Personalized 3D neural culture system for generating human oligodendrocytes and studying myelination in vitro
Human pluripotent stem cells are differentiated in vitro into oligodendro-spheroids comprising oligodendrocytes for use in analysis, screening programs, and the like.
1. A method for producing a 3-dimensional culture of human interacting myelinating oligodendrocytes, myelinated neurons and astrocytes, the method comprising:
(i) inducing human pluripotent human stem cells in suspension culture to a neural fate by culturing intact colonies or aggregates of single cells of the pluripotent stem cells for a period of from 2 to 10 days in medium comprising an effective dose of an inhibitor of BMP and an inhibitor of TGFβ to generate a spheroid of cells;
(ii) differentiating the spheroid of pluripotent human stem cells to a spheroid of neural progenitors by culturing in suspension culture for a period of from 1 to 4 weeks in medium comprising fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF);
(iii) differentiating the spheroid of neural progenitors into a spheroid (hOS) comprising interacting human oligodendrocytes, human neurons, and human astrocytes by culturing in neural media lacking FGF2 and EGF and comprising an effective dose of PDGF-AA, IGF-1, HGF, BDNF, NT3, insulin, cAMP, T3, and biotin;
(iv) maintaining the hOS spheroid for one month or longer in neural medium to derive 3-dimensional cultures that comprise interacting myelinating oligodendrocytes, myelinated neurons and astrocytes.
2. The method of claim 1 , wherein the cells comprise at least one allele associated with a myelin disorder.
3. The method of any of claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.
4. The method of claim 1 , wherein the inhibitor of BMP is dorsomorphin and the inhibitor of TGFβ is SB-431542.
5. The method of claim 4 , wherein the suspension culture of (i) is feeder layer free.
6. The method of claim 1 , wherein the medium in (i) further comprises an effective dose of an inhibitor of wnt, beginning day 2 of culture.
7. The method of claim 1 , further comprising culturing the spheroids in step (i) with sonic hedgehog pathway agonists and retinoic acid; or with sonic hedgehog pathway agonists and Wnt inhibitors.
8. The method of claim 1 , wherein the neural medium in (iv) is lacking growth factors but including ascorbic acid, insulin, cAMP, T3, and biotin.
9. The method of claim 8 , further comprising isolating oligodendrocytes from an oligodendro-spheroid by flow cytometry, magnetic immunoselection, or immunopanning.
10. The method of claim 9 , wherein isolated oligodendrocytes are exposed to a candidate agent or treatment of interest.
11. The method of claim 10 , wherein the oligodendrocytes are combined with neurons or neuronal progenitor cells or glial cells.
12. The method of claim 10 , further comprising the step of determining the effect of the agent on morphologic, genetic or functional parameters.
13. The method of claim 12 , wherein the candidate agent is an immune effector cell or an immune modulatory agent.
14. The method of claim 10 , wherein a panel of oligodendrocytes comprising at least two oligodendrocytes having differing genotypes are isolated and contacted with the candidate agent or treatment of interest.
15. The method of claim 10 , wherein a panel of oligodendrocytes comprising at least two oligodendrocytes under differing culture conditions are isolated and contacted with the candidate agent or treatment of interest.
16. The method of claim 10 , wherein the isolated oligodendrocytes comprise at least one allele associated with a neural disease.