Compositions and methods for generating oligodendrocyte precursors
The present disclosure provides a three-dimensional culture system comprising a biocompatible polymer and a combination of factors in an amount effective to induce differentiation of oligodendrocyte precursors and/or oligodendrocytes from pluripotent stem cells. The present disclosure provides methods of generating oligodendrocyte precursors and/or oligodendrocytes using a three-dimensional culture system of the present disclosure. The present disclosure provides methods to treat neurological diseases and demyelinating diseases.
1. A three-dimensional culture system comprising:
a) biocompatible thermoresponsive polymer that forms a hydrogel at 37° C.; and
b) a combination of factors that promote differentiation of oligodendrocyte precursors from pluripotent stem cells in 20 days or less, wherein the combination of factors consists of a Sonic hedgehog (Shh) signaling pathway agonist and retinoic acid (RA); and
wherein the thermoresponsive polymer comprises:
an N-isopropylacrylamide co-monomer;
an alkyl [meth]acryl[ate/amide] co-monomer, wherein the alkyl is a lower alkyl group;
a PEG acrylamide co-monomer; and
a modifying acryl[ate/amide] co-monomer comprising a linked functional group or a linked modifying agent,
wherein the thermoresponsive polymer is described by formula (II):
wherein:
a, b, c and d are molar fractions of the co-monomers, wherein a >0.8, 0.1>b>0, and 0.2>c>0;
PEG n is a polyethylglycol polymer;
Z 2 is a functional group or a linked modifying agent;
L is a linker;
R 1 is a lower alkyl;
G 1 and G 2 are each independently selected from a polymer segment, a terminal group, a linker and a linked modifying agent.
2. The culture system of claim 1 , wherein:
the Shh signaling pathway agonist is 3,4-dichloro-N-(cis-4-(methylamino)cyclohexyl)-N-(3-pyridin-4-ylbenzyl)benzo[b]thiophene-2-carboxamide, an Shh polypeptide, an Shh protein-polymer conjugate, or a compound of the formula:
3. The culture system of claim 1 , comprising a pluripotent stem cell.
4. The three-dimensional culture system of claim 1 , wherein the Shh signaling pathway agonist is a small molecule.
5. The three-dimensional culture system of claim 1 , wherein the alkyl [meth]acryl[ate/amide] co-monomer is an isobutyl methacrylate co-monomer.
6. The three-dimensional culture system of claim 1 , wherein R 1 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, tert-butyl, cyclopropyl, and cyclobutyl.
7. The three-dimensional culture system of claim 1 , wherein Z 2 is a chemoselective functional group.
8. The three-dimensional culture system of claim 1 , wherein the thermoresponsive polymer is described by the formula (IV):
9. The three-dimensional culture system of claim 1 , wherein G 1 and G 2 are each independently selected from a terminal group, a linker and a linked modifying agent.
10. The three-dimensional culture system of claim 1 , wherein G 1 , G 2 or Z 2 comprise a linked hyaluronic acid that is linked via conjugation to the carboxylic acid group of a hyaluronic acid monomer.
11. The three-dimensional culture system of claim 1 , wherein G 1 , G 2 or Z 2 comprise the following structure:
12. The three-dimensional culture system of claim 11 , wherein Z 2 comprises the following structure:
13. The three-dimensional culture system of claim 1 , wherein G 1 and/or G 2 comprises the following structure: