Method of using human spheroids for drug discovery

Abstract

The present invention discloses, 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. 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.

1 . A method to detect the effect of one or more compounds on spheroids, comprising:
 contacting one or more spheroids of human cells of uniform diameter and one or more test compounds, wherein the spheroids are obtained from cells of an autism patient, cells of a Rett syndrome patient, or cells that are from a model of Rett syndrome; and   detecting the effect of the one or more compounds on one or more of the spheroids.   
     
     
         2 . The method of  claim 1  wherein the one or more spheroids are in wells of a multi-well plate. 
     
     
         3 . The method of  claim 2  wherein each well has one spheroid. 
     
     
         4 . The method of  claim 2  wherein the wells are further contacted with a fluorescent molecule useful to detect calcium, and the amount or change in fluorescence over time is detected in one or more wells. 
     
     
         5 . The method of  claim 4  wherein the amount or change in fluorescence is detecting a quantity of peaks of fluorescence, an amplitude of one or more of the peaks, peak spacing between one or more of the peaks, a width of one or more peaks, or any combination thereof. 
     
     
         6 . The method of  claim 1  wherein the one or more spheroids comprise neurons. 
     
     
         7 . The method of  claim 1  wherein the one or more spheroids comprise neurons and astrocytes. 
     
     
         8 . The method of  claim 1  wherein the one or more spheroids comprise cells from a Rett disease patient 
     
     
         9 . The method of  claim 1  wherein the one or more spheroids comprise microglial cells or oligodendrocytes. 
     
     
         10 . The method of  claim 1  wherein the one or more spheroids have a diameter of about 450 to about 600 microns. 
     
     
         11 . 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. 
     
     
         12 . The method of  claim 3  wherein the fluorescent molecule comprises Calcium 3, Calcium 4, Calcium 5, Calcium 6, Fluo 3, or Fluo4. 
     
     
         13 . The method of  claim 3  wherein the amount of change in fluorescence is compared to the fluorescence in a well with spheroids and the fluorescent molecule but no test compound. 
     
     
         14 . A method or prevent, inhibit or treat one or more symptoms of Rett syndrome in a human, comprising: administering to the patient a composition comprising an effective amount of one or more of a 5-HT 4  receptor selective agonist, a zabicycloalkyl benzimidazolone, a GABA receptor antagonist, a benzodiazepine, a competitive antagonist at the benzodiazepine receptor, an acetazolamide, a selective noradrenaline reuptake inhibitor, an antimuscarinic, a selective serotonin receptor agonist, a compound that enhances release of gonadotropin releasing hormone, a selective serotonin reuptake inhibitor, a branched-chain saturated fatty acid anion, an inhibitor of CYP2C9, an inhibitor of glucuronyl transferase, an inhibitor of histone 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 modulator of glutamate AMPA receptors, an anti-emetic, a serotonin inverse agonist, an inhibitor of monoamine oxidase, or an alkaloid, or any combination thereof. 
     
     
         15 . The method of  claim 14  wherein the composition comprises acetazolamide, atomoxetine (tomoxetine), benzhexol hydrochloride, BIMU-8, eletriptan HBr Salt, iloperidone, trazodone (Beneficat), valproate DPA, baclofen, benzydiamine hydrochloride, bromoindirubin-3-oxime, biperiden, citalopram, clebopride malate, bonepezil, flumazenil, hydroxyzine dichloride, IDRA-21, ondansetron, paroxetine, pinmavanserin, pirlindole mesylate, selegiline hydrochloride, or vinpocetine, or any combination thereof. 
     
     
         16 . The method of  claim 14  wherein the composition is orally administered. 
     
     
         17 . The method of  claim 14  wherein the composition is a sustained release formulation. 
     
     
         18 . The method of  claim 14  wherein administration is intravenous, intra-arterial, subcutaneous, intranasal, intrathecal, intracerebroventricular, intraparenchymal, trans-retinal, intramuscular, transdermal, or rectal. 
     
     
         19 . The method of  claim 14  wherein the composition is a sustained release formulation. 
     
     
         20 . The method of  claim 14  wherein the amount inhibits or treats delayed growth, loss of normal movement, loss of coordination, loss of communication abilities, abnormal hand movements, abnormal eye movements, breathing problems, cognitive disabilities, seizures, scoliosis, irregular heartbeat, or sleep disturbances