Compositions and methods for efficient amplification of retinal progenitors cells

Patent metadata

Field	Value
Publication number	US-12497591-B2
Application number	US-201916976581-A
Country	US
Kind code	B2
Filing date	Mar 6, 2019
Priority date	Mar 7, 2018
Publication date	Dec 16, 2025
Grant date	Dec 16, 2025

Abstract

The disclosure pertains to a defined cell culture medium for the expansion of human retinal progenitors, comprising or consisting of a nutrient medium, a SHH-pathway activator and a GSK3 inhibitor. To the use of the defined cell culture medium for the expansion of retinal progenitors, as well as to an in vitro method for expanding retinal progenitors, comprising: (i) placing a culture of human retinal progenitors in a defined cell culture medium as defined in claims 1 to 8 ; and (ii) culturing the cells in said defined cell culture medium.

First claim

The invention claimed is:

1 . A defined cell culture medium for the expansion of human retinal progenitors while maintaining their multipotency, comprising a nutrient medium, a SHH-pathway activator, a GSK3 inhibitor, FGF2, EGF, and ATP, wherein the defined cell culture medium is devoid of DAPT.

2 . The defined cell culture medium of claim 1 , further comprising a pro-neural supplement, wherein the pro-neural supplement comprises insulin and transferrin.

3 . The defined cell culture medium of claim 2 , wherein the pro-neural supplement comprises:
(a) BSA, transferrin, insulin, progesterone, putrescine, sodium selenite, biotine, 1-carnitine, cortisone or hydrocortisone, ethanolamine, d(+)galactose, glutathione (reduced), linolenic acid, linoleic acid, retinyl acetate, selenium, T3 (triodo-1-thryonine), dl-α-tocopherol (vitamin E), dl-α-tocopherol acetate, catalase, and superoxide dismutase; (b) transferrin, insulin, progesterone, putrescine, and sodium selenite; (c) BSA, transferrin, and insulin; or (d) transferrin, insulin and sodium selenite.

4 . The defined cell culture medium of claim 1 , wherein the SHH-pathway activator is selected from the group consisting of purmorphamine, SHH, smoothened agonist, Hh-Ag 1.5, and zinc finger protein Gli-2.

5 . The defined cell culture medium of claim 4 , wherein the SHH-pathway activator comprises purmorphamine, at a concentration between 1 nM and 3 μM.

6 . The defined cell culture medium of claim 1 , wherein the GSK3 inhibitor is selected from the group consisting of SB-216763, SB-415286, CHIR-98023, CHIR99021, AR-A014418, L803 peptide or its myristoylated form L803-mts, and LiCL.

7 . The defined cell culture medium of claim 6 , wherein the GSK3 inhibitor is CHIR99021 at a concentration between 2 μM and 10 μM.

8 . Cryopreserved retinal progenitor cells (RPCs) in the cell culture medium as defined in claim 1 with DMSO.

9 . A method of expanding retinal progenitors, comprising culturing the retinal progenitors in a defined cell culture medium as defined in claim 1 .

10 . An in vitro method for expanding retinal progenitors, comprising:
(i) placing a culture of human retinal progenitors in a defined cell culture medium as defined in claim 1 ; and (ii) culturing the cells in said defined cell culture medium.

11 . The method according to claim 10 wherein the culture in step (i) and (ii) is adherent.

12 . The method according to claim 10 , wherein the retinal progenitors are passaged at least once.

13 . The method according to claim 10 , wherein the cells obtained in step (ii) have retained their retinal multipotency property.

14 . A method for obtaining photoreceptors or precursors thereof, wherein said method comprises the steps of:
(i) placing a culture of human retinal progenitors in a defined cell culture medium as defined in claim 1 ; (ii) culturing the cells in said defined cell culture medium; and (iii) culturing the cells obtained in step (ii) in a pro-neural medium.

15 . A method for obtaining retinal ganglion cells, wherein said method comprises the steps of:
(i) placing a culture of human retinal progenitors in a defined cell culture medium as defined in claim 1 ; (ii) culturing the cells in said defined cell culture medium; (iii) culturing the cells obtained in step (ii) in a pro-neural medium; and (iv) culturing the cells obtained in step (iii) in a pro-neural medium further comprising DAPT.

Assignees

• Univ Sorbonne
• Centre Nat Rech Scient
• Inst Nat Sante Rech Med

Inventors

• Reichman Sacha
• Goureau Olivier
• Sahel José-Alain

Classifications

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/41

  Hedgehog proteins; Cyclopamine (inhibitor) · CPC title

• C12N2501/33

  Insulin · CPC title

• C12N2501/115

  Basic fibroblast growth factor (bFGF, FGF-2) · CPC title