Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells

a retinal pigment epithelium and reproducible technology, applied in the field of stem cell biology, can solve the problems of degeneration of the retina, loss of visual function, blindness, lack of efficient large-scale production of ipsc- or esc-derived rpe cells, etc., and achieve the effect of varying efficiency and not being scalabl

Inactive Publication Date: 2019-06-06
FUJIFILM CELLULAR DYNAMICS INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]Unfortunately, routine and reproducible production of RPEs from pluripotent cells, such as iPSCs or ESCs, from a starting population of embryoid bodies is problematic, due to the fact that the process of producing embryoid bodies itself is not reproducible, has varying efficiency and is not scalable, which is needed for commercial scale production of RPEs. Disclosed are methods for obtaining a retinal pigment epithelial (RPE) cell population that avoids the requirement for using embryoid bodies and instead employ cell suspension populations, preferably single cell suspensions, of pluripotent stem cells, as opposed to using embryoid bodies. In certain embodiments, the starting cell population of pluripotent stem cells may be, for example, embryonic stem cells or induced pluripotent stem cells.

Problems solved by technology

A disorder or injury to the RPE cells can result in degeneration of the retina, loss of visual function, and blindness.
There is a lack of methods for efficient large-scale production of iPSC- or ESC-derived RPE cells needed for therapeutics, screening assays, models of retinal disease, and RPE biology research
Unfortunately, routine and reproducible production of RPEs from pluripotent cells, such as iPSCs or ESCs, from a starting population of embryoid bodies is problematic, due to the fact that the process of producing embryoid bodies itself is not reproducible, has varying efficiency and is not scalable, which is needed for commercial scale production of RPEs.

Method used

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  • Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells
  • Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells
  • Method for reproducible differentiation of clinical-grade retinal pigment epithelium cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

on of Starting Pluripotent Stem Cell Population

[0176]A starting population of RPE cells can be derived from pluripotent stem cells such as ES cells and iPSCs. In exemplary methods, the RPE cells were derived from human iPSCs reprogrammed from somatic cells by methods known in the art such as U.S. Pat. Nos. 8,546,140, 8,741,648, 8,691,574, Published U.S. Patent Application No. 20090246875, Published U.S. Pat. No. 8,278,104, Published U.S. Pat. Nos. 9,005,967, 8,058,065, 8,129,187, PCT Publication NO. WO 2007 / 069666 A1, U.S. Pat. Nos. 8,183,038 and 8,268,620, which are incorporated herein by reference. In one exemplary method, nuclear programming factors Oct4, Sox2, c-Myc and Klf4 were used to produce pluripotent stem cells from a somatic cell. In another exemplary method, nuclear programming factors Oct4, Sox2, Nanog, Lin28, L-Myc, and SV40 Large T-antigen were used to produce pluripotent stem cells from a somatic cell.

[0177]The iPSCs were grown without mouse or human feeder layers i...

example 2

iation of iPSCs into RPE Cells

[0180]Once the single cell iPSCs seeded at the appropriate cells density were cultured for about 2 days as in Example 1, they were cultured in various differentiation media for deriving RPE cells. On day 3, the E8™ medium was aspirated and room temperature Retinal Induction Medium (RIM) (e.g., Table 3) was added. Briefly, the RIM comprised DMEM and F12 at about a 1:1 ratio, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, the RIM comprised a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor and insulin growth factor 1 (IGF1). Each day the media was aspirated and fresh RIM was added to the cells. The cells were cultured in the RIM for about two to four days.

[0181]The cells were next cultured in Retinal Differentiation Medium (RDM) for about seven to fourteen days. Briefly, the RDM (Table 2) comprised DMEM and F12 at about a 1:1 ratio,...

example 3

n of RPE Cells

[0184]For continued maturation of the RPE cells produced in Example 2, the cells were dissociated in a cell dissociation enzyme such as TRYPLE™ and reseeded on a degradable scaffold assembly in a specialized SNAPWELL™ design for 1-2 weeks in the RPE-MM with a MEK inhibitor such as PD325901. This resulted in differentiated, polarized, and confluent monolayers of functional RPE cells (FIG. 1D) which can be cryopreserved at this stage in xenofree CS10 medium.

[0185]The mature RPE cells were further developed into functional RPE cell monolayers that function as an intact RPE tissue by continued culture in the RPE-MM with additional small molecules such as primary cilium inducers like PGE2 or aphidicolin. Without being bound by theory, these primary cilium inducers suppress the canonical WNT pathway, induce cell cycle exit in the cells, and induce apical-basal polarization in the RPE monolayer. RPE maturity can alternatively be induced by canonical WNT pathway inhibitors suc...

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Abstract

Provided herein are methods of producing an RPE cell population from a starting cell suspension, such as a single cell suspension, of pluripotent stem cells (PSCs). Such a method may comprise culturing the starting single cell suspension of PSCs in differentiation media to produce human RPE cells.

Description

[0001]This claims the priority benefit of U.S. provisional application No. 62 / 215,579, filed Sep. 8, 2015, the entire contents of which are incorporated herein by reference.PARTIES TO JOINT RESEARCH AGREEMENT[0002]The present invention was made as a result of activities undertaken within the scope of a joint research agreement that was in effect at the time the present invention was made. The parties to said joint research agreement are The Government of the United States of America, U.S. Department of Health and Human Services, as represented by the National Eye Institute, an institute of the National Institutes of Health and Cellular Dynamics International, Inc.BACKGROUND1. Field[0003]This disclosure relates generally to the field of stem cell biology. More particularly, it concerns methods of efficient production of stem cell-derived retinal pigment epithelial cell populations for use as a cell therapy.2. Description of Related Art[0004]The retina is a light-sensitive layer of ti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/079
CPCC12N5/0621C12N2501/105C12N2501/15C12N2501/16C12N2501/415C12N2506/45C12N2533/52C12N2533/50C12N2500/30C12N2501/155
Inventor BHARTI, KAPILCHASE, LUCASXUEZHU, FENGJHA, BALENDU SHEKHAR
Owner FUJIFILM CELLULAR DYNAMICS INC
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