# US 2018/0021383 A1 · Production of midbrain dopaminergic neurons and methods for the use thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0021383 A1 - Document ID: US20180021383A1 - Status: Published application - Kind: A1 - Application number: 15/614,755 - Filed: 2017-06-06 - Published: 2018-01-25 - Earliest claimed: 2014-03-21 - Assignees: Cellular Dynamics International, Inc. - Applicants: Cellular Dynamics International, Inc. - Inventors: Matt GEORGE; Carrie CHAVEZ; Chris McMAHON; Wen Bo WANG; Lucas CHASE; Brad SWANSON - Web page: https://patentgrep.com/patent/US20180021383A1 ## CPC - A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue - A61J 1/00: Containers specially adapted for medical or pharmaceutical purposes (capsules or the like for oral use A61J3/07; specially adapted for surgical or diagnostic appliances or instruments A61B50/30; containers for radioactive substances G21F5/00) - A61P 25/16: Anti-Parkinson drugs - B65D 25/205: {Means for the attachment of labels, cards, coupons or the like; (cards or coupons for the other types of rigid or semi-rigid containers B65D25/36 takes precedence)} - C12N 5/0619: {Neurons} - C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)} - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - G01N 33/5058: {Neurological cells} - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor - C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor) - C12N 2501/415: Wnt; Frizzeled - C12N 2501/727: Kinases (EC 2.7.) - C12N 2506/45: from artificially induced pluripotent stem cells - C12Q 2600/136: Screening for pharmacological compounds - C12Q 2600/158: Expression markers - C12Y 114/16002: Tyrosine 3-monooxygenase (1.14.16.2) - G01N 2333/4706: stimulating, promoting or activating activity - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61J: CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - B65D: CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Y: ENZYMES ## Abstract Methods are provided for efficient production of midbrain dopaminergic (DA) neurons. In some aspects, methods involve differentiation and selection of DA neurons for a transgenic pluripotent cell population (e.g., cells comprising a selectable marker gene). Cell populations produced by the instant methods and methods of their use are likewise provided. ## Claims ### Claim at position 1 1-44. (canceled) ### Claim 45 A method for providing an enriched population of midbrain dopaminergic (DA) neurons comprising: differentiating cells of a population of human induced pluripotent cells to provide a neural lineage cell population; further differentiating cells of the neural lineage cell population to generate a cell population which includes midbrain neurons; and purifying cells from said cell population using a transgenic screenable or selectable marker under the control of a pan-neural promoter expressed by cells of the cell population, to provide an enriched population of midbrain DA neurons; wherein the differentiation differentiation steps are carried out in a chemically defined medium that is free of feeder cells and feeder cell extracts and at least 80% of cells in the enriched population are positive for both Lmx1 and FoxA2 expression. ### Claim 46 (depends on 45) The method of claim 45, wherein the enriched population of midbrain dopaminergic (DA) neurons comprise mammalian midbrain dopaminergic (DA) neuronal cells, at least about 85% the neuronal cells being positive for both LIM homeobox transcription factor 1 (Lmx1) and forkhead box A2 (FoxA2) expression. ### Claim 47 (depends on 46) The method of claim 46, wherein the neuronal cells comprise at least 500,000 cells. ### Claim 48 (depends on 47) The method of claim 47, wherein the neuronal cells comprise at least 1 million cells. ### Claim 49 (depends on 46) The method of claim 46, wherein at least about 80% of cells in the enriched population are positive for TH expression. ### Claim 50 (depends on 45) The method of claim 45, wherein the pan-neural promoter is a TuJ-1, Map-2, Dcx, Synapsin, enolase 2, glial fibrillary acidic protein, or tubulin alpha-1A chain promoter. ### Claim 51 (depends on 50) The method of claim 50, wherein the pan-neural promoter is the Map-2 promoter. ### Claim 52 (depends on 45) The method of claim 45, wherein differentiating cells into a neural lineage cell population comprises culturing a population of pluripotent cells in a medium comprising a BMP signaling inhibitor; a TGFβ signaling inhibitor; an activator of Sonic hedgehog (SHH) signaling; and an activator of Wnt signaling. ### Claim 53 (depends on 52) The method of claims 52, comprising culturing a population of pluripotent cells in a medium further comprising a MEK inhibitor and/or FGF8. ### Claim 54 (depends on 45) The method of claim 45, further defined as comprising: (a) obtaining a population of pluripotent cells; (b) culturing the population of cells in media comprising: a BMP signaling inhibitor; a TGFβ signaling inhibitor; an activator of Sonic hedgehog (SHH) signaling; and an activator of Wnt signaling; (c) transferring the cell population to a suspension culture in a media comprising a BMP signaling inhibitor; an activator of SHH signaling; and an activator of Wnt signaling, thereby forming cell aggregates; (d) dissociating cell aggregates and seeding the dissociated cells into a matrix culture to provide a neural lineage cell population; (e) further differentiating the neuronal lineage cell population in a maturation media comprising neuronal maturation factors to generate a cell population which includes midbrain neurons; and (f) purifying the cells using a marker, said marker being expressed from a pan-neural promoter, to provide an enriched population of midbrain neurons. ### Claim 55 A method for providing an enriched population of midbrain dopaminergic (DA) neurons comprising: obtaining a population of human induced pluripotent cells; differentiating the cells into a neural lineage cell population in a medium comprising a MEK inhibitor; and further differentiating cells of the neural lineage cell population to provide an enriched population of midbrain DA neurons; and wherein said differentiation steps are carried out in chemically defined medium that is free of feeder cells and feeder cell extracts and at least 80% of cells in the enriched population are positive for both Lmx1 and FoxA2 expression. ### Claim 56 (depends on 55) The method of claim 55, further defined as comprising: (a) obtaining a population of pluripotent cells; (b) culturing the population of cells in media comprising: a BMP signaling inhibitor; a TGFβ signaling inhibitor; an activator of Sonic hedgehog (SHH) signaling; an activator of Wnt signaling and not containing exogenously added FGF8b; (c) transferring the cell population to a suspension culture in a media not containing exogenously added FGF8b and comprising a BMP signaling inhibitor; an activator of SHH signaling; an activator of Wnt signaling; a MEK inhibitor; and not containing exogenously added FGF8b, thereby forming cell aggregates; (d) dissociating cell aggregates and seeding the dissociated cells into a matrix culture; and (e) further differentiating the cells to generate an enriched population of midbrain DA neurons. ### Claim 57 (depends on 45, 55) The method of claim 45 or 55, wherein the cells are human cells. ### Claim 58 (depends on 45, 55) The method of claim 45 or 55, wherein the cells are from a patient with a symptom of Parkinson's disease. ### Claim 59 (depends on 45, 55) The method of claim 45 or 55, wherein the method does not comprise purification of cells using a DA-specific marker. ### Claim 60 (depends on 45, 55) The method of claim 45 or 55, wherein enriched population of midbrain DA neurons comprises at least 1×102, 1×103, 1×104, 1×105, or 1×106 midbrain DA neuron cells.