Method of producing human retinal pigment epithelial cells

• US 10,519,422 B2
• Filed: 02/29/2012
• Issued: 12/31/2019
• Est. Priority Date: 02/29/2012
• Status: Active Grant

• Current Assignee: Riken
• Original Assignee: Riken
• Inventors: Takahashi, Masayo, Okamoto, Satoshi, Sakai, Noriko
• Primary Examiner(s): Bowers, Erin M.
• Application Number:
  US13/408,642
• Publication Number:
  US 20130224156A1
• Time in Patent Office: 2,862 Days
• Field of Search: None
• 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: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0621 Eye cells, e.g. cornea, iris pigmented cells photoreceptors C12N5/062

Abstract

The invention relates to a method of producing a retinal pigment epithelial cell from a human pluripotent stem cell, and a method of treating or preventing a retinal disease by using the produced cell. The retinal pigment epithelial cell is prepared by (a) inducing differentiation of a human pluripotent stem cell into a pigment cell by adhesion cultivation of a human pluripotent stem cell in a medium containing a Nodal signal inhibitor and a Wnt signal inhibitor in the absence of a feeder cell to give a culture containing the pigment cell, (b) subjecting the obtained culture to further adhesion culture to give a culture containing a pigment cell colony, and (c) isolating the pigment cell from the obtained culture and culturing the cell to give a retinal pigment epithelial cell.

Claims

1. A method of producing a retinal pigment epithelial cell from a human pluripotent stem cell, which comprises

(a) a step of providing isolated human pluripotent stem cells, wherein the isolated human pluripotent stem cells are dispersed by a cell dissociation solution,

(b) a step of inducing differentiation of the isolated human pluripotent stem cells into pigment cells by adhesion cultivation of the isolated and dispersed human pluripotent stem cells of step (a) in a medium containing a Nodal signal inhibitor and a Wnt signal inhibitor at a concentration sufficient to induce differentiation of the human pluripotent stem cells into pigment cells in the absence of feeder cells to yield a culture containing the pigment cells,

(c) a step of subjecting the obtained culture of step (b) to further adhesion culture to yield a culture containing a pigment cell colony, and

(d) a step of isolating the pigment cell from the obtained culture of step (c) and culturing the cell to yield the retinal pigment epithelial cell.

2. The method according to claim 1, wherein the adhesion culture in step (b) is performed using a culture vessel coated with gelatin, or a culture vessel coated with polylysine and gelatin.

3. The method according to claim 1, wherein the Nodal signal inhibitor is SB-431542, and the Wnt signal inhibitor is CKI-7.

4. The method according to claim 1, further comprising adding a serum and/or a serum alternative while stepwisely reducing the concentration(s) thereof in step (b).

5. The method according to claim 1, wherein the medium in step (b) is a serum-free medium.

6. The method according to claim 1, wherein step (d) comprises

(d-1) a step of subjecting the obtained culture of step (c) to floating culture and

(d-2) a step of isolating the pigment cell from the obtained floating aggregate, subjecting the pigment cell to adhesion culture, and to selective passage to give a retinal pigment epithelial cell.

7. The method according to claim 1, wherein the adhesion cultivation in step (b) is performed by using a culture vessel having a coated layer containing an extracellular matrix.