Method for producing retinal pigment epithelial cells
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Publication Number
US-12221625-B2
Publication Date
2025-02-11
Expiration Date
Abstract
The invention provides a method for producing a retinal pigment epithelial cell by (1) maintaining and/or expanding human pluripotent stem cells comprising culturing the human pluripotent stem cells in the absence of a feeder cell in a medium comprising a factor for maintaining an undifferentiated state, (2) a first step for culturing the maintained and/or expanded human pluripotent stem cells in a medium comprising a MEK inhibitor in the absence of feeder cells for a period of not less than 2 days and not more than 30 days, wherein the culture condition in the first step is a condition sufficient for inducing gene expression of at least one eye field transcription factor, and (3) a second step for culturing the cells obtained in the first step in the presence of a Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form a retinal pigment epithelial cell.
Core Innovation
The invention provides a production method of a human retinal pigment epithelial cell using human pluripotent stem cells in a feeder-free culture system. Human pluripotent stem cells are maintained and/or expanded in a medium comprising a factor for maintaining an undifferentiated state in the absence of feeder cells.
The method further includes culturing the maintained and/or expanded human pluripotent stem cells in feeder-free condition in a medium comprising a MEK inhibitor. This first step is carried out for a period of not less than 2 days and not more than 30 days, under a culture condition sufficient for inducing gene expression of at least one eye field transcription factor, exemplified by PAX6, LHX2, and SIX3.
A second step cultures the cells obtained in the first step in the presence of a Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form a retinal pigment epithelial cell. The approach is described as yielding RPE cells with markedly higher RPE formation and characteristic brown-black cobblestone morphology, and the produced RPE cells are connected to toxicity/efficacy evaluation using the produced RPE cells.
Claims Coverage
The document provides one independent claim defining a feeder-free, two-step production method for human retinal pigment epithelial cells, with maintenance and/or expansion of human pluripotent stem cells without feeder cells and induction of eye field transcription factor gene expression.
Feeder-free maintenance and/or expansion of human pluripotent stem cells
Maintaining and/or expanding human pluripotent stem cells by culturing the human pluripotent stem cells in the absence of a feeder cell in a medium comprising a factor for maintaining an undifferentiated state.
MEK-inhibitor first step inducing eye-field transcription factor gene expression
Culturing the maintained and/or expanded human pluripotent stem cells in a medium comprising a MEK inhibitor in the absence of feeder cells for a period of not less than 2 days and not more than 30 days, wherein the culture condition in the first step is a condition sufficient for inducing gene expression of at least one eye field transcription factor.
Nodal and/or Wnt pathway inhibitor second step to form RPE cells
Culturing the cells obtained in the first step in the presence of a Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form a retinal pigment epithelial cell.
Across the independent claim, retinal pigment epithelial cell production is achieved through feeder-free maintenance and/or expansion of human pluripotent stem cells, a MEK inhibitor first step sufficient to induce eye field transcription factor gene expression for a defined 2–30 day period, and a second step using Nodal signal transduction pathway inhibitor and/or a Wnt signal transduction pathway inhibitor to form retinal pigment epithelial cells.
Stated Advantages
Markedly higher RPE formation.
Characteristic brown-black cobblestone morphology.
Documented Applications
Toxicity or efficacy evaluation using produced retinal pigment epithelial cells.
Therapeutic use for retinal diseases (e.g., age-related macular degeneration).
Diagnostic use connected to produced retinal pigment epithelial cells.
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