Method for producing retinal pigment epithelial cells
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11649432-B2
Publication Date
2023-05-16
Expiration Date
Abstract
The present invention provides a method for more efficiently producing retinal pigment epithelial cells from pluripotent stem cells. The method of the present invention for producing retinal pigment epithelial cells includes the following steps:(1) a first step for culturing a pluripotent stem cell in a medium comprising an FGF receptor inhibitor and/or an MEK inhibitor for a period of not more than 30 days, and(2) a second step for culturing the cell 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 relates to a production method of a human retinal pigment epithelial cell from human pluripotent stem cells by using a two-step culturing strategy. Human pluripotent stem cells are maintained and/or expanded in the absence of feeder cells in a medium comprising a factor for maintaining an undifferentiated state, and this is followed by a defined first culturing step intended to induce eye field transcription factor gene expression.
In the first step, the maintained and/or expanded human pluripotent stem cells are cultured in a medium comprising an FGF receptor inhibitor in the absence of feeder cells for a period of not less than 2 days and not more than 30 days. The culture condition in the first step is sufficient for inducing gene expression of at least one eye field transcription factor, and the first step excludes the presence of a MEK inhibitor.
In the second step, cells obtained in the first step are cultured 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 produces retinal pigment epithelial cells efficiently by combining an FGF receptor inhibitor-based first step with a subsequent Nodal and/or Wnt inhibitor-based second step for retinal pigment epithelial formation.
Claims Coverage
The independent claim in the provided content is a single independent method claim. It includes four core inventive constraints: feeder-free maintenance in an undifferentiated state medium, an FGF receptor inhibitor first step with a defined time window, an explicit exclusion of MEK inhibitor during the first step while inducing eye field transcription factor gene expression, and a second step using Nodal and/or Wnt signal transduction pathway inhibitors to form retinal pigment epithelial cells.
Feeder-free maintenance and/or expansion in undifferentiated state
A maintenance and/or expanding step of 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.
FGF receptor inhibitor first step excluding MEK inhibitor and inducing eye field transcription factors
A first step culturing the maintained and/or expanded human pluripotent stem cells in a medium comprising an FGF receptor 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 sufficient for inducing gene expression of at least one eye field transcription factor, wherein the first step excludes the presence of a MEK inhibitor.
Nodal and/or Wnt inhibitor second step to form retinal pigment epithelial cells
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.
The claim coverage centers on a sequential, feeder-free differentiation workflow in which a MEK-excluded FGF receptor inhibitor step induces eye field transcription factor gene expression over a defined 2 to 30 day period, followed by a Nodal and/or Wnt pathway inhibitor step that forms retinal pigment epithelial cells.
Stated Advantages
Improved RPE characteristics and gene marker induction with the two-step inhibitor strategy.
Documented Applications
Toxicity/efficacy evaluation reagents using the described retinal pigment epithelial cells.
Therapeutic RPE cell/pharmaceutical compositions.
Interested in licensing this patent?