Methods and compositions for the production of oligodendrocyte progenitor cells
Provided herein are pluripotent stem cells comprising particular combinations of transcription factor for the production of oligodendrocyte progenitor cells (OPCs). Also provided herein are methods of producing the pluripotent stem cells and methods of using the pluripotent stem cells to produce (OPCs) and oligodendrocytes.
1. A method comprising:
introducing into pluripotent stem cells at least four transcription factors selected from (a) oligodendrocyte 2 (OLIG2), (b) SRY-box 9 (SOX9), (c) NKX homeobox 6.1 (NKX6.1), (d) NKX homeobox 6.2 (NKX6.2), and (e) octamer-binding 4 (OCT4), wherein each of the at least four transcription factors is encoded by a coding sequence operably linked to an inducible promoter; and
culturing the pluripotent stem cells in the presence of an inducing agent that activates the inducible promoter, thereby activating transcription of the at least four transcription factors and producing a cell population comprising oligodendrocyte progenitor cells (OPCs).
2. The method of claim 1 , wherein the at least four transcription factors comprise OCT4, SOX9, NKX6.1 and NKX6.2.
3. The method of claim 2 , wherein the at least four transcription factors comprise OLIG2, OCT4, SOX9, NKX6.1 and NKX6.2.
4. The method of claim 1 , wherein the culturing comprises culturing the pluripotent stem cells for 1 to 10 days.
5. The method of claim 1 , wherein at least 20% of the cells of the cell population express O4 after 4-10 days of the culturing.
6. The method of claim 5 , wherein at least 40% of the cells of the cell population express O4 after 4-10 days of the culturing.
7. The method of claim 1 , further comprising culturing the OPCs with another cell type to produce a myelinated organoid.
8. The method of claim 1 , wherein each of the at least four transcription factors is introduced on an engineered nucleic acid comprising a nucleotide sequence encoding the respective transcription factor.
9. The method of claim 8 , wherein at least two of the at least four transcription factors are encoded by the same engineered nucleic acid.
10. The method of claim 1 , wherein at least 20% of the cells of the cell population comprising OPCs express O4.
11. The method of claim 10 , wherein at least 20% of the cells of the cell population comprising OPCs express O4 within four days following the inducing.
12. The method of claim 11 , wherein at least 40% of the cells of the cell population comprising OPCs express O4 within four days following the inducing.
13. The method of claim 1 , wherein at least one engineered nucleic acid encoding a cytokine selected from IL-10 and IFNβ is introduced into the pluripotent stem cells.
14. The method of claim 13 , wherein the cells of the cell population comprising OPCs secrete IL-10 within three, four, five or seven days following the inducing.
15. The method of claim 14 , wherein a level of IL-10 secreted by cells of the cell population comprising OPCs is at least 50-fold higher than control cells, wherein the control cells are (a) naturally occurring OPCs, (b) OPCs that are not engineered to express IL-10, and/or (c) OPCs that are engineered to express IL-10 under an inducible promoter and are cultured in the absence of an inducing agent.
16. The method of claim 1 , wherein the inducible promoter is a doxycycline inducible promoter and the inducing agent is doxycycline.
17. The method of claim 1 , wherein the at least four transcription factors comprise OCT4, OLIG2, NKX6.1, and SOX9.
18. The method of claim 1 , wherein the at least four transcription factors comprise OCT4, OLIG2, NKX6.2, and SOX9.
19. The method of claim 1 , wherein the at least four transcription factors comprise OLIG2, SOX9, NKX6.1, and NKX6.2.
20. The method of claim 1 , wherein the at least four transcription factors comprise OCT4, OLIG2, NKX6.1, and NKX6.2.