In vitro production of medial ganglionic eminence precursor cells
Methods and systems for generating MGE precursor cells in vitro as well as compositions of enriched MGE precursor cells are provided. The methods and systems provide efficient production of MGE precursors. The methods and systems disclosed herein provide functional MGE precursors which differentiate into functional GABAergic interneurons.
1. A method comprising:
a) providing primate pluripotent stem (pPS) cells in a culture medium;
b) introducing to the culture medium, factors comprising:
i) an activator of the sonic hedgehog (shh) pathway,
ii) a SMAD inhibitor, and
iii) a wnt pathway inhibitor,
to produce a cell culture enriched in MGE precursor cells;
c) culturing the MGE precursor cells to produce PSA-NCAM-negative or DLX2-negative cells; and
d) enriching for the PSA-NCAM-negative or DLX2-negative cells.
2. The method of claim 1 , wherein step c) comprises culturing the MGE precursor cells in absence of a Notch pathway inhibitor.
3. The method of claim 1 , wherein the MGE precursor cells express Nkx2.1.
4. The method of claim 1 , wherein the MGE precursor cells express OLIG2.
5. The method of claim 1 , wherein the human pluripotent stem cells comprise human induced pluripotent stem cells.
6. The method of claim 1 , wherein the human pluripotent stem cells comprise human embryonic stem cells.
7. The method of claim 1 , wherein the MGE precursor cells are genetically modified.
8. The method of claim 7 , wherein the MGE precursor cells express a fluorescent protein.
9. The method of claim 1 , wherein the primate pluripotent stem cells are cultured in the absence of a feeder layer.
10. The method of claim 1 , wherein the primate pluripotent stem cells are cultured in suspension.
11. The method of claim 1 , wherein the culture medium comprises two or more inhibitors of SMAD.
12. The method of claim 1 , wherein the culture medium further comprises an apoptosis inhibitor.
13. The method of claim 1 , wherein the inhibitor of apoptosis is an inhibitor of Rho-associated kinase (ROCK).
14. The method of claim 1 , wherein a neural supplement is added to the culture medium.
15. The method of claim 1 , wherein a neurotrophic factor is added to the culture medium.
16. The method of claim 1 , wherein a growth factor is added to the culture medium.
17. The method of claim 1 , wherein enriching for the PSA-NCAM negative cells comprises isolating PSA-NCAM-negative cells.
18. The method of claim 1 , comprising enriching for the PSA-NCAM-negative cells by removing PSA-NCAM-positive cells.
19. The method of claim 1 , comprising enriching for DLX2-negative cells by isolating DLX2-negative cells.
20. The method of claim 1 , comprising enriching for the DLX2-negative cells by removing DLX2-positive cells.
21. The method of claim 1 , comprising enriching for the DLX2-negative cells.
22. The method of claim 1 , wherein the PSA-NCAM-negative cells are progenitor cells.
23. The method of claim 1 , further comprising transplanting the PSA-NCAM-negative cells into nervous system of a mammal.
24. The method of claim 1 , wherein step c) comprises culturing the MGE precursor cells to produce PSA-NCAM-negative cells.
25. The method of claim 24 , comprising enriching for the PSA-NCAM-negative cells by removing PSA-NCAM-positive cells.
26. The method of claim 24 , wherein the culture medium comprises two or more inhibitors of SMAD.
27. The method of claim 26 , wherein the enriching comprises isolating PSA-NCAM-negative cells.