections between the sensory assembloid subregions, an extracellular recording system was developed to record from all four parts simultaneously. Spontaneous and synchronous activity was found across all regions of the assembloid, implying functional connections between the individual organoids. This activity was analyzed by calculating a synchrony index. This index calculated the probability of aligned activity at spinal, thalamic, and cortical subregions compared to the sensory neuron subregion activity. When compared with a random timepoint, all other assembloid subregions were significantly aligned to the hSeO. In addition, it was found that neurons in assembloids exhibited synchronous activity across all four regions. Peak correlation for all the combinations of the four regions were significantly higher in assembloids than in individual, non-assembled neural organoids. Thus, experiments using two orthogonal probing methods suggest we have developed a successful protocol to model the human ascending sensory pathway in vitro.

[0133] To demonstrate the emergent property in the human sensory ascending pathway assembloids that are not found in the single regionalized neural organoids, neuronal responses to the pain-inducing chemical ct|3-MeATP was investigated, in all four regions by large field-of-view confocal live time-lapse imaging. In single organoids, only neurons in hSeO showed calcium raise following application of ap-MeATP. Interestingly, in the human sensory ascending pathway assembloids, some cortical, thalamic, and dorsal spinal neurons alsoshowed calcium raise by ap-MeATP. This experiment suggests that the sensory information has been transmitted from sensory neurons to dorsal spinal, thalamic, and cortical neurons.

[0134] To further verify whether specific stimulation of sensory neurons in hSeO elicited responses in other regions of the assembloid, 405 nm photo-stimulation was used in the presence of MNI-caged glutamate to trigger rapid and localized release of glutamate in a specific area. Photo-stimulation of hSeO was performed within hASA and detected a robust increase in calcium levels, not only in hSeO but also in all other regions. These experiments suggest transmission of somatosensory signals within the assembloid from sensory neurons through spinal and thalamic neurons to cortical neurons.

[0135] In addition to pain-inducing chemical ap-MeATP, the hSeO and sensory ascending pathway assembloids were tested to determine if they respond to other sensory modalities, such as mechanical pressure. Initially, the sensory neurons in hSeO were tested to determine if calcium activity was induced by mechanical stimulation, and how calcium activities in hSeO could be influenced by varying intensities of mechanical stimulation through the increment of flow speed of a solution. It was found that not only the amplitudes of calcium activities in the single neurons were increased, but also the proportion of active neurons were increased by higher mechanical stimulations. Next, the response to mechanical stimulation in hSeO was investigated to determine if it can be transmitted to upper regions of the assembloids. It was found that neurons in all four regions showed calcium activity by mechanicals simulation, which indicates information transmission through this sensory ascending pathway in vitro. It was confirmed that these responses were mediated by the mechanosensitive ion channel PIEZO2-dependent using a specific blocker, GsMTx4. These experiments suggest that the sensory ascending pathway assembloids can model both chemical and also mechanical sensory stimuli and responses.

[0136] Finally, it was demonstrated that the sensory ascending pathway assembloids can be used to study mechanisms and therapeutic approaches for neurologic and psychiatric disorders. Pathogenic variants in the gene SCN9A, encoding the voltage-gated sodium channel Nav1.7, can cause pain insensitivity if the variant leads to loss of function or hyperalgesia if there the mutation is a gain of channel mutation. It was found that SCN9A was highly expressed in hSeO. When we disrupted SCN9A via CRISPR-mediated gene editing in hiPS cells and then derived hSeO, we confirmed reduced mRNA and protein expression. hSeO lacking SCN9A demonstrated reduced spontaneous calcium activity as detected by a genetically encoded GCaMP calcium indicator. These data are consistent with diminished excitability of sensory neurons in the absence of this key sodium channel. In addition, we generated the sensory ascending pathway assembloids from control and SCN9A knock-out (KO) hiPS cells, to demonstrate whether the synchronized activity in assembloid has been affected by SCN9A KO. We found that the synchronized activity has been disrupted in SCN9AKO assembloids, and the correlation coefficient of calcium activity across all four regions were significantly reduced by SCN9A KO. This experiment suggests that our sensory ascending pathway assembloids successfully recapitulate one of the known phenotypes, hypoexcitability of sensory neurons following loss of SCN9A. In addition, the sensory ascending pathway assembloids revealed, for the first time, disruptions in the emergent synchrony features of the ascending somatosensory.

[0137] Taken together, anatomical and functional connectivity was demonstrated in sensory ascending pathway assembloids using timelapse axon projection imaging, trans-synaptic tracing, and live-cell calcium imaging. This new platform has the potential to uncover the fundamental principles of human sensory ascending pathway development, to revolutionize disease modeling and discover therapeutics.Methods

[0138] Generation of regionalized neural organoids, including human sensory organoids and human dorsal hindbrain / cervical spinal cord organoid. Human induced pluripotent stem (hiPS) cells were cultured on vitronectin-coated plates (5 ig ml-1 , Thermo Fisher Scientific, A14700) in Essential 8 medium (Thermo Fisher Scientific, A1517001 ). Cells were passaged every 4 or 5 days with UltraPure™ 0.5 mM EDTA, pH 8.0 (Thermo Fisher Scientific, 15575020). For the generation of regionalized neural organoids, hiPS cells were incubated with Accutase® (Innovative Cell Technologies, AT-104) at 37°C for 7-10 min and dissociated into single cells. Optionally, 1-2 day before organoids formation, hiPS cells can be exposed to 1 % dimethylsulfoxide (DMSO) (MilliporeSigma, D2650) in Essential 8 medium. To obtain uniformly sized organoids, AggreWell-800 (STEMCELL Technologies, 34815) containing 300 microwells was used. Approximately 2 or 3 x 106 single cells were added per AggreWell-800 well in Essential 8 medium supplemented with the ROCK inhibitor Y27632 (10 pM, Selleckchem, S1049), centrifuged at 100g for 3 min to capture the cells in the microwells, and incubated at 37°C with 5% CO2. After 24 hours, organoids consisting of approximately 6,666 or 10,000 cells were collected from each microwell by pipetting medium in the well up and down with a cut P1000 pipet tip and transferred into ultra-low attachment plastic dishes (Corning, 3262). in Essential 6 medium (Thermo Fisher Scientific, A1516401 ). supplemented with two SMAD pathway inhibitors - dorsomorphin (2.5 pM, Sigma-Aldrich, P5499) and SB- 431542 (10 pM, R&D Systems, 1614).

[0139] To generate hSeO, from day 1 to day 6 in suspension, organoids were maintained in Essential 6 medium (Thermo Fisher Scientific, A1516401 ), supplemented with SB-431542 (10 pM, R&D Systems, 1614). From day 1 to day 4, BMP4 (5 ng / ml, PeproTech, 120-05ET) was added. From day 3 to day 5, CHIR (600 nM, Selleckchem, S1263) was added. On day 7 in suspension, organoids were transferred to neural medium containing Neurobasal™-A Medium(Thermo Fisher Scientific, 10888022), B-27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1 :100, Thermo Fisher Scientific, 35050079), Penicillin-Streptomycin (1 :100, Thermo Fisher Scientific, 15070063), and supplemented with SB-431542 (10 pM, R&D Systems, 1614). From day 9, BDNF (20 ng / ml, PeproTech, 450-02), GDNF (25 ng / ml PeproTech, 450-10), and NGF (25 ng / ml, PeproTech, 450-01 ) were supplemented until the end of the experiment. From day 9 to day 14, medium was supplemented with SB-431542 (10 JJ.M, R&D Systems, 1614). From day 15 to day 20, DAPT (2.5 pM, STEMCELL Technologies, 72082) was included.

[0140] To generate hdSpO, from day 1 to day 6, organoids were maintained Essential 6 medium (Thermo Fisher Scientific, A1516401 ) supplemented with SB-431542 (10 pM, R&D Systems, 1614) and dorsomorphin (2.5 pM, Sigma-Aldrich, P5499). From day 5 to day 6, CHIP (3 y.M, Selleckchem, S1263) was added. On day 7, organoids were transferred to a neural medium containing Neurobasal™-A Medium (Thermo Fisher Scientific, 10888022), B- 27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1 :100, Thermo Fisher Scientific, 35050079), Penicillin-Streptomycin (1 :100, Thermo Fisher Scientific, 15070063), and supplemented with EGF (20 ng / ml, R&D Systems, 236-EG), Retinoic acid (0.1 .M, Sigma-Aldrich, R2625), and CHIR (3 y.M, Selleckchem, S1263). On day 21 , the media was supplemented with BDNF (20 ng / ml, PeproTech, 450-02), N6, 2’-O-Dibutyryladenosine 3', 5’ -cyclic monophosphate sodium salt (cAMP; 50 jiM, Millipore Sigma, D0627), L-Ascorbic Acid 2-phosphate Trisodium Salt (AA; 200 |uM, Wako, 323-44822), IGF-1 (10 ng / ml, PeproTech, 100-11 ) until the end of experiments. From day 21 to day 26, DAPT (2.5 pM, STEMCELL Technologies, 72082) was added.

[0141] Generation of Assembloids. For two-part assembloids, the organoids of interest were fused by placing them in proximity in 1 .5 ml Eppendorf tubes for 2-3 days in an incubator. For sensory ascending pathway assembloids, four organoids were integrated by placing them in proximity in tilted 6-well ultra-low attachment plates for 1 week in an incubator. After assembly, the assembloids were maintained in ultra-low attachment plates in media containing growth factors and small molecules described above.

[0142] Live Calcium Imaging. To achieve single-cell resolution live calcium imaging in intact 3D organoids or assembloids without significant sample movement during chemical injection, we acutely attached samples to Ethyleneimine polymer (PEI)-coated coverslips before imaging. Autoclaved 12 mm coverslips were coated with 0.01875% PEI (Sigma, #03880) in water for at least 1 hour at 37°C and then washed three times with water before use. An organoid or assembloid was carefully positioned on top of a PEI-coated coverslip, and an L- shaped tubing-type Chamlide CMB chamber (Live Cell Instrument, CM-B12-1 PB) was assembled. This chamber was connected to a peristaltic pump (Harvard Apparatus, #702027)to establish continuous suction through the outlet hole. To prevent sample drying, a vehicle solution was applied immediately after assembly. Calcium activity, with or without various stimuli, was imaged using a confocal microscope.

[0143] Viral Labeling and Rabies Tracing. Two or three organoids were placed in a 1.5 ml Eppendorf tube containing 200 pl media with the desired virus(es) and incubated overnight. Fresh medium was added the following day, and organoids were transferred to ultra-low attachment plates the next day. For retrograde tracing, organoids representing the presynaptic part were labeled with AAV-DIO-tdTomato and organoids representing the postsynaptic part were separately labeled with both rabies-AG-Cre-GFP and AAV-EF1 a-CVS-G. Two days after viral infection, organoids were assembled. After 3 weeks of integration, assembloids were fixed with 4% paraformaldehyde and processed for immunocytochemistry.

[0144] Single Cell RNA sequencing. Organoids were dissociated to obtain single cell suspension using papain enzyme solution. Organoids were gently triturated to achieve a single cell suspension in a protease inhibitor solution. Cells were resuspended in 0.04% BSA / PBS. cDNA libraries were generated with the Chromium Single Cell 3’ Reagent Kits v3.1. Each library was sequenced using the Illumina NovaSeq. Downstream analyses were performed using the R package Seurat and Cell Ranger software.

[0145] Extracellular Recording. Organoids or assembloids were embedded into 3% low- melting gel agarose and transferred into artificial cerebrospinal fluid (aCSF) containing 124 mM NaCI, 3 mM KCI, 1 .25 mM NaH2PO4, 1 .2 mM MgSO4, 1 .5 mM CaCI2, 26 mM NaHCO3 and 10 mM D-(+)-glucose with addition of GlutaMAX (Gibco). Embedded organoids or assembloids were placed on a perfusion recording chamber and continuously perfused with aCSF (bubbled with 95% 02 and 5% CO2) at 37°C. To verify the regional identity in assembloids, tdTomato was expression in one of the regions under human SYN1 promoter. Acute 32 channel P-1 probe with 2 shanks (Cambridge NeuroTech) was used for recording.

[0146] CRISPR Engineering. gRNAs were designed to target the exons of the target genes. For each gene, the hiPS cells were nucleofected with SpCas9 and gRNAs. Approximately 2- 3 days after nucleofection, hiPS cells were sorted into the wells of 96-well plates, expanded and frozen. In parallel, cell pellets were also collected for sequencing. After validating their mutation profiles, the knockout hiPS cells clones were recovered from the 96-well plates.Example 2Introduction

[0147] The ascending somatosensory pathways are responsible for conveying environmental and bodily sensory information from peripheral organs to the central nervous system (CNS). Somatosensory neurons, whose cell bodies reside in dorsal root ganglia (DRG) and trigeminal ganglia (TG), are pseudo-unipolar cells that innervate peripheral organs including the skin todetect somatosensory signals and transmit them to the spinal cord and hindbrain. Neurons in the dorsal spinal cord or hindbrain monosynaptically transmit this information to various brain structures, including the thalamus, which further relays these signals to the cerebral cortex. Genetic or environmental disruptions to these pathways can lead or contribute to various neurological disorders, including chronic pain and autism spectrum disorder, and impact both heightened and diminished sensory functions. Despite the clinical significance, how ascending sensory pathways form during human development and how functional defects arise in pathological conditions during development are still poorly understood. Challenges in bench to bedside translation of pain and sensory research are thought to be related, at least in part, to differences across species. Crucially, the discovery of novel treatments and their translation is hindered by the lack of reliable human experimental models reconstituting the ascending sensory pathway. In fact, concomitantly probing all components of these circuits via imaging or recording has not yet been achieved in animal models, which limits our understanding of the consequences of various sensory manipulations at the circuit level. Therefore, there is a critical need to develop human multi-cellular models that model the circuit and integrate the components of the human sensory ascending pathway in vitro to model disorders of sensory processing and to develop effective therapies.Results

[0148] The first four-part model of the human ascending somatosensory was developed using assembloids (FIG. 1). The method involves differentiating human induced pluripotent stem (hiPS) cells into regionalized neural organoids resembling the components of the human sensory ascending pathway: human sensory organoids (hSeO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO), human diencephalon organoids (hDiO) and human cortical organoids (hCO). To characterize the cellular identity within hSeO, the pattern of expression of several canonical markers was inspected using scRNA-seq data. It was found that approximately 30-40% of neurons (STMN2+) expressed the peripheral neuron-specific markers POU4F1 and SIX1, which was independently confirmed in other batches of organoids by RT-qPCR. Co-localization of VGLUT2 and NeuN in immunostaining experiments demonstrated the excitatory neuronal identity of hSeO neurons. It was also found that a substantial proportion of cells expressing neural crest cell markers, such as SOX10 and FOXD3, which is consistent with a neural crest origin of peripheral sensory neurons (FIG 2).

[0149] Next, the cellular identity of neurons was investigated in hdSpO using scRNA-seq data. Similar to ventral part of hindbrain / cervical spinal cord organoids, hdSpO displayed high neuronal diversity. Relevant to the ascending somatosensory pathway, clusters expressing the projection neuronal markers PHOX2A, LBX1, and TACP1 (encoding NK1 R) were found. It was also confirmed that the expression of TACR1 and LBX1 by RT- qPCR in hdSpO. Cellularclusters in hdSpO were annotated using combinatorial markers expression, as previously described. Overlap of PHOX2A+ neurons with dl5 cluster was found, which is consistent with previous reports. Label transfer analysis of scRNA-seq data from the developing human spinal cord dl5 cells was performed onto the hdSpO scRNA-seq dataset. It was found that the SLC17A6+ / PHOX2A+ / TACR1+ neuron cluster demonstrated the highest prediction score values for the developing human dl5 cells, indicating that the hdSpO contains a neuronal cluster resembling the transcriptomic identity of human dl5 cells. The cells in dl5 also express the dl5 marker gene LMX1B but not the dl4 / 6 marker gene PAX2. Immunostaining for PAX2 and PHOX2A indicated that the distinct types of spinal neurons in hdSpO were intermingled without a clear spatial organization (FIG 3).

[0150] A unique feature of sensory neurons is their ability to respond to environmental stimuli. To verify whether neurons in hSeO respond to sensory stimuli, a live imaging system of intact 3D organoids expressing genetically-encoded calcium indicator was established using a chamber equipped to inject chemicals. Consistent with high abundance of RNA encoding calcium-permeable P2X purinoceptor 3, P2RX3, and capsaicin receptor TRPV1, bath application of their agonists, a,p-methyleneATP (ap-MeATP) and capsaicin respectively, induced calcium transients. Interestingly, the dynamics of the responses were similar between hSeO and intact ex vivo primary mouse DRG; ap-MeATP induced a fast calcium spike while capsaicin induced a slow and prolonged calcium transient, which is consistent with previous pharmacological studies using DRG neurons isolated from rat or mouse, as well as induced nociceptors in 2D culture (FIG. 4).

[0151] There are species-specific differences in the expression and function of some receptors on sensory DRG neurons, which may be responsible for sensory processing differences and challenges in translation from animal models to humans. For instance, the 2’,3’-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) is a potent antagonist of the rodent P2X3 receptor; however, a primate-specific amino acid substitution in the P2X3 receptor is thought to modify sensitivity to TNP-ATP in the macaque DRG. Consistent with previous reports, it was found that TNP-ATP successfully blocked calcium responses in ex vivo mouse DRG in a dose-dependent manner. When applied to human sensory neurons in hSeO, however, TNP- ATP had no significant effect, which is suggestive of differences in responsiveness across species. These results are consistent with the previous report showing the importance of primate-specific amino acid changes at two residues of the P2X3 receptor- A197 and T202, which influence TNP-ATP antagonism (FIG. 5).

[0152] The specificity of projections was examined between pairs of regionalized organoids. First, it was investigated whether the sensory neurons in hSeO could send projections to spinal neurons in hSeO-hdSpO assembloids. hSeO were virally labeled with hSYN1 ::EYFP and integrated them with hdSpO to form hSeO-hdSpO assembloids. It was found that the area ofEYFP fluorescence on the hdSpO side, corresponding to projections from hSeO neurons, increased progressively over 28 days after fusion (daf). These EYFP+ sensory projections colocalized with the axonal marker NF-H. Rabies virus-mediated retrograde tracing combined with Cre recombination was used in hSeO-hdSpO assembloids. At daf 21 , tdTomato+ / GFP+ cells were observed on the hSeO side, indicating successful retrograde tracing of rabies-Cre- GFP resulting in the recombination and expression of DIO-tdTomato. These tdTomato+ / GFP+ cells were not detected in unassembled organoids which were kept in the same cell culture well and media, confirming projection-dependent retrograde transmission. To investigate the specificity of these projections, the sensory neuronal marker BRN3A was co-immunostained and found that nearly 80% of tdTomato-i- cells were also BRN3A+. To further verify functional connectivity between hSeO and hdSpO, optogenetic stimulation was combined with wholecell electrophysiological recordings. Upon 460 nm light stimulation of channelrhodopsin 2 (ChR2)-expressing hSeO, optically evoked excitatory postsynaptic currents (oEPSCs) were observed, with a success rate of 8 out of 30 recorded cells. Responsive neurons exhibited an average oEPSC amplitude of approximately 52 pA and an onset delay of 32 ms. These results demonstrate that hSeO sensory neurons can form functional connections with neurons in hdSpO (FIG. 6).

[0153] A similar approach to investigate spinothalamic projections in hdSpO-hDiO assembloids was taken. A significant increase of spinothalamic projections was observed at daf 28. Using rabies virus-mediated retrograde tracing, it was found that approximately 40% of tdTomato+ cells co-expressed the spinal cord projection neuronal marker NK1 R+. Colocalization of tdTomato and PHOX2A was also observed. In addition, it was examined whether hdSpO neurons preferentially project to hDiO compared to hSeO by utilizing three- part hSeO-hdSpO-hDiO assembloids. Quantification of EYFP+ spinal projections within the same assembloid, in both the hDiO and hSeO regions, revealed more projections in hDiO, indicating a preferential for hdSpO targeting the hDiO. These studies, in combination with our previous experiments on thalamocortical projections in hCO-hDiO assembloids, confirmed that the three pairs of assembloids (hCO-hDiO, hDiO-hdSpO, hdSpO-hSeO) built from the four regionalized neural organoids intrinsically recapitulate some of the basic inter-regional projections (FIG. 7).

[0154] The functionality of these connections was investigated in three-part hSeO-hdSpO- hDiO assembloids by combining rabies virus-mediated retrograde tracing, recombinase Flpo- dependent expression of GCaMP6f, and live calcium imaging following a|3-MeATP treatment. AAV-EF1 a::fDIO-GCaMP6f-infected hdSpO were assembled with hSeO to generate hSeO- hdSpO. At daf 43, hDiO were infected with rabies-FIpo-dsRedExpress and AAV-G and fused these onto the hdSpO side of the hSeO-hdSpO assembloid. At daf 70, expression of f DIO- GCaMP6f was detected, which is suggestive of transmission of rabies-FIpo- dsRedExpressfrom hDiO into hdSpO. These GCaMP6f+ neurons demonstrated increased calcium activity after ap-MeATP exposure, thereby indicating signal transmission from sensory neurons to spinal neurons. These calcium transients were blocked by the glutamate receptor antagonists NBQX and APV. These results imply that chemical stimulation of sensory neurons in hSeO elicited calcium activity that was transmitted to, and induced calcium increase in connected rabies-FIpo-labeled spinothalamic projection neurons in hdSpO. Moreover, hdSpO neurons projected axons into hDiO, as evidenced by rabies tracing (FIG. 8).

[0155] The 4 organoids were combined via physical proximity to generate a human sensory ascending pathway assembloid that was assembled by 7 days after fusion. This was achieved by linear assembly. Each organoid was labeled with a distinct neuron-specific promoter-driven reporter prior to fusion to be able to visually confirm regional identity (FIG. 9).

[0156] Functional connectivity within hASA was examined. In one continuous imaging session, hASA were serially treated with either vehicle or a[3-MeATP while recording jGCaMP8s signal at single cell resolution. The adjusted AF / Fbase was calculated by subtracting the response to vehicle from the response to ap-MeATP. It was found that ap- MeATP exposure significantly elevated calcium levels in hASA neurons in all 4 regions at daf 74-78. In contrast, in non-assembled organoids, only neurons in hSeO had a significant calcium rise after application of ap-MeATP, while neurons in other organoids did not (FIG. 10).

[0157] To further verify whether specific stimulation of sensory neurons in hSeO elicited responses in other regions of the assembloid, 405 nm photo-stimulation was used in the presence of MNI-caged glutamate to trigger rapid and localized release of glutamate in a specific area. Photo-stimulation of hSeO was performed within hASA and detected a robust increase in calcium levels, not only in hSeO but also in all other regions, at daf 74-99. These experiments suggest transmission of somatosensory signals within the assembloid from sensory neurons through spinal and thalamic neurons to cortical neurons (FIG. 11).

[0158] It was next explored whether the four-part assembly is associated with emergent network properties. Baseline calcium activity was recorded at single cell resolution and compared hASA with four non-assembled organoids placed in close proximity just before imaging. Remarkably, it was found that neurons in assembloids exhibited synchronous activity across all four region. Scaled correlation analysis (SCA) was applied to minimize effects of low frequency noise signals. Representative SCA correlograms from all possible pairs between hSeO and hCO neurons in an assembloid showed that the correlation peaks were distributed around 0 sec, while representative SCA correlograms from unfused organoids had equal distribution of correlation peaks. Peak correlation for all the combinations of the four regions were significantly higher in assembloids than in individual, non-assembled neural organoids. Activity patterns were further investigated through concomitant extracellular recordings with 32 electrodes in each of the four hASA parts. Spontaneous and synchronousactivity was found across the entire assembloid. Co-activation indexes were calculated based on cross-correlograms. When the co-activation index aligned with activity of hSeO was compared, it was found that significantly higher values than alignment to random timepoints. Taken together, these results demonstrate that the assembly of intact 3D human regionalized neural organoids resembling the human DRG / TG, dorsal spinal cord / hindbrain, thalamus, and cerebral cortex enables the formation of connections capable of synchronized activity across the pathway and in response to somatosensory like (chemical sensory) stimuli (FIG. 12).

[0159] In addition to pain-inducing chemical ap-MeATP, the hSeO and sensory ascending pathway assembloids were tested to determine if they respond to other sensory modalities, such as mechanical pressure. Initially, it was explored whether the sensory neurons in hSeO show calcium activity by mechanical stimulation, and how calcium activities in hSeO could be influenced by varying intensities of mechanical stimulation through the increment of flow speed of a solution. It was found that not only the amplitudes of calcium activities in the single neurons were increased, but also the proportion of active neurons were increased by higher mechanical stimulations. Next, it was investigated if the response to mechanical stimulation in hSeO can be transmitted to upper regions of the assembloids. Neurons in all four regions were found to show calcium activity by mechanicals simulation, which is suggestive of information transmission through this sensory ascending pathway in vitro. It was confirmed that these responses were mediated by the mechanosensitive ion channel PIEZO2-dependent using a specific blocker, GsMTx4. These experiments suggest that our sensory ascending pathway assembloids can model both chemical and also mechanical sensory stimuli and responses (FIG. 13).

[0160] Lastly, whether hASA could be used to identify functional defects in models of neurologic and psychiatric disorders was studied. Pathogenic variants in the gene SCN9A, which encodes the voltage-gated sodium channel NaV1 .7, cause pain insensitivity if the variant leads to loss of channel function, or hyperalgesia if the variant causes gain-of-function. While defects in channel conductance and excitability of sensory neurons have been proposed as disease mechanisms in cellular and animal models, the circuit-level consequences have not yet been explored in a human model. Expression of SCN9A was verified and found high levels in hSeO. CRISPR-mediated gene editing was applied to induce one or more frame-shift variants in the SCN9A gene. It was confirmed that hSeO derived from pooled SCN9A KO iPS cell lines exhibited decreased SCN9A mRNA and NaV1.7 protein levels. The expression of the sensory neuron marker BRN3A was not affected by SCN9A KO. Consistent with previous reports of hypo-excitability of sensory neurons in the absence of this key sodium channel, it was found that SCN9A KO hSeO displayed reduced frequency of spontaneous calcium activity. hASA were generated from isogenic control and SCN9A KO hiPS cells in order to explore the circuit level consequences. Spontaneous activity patterns was recorded usingjGCaMP8s in all regions from control and SCN9A KO hASA. It was discovered that the synchronous pattern of activity across hASA was reduced in SCN9A KO, although nonsynchronized events were found in all regions. SCA analysis was performed and found a significant reduction in correlation, indicating that SCN9A KO impaired the emergent synchronization across the assembloid (FIG. 14).

[0161] In addition, the circuit-level consequences of the T1464I SCN9A gain-of-function variant were investigated, which is associated with paroxysmal extreme pain disorder. CRISPR / Cas9 gene editing was applied to introduce the heterozygous SCN9A T1464I into a hiPS cell line. It was confirmed mixed gDNA of control and T1464I SCN9A by Sanger sequencing. Calcium activity imaging of control and SCN9A T1464I hSeO demonstrated that the gain-of-function mutation produced hyperexcitability of sensory neurons. To investigate the effects of SCN9A T1464I variant on the emergent synchronized activity of the sensory ascending pathway, hASA were derived from control and SCN9A T1464I hiPS cell lines and imaged spontaneous calcium activity. Interestingly, SCA analysis revealed higher peak correlation values across the pairs in hASA, suggesting that SCN9A T1464I resulted in the hypersynchrony (FIG. 15).

[0162] Taken together, anatomical and functional connectivity was demonstrated in sensory ascending pathway assembloids using timelapse axon projection imaging, trans-synaptic tracing, and live-cell calcium imaging. This new platform has the potential to uncover the fundamental principles of human sensory ascending pathway development, to revolutionize disease modeling and discover therapeutics.Methods

[0163] Generation of regionalized neural organoids, including human sensory organoids and human dorsal hindbrain / cervical spinal cord organoid. Human induced pluripotent stem (hiPS) cells were cultured on vitronectin-coated plates (5 ig ml-1 , Thermo Fisher Scientific, A14700) in Essential 8 medium (Thermo Fisher Scientific, A1517001). Cells were passaged every 4 or 5 days with UltraPure™ 0.5 mM EDTA, pH 8.0 (Thermo Fisher Scientific, 15575020). For the generation of regionalized neural organoids, hiPS cells were incubated with Accutase® (Innovative Cell Technologies, AT-104) at 37°C for 7-10 min and dissociated into single cells. Optionally, 1-2 day before organoids formation, hiPS cells can be exposed to 1% dimethylsulfoxide (DMSO) (MilliporeSigma, D2650) in Essential 8 medium. To obtain uniformly sized organoids, AggreWell-800 (STEMCELL Technologies, 34815) containing 300 microwells was used. Approximately 2 or 3 x 106single cells were added per AggreWell-800 well in Essential 8 medium supplemented with the ROCK inhibitor Y27632 (10 pM, Selleckchem, S1049), centrifuged at 100g for 3 min to capture the cells in the microwells, and incubated at 37°C with 5% CO2. After 24 hours, organoids consisting of approximately6,666 or 10,000 cells were collected from each microwell by pipetting medium in the well up and down with a cut P1000 pipet tip and transferred into ultra-low attachment plastic dishes (Corning, 3262). in Essential 6 medium (Thermo Fisher Scientific, A1516401 ). supplemented with two SMAD pathway inhibitors - dorsomorphin (2.5 .M, Sigma-Aldrich, P5499) and SB- 431542 (10 p.M, R&D Systems, 1614).

[0164] To generate hSeO, from day 1 to day 6 in suspension, organoids were maintained in Essential 6 medium (Thermo Fisher Scientific, A1516401 ), supplemented with SB-431542 (10 p.M, R&D Systems, 1614). From day 1 to day 4, BMP4 (5 ng / ml, PeproTech, 120-05ET) was added. From day 3 to day 5, CHIR (600 nM, Selleckchem, S1263) was added. On day 7 in suspension, organoids were transferred to neural medium containing Neurobasal™-A Medium (Thermo Fisher Scientific, 10888022), B-27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1 :100, Thermo Fisher Scientific, 35050079), Penicillin-Streptomycin (1 :100, Thermo Fisher Scientific, 15070063), and supplemented with SB-431542 (10 pM, R&D Systems, 1614). From day 9, BDNF (20 ng / ml, PeproTech, 450-02), GDNF (25 ng / ml PeproTech, 450-10), and NGF (25 ng / ml, PeproTech, 450-01 ) were supplemented until the end of the experiment. From day 9 to day 14, medium was supplemented with SB-431542 (10 pM, R&D Systems, 1614). From day 15 to day 20, DAPT (2.5 pM, STEMCELL Technologies, 72082) was included.

[0165] To generate hdSpO, from day 1 to day 6, organoids were maintained Essential 6 medium (Thermo Fisher Scientific, A1516401 ) supplemented with SB-431542 (10 pM, R&D Systems, 1614) and dorsomorphin (2.5 pM, Sigma-Aldrich, P5499). From day 5 to day 6, CHIR (3 pM, Selleckchem, S1263) was added. On day 7, organoids were transferred to a neural medium containing NeurobasalTM-A Medium (Thermo Fisher Scientific, 10888022), B- 27™ Supplement, minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX™ Supplement (1 :100, Thermo Fisher Scientific, 35050079), Penicillin-Streptomycin (1 :100, Thermo Fisher Scientific, 15070063), and supplemented with EGF (20 ng / ml, R&D Systems, 236-EG), Retinoic acid (0.1 pM, Sigma-Aldrich, R2625), and CHIR (3 pM, Selleckchem, S1263). On day 21 , the media was supplemented with BDNF (20 ng / ml, PeproTech, 450-02), N6, 2’-O-Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP; 50 pM, Millipore Sigma, D0627), L-Ascorbic Acid 2-phosphate Trisodium Salt (AA; 200 pM, Wako, 323-44822), IGF-1 (10 ng / ml, PeproTech, 100-11 ) until the end of experiments. From day 21 to day 26, DAPT (2.5 pM, STEMCELL Technologies, 72082) was added.

[0166] Generation of Assembloids. For two-part assembloids, the organoids of interest were fused by placing them in proximity in 1 .5 ml Eppendorf tubes for 2-3 days in an incubator. For sensory ascending pathway assembloids, four organoids were integrated by placing them in proximity in tilted 6-well ultra-low attachment plates for 1 week in an incubator. Afterassembly, the assembloids were maintained in ultra-low attachment plates in media containing growth factors and small molecules described above.

[0167] Live Calcium Imaging. To achieve single-cell resolution live calcium imaging in intact 3D organoids or assembloids without significant sample movement during chemical injection, we acutely attached samples to Ethyleneimine polymer (PEI)-coated coverslips before imaging. Autoclaved 12 mm coverslips were coated with 0.01875% PEI (Sigma, #03880) in water for at least 1 hour at 37°C and then washed three times with water before use. An organoid or assembloid was carefully positioned on top of a PEI-coated coverslip, and an L- shaped tubing-type Chamlide CMB chamber (Live Cell Instrument, CM-B12-1 PB) was assembled. This chamber was connected to a peristaltic pump (Harvard Apparatus, #702027) to establish continuous suction through the outlet hole. To prevent sample drying, a vehicle solution was applied immediately after assembly. Calcium activity, with or without various stimuli, was imaged using a confocal microscope.

[0168] Viral Labeling and Rabies Tracing. Two or three organoids were placed in a 1.5 ml Eppendorf tube containing 200 pl media with the desired virus(es) and incubated overnight. Fresh medium was added the following day, and organoids were transferred to ultra-low attachment plates the next day. For retrograde tracing, organoids representing the presynaptic part were labeled with AAV-DIO-tdTomato and organoids representing the postsynaptic part were separately labeled with both rabies-AG-Cre-GFP and AAV-EF1 a-CVS-G. Two days after viral infection, organoids were assembled. After 3 weeks of integration, assembloids were fixed with 4% paraformaldehyde and processed for immunocytochemistry.

[0169] Single Cell RNA sequencing. Organoids were dissociated to obtain single cell suspension using papain enzyme solution. Organoids were gently triturated to achieve a single cell suspension in a protease inhibitor solution. Cells were resuspended in 0.04% BSA / PBS. cDNA libraries were generated with the Chromium Single Cell 3’ Reagent Kits v3.1. Each library was sequenced using the Illumina NovaSeq. Downstream analyses were performed using the R package Seurat and Cell Ranger software.

[0170] Extracellular Recording. Organoids or assembloids were embedded into 3% low- melting gel agarose and transferred into artificial cerebrospinal fluid (aCSF) containing 124 mM NaCI, 3 mM KCI, 1 .25 mM NaH2PO4, 1 .2 mM MgSO4, 1 .5 mM CaCI2, 26 mM NaHCO3 and 10 mM D-(+)-glucose with addition of GlutaMAX (Gibco). Embedded organoids or assembloids were placed on a perfusion recording chamber and continuously perfused with aCSF (bubbled with 95% 02 and 5% CO2) at 37°C. To verify the regional identity in assembloids, tdTomato was expression in one of the regions under human SYN1 promoter. Acute 32 channel P-1 probe with 2 shanks (Cambridge NeuroTech) was used for recording.

[0171] CRISPR Engineering. gRNAs were designed to target the exons of the target genes. For each gene, the hiPS cells were nucleofected with SpCas9 and gRNAs. Approximately 2-3 days after nucleofection, hiPS cells were sorted into the wells of 96-well plates, expanded and frozen. In parallel, cell pellets were also collected for sequencing. After validating their mutation profiles, the knockout hiPS cells clones were recovered from the 96-well plates.

[0172] Notwithstanding the appended claims, the disclosure is also defined by the following clauses:1 . A method for producing human sensory organoids in vitro, the method comprising:(a) inducing a human pluripotent stem cell in suspension culture to a neural fate to generate a neural organoid;(b) differentiating the neural organoid into a human sensory organoid (hSeO); and(c) maintaining the hSeO for an extended period of time in neural medium such that the hSeO comprises human sensory neurons, Schwann cells and neural crest cells.2. The method of clause 1 , wherein the human sensory neurons express one or more genes selected from the group consisting of SOX2, SOX10, STMN2, PRPH, BRN3A, SIX1 , P2RX3, and SCN9A.3. The method of clauses 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells.4. The method of any of clauses 1 -3, wherein inducing the human pluripotent stem cell in suspension culture to the neural fate in step (a) comprises culturing in a medium comprising one or more SMAD inhibitors, and an inhibitor of GSK-3, and BMP4.5. The method of clause 4, wherein the one or more SMAD inhibitor is selected from the group consisting of dorsomorphin, A 83-01 , DMH-1 , K 02288, ML 347, SB 505124, SB- 431542, LDN-193189, Galunisertib, LY2109761 , SD-208, EW-7197, Kartogenin, DMH1 , LDN- 212854, ML347, LDN-193189 HCI, SB505124; Pirfenidone, RepSo, K02288, Hesperetin; GW788388, and LY364947.6. The method of clauses 4 or 5, wherein the inhibitor of GSK is selected from the group consisting of TWS1 19, CHIR98014, SB216763, CHIR99021 , and GSK inhibitor IX (BIO).7. The method of any of clauses 4-6, wherein step (a) comprises culturing the human pluripotent stem cells in a medium comprising the one or more SMAD inhibitors for a period of 6 to 8 days, adding the BMP4 to the medium on day 1 to day 4, and adding the GSK inhibitor on day 3 to day 5.8. The method of clause 7, further comprising transferring the human pluripotent stem cells to a medium comprising one or more SMAD inhibitors inhibitor on day 7 to day 9.9. The method of clause 8, wherein differentiating the neural organoid into a human sensory organoid of step (b) comprises adding brain-derived neurotrophic factor (BDNF), Glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to the medium on day 9; and removing the one or more SMAD inhibitors from the medium on day 15.10. The method of clause 9, further comprising adding a gamma secretase inhibitor to the medium.1 1 . The method of clause 10, wherein the gamma secretase inhibitor is selected from the group consisting of dibenzazepine, LY41 1575, and DAPT.12. The method of any of clauses 1 -1 1 , wherein the sensory neurons are capable of being stimulated by noxious or mechanical stimuli.13. The method of any of clauses 1 -12, wherein the sensory neurons comprise at least one allele associated with a sensory disorder.14. The method of clause 13, wherein the sensory disorder is selected from the group consisting of small fiber neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.15. An in vitro generated sensory organoid produced by the method of any of clauses 1 - 14.16. A method for producing human dorsal hindbrain / cervical spinal cord organoids in vitro, the method comprising:(a) inducing a human pluripotent stem cell in suspension culture to a neural fate to generate a neural organoid;(b) differentiating the neural organoid into a human dorsal hindbrain / cervical spinal cord organoid (hdSpO); and(c) maintaining the hdSpO for an extended period of time in neural medium such that the hdSpO comprises spinothalamic neurons.17. The method of clause 16, wherein the dorsal spinal cord neurons, including projection neurons, that express one or more genes selected from the group consisting of SOX2, SLC17A6, LBX1 , STMN2, GAD1 and TACR1 .18. The method of any of clauses 16 or 17, wherein the pluripotent stem cells are induced pluripotent stem cells.19. The method of any of clauses 16-18, wherein inducing the human pluripotent stem cell in suspension culture to the neural fate in step (a) comprises culturing in a medium comprising two or more SMAD inhibitors, and an inhibitor of GSK-3.20. The method of clause 19, wherein the two or more SMAD inhibitors are selected from the group consisting of dorsomorphin, A 83-01 , DMH-1 , K 02288, ML 347, SB 505124, SB- 431542, LDN-193189, Galunisertib, LY2109761 , SD-208, EW-7197, Kartogenin, DMH1 , LDN-212854, ML347, LDN-193189 HCI, SB505124; Pirfenidone, RepSo, K02288, Hesperetin; GW788388, and LY364947.21. The method of clauses 19 or 20, wherein the inhibitor of GSK is selected from the group consisting of TWS119, CHIR98014, SB216763, CHIR99021 , and GSK inhibitor IX (BIO).22. The method of any of clauses 19-21 , wherein step (a) comprises culturing the human pluripotent stem cells in a medium comprising the two or more SMAD inhibitors for a period of 6 to 8 days and adding the GSK inhibitor to the medium on day 5 to day 6.23. The method of clause 22, further comprising transferring the human pluripotent stem cells to a medium comprising epidermal growth factor (EGF), retinoic acid, and the GSK inhibitor on day 7.24. The method of any of clauses 16-23, wherein differentiating the neural organoid into the hdSpO of step (b) comprises adding brain-derived neurotrophic factor (BDNF), N6, 2’-O- Dibutyryladenosine 3’, 5’ -cyclic monophosphate sodium salt (cAMP), L-Ascorbic Acid 2- phosphate Trisodium Salt, and insulin-like growth factor (IGF-1 ) to the medium on day 21 .25. The method of clause 24, further comprising adding a gamma secretase inhibitor to the medium.26. The method of clause 25, wherein the gamma secretase inhibitor is selected from the group consisting of dibenzazepine, LY411575, and DAPT.27. The method of any of clauses 16-26, wherein the dorsal spinal cord neurons comprise at least one allele associated with a sensory disorder.28. The method of clause 27, wherein the sensory disorder is selected from the group consisting of small fibre neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.29. An in vitro generated human dorsal hindbrain / cervical spinal cord organoid produced by the method of any of clauses 16-28.30. A method for producing a human ascending somatosensory assembloid in vitro, the method comprising:(i) inducing in a human pluripotent stem cell suspension culture a neural fate to generate a neural organoid;(ii) differentiating the neural organoid into two or more of: human cortical organoids (hCO), human diencephalon organoids (hDiO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO) and human sensory organoid (hSeO); and(iii) culturing the two or more of hCO, hDiO, hdSpO and hSeO under conditions permissive for organoid fusion into a linear assembloid while maintaining for an extended period of time in neural medium; wherein an integrated structure is differentiated comprising interacting neurons that form a linear circuit.31 . The method of clause 30, wherein the assembloid comprises hCO-hDiO-hdSpO- hSeO, hCO-hDiO-hdSpO, hDiO-hdSpO-hSeO, or hdSpO-hSeO.32. The method of clauses 30 or 31 , wherein the assembloid comprises hCO-hDiO- hdSpO-hSeO.33. The method of any of clauses 30-32, wherein the neurons in the assembloid comprise at least one allele associated with a sensory disorder.34. The method of clause 33, wherein the sensory disorder is selected from the group consisting of small fiber neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.35. The method of any of clauses 30-34, wherein step (iii) comprises culturing the two or more of hCO, hDiO, hdSpO and hSeO in close proximity for a period of 2 to 10 days.36. The method of any of clauses 30-35, wherein the culturing is performed on a tilt.37. The method of any of clauses 30-36, wherein one or more of the neurons in the assembloid comprise a calcium sensor.38. An in vitro generated human ascending somatosensory assembloid produced by the method of any of clauses 30-37.39. A method determining the effect of a candidate agent on a human ascending somatosensory pathway, the method comprising: contacting the candidate agent with one or a panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembloids differentiated from induced human pluripotent stem (hiPS) cells according to the method of any of clauses 30-37, or a population of cells isolated therefrom; and determining the effect of the agent on morphologic, genetic or functional parameters.40. The method of clause 39, wherein the neurons in the assembloids comprise at least one allele associated with a sensory or pain disorder.41 . The method of clause 40, wherein the sensory disorder is selected from the group consisting of small fiber neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phasesyndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.42. The method of any of clauses 39-41 , wherein the functional parameters are one or more of intrinsic electrophysiological properties, action potential propagation in response to a stimuli, spontaneous action potential propagation, synaptic integration, and calcium signaling.43. The method of any of clauses 39-42, further comprising contacting the panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembled organoids with a noxious or mechanical stimulus at the same time, before, or after contacting the panel with the candidate agent.44. The method of clause 43, wherein the noxious stimulus are capsaicin or a|3-MeATP.45. The method of clause 42 or 43, further comprising contacting the panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembled organoids with an inflammatory mediator at the same time, before, or after contacting the panel with the noxious stimuli.46. The method of clause 45, wherein the inflammatory mediator is selected from the group consisting of: extracellular protons, arachidonic acid, serotonin, nerve growth factor (NGF), prostaglandins (PG), bradykinin (BK), adenosine triphosphate (ATP) and histamine.

Claims

THAT WHICH IS CLAIMED IS:1 . A method for producing human sensory organoids in vitro, the method comprising:(a) inducing a human pluripotent stem cell in suspension culture to a neural fate to generate a neural organoid;(b) differentiating the neural organoid into a human sensory organoid (hSeO); and(c) maintaining the hSeO for an extended period of time in neural medium such that the hSeO comprises human sensory neurons, Schwann cells, and neural crest cells.

2. The method of claim 1 , wherein the human sensory neurons express one or more genes selected from the group consisting of SOX2, SOX10, STMN2, PRPH, BRN3A, SIX1 , P2RX3, and SCN9A.

3. The method of claim 1 or 2, wherein inducing the human pluripotent stem cell in suspension culture to the neural fate in step (a) comprises culturing in a medium comprising one or more SMAD inhibitors, and an inhibitor of GSK-3, and BMP4, wherein: the one or more SMAD inhibitor is selected from the group consisting of dorsomorphin, A 83-01 , DMH-1 , K 02288, ML 347, SB 505124, SB-431542, LDN-193189, Galunisertib, LY2109761 , SD-208, EW-7197, Kartogenin, DMH1 , LDN-212854, ML347, LDN-193189 HCI, SB505124; Pirfenidone, RepSo, K02288, Hesperetin; GW788388, and LY364947, and the inhibitor of GSK is selected from the group consisting of TWS119, CHIR98014, SB216763, CHIR99021 , and GSK inhibitor IX (BIO).

4. The method of any of claims 1 -3, wherein step (a) comprises culturing the human pluripotent stem cells in a medium comprising the one or more SMAD inhibitors for a period of 6 to 8 days, adding the BMP4 to the medium for a period of 3 to 6 days, adding the GSK inhibitor for a period of 2 to 6 days, and transferring the human pluripotent stem cells to a medium comprising one or more SMAD inhibitors inhibitor after day 7.

5. The method of any of claims 1 -4, wherein differentiating the neural organoid into a human sensory organoid of step (b) comprises adding brain-derived neurotrophic factor (BDNF), Glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF) to the medium following day 7 to 9; and optionally adding a gamma secretase inhibitor to the medium, wherein the gamma secretase inhibitor is selected from the groupconsisting of dibenzazepine, LY411575, and DAPT.

6. A method for producing a human ascending somatosensory assembloid in vitro, the method comprising:(i) inducing in a human pluripotent stem cell suspension culture a neural fate to generate a neural organoid;(ii) differentiating the neural organoid into two or more of: human cortical organoids (hCO), human diencephalon organoids (hDiO), human dorsal hindbrain / cervical spinal cord organoids (hdSpO) and human sensory organoid (hSeO); and(iii) culturing the two or more of hCO, hDiO, hdSpO and hSeO under conditions permissive for organoid fusion into a linear assembloid while maintaining for an extended period of time in neural medium; wherein an integrated structure is differentiated comprising interacting neurons that form a linear circuit.

7. The method of claim 6, wherein the assembloid comprises hCO-hDiO- hdSpO-hSeO, hCO-hDiO-hdSpO, hDiO-hdSpO-hSeO, or hdSpO-hSeO.

8. The method of claims 6 or 7, wherein the assembloid comprises hCO-hDiO- hdSpO-hSeO.

9. The method of any of claims 6-8, wherein the neurons in the assembloid comprise at least one allele associated with a sensory disorder.

10. The method of claim 9, wherein the sensory disorder is selected from the group consisting of small fiber neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.11 . The method of any of claims 6-10, wherein step (iii) comprises culturing the two or more of hCO, hDiO, hdSpO and hSeO in close proximity for a period of 2 to 10 days.

12. The method of any of claims 6-11 , wherein the culturing is performed on a tilt.

13. The method of any of claims 6-14, wherein one or more of the neurons in theassembloid comprise a calcium sensor.

14. A method determining the effect of a candidate agent on a human ascending somatosensory pathway, the method comprising: contacting the candidate agent with one or a panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembloids differentiated from induced human pluripotent stem (hiPS) cells, or a population of cells isolated therefrom; and determining the effect of the agent on morphologic, genetic or functional parameters.

15. The method of claim 14, wherein the neurons in the assembloids comprise at least one allele associated with a sensory or pain disorder.

16. The method of claim 15, wherein the sensory disorder is selected from the group consisting of small fiber neuropathy, sodium-channelopathy, familial hemiplegic migraine type 1 and 2, hereditary sensory and autonomic neuropathy type l-IV, primary erythromelalgia, paroxysmal, fibromyalgia, congenital insensitivity to pain, familial advanced sleep phase syndrome, familial episodic pain syndrome, familial cold autoinflammatory syndrome, congenital insensitivity to pain with anhidrosis, Marsili syndrome, hereditary sensory neuropathy, and autism spectrum disorder.

17. The method of any of claims 14-16, wherein the functional parameters are one or more of intrinsic electrophysiological properties, action potential propagation in response to a stimuli, spontaneous action potential propagation, synaptic integration, and calcium signaling.

18. The method of any of claims 14-17, further comprising contacting the panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembled organoids with a noxious or mechanical stimulus at the same time, before, or after contacting the panel with the candidate agent.

19. The method of claim 18, wherein the noxious stimulus are selected from the group consisting of: capsaicin, ap-MeATP, Zymosan, Bradykinin, MSP-3, menthol, and AITC.

20. The method of claim 18 or 19, further comprising contacting the panel of functionally integrated human hDiO-hdSpO-hSeO or hCO-hDiO-hdSpO-hSeO assembled organoids with an inflammatory mediator at the same time, before, or after contacting the panel with the noxious stimuli.

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