Modalities for the treatment of degenerative diseases of the retina

• US 9,045,732 B2
• Filed: 04/08/2013
• Issued: 06/02/2015
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Ocata Therapeutics, Inc. (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina V., Lanza, Robert P.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/858,497
• Publication Number:
  US 20130316451A1
• Time in Patent Office: 785 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: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61K 35/36 Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells islets of Langerhans A61K35/39
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/02 from embryonic cells
• 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

Abstract

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.

Claims

1. A method of producing a human RPE preparation suitable for transplantation into the eye of a human patient in need thereof comprising:

(a) differentiating in vitro human pluripotent cells that express Oct-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-I-60, and TRA-I-81 under conditions that do not maintain the undifferentiated state of the pluripotent cells to obtain a heterogeneous population of human cells including pigmented cells comprising brown pigment dispersed in their cytoplasm;

(b) isolating and culturing said pigmented cells from the heterogeneous cell population to obtain a monolayer comprising cells having a cobblestone, polygonal, epithelial-like appearance and brown pigment dispersed in their cytoplasm, and expressing RPE65 and bestrophin, and

(c) isolating and purifying the RPE cells to form a composition suitable for human therapy.

4. The method of claim 1, wherein the composition suitable for human therapy of step (c) comprises human RPE cells disposed on a matrix or substrate.

5. The method of claim 1, wherein composition suitable for human therapy of step (c) comprises a suspension of human RPE cells.

10. The method of claim 1, further comprising repassaging the RPE cells of step (c) one or more times.

2. A method of producing a composition comprising isolated human RPE cells suitable for human therapy, comprising:

(a) allowing human pluripotent cells to overgrow and form a thick multilayer of cells;

(b) culturing the pluripotent cells of step (a) under adherent conditions that do not maintain the undifferentiated state of the pluripotent cells for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm; and

(c) isolating and culturing the pigmented cells to form a monolayer comprising cells having a cobblestone, polygonal, epithelial-like appearance and brown pigment dispersed in their cytoplasm, and expressing RPE65 and bestrophin, thereby obtaining human RPE cells.

6. The method of claim 2, wherein the monolayer of step (c) is disposed on a matrix or substrate to form a monolayer of human RPE cells suitable for human therapy.

7. The method of claim 2, wherein the monolayer of step (c) is dissociated to form a suspension of human RPE cells suitable for human therapy.

11. The method of claim 2, further comprising dispersing cells in the monolayer of step (c).

12. The method of claim 2, wherein the monolayer of step (c) is repassaged one or more times, comprising dispersing cells in the monolayer.

3. A method of producing a composition comprising isolated human RPE cells suitable for human therapy, comprising:

(a) providing a multilayer culture of human pluripotent cells;

(b) culturing the multilayer culture of pluripotent cells of step (a) under adherent conditions that do not maintain the undifferentiated state of the pluripotent cells for a sufficient time for the appearance of pigmented cells comprising brown pigment dispersed in their cytoplasm; and

(c) isolating and culturing the pigmented cells to form a monolayer comprising cells having a cobblestone, polygonal, epithelial-like appearance and brown pigment dispersed in their cytoplasm, and expressing RPE65 and bestrophin, thereby obtaining human RPE cells.

8. The method of claim 3, wherein the monolayer of step (c) is disposed on a matrix or substrate to form a monolayer of human RPE cells suitable for human therapy.

9. The method of claim 3, wherein the monolayer of step (c) is dissociated to form a suspension of human RPE cells suitable for human therapy.

13. The method of claim 3, further comprising dispersing cells in the monolayer of step (c).

14. The method of claim 3, wherein the monolayer of step (c) is repassaged one or more times, comprising dispersing cells in the monolayer.