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 of providing a cell culture enriched for GABAergic neuronal precursors, the 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; and
c) introducing a Notch pathway inhibitor to the cell culture enriched in MGE precursor cells;
wherein steps a) through c) result in a cell culture enriched in GABAergic neuronal precursors.
2. The method of claim 1 , wherein the GABAergic neuronal precursors co-express NKX2.1-GFP and i12b-RFP.
3. The method of claim 1 , wherein the GABAergic neuronal precursors have downregulated expression of NKX2.1.
4. The method of claim 1 , wherein the pPS cells are provided in a serum-free culture medium.
5. The method of claim 1 , wherein the pPS cells are human induced pluripotent stem cells.
6. The method of claim 1 , wherein the pPS cells are human embryonic stem cells.
7. The method of claim 1 , wherein the pPS cells are cultured in the absence of a feeder layer.
8. The method of claim 1 , wherein the pPS cells are cultured in suspension.
9. The method of claim 1 , wherein two or more inhibitors of SMAD are introduced to the cells cultured to enrich for MGE precursor cells.
10. The method of claim 1 , wherein an apoptosis inhibitor is introduced to the cells cultured to enrich for MGE precursor cells.
11. The method of claim 10 , wherein the apoptosis inhibitor is an inhibitor of Rho-associated kinase (ROCK).
12. The method of claim 1 , wherein a neural supplement is added to the culture.
13. The method of claim 1 , wherein an anti-mitotic compound is added to the cell culture enriched in MGE precursor cells to enrich for GABAergic neuronal precursors.
14. The method of claim 1 , wherein a neurotrophic factor is added to the culture to enrich for GABAergic neuronal precursors.
15. The method of claim 1 , further comprising adding a cryoprotectant to the cell culture enriched in GAB Aergic neuronal precursors.
16. The method of claim 1 , wherein the pPS cells are genetically modified.
17. The method of claim 16 , wherein the pPS cells express a fluorescent protein.
18. The method of claim 1 , further comprising isolating the GABAergic neuronal precursors.
19. The method of claim 18 , wherein the isolating comprises using mechanical means.
20. The method of claim 18 , wherein the isolating comprises using an affinity reagent that binds to the GABAergic neuronal precursors.
21. The method of claim 18 , wherein the isolating comprises using enzymatic means.