Neural progenitor cell populations
This invention provides populations of neural progenitor cells, differentiated neurons, glial cells, and astrocytes. The populations are obtained by culturing stem cell populations (such as embryonic stem cells) in a cocktail of growth conditions that initiates differentiation, and establishes the neural progenitor population. The progenitors can be further differentiated in culture into a variety of different neural phenotypes, including dopaminergic neurons. The differentiated cell populations or the neural progenitors can be generated in large quantities for use in drug screening and the treatment of neurological disorders.
1. A method of producing a culture comprising dopaminergic neurons, comprising:
a) initiating differentiation of human embryonic stem (hES) cells;
b) culturing the initiated cells from a) in a medium comprising basic fibroblast growth factor (bFGF), optionally further comprising one or more factors selected from neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), sonic hedgehog (SHH) and fibroblast growth factor 8(FGF8) for a first culture period; and
c) culturing the cells from b) in a medium comprising neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), or alternatively comprising cyclic adenosine monophosphate (cAMP) and ascorbic acid for a second culture period sufficient to produce a culture comprising dopaminergic neurons.
2. The method of claim 1 , wherein the hES cells express SSEA3, SSEA4, Tra-1-60, and Tra-1-81.
3. The method of claim 1 , wherein the first culture period is for about six days.
4. The method of claim 1 , wherein the medium used for the second culture period comprises NT-3 and BDNF.
5. The method of claim 1 , wherein the medium used for the second culture period comprises cAMP and ascorbic acid.
6. The method of claim 1 , wherein the medium used for the second culture period does not comprise an added mitogen.
7. The method of claim 1 , wherein the second culture period is for about six days.
8. The method of claim 1 , wherein the culture comprising dopaminergic neurons comprises MAP-2 positive cells.
9. The method of claim 8 , wherein the culture comprising dopaminergic neurons comprises at least 21% MAP-2 positive cells.
10. The method of claim 9 , wherein the culture comprising dopaminergic neurons comprises from 21% to 37% MAP-2 positive cells.
11. The method of claim 8 , wherein the MAP-2 positive cells comprise TH positive cells.
12. The method of claim 11 , wherein at least 3.5% of the MAP-2 positive cells are TH positive.
13. The method of claim 12 , wherein from 3.5% to 8.7% of the MAP-2 positive cells are TH positive.