Method for induction/differentiation into photoreceptor cell

• US 9,133,435 B2
• Filed: 01/11/2008
• Issued: 09/15/2015
• Est. Priority Date: 01/18/2007
• Status: Expired due to Fees

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: Ikeda, Hanako, Takahashi, Masayo, Osakada, Fumitaka, Mandai, Michiko
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US12/523,444
• Publication Number:
  US 20100105137A1
• Time in Patent Office: 2,804 Days
• US Class Current: 1/1
• 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 photoreceptor precursors, comprising

(a) culturing mammalian embryonic stem cells as suspended aggregates in a medium such that the ES cells differentiate into retinal progenitor cells indicated by the expression of a retinal progenitor cell maker selected from the group of Pax6, Rx and Mitf;

(b) culturing the retinal progenitor cells differentiated from the embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor to produce photoreceptor precursors indicated by the expression of the photoreceptor precursor marker selected from the group Crx and rhodopsin;

(c) isolating photoreceptor precursors from the culture.

2. The method of claim 1, wherein the gamma secretase inhibitor is N—

[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

5. The method of claim 1, wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.

17. The method of claim 5, wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242.

18. The method of claim 17, wherein the Nodal signal inhibitor is Lefty-A or SB-431542.

19. The method of claim 5, wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).

20. The method of claim 19, wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.

21. The method of claim 5, wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).

22. The method of claim 21, wherein the mammalian embryonic stem cells are human embryonic stem cells.

23. The method of claim 2, wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least 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 Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-1-yl}benzamide (D4476).

25. The method of claim 24, wherein the Nodal signal inhibitor is Lefty-A or SB-431542, and wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.

26. The method of claim 1, wherein the mammalian embryonic stem cells are human embryonic stem cells.

3. A method of producing cone photoreceptors, comprising

(a) culturing mammalian embryonic stem cells as suspended aggregates in a medium such that the ES cells differentiate into retinal progenitor cells indicated by the expression of a retinal progenitor cell maker selected from the group of Pax6, Rx and Mitf;

(b) culturing the retinal progenitor cells differentiated from the embryonic stem cells under adhesive conditions in the presence of a gamma secretase inhibitor to produce photoreceptor precursors indicated by the expression of the photoreceptor precursor marker selected from the group Crx and rhodopsin;

(c) culturing the photoreceptor precursor cells under adhesive conditions in a medium comprising a gamma secretase inhibitor and one of aFGF, bFGF, taurine, Shh or RA to produce cone photoreceptors indicated by the expression of a photoreceptor maker selected from the group rhodopsin, red/green opsin, blue opsin and Recoverin; and

(d) isolating cone photoreceptors from the culture.

4. The method of claim 3, wherein the gamma secretase inhibitor is N—

[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).

6. The method of claim 3, wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.

7. The method of claim 6, wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242.

8. The method of claim 7, wherein the Nodal signal inhibitor is Lefty-A or SB-431542.

9. The method of claim 6, wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).

10. The method of claim 9, wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.

11. The method of claim 6, wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{442,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).

12. The method of claim 11, wherein the mammalian embryonic stem cells are human embryonic stem cells.

13. The method of claim 4, wherein the culturing of the embryonic stem cells as suspended aggregates is performed in a serum-free medium containing at least one inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor.

14. The method of claim 13, wherein the Nodal signal inhibitor is Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, or SB-431242, and wherein the Wnt signal inhibitor is Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide (CKI-7), or 4-{4-(2,3-dihydrobenzo[1,4]dioxyn-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide (D4476).

15. The method of claim 14, wherein the Nodal signal inhibitor is Lefty-A or SB-431542, and wherein the Wnt signal inhibitor is Dkk1, CKI-7, or D4476.

16. The method of claim 3, wherein the mammalian embryonic stem cells are human embryonic stem cells.