Retinal pigment epithelial cells differentiated from embryonic stem cells with nicotinamide and activin A

• US 8,956,866 B2
• Filed: 04/27/2008
• Issued: 02/17/2015
• Est. Priority Date: 04/18/2007
• Status: Active Grant

• Current Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Original Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Inventors: Idelson, Masha, Alper-Pinus, Ruslana, Obolensky, Alex, Banin, Eyal, Reubinoff, Benjamin
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/450,943
• Publication Number:
  US 20110027333A1
• Time in Patent Office: 2,487 Days
• US Class Current: 435/377
• 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 35/48 Reproductive organs
• CPC Class Codes: A61K 9/0051 Ocular inserts, ocular implants
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• 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: C12N 2500/38 Vitamins
• 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 2501/115 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 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention concerns RPE cells obtainable by directed differentiation from stem cell, particularly, human stem cells. It has been specifically found that culturing stem cells in the presence of one or more member of the TGFβ superfamily, such as Activin A) induced directed differentiation into mature and functional RPE cells. This was evidenced by the expression of markers specific to mature RPE cells, including MiTF-A, RPE65 or Bestrophin). In accordance with one particular embodiment, the cells are a priori cultured with nicotinamide (NA) which was found to augment the cells'"'"' response to the inductive effect of the one or more member of the TGFβ superfamily. The invention also provides methods of performing the directed differentiation, as well as methods for use of the resulting RPE cells.

Claims

1. A method for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) fate, the method comprising:

(a) culturing human pluripotent stem cells in a medium supplemented with nicotinamide (NA) to generate differentiating cells; and

subsequently

(b) culturing said differentiating cells in a medium supplemented with activin A under conditions which induce said differentiating cells to differentiate into RPE cells.

2. The method of claim 1, wherein said culturing in said medium supplemented with NA is effected for at least two days prior to culturing in said medium supplemented with said activin A.

3. The method of claim 1, wherein said cells are cultured in a suspension.

4. The method of claim 1, wherein said human pluripotent stem cells are human embryonic stem cells.

5. The method of claim 1, wherein culturing said differentiating cells in said medium supplemented with nicotinamide is effected for about two weeks.

6. The method of claim 1, wherein culturing said differentiating cells in said medium supplemented with activin A in step (a) is effected for about two weeks.

7. The method of claim 1, wherein a concentration of said nicotinamide is about 10 mM.

8. The method of claim 1, wherein a concentration of said activin A is between 20-180 ng/ml.

9. The method of claim 1, wherein said medium supplemented with activin A is further supplemented with nicotinamide.