Method of using human spheroids for drug discovery

Abstract

To provide, in one embodiment, a method of using human-induced pluripotent stem cells to generate three-dimensional human organ tissue for therapeutic drug toxicity and discovery.SOLUTION: In one embodiment, a high throughput microtiter plate is loaded with both wild type and Rett disease 3D spheroids and exposed to a drug library, and activity is measured and analyzed for disease rescue to wild type cell behavior.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 800,430, filed February 2, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] The use of mice and other animals as tools in biomedical research is well established. Their existence offers the ability to assess disease pathogenesis and therapeutic profiling in a low-cost, easy-to-maintain, and rapidly propagating mammalian model. Over the years, there has been a shift from forward genetics, which studies spontaneous and chemically induced mouse models, to reverse genetics, which studies gene function through genetically engineered knockouts. By studying both constitutive and tissue-specific loss-of-function mutations through standard genetic engineering techniques, a vast amount of information has been obtained regarding gene function, pathways, and disease pathophysiology. However, progress has been hindered by the costs associated with traditional genetic engineering, much of which is related to the time required to target embryonic stem (ES) cells, establish germline transmission, and breed away from selectable markers. Furthermore, a lack of efficiency in ES cells derived from different inbred strains has limited the exploration of phenotypes in different genetic backgrounds. This limitation is an important consideration when comparing disease models from a single inbred background to heterogeneous patient populations, where disease penetrance may differ.

[0003] Animal studies provide invaluable information in the development and testing of human drugs that cannot be obtained through test-tube or petri dish experiments alone. They provide information on how a drug is absorbed and distributed throughout the living animal's body, and how it affects target and other tissues. They also inform how the body processes and eliminates the drug. For most drugs, this is primarily accomplished by the liver and kidneys. These studies help determine whether a drug should proceed to human trials and, if so, what a reasonable starting dose for humans should be. However, due to species differences, what is effective and safe in nonhuman animals may not be so in humans.

[0004] Scientific journal publications about animal studies typically include a disclaimer that "the effect has only been demonstrated in animals and may not be replicated in humans." And for very good reason. The review looked at studies in which medical interventions were tested in non-human animals, and whether the results were replicated in human trials.

[0005] Of the most cited nonhuman animal studies in prestigious scientific journals such as Nature and Cell, only 37% were replicated in subsequent human randomized trials, and 18% were contradictory in human trials. It is safe to assume that less cited nonhuman animal studies in fewer journals have even lower success rates. Another review found that the therapeutic effects (benefits or harms) of six medical interventions performed in humans and nonhuman animals were similar for only half of the interventions. In other words, the results of nonhuman animal and human trials were often inconsistent.

[0006] Discrepancies between non-human animal and human trials can cause major problems. Developing a drug to the non-human animal clinical trial stage is already incredibly expensive, but bringing it to human clinical trials adds enormous costs, often tens of millions of dollars. If a promising drug fails to impress in human trials, it can mean a lot of wasted money, time, and effort.

[0007] Far more problematic, however, are drugs that appear safe in nonhuman animal tests but are found to be unsafe in humans. The consequences can be tragic. For example, thalidomide (a drug used to treat morning sickness) does not cause birth defects when administered to pregnant rats and mice, but in humans it caused an international epidemic of birth defects, including severe limb deformities, in the 1950s and 1960s. Summary of the Invention

[0008] The present disclosure provides the use of human models using tissues generated from human iPSC cell lines, thereby providing an alternative to animal models for drug safety and therapeutic testing. Cell lines that model the disease of interest are selected for preclinical drug discovery and toxicity testing. The disclosed human tissue disease models can be generated for most organs, including the brain, heart, lungs, kidneys, liver, pancreas, spleen, skin, eyes, muscle, and / or bone. Assays can use any one or any combination of tissue models. In one embodiment, the model is a model of autism spectrum disorder, and thus, therapeutic compounds identified in the screen can be used to prevent, suppress, or treat one or more symptoms of autism spectrum disorder or a similar disorder, such as Rett syndrome. Autism spectrum disorder (ASD) is a neurological and developmental disorder that begins early childhood and persists throughout a person's life. It affects how humans behave, interact with others, communicate, and learn. Thus, drug screening assays with a variety of different tissues can identify one or more compounds that are beneficial for treating one or more symptoms in one or more organs or tissues of patients with ASD.

[0009] For example, Rett Syndrome is characterized by neurological and developmental symptoms including, but not limited to, growth retardation, loss of normal movement and coordination, loss of communication skills, abnormal hand and eye movements, respiratory problems, cognitive impairment, seizures, scoliosis, irregular heartbeat, and sleep disorders, thus affecting many organ systems. Thus, drug screening assays with a variety of different tissues can identify one or more compounds that are beneficial for treating one or more symptoms in one or more organs or tissues of Rett Syndrome patients.

[0010] In one embodiment, the method provides induced pluripotent stem cell (iPSc) generated human disease tissue models for the testing and discovery of therapeutic drug compounds.

[0011] In one embodiment, the method provides an iPSC-generated human tissue model for testing and discovery of therapeutic compounds having a three-dimensional structure (eg, a spheroid or three-dimensional spheroid form factor).

[0012] In one embodiment, the method provides iPSC-generated human tissue models for testing and discovery of therapeutic compounds that have three-dimensional structures and are formatted into high-throughput arrays, e.g., high-throughput microtiter arrays.

[0013] In one embodiment, the method provides an iPSC-generated human tissue model for testing and discovery of therapeutic compounds having three-dimensional structure, capable of generating functional information regarding tissue response, e.g., in diseased versus control tissue, when exposed to a therapeutic compound.

[0014] In one embodiment, the method provides an iPSC-generated human tissue model with three-dimensional structure for the testing and discovery of therapeutic compounds that can replace the use of non-human animals in preclinical and clinical trials.

[0015] In one embodiment, the present disclosure provides optical assays, such as functional FLIPR assays or high-content high magnification optical microscopy, of 3D human cell spheroids, e.g., mixed population human cell neuron spheroids. In one embodiment, spheroids are cultured for 6-14 weeks prior to testing to induce robust synchronized synaptic networks to mimic, for example, mature human brain function. These mixed populations of spheroids fired predictably and consistently over time. In one embodiment, the present disclosure provides high-throughput optical assays of mixed populations of human cell 3D spheroids using FLIPR and calcium uptake fluorescence oscillations. Oscillations can be modulated with compounds, and oscillatory firing can be altered with agonists or antagonists. In one embodiment, the cells are derived from a patient with Rett syndrome or a model of Rett syndrome.

[0016] In one embodiment, the present disclosure provides an optical method for detecting the effect of one or more compounds on spheroids, e.g., from Rett syndrome patient cells or from a Rett syndrome model. The method includes contacting one or more test compounds with a tissue culture plate, e.g., a plate having wells containing one or more spheroids (e.g., spheroids of uniform diameter) of Rett syndrome patient cells or human cells from a Rett syndrome model; and optically detecting the amount or change in spheroid oscillation. In one embodiment, the plate is a multiwell plate. In one embodiment, the spheroids are further contacted with a fluorescent molecule useful for detecting calcium, and the amount or change in fluorescence over time is detected. In one embodiment, the amount or change in fluorescence is detected via the amount of fluorescence peaks, the amplitude of one or more peaks, the peak spacing between one or more peaks, the width of one or more peaks, or any combination thereof. In one embodiment, the spheroids comprise neurons. In one embodiment, the spheroids comprise neurons and astrocytes. In one embodiment, the spheroids comprise cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the spheroids comprise microglial cells or oligodendrocytes. In one embodiment, the spheroids comprise pericytes and endothelial cells. In one embodiment, the spheroids comprise endothelial cells, microglial cells, neurons, oligodendrocytes, or any combination thereof. In one embodiment, the cells are differentiated cells. In one embodiment, the progenitor cells are progenitor cells for neurons, astrocytes, cardiac cells, hepatic cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the one or more spheroids have a diameter of about 500 to about 600 microns. In one embodiment, the one or more spheroids have a diameter of about 450 to about 500 microns. In one embodiment, the one or more spheroids are cultured for at least 4 to 6 weeks before contacting with one or more test compounds.In one embodiment, the fluorescent molecule comprises Calcium 3, Calcium 4, Calcium 5, Calcium 6, Fluo 3, or Fluo 4, or a combination thereof. In one embodiment, the one or more spheroids are further contacted with a cell membrane impermeant quencher. In one embodiment, the change in fluorescence is compared to the fluorescence of the one or more spheroids and the fluorescent molecule, but without the test compound. In one embodiment, in a multi-well plate, each well contains one spheroid.

[0017] Also provided are multiwell plates containing one or more mixed human cell spheroids per well, e.g., derived from cells of a Rett syndrome patient or a model of Rett syndrome. In one embodiment, the spheroids comprise neurons and astrocytes. In one embodiment, the spheroids comprise cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the spheroids comprise microglial cells or oligodendrocytes. In one embodiment, the spheroids comprise pericytes and endothelial cells. In one embodiment, the spheroids comprise endothelial cells, microglial cells, neurons, oligodendrocytes, or any combination thereof. In one embodiment, the spheroids comprise progenitor cells of neurons, astrocytes, cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. [Brief explanation of the drawings]

[0018] [Figure 1] Microscopic images of cortical brain spheroids in a high-throughput format, a single well, and spheroid size distribution are shown. [Figure 2] Shown is the calcium oscillation activity of both wild-type (WT) and Rett (RTT) spheroids captured from different wells. [Figure 3-1]Multiparametric plots of spheroid calcium oscillation activity for WT and RTT spheroids before and after drug exposure for BIMU-8 and flumazenil are shown. [Figure 3-2] Multiparametric plots of spheroid calcium oscillation activity for WT and RTT spheroids before and after drug exposure for BIMU-8 and flumazenil are shown. [Figure 4-1] 1 shows multiparametric plots of spheroid calcium oscillation activity for WT and RTT spheroids before and after exposure to vorinostat and epigallocatechin. [Figure 4-2] 1 shows multiparametric plots of spheroid calcium oscillation activity for WT and RTT spheroids before and after exposure to vorinostat and epigallocatechin. [Figure 5] We show calcium oscillatory activity in WT and RTT spheroids after exposure to flumazenil and BIMU-8 drug molecules, demonstrating that oscillatory activity is restored to normal behavior in WT. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description definition In describing the present invention, the following terminology will be used in accordance with the definitions set out below.

[0020] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0021] As used herein, the term "about" means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. For example, in one embodiment, the term "about" is used herein to modify a numerical value above and below a stated value by a variance of 20%. When referring to a numerical value or range, the term "about" takes into account some degree of variability in the value or range, for example, within 10% or within 5% of the stated limit of the stated value or range.

[0022] The terms "disease" and "disorder" and "syndrome" are used interchangeably.

[0023] The phrase "effective amount," when used to describe treatment of an individual afflicted with a disorder, refers to an amount of a compound or composition effective to prevent, inhibit, or otherwise treat one or more symptoms of the disease or disorder.

[0024] As used herein, "substantially" means completely or nearly completely, e.g., a composition that is "substantially free" of a component either does not contain the component or contains such a small amount that the relevant functional properties of the composition are not affected by the presence of the small amount, or a compound is "substantially pure" if only negligible impurities are present.

[0025] Administration of the composition may be for either "prophylactic" or "therapeutic" purposes. When provided prophylactically, the composition is provided before the onset of symptoms or clinical signs of disease. Prophylactic administration of the composition serves to prevent or alleviate subsequent symptoms or clinical signs. When provided therapeutically, the composition is provided upon detection of symptoms or clinical signs of disease.

[0026] Thus, the compositions can be provided either prior to the onset of the disease or condition (to prevent or alleviate symptoms) or after the onset of clinical signs of the condition or disease.

[0027] A composition is said to be "pharmacologically acceptable" if its administration is tolerated by a recipient mammal, such as a human. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.

[0028] The "protection" provided need not be absolute, i.e., there is a statistically significant improvement compared to a control population or set of mammals, but need not be complete prevention or eradication. Protection may be limited to mitigating the severity or rate of onset of clinical signs of a condition or disease.

[0029] "Treating" or "treatment," as used herein, refers to alleviating symptoms associated with a disorder or disease, or preventing further progression or worsening of those symptoms, or preventing or prophylaxis of a disease or disorder, or curing a disease or disorder. Similarly, as used herein, an "effective amount" or "therapeutically effective amount" of a compound described herein refers to an amount of compound that, in whole or in part, alleviates symptoms associated with a disorder or condition, or stops or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis of a disorder or condition. In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of a compound of the invention outweigh the therapeutically beneficial effects.

[0030] "Salts," as they are known in the art, include organic compounds, such as carboxylic acids, sulfonic acids, or amines, in ionic form in combination with a counterion. For example, acids in their anionic form can form salts with metal cations, such as sodium and potassium; ammonium salts, such as NH4, or cations of various amines, including tetraalkylammonium salts, such as tetramethylammonium, or other cations, such as trimethylsulfonium. "Pharmaceutically acceptable" or "pharmacologically acceptable" salts are salts approved for human consumption and formed from generally non-toxic ions, such as chloride or sodium salts. "Zwitterions" are internal salts that can form within molecules with at least two ionizable groups, one forming an anion and the other forming a cation, which serve to balance each other. For example, amino acids, such as glycine, can exist in zwitterionic form. "Zwitterions" are salts within the meaning of this specification. The compounds of the present invention can be in the form of salts. The term "salt" encompasses addition salts of free acids or free bases of the compounds of the present invention. The salts may be "pharmaceutically acceptable salts." The term "pharmaceutically acceptable salt" refers to salts that have toxicity profiles within a range that makes them useful in pharmaceutical applications. Nonetheless, salts that are not pharmaceutically acceptable may have properties, such as high crystallinity, that make them useful in the practice of the present invention, e.g., useful during the synthesis, purification, or formulation of the compounds of the present invention.

[0031] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from the aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic acid classes of organic acids, and examples of such organic acids include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, maleic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), and the like. (pamoic)], methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

[0032] Suitable pharmaceutically acceptable base addition salts of compounds include, for example, metal salts containing alkali metals, alkaline earth metals, and transition metal salts such as calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. Although pharmaceutically unacceptable salts are generally not useful as pharmaceuticals, such salts may be useful, for example, as intermediates in the synthesis of compounds and in their purification, for example, by recrystallization. All of these salts can be prepared by conventional means from the corresponding compounds, for example, by reacting the appropriate acid or base with the compound. The term "pharmaceutically acceptable salt" refers to a non-toxic inorganic or organic acid and / or base addition salt. See, for example, Lit et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217, incorporated herein by reference.

[0033] A "hydrate" is a compound that exists in a composition with water molecules. The composition may contain a stoichiometric amount of water, such as a monohydrate or dihydrate, or may contain a random amount of water. As used herein, the term "hydrate" refers to a solid form. That is, a compound in an aqueous solution may be hydrated, but is not a hydrate as the term is used herein.

[0034] A "solvate" is a similar composition except that a solvent other than water replaces the water. For example, methanol or ethanol may form an "alcohol acid salt," which may also be stoichiometric or non-stoichiometric. As used herein, the term "solvate" refers to a solid form. That is, a compound in a solvent solution may be solvated, but is not a solvate as the term is used herein.

[0035] Exemplary Assay Methods In one embodiment, the present disclosure provides human induced pluripotent stem cells (iPSCs), which are subsequently differentiated into human cortical neurons containing neurons and astrocytes in an approximately 50:50 ratio. This ratio can be adjusted on a case-by-case basis, ranging from 1% to 99% of either neurons or astrocytes, depending on the specific disease of interest. 3D spheroids are composed of approximately 50:50 neurons and astrocytes in a ratio of + / - 10%. Methods for differentiating and forming 3D spheroids, as well as functional testing using FLIPR optical technology, are described in pending patent application Ser. No. 62 / 532667. In the examples of the present invention, a disease model cell line known to carry the gene responsible for Rett syndrome is used.

[0036] Rett syndrome (RTT) is a unique postnatal neurological disorder first recognized in infancy, most commonly seen in girls but rarely in boys. Rett syndrome is most commonly misdiagnosed as autism, cerebral palsy, or nonspecific developmental delay. Rett syndrome is caused by mutations on the X chromosome in a gene called MECP2. More than 200 different mutations can be found in the MECP2 gene. Most of these mutations are found in eight distinct "hotspots." Rett syndrome affects all races and ethnicities and occurs in 1 in 10,000 female births worldwide. Rett syndrome is a postnatal neurological disorder; it is not a degenerative disease. Rett syndrome causes problems with brain function involved in cognition, sensation, emotion, movement, and autonomic function. These problems include learning, speech, sensory sensations, mood, movement, breathing, cardiac function, and even chewing, swallowing, and digestion.

[0037] Symptoms of Rett syndrome appear by 6 to 18 months of age, after an initial period of apparently normal or near-normal development, with delayed or stagnant skills. This is followed by a period of regression as she loses communication skills and purposeful hand use. Soon, stereotyped hand movements, such as handwashing, gait disturbances, and a slowing of normal head growth rate become evident. Other problems may include seizures and irregular breathing patterns while she is awake. Early on, there may be periods of isolation or withdrawal when she becomes irritable and cries a lot. Over time, motor problems may increase, but irritability typically decreases, and eye contact and communication improve.

[0038] Rett syndrome is confirmed by a simple blood test to identify MECP2 mutations. However, because MECP2 mutations are also found in other disorders, the presence of an MECP2 mutation alone is not sufficient for diagnosing Rett syndrome. Diagnosis requires the presence of the mutation (molecular diagnosis) or confirmation of diagnostic criteria (clinical diagnosis based on observable signs and symptoms), or both. Rett syndrome can present with a wide range of disorders, from mild to severe. The course and severity of Rett syndrome depend on the location, type, and severity of the mutation and X-chromosome inactivation. Therefore, two girls of the same age with the same mutation may appear completely different.

[0039] In this disclosure, both wild-type (WT) and Rett Disease (RTT) cortical brain human tissue spheroids were prepared for the purpose of high-throughput screening to identify therapeutic compounds that can modulate brain activity to return or rescue disease to near-normal function. For example, the cell lines used (control or "WT" and disease "RTT") may be derived from families with and without the Rett Disease phenotype.

[0040] The methods disclosed herein, in one embodiment, may include generating 3D spheroids in a 384-well round-bottom microtiter plate. See, for example, U.S. Patent Application No. 62 / 532,667, the disclosure of which is incorporated herein. Once the spheroids are generated and allowed to mature for at least six weeks, testing of potential therapeutic compounds can begin. In the present disclosure, 6-14 weeks of maturation is the maturity range for testing, e.g., 8-10 weeks of maturation.

[0041] Referring to Figure 1, the image on the left shows a 384-well microtiter plate, each well loaded with human tissue microspheres approximately 600 microns in diameter, each containing approximately 10,000 cells. The number of microtiter wells may range from 28 to 1,536 wells per plate, or from 96 to 384 wells. The number of cells forming each spheroid may range from 2,500 to 50,000 cells, or from 5,000 to 15,000 cells. The image on the right shows the uniformity across the microspheres within a single well. Uniformity is critical in this invention to obtain highly reproducible results and limit well-to-well and plate-to-plate variability. The image below is a plot showing the average spheroid size across a row of microtiter wells. Spheroid sizes may range from 100 microns to 5 mm, or from 400 to 800 microns.

[0042] Referring to Figure 2, this figure shows examples of both WT and RTT spheroids from randomly selected wells before exposure to a therapeutic agent. The oscillations being measured are due to calcium flux into and out of the cells under ambient conditions. The oscillations are spontaneous and not generated by external factors. Measurements are performed in real time, and the data are captured using a technology known in the art called FLIPR. As can be seen in the image, the WT spheroids have very regular, periodic peak intensities from the randomly selected wells. However, the RTT disease spheroid model on the right is very unstable and somewhat unpredictable. This finding is consistent with the erratic behavior seen in patients with the Rett phenotype and described above. When analyzing data, it is important to understand that multiple factors contribute to the signal recorded by the FLIPR software. Therefore, in this disclosure, seven parameters are recorded to determine whether the data generated from the spheroids is meaningful. The measured parameters include, for example, peak count, peak width, mean peak spacing, peak spacing standard deviation, peak decay time, peak rise time, and / or peak amplitude. By comparing WT spheroid multiparameter data with RTT data before and after drug exposure, it is possible to determine whether promising therapeutic candidates are effective in reverting disease state to a WT normal state.

[0043] In this disclosure, a SMART library containing 298 compounds was used on both wild-type and rett-transplant (RTT) mature spheroids over several weeks, and FLIPR data was collected and analyzed for potential therapeutic effects. The SMART (Selected Molecular Agents for Rett Therapy) library of compounds has been thoroughly curated using modern bioinformatics methods, with a strict focus on Rett syndrome and its biological causes. The library is currently housed at the University of Illinois at Chicago. The Rettsyndrome.org Science Advisory Board has also recommended many of the compounds currently included in the library, purchased or prepared. With the goal of saving both time and resources and accelerating drug discovery for Rett syndrome in mind, the compounds in the SMART library are readily available to researchers working on Rett syndrome research.

[0044] Referring to Figure 3, this shows the results for WT and RETT spheroids exposed to vehicle, which has no effect on the spheroids in the top two images. In the middle and bottom of the figure are RTT spheroids exposed to 1 micromolar doses of BIMU-8 and flumazenil from the SMART library. As can be seen in Figure 3, both BIMU-8 and flumazenil had a significant effect in reversing or restoring the RTT pathology generated for WT spheroids to near-normal, as shown in the top left of Figure 3.

[0045] This is in stark contrast to Figure 4, where WT and RTT exposure to 1 micromolar concentrations of vorinostat and epigallocatechin showed no effect on restoring or reversing spontaneous calcium oscillations in spheroids (multivariate plot below). In fact, the drugs exacerbated the condition.

[0046] Referring to Figure 5, this shows spontaneous calcium peaks captured and plotted from random microtiter wells of WT, RTT, and RTT spheroids exposed to flumazenil and BIMU-8 over a two-week exposure period. As can be seen in the bottom two images, the drug candidate nearly restored the WT state of peak frequency and amplitude behavior of the cell spheroids. Based on these results and multivariate analysis, the assay identified approximately 10% of the SMART library as therapeutic candidates and potential drugs for preclinical or clinical trials. Therapeutic agents for Rett syndrome targeted by the SMART library include, but are not limited to, acetazolamide, atomoxetine (tomoxetine), benzhexol hydrochloride, BIMU-8, eletriptan HBr Salt, iloperidone, trazodone (Beneficat), valproic acid (DPA), baclofen, benzydiamine hydrochloride, bromoindirubin-3-oxime, biperiden, citalopram, clebopride malate, donepezil, flumazenil, hydroxyzine dichloride, IDRA-21, ondansetron, paroxetine, pimavanserin, pirlindole mesylate, selegiline hydrochloride, and vinpocetine. These promising therapeutics and their derivatives are also good candidates for the treatment of Rett syndrome. Compounds that are effective in Rett syndrome may also be effective in other autistic conditions.

[0047] Spheroids, such as those formed from two or more different cell types, can be prepared using any suitable medium, optionally containing one or more different growth factors, and any suitable conditions. For example, spheroids formed from neurons and astrocytes can be prepared in one embodiment using one or more of the following media and / or conditions: BrainPhys™ Neuronal Medium (StemCell Tech; Catalog No. 05792; StemCell Technologies) supplemented with 1×SM1 Neuronal Supplement, 20 ng / mL BDNF (Cat. No. 78005; StemCell Technologies), 20 ng / mL GDNF (Cat. No.; StemCell Technologies), and penicillin / streptomycin (Cat. No. SV30010; GE Healthcare Life Sciences). Cells are maintained at 37°C in a 5% CO2 and humidified incubator.

[0048] The present subject matter includes contacting a plate, e.g., a multiwell plate having wells containing one or more spheroids of human cells of uniform diameter, a fluorescent molecule useful for detecting calcium, and one or more test compounds; and optionally optically detecting the amount or change in fluorescence over time in each well. In various examples, the method measures the amount of fluorescence peaks, the amplitude of one or more peaks, the peak spacing between one or more peaks, the width of one or more peaks, or any combination thereof. In various examples, the method includes producing spheroids containing neurons, or spheroids containing neurons and astrocytes, or spheroids containing cardiac cells, hepatic cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells, or spheroids containing cancer cells. In various examples, the spheroids may contain multiple different cell types. In some examples, the cells are derived from human iPS cells. In some examples, the cells are differentiated cells. In some examples, the cells are progenitor cells. In some examples where progenitor cells are used, the progenitor cells are progenitor cells for neurons, astrocytes, cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells.

[0049] In various of the methods described above, the spheroids may have a diameter of about 500 to about 600 microns or about 450 to about 500 microns. In various of the methods described above, the spheroids may be cultured for at least 6 weeks. In various of the methods described above, the fluorescent molecule comprises Calcium 3, Calcium 4, Calcium 5, Calcium 6, Fluo 3, or Fluo 4.

[0050] In one embodiment, an optical assay is provided, such as a functional FLIPR assay or high-content, high-magnification optical microscopy of 3D human cell spheroids, e.g., spheroids formed from a mixed population of neurons, oligodendrocytes, microglial cells, endothelial cells, or any combination thereof.

[0051] In one embodiment, a multi-well optical assay, such as a functional FLIPR assay, of 3D mixed population human cell spheroids in a multi-well format, for example, 96, 384, or 1536 microplate wells, for example, in a round bottom well format, is provided.

[0052] Additionally, optical assays, e.g., functional FLIPR assays, of 3D mixed population spheroids are provided, where the spheroids within each microplate well are uniform in size, e.g., diameter + / - 50 or + / - 25 microns. In one embodiment, FLIPR generates real-time functional data on 3D neuron-based cell spheroids that are highly consistent within a microplate, e.g., well-to-well and plate-to-plate.

[0053] In one embodiment, the present disclosure provides optical assays, including functional FLIPR assays, of 3D mixed population spheroids responding in real time to agonist or antagonist drug challenges.

[0054] In one embodiment, the present disclosure provides optical assays, such as functional FLIPR assays, of 3D mixed population spheroids derived from human primary cells, iPSCs, differentiated cells, or other human cell lines.

[0055] Exemplary Treatment Methods and Compositions The present disclosure provides methods for preventing or alleviating, e.g., suppressing or treating, in a human, one or more symptoms associated with a disorder, such as autism spectrum disorder or Rett syndrome. In some embodiments, methods are provided for suppressing or treating a neurological or developmental symptom of the disease.

[0056] Also provided are methods for reducing the risk, progression, or onset of diseases characterized by growth retardation, loss of normal movement and coordination, loss of ability to communicate, abnormal hand and eye movements, breathing problems, cognitive impairment, seizures, scoliosis, irregular heartbeat, or sleep disorders.

[0057] Further provided are methods for reducing the risk, reducing the severity, or slowing the progression or onset of Rett's Disease.

[0058] In one embodiment, the composition administered comprises a 5-HT4 receptor selective agonist. In one embodiment, the composition administered comprises a bicycloalkylbenzimidazolone. In one embodiment, the composition administered comprises a GABA receptor antagonist. In one embodiment, the composition administered comprises a benzodiazepine. In one embodiment, the composition administered comprises a competitive antagonist at the benzodiazepine receptor. In one embodiment, the composition administered comprises acetazolamide. In one embodiment, the composition administered comprises a selective noradrenaline reuptake inhibitor. In one embodiment, the composition administered comprises an antimuscarinic agent. In one embodiment, the composition administered comprises a selective serotonin receptor agonist. In one embodiment, the composition administered comprises a compound that promotes the release of gonadotropin-releasing hormone. In one embodiment, the composition administered comprises a selective serotonin reuptake inhibitor. In one embodiment, the composition administered comprises a branched-chain saturated fatty acid anion. In one embodiment, the administered composition comprises an inhibitor of CYP2C9, an inhibitor of glucuronyltransferase, an inhibitor of histone deacetylase, or an inhibitor of epoxide hydrolase. In one embodiment, the administered composition comprises a gamma-aminobutyric acid (GABA) agonist. In one embodiment, the administered composition comprises a locally acting nonsteroidal anti-inflammatory drug (NSAID), e.g., with local anesthetic and analgesic properties. In one embodiment, the administered composition comprises a biindole, e.g., indirubin. In one embodiment, the administered composition comprises an anticholinergic. In one embodiment, the administered composition comprises a dopamine antagonist with antiemetic or prokinetic properties. In one embodiment, the administered composition comprises a selective acetylcholinesterase inhibitor. In one embodiment, the administered composition comprises an antihistamine. In one embodiment, the administered composition comprises a benzothiadiazine. In one embodiment, the administered composition induces positive allosteric modulation of glutamate AMPA receptors. In one embodiment, the composition administered comprises an anti-mimetic.In one embodiment, the composition administered comprises a selective serotonin inverse agonist. In one embodiment, the composition administered comprises an inhibitor of monoamine oxidase. In one embodiment, the composition administered comprises a reversible inhibitor of monoamine oxidase, such as a selective, reversible inhibitor of monoamine oxidase A. In one embodiment, the composition administered comprises an alkaloid. In one embodiment, the composition administered comprises a vinca alkaloid.

[0059] In one embodiment, the composition includes BIMU-8. In one embodiment, the composition includes flumazenil. In one embodiment, the composition includes acetazolamide. In one embodiment, the composition includes N-methylacetazolamide. In one embodiment, the composition includes atomoxetine (tomoxetine). In one embodiment, the composition includes benzhexol hydrochloride. In one embodiment, the composition includes eletriptan. In one embodiment, the composition includes iloperidone. In one embodiment, the composition includes trazodone. In one embodiment, the composition includes valproate. In one embodiment, the composition includes baclofen. In one embodiment, the composition includes benzydiamine hydrochloride. In one embodiment, the composition includes bromoindirubin-3-oxime. In one embodiment, the composition includes yperiden. In one embodiment, the composition includes citalopram. In one embodiment, the composition includes clebopride malate. In one embodiment, the composition includes donepezil or an analog thereof. See, for example, Saglik et al. (Eur J Med Chem. 2016 Nov 29;124:1026-1040. doi: 10.1016 / j.ejmech.2016.10.042), the disclosure of which is incorporated by reference in its entirety. In one embodiment, the composition comprises hydroxyzine dichloride. In one embodiment, the composition comprises IDRA-21. In one embodiment, the composition comprises ondansetron, dolasterone, or palonosetron. In one embodiment, the composition comprises paroxetine. In one embodiment, the composition comprises pimavanserin. In one embodiment, the composition comprises pirlindole mesylate. In one embodiment, the composition comprises selegiline hydrochloride. In one embodiment, the composition comprises vinpocetine.

[0060] Pharmaceutical Composition Pharmaceutical compositions comprising one or more of the compounds described herein suitable for administration, for example, nasal, parenteral, or oral administration, including intrathecal, intraventricular, or intraparenchymal delivery to the central nervous system, such as by intravenous, intramuscular, topical, or subcutaneous routes, or by any other route of administration that allows the drug to be delivered to the body or specific organs and tissues of the body, optionally further comprising sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further comprise adjuvants or excipients as known in the art. Compositions comprising one or more of the compounds described herein are generally presented in the form of individual doses (unit doses).

[0061] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain adjuvants or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration generally include liposome solutions containing liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as refinements. In addition to inert diluents, such compositions may also contain adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

[0062] When compositions having one or more of the compounds described herein are used for administration to an individual, they may further include salts, buffers, adjuvants, or other substances that are desirable to improve the effectiveness of the composition.

[0063] In one embodiment, the pharmaceutical composition is part of a controlled release system, e.g., one having a pump or made of a polymeric material (see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Langer & Peppas, J. Macromol. Sci. Rev. Macromol. Chem., 23:61 (1983); see also Levy et al., Science, 228:190 (1985); During et al., Ann. Neurol., 25:351 (1989); Howard et al., J. Neurosurg., 71:105 (1989)). Other controlled release systems are discussed in the review by Langer (Science, 249:1527 (1990)).

[0064] Pharmaceutical compositions containing one or more of the compounds described herein comprise a therapeutically effective amount of the compound, e.g., one identified by a screening method, and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. These compositions can be formulated as suppositories. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions contain a therapeutically effective carrier in a suitable amount to provide a form for proper administration to the patient. The formulation should follow the method of administration.

[0065] The compositions can be administered systemically, for example, orally in combination with a pharmaceutically acceptable vehicle, such as an inert diluent. For oral administration, the compounds can be combined with one or more excipients and used in the form of ingestible capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions should contain at least 0.1% of the active compound. Of course, the percentage of the compositions and preparations may be varied and may conveniently be between about 2 and about 60% of the weight of a given unit dosage form. The amount of active compound in such useful compositions is such that an effective dosage level will be obtained.

[0066] The compositions may also contain binders such as tragacanth gum, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose, or aspartame, or flavorings such as peppermint, oil of wintergreen, or cherry flavoring. Various other ingredients may also be present. For example, a syrup or elixir may contain the compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, coloring agents, and flavorings such as cherry or orange flavoring. Of course, any material used in preparing any unit dosage form, including sustained-release formulations or devices, must be pharmaceutically acceptable and substantially non-toxic in the amounts used.

[0067] The composition can also be delivered by intravenous, intraperitoneal, intraarterial, intrathecal, intraparenchymal or intracerebroventricular infusion or injection, or any other administration route suitable or appropriate for drug delivery in liquid formulations.The compound solution can be prepared in water or a suitable buffer, optionally mixed with a non-toxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, as well as oils.Under normal storage and use conditions, these formulations contain preservatives to prevent the growth of undesirable microorganisms.

[0068] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the ultimate dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of undesirable microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride.

[0069] Sterile injectable solutions are prepared by incorporating the compound in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by irradiation, steam (heat) or filter sterilization, or any other preparation method that renders the formulation essentially free of bacterial and / or viral contaminants.

[0070] Useful liquid carriers include water, alcohols, or glycols, or water-alcohol / glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for specific applications. The resulting liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0071] Useful dosages of the compositions can be determined by comparing their in vitro activity, and in vivo activity in animal models.

[0072] Illustrative Embodiments In one embodiment, a method for detecting the effect of one or more compounds on spheroids is provided. In one embodiment, the method includes contacting one or more spheroids of uniform diameter human cells with one or more test compounds, where the spheroids are obtained from cells of an autism patient, cells of a Rett syndrome patient, or cells from a model of Rett syndrome. The effect of the one or more compounds on the one or more spheroids is detected, e.g., measured, optionally in comparison with corresponding wild-type cells. In one embodiment, the one or more spheroids are located within wells of a multiwell plate. In one embodiment, each well contains one spheroid. In one embodiment, the wells are further contacted with a fluorescent molecule useful for detecting calcium, and the amount or change in fluorescence over time is detected in one or more wells. In one embodiment, the amount or change in fluorescence is detected by detecting the amount of a fluorescence peak, the amplitude of one or more peaks, the peak spacing between one or more peaks, the width of one or more peaks, or any combination thereof. In one embodiment, the one or more spheroids comprise neurons. In one embodiment, the one or more spheroids comprise neurons and astrocytes. In one embodiment, the one or more spheroids comprise cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the one or more spheroids comprise cells or oligodendrocytes. In one embodiment, the one or more spheroids comprise pericytes and endothelial cells. In one embodiment, the one or more spheroids comprise endothelial cells, microglial cells, neurons, oligodendrocytes, or any combination thereof. In one embodiment, the cells are progenitor cells. In one embodiment, the progenitor cells are progenitor cells for neurons, astrocytes, cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the one or more spheroids have a diameter of about 500 to about 600 microns. In one embodiment, the one or more spheroids have a diameter of about 450 to about 500 microns.In one embodiment, the one or more spheroids are cultured for at least six weeks before contacting with the one or more test compounds. In one embodiment, the fluorescent molecule comprises Calcium 3, Calcium 4, Calcium 5, Calcium 6, Fluo 3, or Fluo 4. In one embodiment, the method further comprises contacting the well with a cell membrane impermeant quencher. In one embodiment, the change in fluorescence is compared to the fluorescence in a well containing the spheroids and fluorescent molecule but no test compound.

[0073] In one embodiment, a method is provided for preventing, suppressing, or treating one or more symptoms of autism spectrum disorder in a human. In one embodiment, a method is provided for preventing, suppressing, or treating one or more symptoms of Rett's disease in a human. The method, in one embodiment, comprises administering to a human an effective amount of one or more of a 5-HT4 receptor selective agonist, a zabicycloalkyl benzimidazolone, a GABA receptor antagonist, a benzodiazepine, a competitive antagonist at the benzodiazepine receptor, acetazolamide, a selective noradrenaline reuptake inhibitor, an antimuscarinic agent, a selective serotonin receptor agonist, a compound that enhances the release of gonadotropin-releasing hormone, a selective serotonin reuptake inhibitor, a branched chain saturated fatty acid anion, an inhibitor of CYP2C9, an inhibitor of glucuronyltransferase, a histone dehydrogenase inhibitor, a steroid hormone receptor antagonist ... The method includes administering to a human a composition comprising an inhibitor of acetylcholine deacetylase, an inhibitor of epoxide hydrolase, a gamma-aminobutyric acid (GABA) agonist, a nonsteroidal anti-inflammatory drug (NSAID), a biindole, an anticholinergic, a dopamine antagonist, an acetylcholinesterase inhibitor, an antihistamine, a benzothiadiazine, a glutamate AMPA receptor modulator, an antiemetic, a serotonin inverse agonist, an inhibitor of monoamine oxidase, or an alkaloid. In one embodiment, the composition comprises acetazolamide, atomoxetine (tomoxetine), benzhexol hydrochloride, BIMU-8, eletriptan HBr Salt, iloperidone, trazodone (Beneficat), valproic acid DPA, baclofen, benzydiamine hydrochloride, bromoindirubin-3-oxime, biperiden, citalopram, clebopride malate, donepezil, flumazenil, hydroxyzine dichloride, IDRA-21, ondansetron, paroxetine, pimavanserin, pirlindole mesylate, selegiline hydrochloride, or vinpocetine. In one embodiment, the composition is administered orally.In one embodiment, the composition is a sustained release formulation. In one embodiment, administration is intravenous, intraarterial, subcutaneous, intranasal, intrathecal, intraventricular, intraparenchymal, transretinal, intramuscular, transdermal, or rectal. In one embodiment, the composition is a sustained release formulation. In one embodiment, the amount prevents or treats growth retardation, loss of normal movement, loss of coordination, loss of communication ability, abnormal hand movements, abnormal eye movements, breathing disorders, cognitive impairment, seizures, scoliosis, irregular heartbeat, or sleep disorders.

[0074] Also provided is a multiwell plate containing one or more mixed human cell spheroids per well. In one embodiment, the spheroids are obtained from cells of an autism patient, a Rett syndrome patient, or cells from a model of Rett syndrome. In one embodiment, the one or more spheroids comprise neurons and astrocytes. In one embodiment, the one or more spheroids comprise cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the one or more spheroids comprise microglial cells or oligodendrocytes. In one embodiment, the one or more spheroids comprise pericytes and endothelial cells. In one embodiment, the one or more spheroids comprise endothelial cells, microglial cells, neurons, oligodendrocytes, or any combination thereof. In one embodiment, the one or more spheroids comprise progenitor cells of neurons, astrocytes, cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells. In one embodiment, the wells contain spheroids formed from a variety of cells.

[0075] The above discussion is intended to illustrate the principles and various embodiments of the present invention. Once the above disclosure is fully appreciated, many variations and modifications will become apparent to those skilled in the art, such as using different cell types, e.g., heart, liver, kidney, lung, skin, pancreas, spleen, bone, in 3D spheroid formation factors. It is intended that the following claims be construed to encompass all such variations and modifications.

[0076] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described in connection with certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments, and that the specific details herein can be varied considerably without departing from the underlying principles of the invention.

Claims

1. 1. A method for detecting the effect of one or more compounds on spheroids to reverse or restore calcium oscillation activity, said method comprising: contacting one or more spheroids of uniform diameter human cells with one or more test compounds, wherein the spheroids are obtained from cells of an autism patient, cells of a Rett Syndrome patient, or cells derived from a model of Rett Syndrome; and detecting the effect of one or more compounds on one or more spheroids in reversing or restoring said calcium oscillation activity. A method comprising:

2. 10. The method of claim 1, wherein the one or more spheroids are in a well of a multi-well plate.

3. The method of claim 2, wherein each well contains one spheroid.

4. 4. The method of claim 2 or 3, wherein the wells are further contacted with a fluorescent molecule useful for detecting calcium, and the amount or change in fluorescence over time is detected in one or more wells.

5. 5. The method of claim 4, wherein the amount or change of the fluorescence is detected by detecting the amount of a fluorescence peak, the amplitude of one or more of the peaks, the peak interval between one or more of the peaks, the width of one or more of the peaks, or a combination thereof.

6. The method of any one of claims 1 to 3, wherein the one or more spheroids comprise neurons.

7. The method of any one of claims 1 to 3, wherein the one or more spheroids comprise neurons and astrocytes.

8. The method of any one of claims 1 to 3, comprising one or more spheroids comprising cardiac cells, liver cells, kidney cells, pancreatic cells, lung cells, endothelial cells or epithelial cells.

9. The method of any one of claims 1 to 3, wherein the one or more spheroids comprise cells derived from a patient with Rett disease.

10. The method of any one of claims 1 to 3, wherein the one or more spheroids comprise microglial cells or oligodendrocytes.

11. The method of any one of claims 1 to 3, comprising the one or more spheroids comprising pericytes and endothelial cells.

12. The method of any one of claims 1 to 3, wherein the one or more spheroids comprise endothelial cells, microglial cells, neurons, oligodendrocytes, or any combination thereof.

13. The method of any one of claims 1 to 3, wherein the cells are progenitor cells.

14. 14. The method of claim 13, wherein the progenitor cells are progenitor cells for neurons, astrocytes, cardiac cells, hepatic cells, kidney cells, pancreatic cells, lung cells, endothelial cells, or epithelial cells.

15. 4. The method of any one of claims 1 to 3, wherein the one or more spheroids have a diameter of 500 to 600 microns.

16. 4. The method of any one of claims 1 to 3, wherein the one or more spheroids have a diameter of 450 to 500 microns.

17. 4. The method of claim 1, wherein the one or more spheroids are cultured for at least 4 to 6 weeks before contacting with the one or more test compounds.

18. 5. The method of claim 4, wherein the fluorescent molecule comprises Calcium 3, Calcium 4, Calcium 5, Calcium 6, Fluo 3, or Fluo 4.

19. 4. The method of claim 3, further comprising contacting the well with a cell membrane impermeant quencher.

20. 5. The method of claim 4, wherein the change in fluorescence is compared to the fluorescence in a well containing spheroids and the fluorescent molecules but not the test compound.

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