METHOD FOR INDUCTION/DIFFERENTIATION INTO PHOTORECEPTOR CELL

• US 20100105137A1
• Filed: 01/11/2008
• Published: 04/29/2010
• Est. Priority Date: 01/18/2007
• Status: Active Grant

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: Ikeda, Hanako, Mandai, Michiko, Takahashi, Masayo, Osakada, Fumitaka
• Granted Patent: US 9,133,435 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/33 other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/08 from cells of the nervous system
• CPC Class Codes: C12N 2533/00 Supports or coatings for cell culture, characterised by material
• CPC Class Codes: C12N 5/062 Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

The present invention provides a method of producing primate retinal progenitor cells, comprising culturing primate embryonic stem cells as suspended aggregates in a serum-free medium, and obtaining retinal progenitor cells from the culture. The present invention further provides a method of producing photoreceptor precursor cells, comprising culturing isolated retinal progenitor cells differentiated from embryonic stem cells, under adhesive conditions, in the presence of a gamma secretase inhibitor, and obtaining a photoreceptor precursor from the culture.

Claims

1. A method of producing primate retinal progenitor cells, comprising culturing primate embryonic stem cells as suspended aggregates in a serum-free medium, and obtaining retinal progenitor cells from the culture.

2. The method of claim 1, wherein the cultivation of the suspended aggregates is performed in the serum-free medium over the entire period thereof.

3. The method of claim 1, wherein the serum-free medium contains at least any one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.

4. The method of claim 3, wherein the serum-free medium contains a Nodal signal inhibitor and a Wnt signal inhibitor.

5. The method of claim 3, wherein the Nodal signal inhibitor is Lefty-A or SB-431542.

6. The method of claim 3, wherein the Wnt signal inhibitor is Dkk1, CKI-7 or D4476.

7. The method of claim 1, wherein each aggregate at the start of cultivation is configured with 2 to 50 embryonic stem cells.

8. The method of claim 1, wherein the suspended aggregates are cultured for at least 3 days.

9. The method of claim 1, wherein the retinal progenitor cells are neural retinal progenitor cells or retinal pigment epithelium progenitor cells.

10. The method of claim 1, further comprising culturing the cultured cells under adhesive conditions after cultivation of the suspended aggregates.

23. The method of claim 2, wherein the serum-free medium contains at least any one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.

24. The method of claim 23, wherein the serum-free medium contains a Nodal signal inhibitor and a Wnt signal inhibitor.

25. The method of claim 23, wherein the Nodal signal inhibitor is Lefty-A or SB-431542.

26. The method of claim 23, wherein the Wnt signal inhibitor is Dkk1, CKI-7 or D4476.

11. A method of producing primate retinal pigment epithelial cells, comprising culturing primate embryonic stem cells as suspended aggregates in a serum-free medium, further culturing the cultured cells under adhesive conditions, and obtaining retinal pigment epithelial cells from the culture.

12. A method of producing primate photoreceptors or a precursor thereof, comprising culturing primate embryonic stem cells as suspended aggregates in a serum-free medium, further culturing the cultured cells under adhesive conditions, and obtaining photoreceptors or a precursor thereof from the culture.

13. The method of claim 12, wherein the cultivation under adhesive conditions is performed in a medium containing at least one factor selected from the group consisting of retinoic acid and taurine.

14. The method of claim 13, wherein the medium contains retinoic acid and taurine.

15. The method of claim 13, wherein at least one factor selected from the group consisting of retinoic acid and taurine is added to the culture under adhesive conditions at a stage after the emergence of photoreceptor precursors in the culture.

16. A method of producing photoreceptor precursors, comprising culturing isolated retinal progenitor cells differentiated from embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor, and obtaining photoreceptor precursors from the culture. DAPT.

17. The method of claim 16, wherein the gamma secretase inhibitor is DAPT.

18. A method of producing cone photoreceptors, comprising culturing isolated retinal progenitor cells differentiated from embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor, and obtaining cone photoreceptors from the culture.

19. The method of claim 18, wherein the gamma secretase inhibitor is DAPT.

20. A method of producing rod photoreceptors, comprising culturing isolated retinal progenitor cells differentiated from embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor, further culturing the cultured cells under adhesive conditions in the presence of at least any one factor selected from the group consisting of an FGF, an shh signal promoter, retinoic acid and taurine, and obtaining rod photoreceptors from the culture.

21. The method of claim 20, wherein the further cultivation is performed in the presence of a gamma secretase inhibitor.

22. The method of claim 21, wherein the gamma secretase inhibitor is DAPT.