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 primate medial ganglionic eminence (MGE) precursor cells, the method comprising:
providing primate pluripotent stem cells in a serum-free culture medium;
introducing to the culture medium factors comprising:
a) an activator of the sonic hedgehog (shh) pathway;
b) a SMAD inhibitor;
c) a wnt pathway inhibitor; and
d) a neural inducing supplement,
wherein the serum-free culture medium lacks Knockout Serum Replacement (KSR);
wherein the introduction of a) through d) provides a cell culture enriched in MGE precursor cells compared to a cell culture untreated by this combination of factors, and wherein the MGE precursor cells express NKX2.1 and do not detectably express PAX6.
2. The method of claim 1 , wherein the primate pluripotent stem cells are human induced pluripotent stem cells.
3. The method of claim 1 , wherein the primate pluripotent stem cells are human embryonic stem cells.
4. The method of claim 1 , wherein the primate pluripotent stem cells are cultured in the absence of a feeder layer.
5. The method of claim 1 , wherein the primate pluripotent stem cells are cultured in suspension.
6. The method of claim 1 , wherein the culture medium comprises two or more inhibitors of SMAD.
7. The method of claim 1 , wherein the culture medium further comprises an apoptosis inhibitor.
8. The method of claim 7 , wherein the inhibitor of apoptosis is an inhibitor of Rho-associated kinase (ROCK).
9. The method of claim 1 , further comprising adding a cryoprotectant to the cell culture enriched in MGE precursor cells.
10. The method of claim 1 , wherein the primate pluripotent stem cell culture provided is genetically modified.
11. The method of claim 10 , wherein the primate pluripotent stem cell culture provided expresses a fluorescent protein.
12. The method of claim 1 , further comprising isolating the MGE precursor cells from the cell culture.
13. The method of claim 12 , wherein the isolating comprises using mechanical means.
14. The method of claim 12 , wherein the isolating comprises using an affinity reagent that binds to the MGE precursor cells.
15. The method of claim 12 , wherein the isolating comprises using enzymatic means.