Derivation of midbrain dopaminergic neurons from embryonic stem cells
First Claim
19. A method of culturing dopaminergic neuronal cells, comprising:
- a. generating embryoid bodies from the suspension of single embryonic stem cells;
b. selecting central nervous system precursor cells;
c. expanding the central nervous system precursor cells by culturing the central nervous system precursor cells in an expansion medium that comprises at least one neurologic agent and lacks 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; and
d. differentiating the expanded nervous system precursor cells in an expansion medium that lacks the neurologic agent to form a culture of differentiated neuronal cells that comprises at least about 30% dopaminergic neurons.
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Abstract
The invention provides a method of culturing cells. The method generally includes live stages: (1) expansion of ES cells; (2) generation of embryoid bodies; (3) selection of CNS precursor cells; (4) expansion of CNS precursor cells; and (5) differentiation of CNS precursor cells. During the expansion phase, the CNS precursor cells are cultured in a media which includes at least one neurologic agent such as bFGF, SHH, and FGF-8. The expanded CNS precursors are differentiated by withdrawal of at least one neurologic agent, typically, bFGF. Preferably, the differentiation media includes ascorbic acid. The method of the invention can be used to culture a variety of cells, preferably neuronal cells, including, but not limited to dopaminergic neuron cells, cholinergic neuronal cells and serotonergic neuron cells. The invention also provides a method for treating a neurological disorder, such as Parkinson'"'"'s disease, a method of introducing a gene product into a brain of a patient, and an assay for neurologically active substances. The invention further provides a cell culture which includes differentiated neuron cells, of which at least about 20 % of the differentiated neurons are dopaminergic neurons.
91 Citations
46 Claims
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19. A method of culturing dopaminergic neuronal cells, comprising:
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a. generating embryoid bodies from the suspension of single embryonic stem cells;
b. selecting central nervous system precursor cells;
c. expanding the central nervous system precursor cells by culturing the central nervous system precursor cells in an expansion medium that comprises at least one neurologic agent and lacks 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid; and
d. differentiating the expanded nervous system precursor cells in an expansion medium that lacks the neurologic agent to form a culture of differentiated neuronal cells that comprises at least about 30% dopaminergic neurons. - View Dependent Claims (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 32, 33, 44, 45, 46)
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21. A method of treating a patient with neurological disorder, comprising the steps of:
administering a culture of differentiated neuronal cells to the patient wherein the culture of differentiated neuronal cells comprises at least about 30% dopaminergic neurons. - View Dependent Claims (22, 23, 24, 25, 26, 27)
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28. A method of introducing a gene product into a brain of a patient, comprising:
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a. transfecting embryonic stem cells;
b. culturing the transfected embryonic stem cells to provide a culture of differentiated neuronal cells comprising at least about 30% dopaminergic neurons; and
c. administering said differentiated transformed neuronal cells into a patient in need thereof. - View Dependent Claims (29, 30)
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31. An assay for a substance, comprising:
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a. providing a culture of differentiated neuronal cells comprising at least 30% dopaminergic neurons;
b. exposing said differentiated neuronal cells to the substance; and
c. observing the effect of the substance on the differentiated neuronal cells.
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- 34. A cell culture comprising about 50% to about 85% neurons which comprise between about 20% and 40% dopaminergic neurons and between about 1% to about 3% astrocytes.
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38. A method of culturing neurons from embryonic stem cells, comprising:
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a. expanding undifferentiated the embryonic stem cells on a surface that inhibits differentiation cells in the presence of Leukemia Inhibitory Factor (LIF);
b. disengaging the embryonic stem cells from the surface in clusters;
c. dissociating the clusters of embryonic stem cells to obtain a population which includes a majority of individual cells;
d. generating embryoid bodies in suspension;
e. culturing the embryoid bodies on a coated surface in serum free medium to select for Central Nervous System (CNS) precursor cells;
f. expanding the CNS precursor cells by culturing the cells in an expansion medium that comprises at least one neurologic agent selected from SHH, FGF8, EFG and bFGF; and
g. differentiating the expanded CNS precursor cells to form neurons by withdrawing the at least one neurologic agent from the culture. - View Dependent Claims (39, 40, 41, 42, 43)
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46-1. The method of claim 19, wherein the expansion media comprises fibroblast growth factor-8 and sonic hedgehog protein.
Specification