초록 ▼

The present invention relates to a method for identifying molecules promoting or inhibiting dopaminergic neuronal differentiation and/or death of dopaminergic neurons in a three-dimensional cell culture. Furthermore, the present invention relates to a method for producing dopaminergic neurons in a t

The present invention relates to a method for identifying molecules promoting or inhibiting dopaminergic neuronal differentiation and/or death of dopaminergic neurons in a three-dimensional cell culture. Furthermore, the present invention relates to a method for producing dopaminergic neurons in a three-dimensional cell culture. In addition, the present invention relates to a method for segmenting an image of a cell culture.

대표청구항 ▼

1. A method for identifying molecules promoting or inhibiting dopaminergic neuronal differentiation and/or death of dopaminergic neurons in a three-dimensional cell culture, the method comprising a) differentiating neuroepithelial stem cells (NESCs) in a differentiation medium, wherein the different

1. A method for identifying molecules promoting or inhibiting dopaminergic neuronal differentiation and/or death of dopaminergic neurons in a three-dimensional cell culture, the method comprising a) differentiating neuroepithelial stem cells (NESCs) in a differentiation medium, wherein the differentiation medium comprises (i) a sonic hedgehog (SHH)-pathway activator;(ii) at least two different neurotrophins, and(iii) an antioxidant;b) further differentiating the cells obtained in a) in a differentiation medium, wherein the differentiation medium comprises (i) at least two different neurotrophins, and(ii) an antioxidant; andc) adding a molecule of interest to the differentiation medium in a) and/or b),wherein an increase of the differentiation into dopaminergic neurons compared to a control indicates that the molecule of interest promotes dopaminergic neuronal differentiation and/or inhibits death of dopaminergic neurons and wherein a decrease of the differentiation into dopaminergic neurons compared to a control indicates that the molecule of interest inhibits dopaminergic neuronal differentiation and/or induces death or neurodegeneration of dopaminergic neurons. 2. The method of claim 1, wherein the differentiation into dopaminergic neurons is measured by measuring the expression of tyrosine hydroxylase (TH) or by measuring the expression of TH among class III β-tubulin (Tujl-)positive neurons. 3. The method of claim 1, wherein the death of dopaminergic neurons is measured by measuring fragmentation of TH-positive neurons. 4. The method of claim 1, wherein the NESC is obtained from an induced pluripotent stem cell (iPSC). 5. The method of claim 4, wherein the iPSCs have been obtained from a peripheral blood Mononuclear Cells (PBMCs) from blood, keratinocyte, T-cell, CD34+ cell, myeloid cell, or a renal epithelial cell or fibroblasts. 6. The method of claim 4, wherein the iPSCs have been obtained from a subject suffering from a neurodegenerative disease. 7. The method of claim 6, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and frontotemporal dementia. 8. The method of claim 1, wherein the differentiation medium in b) does not comprise a SHH-pathway activator. 9. The method of claim 1, wherein the differentiation medium does not comprise fibroblast growth factor 8 (FGF8). 10. The method of claim 1, wherein molecule of interest is a siRNA, miRNA, binding molecule, small molecule or compound. 11. A method for producing dopaminergic neurons in a three-dimensional cell culture, the method comprising a) contacting neuroepithelial stem cells (NESCs) with a matrix and/or a scaffold and optionally further a maintenance medium;b) plating the NESCs in a container, wherein the container does not comprise a mean for generating fluid flow, thereby forming a three dimensional gel comprising NESCs;c) differentiating NESCs obtained in b) in a differentiation medium, wherein the differentiation medium comprises (i) a SHH-pathway activator;(ii) at least two different neurotrophins; and(iii) an antioxidant;d) further differentiating the cells obtained in c) in a differentiation medium, wherein the differentiation medium comprises (i) at least two different neurotrophins; and(ii) an antioxidant;thereby differentiating said NESCs into dopaminergic neurons. 12. The method of claim 11, wherein the container does not comprise an electronic device and/or mechanic element. 13. The method of claim 12, wherein the electronic device is a pump. 14. A dopaminergic neuron obtainable by a method of claim 11. 15. The dopaminergic neuron of claim 14 for use in the treatment of a disease. 16. The method of claim 11, wherein the three-dimensional cell culture is not an organoid culture. 17. The method of claim 11, wherein the three-dimensional cell culture is not a midbrain organoid culture. 18. The method of claim 11, wherein in the three-dimensional cell culture at least about 25%, of all cells are neurons. 19. The method of claim 11, wherein in the three-dimensional cell culture less than about 50% of all neurons are dopaminergic neurons.