Methods For Producing Enriched Populations of Human Retinal Pigment Epithelium Cells

• US 20110117062A1
• Filed: 08/17/2010
• Published: 05/19/2011
• Est. Priority Date: 01/23/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: Klimanskaya, Irina V., Lanza, Robert
• Granted Patent: US 9,649,340 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• 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 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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

9. (canceled)

11-17. -17. (canceled)

19. Isolated RPE cells spontaneously differentiated from human embryonic stem cells, wherein said isolated RPE cells are free from cells that express Oct4, Sox2, or TDGF1, and wherein said isolated RPE cells can be maintained in culture in RPE medium, wherein said RPE medium consist of 50% MEF medium and 50% EB medium, wherein said MEF medium consists of high glucose DMEM, 2 mM GlutaMAX I, and 500 μ

g/ml Penicillin, 500 μ

g/ml streptomycin and 16% fetal calf serum (FCS), and wherein said EB medium consists of knockout high glucose DMEM, 500 μ

g/ml Penicillin, 500 μ

g/ml streptomycin, 1% non-essential amino acids solution, 2 mM GlutaMAX I, 0.1 mM beta-mercaptoethanol, 4 ng/ml bFGF, 1 ng/ml human LIF, 8.4% of Serum Replacement and 8.4% PLASMANATE®

.

1. A method of treating or preventing retinal degeneration, comprising introducing the isolated RPE cells of claim 19 into an individual in need thereof.

2. The method of claim 1, wherein the condition of retinal degeneration is selected from the group consisting of at least one of:

retinitis pigmentosa and macular degeneration.

3. The method of claim 1, which comprises transplantation of the cell by vitrectomy surgery into the subretinal space of the eye of said individual.

4. The method of claim 3, wherein the cells are transplanted in a suspension, matrix, or substrate.

5. The method of claim 2, wherein the retinitis pigmentosa is associated with an animal model.

6. The method of claim 5, where in the animal model is selected from the group consisting of:

rd mouse, RPE-65 knockout mouse, tubby-like mouse, RCS rat, Abyssinian cat, cone degeneration “

cd”

dog, progressive rod-cone degeneration “

prcd”

dog, early retinal degeneration “

erd”

dog, rod-cone dysplasia 1, 2 &

3 “

rcd1, rcd2 and rcd3”

dogs, photoreceptor dysplasia “

pd”

dog, and Briard “

RPE-65”

dog.

7. The method of claim 6, wherein the outcome of the therapy in the animal model is evaluated using one or more of behavioral tests, fluorescent angiography, histology, and functional testing such as measuring the ability of the cells to perform phagocytosis (photoreceptor fragments), vitamin A metabolism, tight junctions conductivity, or evaluation using electron microscopy.

8. The isolated RPE cells of claim 19 which are prepared by a method for the spontaneous differentiation of hES cells into cells RPE cells, said method comprising:

a) allowing hES cell cultures to overgrow on MEF and form a thick multilayer of cells, or forming an embryoid body (EB) from hES cells;

b) culturing the multilayer of cells or EB 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 from the resultant cell cultures, thereby obtaining human RPE cells.

10. The isolated RPE cells of claim 8, where in the step of isolating the pigmented cells comprises contacting the cultured cells of step (b) with an enzyme selected from the group consisting of one or more of trypsin, collagenase, and dispase.

18. A method for the treatment of Parkinson'"'"'s disease comprising introducing the isolated RPE cells of claim 19 into an individual in need thereof.

20. The isolated RPE cells of claim 19, wherein said isolated RPE cells are Pax6−

, bestrophin+, CRALBP+, PEDF+, and express RPE65.

21. The isolated RPE cells of claim 19, wherein said isolated RPE cells exhibit a cobblestone, polygonal appearance characteristic of epithelial cells.

22. The isolated RPE cells of claim 8, wherein said culturing in step (b) comprises culturing the hES cells in a medium lacking exogenously added FGF, lacking exogenously added LIF and lacking exogenously added PLASMANATE®

.

23. The isolated RPE cells of claim 8, wherein the duration of culturing in step (b) is at least 6 weeks.

24. The isolated RPE cells of claim 8, wherein the duration of culturing in step (b) is between about 6 weeks and about 8 weeks.

25. The isolated RPE cells of claim 8, wherein the duration of culturing in step (b) is between about 3 months and about 5 months.