Methods and compositions for producing stem cell derived dopaminergic cells for use in treatment of neurodegenerative diseases
AssigneeBiolamina AB
InventorsAgnete Kirkeby, Malin Pernilla Parmar
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Abstract
The present disclosure relates to methods for producing dopaminergic cells and evaluating their functionality. When pluripotent human embryonic stem cells are cultured on plates coated with laminin-111, laminin-121, laminin-521, laminin-421, or laminin-511 in cell culture medium containing a GSK3 inhibitor and a TGF-β inhibitor as well as timely administered fibroblast growth factor, desired neural cells are produced at far higher rates. Useful cell culture kits for producing such dopaminergic cells are also described herein, as are methods of using such cells for stem cell therapy.
Claims12
A method for inducing production of dopaminergic cells, comprising:
- (a) plating pluripotent stem cells on a substrate coated with laminin-111, laminin-121, laminin-521, laminin-421, or laminin-511,
- (b) culturing the plated cells in a first medium comprising a neural induction medium or an N2 medium;
- a rho-associated kinase (ROCK) inhibitor;
- a transforming growth factor beta (TGF-β) inhibitor;
- and a glycogen synthase kinase 3 (GSK3) inhibitor,
- (c) removing the first medium and adding a second medium comprising a neural induction medium or an N2 medium;
- a TGF-β inhibitor, and a GSK3 inhibitor, and not containing a ROCK inhibitor,
- (d) removing the second medium and adding a third medium comprising (i) a neural proliferation medium or an N2 medium;
- and (ii) a TGF-β inhibitor, and optionally also contains sonic hedgehog protein,
- (e) replacing the third medium with a fourth medium comprising (i) a neural proliferation medium (NPM) or an N2 medium;
- and (ii) a fibroblast growth factor (FGF),
- (f) replating the cells on a second plate coated with laminin-111, laminin-121, laminin-521, laminin-421, or laminin-511, and
- (g) culturing the replated cells in a fifth medium comprising a B27 medium;
- a brain derived neurotrophic factor (BDNF);
- ascorbic acid (AA);
- a glial cell line-derived neurotrophic factor (GDNF);
- and a fibroblast growth factor (FGF).
Dependencies are inferred from the claim text.