IP Library › Granted Patent US 12,378,520
Granted Patent B2
US 12,378,520 · App. 17/870,569 · Granted Aug 5, 2025

Method for producing retinal tissue and retina-related cells

Inventors: Tokushige Nakano (Osaka, JP); Yoshiki Sasai (Kobe, JP); Chikafumi Ozone (Wako, JP)
Assignees: SUMITOMO CHEMICAL COMPANY, LIMITED; RIKEN
C12N5/0621A61K35/30A61L27/3804A61L27/3834A61L27/3895A61L2430/16C12N2500/33C12N2500/44C12N2500/90C12N2501/155C12N2501/19C12N2501/385C12N2501/415C12N2501/727C12N2506/02C12N2506/45
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Quick Facts
Patent No.
US 12,378,520
App. No.
17/870,569
Granted
Aug 5, 2025
Kind
B2
Abstract

The present invention provides a method for producing a retinal progenitor cell, including (1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and (2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog signal transduction pathway but containing a substance acting on the BMP signal transduction pathway, thereby obtaining an aggregate containing retinal progenitor cells.

Claims (54)

1. A method for producing a cell population containing retinal progenitor cells, comprising

(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells,

(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh at a concentration exerting an adverse influence on the selective differentiation into retinal progenitor cell and retinal tissue and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells, and

(3) a third step of dispersing the aggregate containing retinal progenitor cells formed in step (2), thereby obtaining a dispersed cell population containing retinal progenitor cells.

2. The method according to claim 1 , comprising a fourth step of bringing the dispersed cell population containing retinal progenitor cells formed in step (3) into contact with an antibody against an antigen expressed in the retinal progenitor cells to increase the proportion of retinal progenitor cells in the cell population.

3. The method according to claim 1 , wherein the retinal progenitor cells express retina and anterior neural fold homeobox (Rax) gene and paired box 6 (Pax6) gene.

4. A method for producing a cell population containing retinal tissue constituting cells, comprising

(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells,

(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh at a concentration exerting an adverse influence on the selective differentiation into retinal progenitor cell and retinal tissue and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells,

(3) a third step of subjecting the aggregate formed in step (2) to floating culture in a serum-free medium or serum-containing medium each being free of any of a substance acting on the Shh signal transduction pathway that can enhance signal transduction mediated by Shh at a concentration exerting an adverse influence on the selective differentiation into retinal progenitor cell and retinal tissue, a substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP and a substance acting on the Wingless-related integration site (Wnt) signal pathway that can enhance signal transduction mediated by Wnt, thereby obtaining an aggregate containing retinal tissues and being substantially free of non-neural head ectoderm, and

(4) a fourth step of dispersing the aggregate containing retinal tissues and being substantially free of non-neural head ectoderm formed in step (3), thereby obtaining a dispersed cell population containing retinal tissue constituting cells.

5. The method according to claim 4 , wherein the cell population obtained in step (4) contains one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell.

6. The method according to claim 5 , comprising a fifth step of bringing the cell population obtained in step (4) into contact with an antibody against an antigen expressed in one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell, to increase the proportion in the cell population of cells expressing the antigen.

7. The method according to claim 5 , wherein each of the retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell expresses one or more markers selected from the group consisting of:

Rax and PAX6 in the retinal progenitor cell,

Rhodopsin and Recoverin in the photoreceptor,

Calbindin in the horizontal cell,

Chx10 and L7 in the bipolar cell,

Calretinin in the amacrine cell,

Tuj1 and Brn3 in the retinal ganglion cell,

Nrl in the rod cell, and

Rxr-gamma in the cone cell.

8. The method according to claim 4 , wherein the pluripotent stem cells are primate pluripotent stem cells or human pluripotent stem cells.

9. The method according to claim 4 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.

10. The method according to claim 4 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is BMP4.

11. The method according to claim 4 , wherein the substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP is added to the medium between day 1 and day 15 from the start of the floating culture in step (1).

12. A method for producing a cell population containing retinal layer-specific neural cells, comprising

(1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells,

(2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog (Shh) signal transduction pathway that can enhance signal transduction mediated by Shh at a concentration exerting an adverse influence on the selective differentiation into retinal progenitor cell and retinal tissue and containing a substance acting on the bone morphogenic protein (BMP) signal transduction pathway that can enhance signal transduction pathway mediated by BMP at a concentration necessary for differentiation induction into retinal cells from day 1 or later from the start of the floating culture in step (1) until a cell expressing retina and anterior neural fold homeobox (Rax) gene appears, thereby obtaining an aggregate containing retinal progenitor cells,

(3) a third step of subjecting the aggregate formed in step (2) to floating culture in a serum-free medium or serum-containing medium each being free of any of a substance acting on the Shh signal transduction pathway that can enhance signal transduction mediated by Shh at a concentration exerting an adverse influence on the selective differentiation into retinal progenitor cell and retinal tissue, a substance acting on the BMP signal transduction pathway that can enhance signal transduction pathway mediated by BMP and a substance acting on the Wingless-related integration site (Wnt) signal pathway that can enhance signal transduction mediated by Wnt until the intended retinal layer-specific neural cells appear, thereby obtaining an aggregate containing retinal tissues containing the intended retinal layer-specific neural cells and being substantially free of non-neural head ectoderm, and

(4) a fourth step of dispersing the aggregate containing retinal layer-specific neural cells and being substantially free of non-neural head ectoderm formed in step (3), thereby obtaining a dispersed cell population containing retinal layer-specific neural cells.

13. The method according to claim 12 , wherein the cell population obtained in step (4) contains one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell.

14. The method according to claim 13 , comprising a fifth step of bringing the cell population obtained in step (4) into contact with an antibody against an antigen expressed in one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell, to increase the proportion in the cell population of cells expressing the antigen.

15. The method according to claim 13 , wherein each of the retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell expresses one or more markers selected from the group consisting of:

Rax and PAX6 in the retinal progenitor cell,

Rhodopsin and Recoverin in the photoreceptor,

Calbindin in the horizontal cell,

Chx10 and L7 in the bipolar cell,

Calretinin in the amacrine cell,

Tuj1 and Brn3 in the retinal ganglion cell,

Nrl in the rod cell, and

Rxr-gamma in the cone cell.

16. A method for producing a cell population containing one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell, comprising

(1) producing a cell population containing retinal progenitor cells by the method according to claim 1 , and

(2) culturing the cell population obtained in step (1).

17. The method according to claim 16 , wherein the culturing in step (2) is performed under adhesion conditions.

18. A method for producing a cell population containing one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell, comprising

(1) producing a cell population containing retinal tissue constituting cells by the method according to claim 4 , and

(2) culturing the cell population obtained in step (1).

19. The method according to claim 18 , wherein the culturing in step (2) is performed under adhesion conditions.

20. A method for producing a cell population containing one or more cells selected from the group consisting of retinal progenitor cell, photoreceptor, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, rod cell and cone cell, comprising

(1) producing a cell population containing retinal layer-specific neural cells by the method according to claim 12 , and

(2) culturing the cell population obtained in step (1).

21. The method according to claim 20 , wherein the culturing in step (2) is performed under adhesion conditions.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 30, 2025
From: NAKANO, TOKUSHIGE; SASAI (LEGAL REPRESENTATIVE OF DECEASED INVENTOR YOSHIKI SASAI), MIYUKI; OZONE, CHIKAFUMI
To: SUMITOMO CHEMICAL COMPANY, LIMITED; RIKEN
Reel/Frame 071875/0604 →
Priority Claims (1)
JP 2013-173285 · Aug 23, 2013 · national
Continuity (3)
Continuation 16692484 · Nov 22, 2019
Continuation 14913628
Related Publication 20220364054A1 · Nov 17, 2022
References Cited (83)
US 7709605B2 · Knopf et al. · 2010 [cited by applicant]
US 7842663B2 · Knopf et al. · 2010 [cited by applicant]
US 8252900B2 · Knopf et al. · 2012 [cited by applicant]
US 8343933B2 · Knopf et al. · 2013 [cited by applicant]
US 8492147B2 · Sasai et al. · 2013 [cited by applicant]
US 8956866B2 · Idelson et al. · 2015 [cited by applicant]
US 9138459B2 · Knopf et al. · 2015 [cited by applicant]
US 9328328B2 · Gamm et al. · 2016 [cited by applicant]
US 9365830B2 · Schulz et al. · 2016 [cited by applicant]
US 9399669B2 · Knopf et al. · 2016 [cited by applicant]
US 9752119B2 · Gamm et al. · 2017 [cited by applicant]
US 10259861B2 · Knopf et al. · 2019 [cited by applicant]
US 10501724B2 · Nakano et al. · 2019 [cited by applicant]
US 20060068468A1 · Knopf et al. · 2006 [cited by applicant]
US 20060281179A1 · Sasai et al. · 2006 [cited by applicant]
US 20080044901A1 · Sasai et al. · 2008 [cited by applicant]
US 20080160617A1 · Sasai et al. · 2008 [cited by applicant]
US 20090005308A1 · Knopf et al. · 2009 [cited by applicant]
US 20090053809A1 · Zander et al. · 2009 [cited by applicant]
US 20100105137A1 · Takahashi et al. · 2010 [cited by applicant]
US 20100267133A1 · Knopf et al. · 2010 [cited by applicant]
US 20110027333A1 · Idelson et al. · 2011 [cited by applicant]
US 20110081719A1 · Gamm et al. · 2011 [cited by applicant]
US 20110092670A1 · Knopf et al. · 2011 [cited by applicant]
US 20110274662A1 · Malcuit et al. · 2011 [cited by applicant]
US 20130065299A1 · Knopf et al. · 2013 [cited by applicant]
US 20130184210A1 · Knopf et al. · 2013 [cited by applicant]
US 20130280802A1 · Schulz et al. · 2013 [cited by applicant]
US 20140308743A1 · Sasai et al. · 2014 [cited by applicant]
US 20140341864A1 · Nakano et al. · 2014 [cited by applicant]
US 20150118749A1 · Idelson et al. · 2015 [cited by applicant]
US 20160108379A1 · Knopf et al. · 2016 [cited by applicant]
US 20160186136A1 · Sasai et al. · 2016 [cited by applicant]
US 20160251616A1 · Nakano et al. · 2016 [cited by applicant]
US 20160251618A1 · Gamm et al. · 2016 [cited by applicant]
US 20160281058A1 · Schulz et al. · 2016 [cited by applicant]
US 20170058016A1 · Knopf et al. · 2017 [cited by applicant]
US 20200071381A1 · Knopf et al. · 2020 [cited by applicant]
US 20200102535A1 · Nakano et al. · 2020 [cited by applicant]
US 20220281951A1 · Knopf et al. · 2022 [cited by applicant]
CN 101688178A · 2010 [cited by applicant]
CN 103555654A · 2014 [cited by applicant]
EP 1302533A1 · 2003 [cited by applicant]
JP 2008507288A · 2008 [cited by applicant]
JP 2010518009A · 2010 [cited by applicant]
JP 2013502234A · 2013 [cited by applicant]
JP 2014501518A · 2014 [cited by applicant]
WO WO2001088100A1 · 2001 [cited by applicant]
WO WO2003042384A1 · 2003 [cited by applicant]
WO WO2005123902A1 · 2005 [cited by applicant]
WO WO2006053629A1 · 2006 [cited by applicant]
WO WO2008129554A1 · 2008 [cited by applicant]
WO WO2009132156A1 · 2009 [cited by applicant]
WO WO2011043591A2 · 2011 [cited by applicant]
WO WO2011055855A1 · 2011 [cited by applicant]
WO WO2013065763A1 · 2013 [cited by applicant]
WO WO2013077425A1 · 2013 [cited by applicant]
U.S. Appl. No. 16/692,484, filed Nov. 22, 2019. [cited by applicant]
Boucherie et al., “Brief Report: Self-Organizing Neuroepithelium from Human Pluripotent Stem Cells Facilitates Derivation of Photoreceptors,” [cited by applicant]
Buckholz et al., “Rapid and Efficient Directed Differentiation of Human Pluripotent Stem Cells Into Retinal Pigmented Epithelium,” [cited by applicant]
Eiraku et al., “Self-organizing optic-cup morphogenesis in three-dimensional culture,” [cited by applicant]
Furuta et al., “BMP4 is essential for lens induction in the mouse embryo,” [cited by applicant]
Idelson et al., “Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells,” [cited by applicant]
Ikeda et al., “In vitro differentiation of telencephalic precursors and neural retinal precursors from ES cells,” [cited by applicant]
La Torre et al., “Production and transplantation of retinal cells from human and mouse embryonic stem cells,” [cited by applicant]
Lamba et al., “Efficient generation of retinal progenitor cells from human embryonic stem cells,” [cited by applicant]
Lancaster et al., “Cerebral organoids model human brain development and microcephaly,” [cited by applicant]
Lang, “Pathways regulating lens induction in the mouse,” [cited by applicant]
Liu, Peipei, “The distribution of BMP-4 in rat retina and optic nerve and its effects on the differentiation of oligodendrocyte precursors,” [cited by applicant]
Loebel et al., “Lineage choice and differentiation in mouse embryos and embryonic stem cells,” [cited by applicant]
Meyer et al., “Modeling early retinal development with human embryonic and induced pluripotent stem cells,” [cited by applicant]
Muranishi et al., “An Essential Role for RAX Homeoprotein and NOTCH-HES Signaling in Otx2 Expression in Embryonic Retinal Photoreceptor Cell Fate Determination,” [cited by applicant]
Nakano et al., “Self-Formation of Optic Cups and Storable Stratified Neural Retina from Human ESCs,” [cited by applicant]
Osakada et al., “Control of neural differentiation from pluripotent stem cells,” [cited by applicant]
Osakada et al., “Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells,” [cited by applicant]
Parekh et al., “A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe,” [cited by applicant]
Trousse et al., “Bmp4 Mediates Apoptotic Cell Death in the Developing Chick Eye,” [cited by applicant]
Watanabe et al., “Directed differentiation of telencephalic precursors from embryonic stem cells,” [cited by applicant]
Yang et al., “Efficient generation of lens progenitor cells and lentoid bodies from human embryonic stem cells in chemically defined conditions,” [cited by applicant]
European Patent Office, Extended European Search Report in European Patent Application No. 14838289.8 (Apr. 4, 2017). [cited by applicant]
Intellectual Property Office of Singapore, Search Report in Singaporean Patent Application No. 11201601294P (Feb. 22, 2017). [cited by applicant]
Japanese Patent Office, International Search Report in International Patent Application No. PCT/JP2014/072065 (Nov. 25, 2014). [cited by applicant]
Japanese Patent Office, Written Opinion of the International Searching Authority in International Patent Application No. PCT/JP2014/072065 (Nov. 25, 2014). [cited by applicant]