Method for obtaining microglia from pluripotent stem 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-11473057-B2
Publication Date
2022-10-18
Expiration Date
Abstract
The purpose of the present invention is to efficiently produce microglia from pluripotent stem cells. Provided is a method for producing microglia from pluripotent stem cells, comprising the following steps: (a) a step of co-culturing a pluripotent stem cell together with a feeder cell for 7 days or longer, and obtaining a blood progenitor cell; (b) a step of co-culturing the blood progenitor cell obtained in step (a) together with a feeder cell in the presence of IL-3 and/or GM-CSF, and obtaining an embryonic monocyte; and (c) a step of, in the presence of M-CSF, co-culturing the embryonic monocyte obtained in step (b) together with an astrocyte, or culturing the embryonic monocyte using an astrocyte supernatant.
Core Innovation
The invention relates to producing microglia from a pluripotent stem cell by sequential co-culturing using feeder cells and cell-to-cell differentiation stages. The method co-cultures a pluripotent stem cell with a feeder cell for 7 days or more to obtain a blood progenitor cell, then co-cultures the blood progenitor cell with a feeder cell to obtain a primitive monocyte. The sequential conversion links hematopoietic intermediate stages toward microglia differentiation.
The method further converts the primitive monocyte into microglia by co-culturing the primitive monocyte with an astrocyte in the presence of M-CSF, or by culturing the primitive monocyte using an astrocyte supernatant in the presence of M-CSF. The described approach includes the use of IL-3 and/or GM-CSF during the blood progenitor to primitive monocyte stage, and the feeder-based stages are configured to support microglia generation from pluripotent sources, particularly human iPS/ES cells.
The document reports differentiation into primitive/embryonic monocytes and microglia, with microglial marker assessment using Iba1 positivity and projection morphology. It also describes microglia identification/separation from macrophage using P2Y12 and projections, and presents the sequential feeder co-culture pathway as efficiently producing microglia from pluripotent cells.
Claims Coverage
The independent claim (clm-00001) covers a three-stage sequential co-culture workflow with three inventive features: generating blood progenitor cells from a pluripotent stem cell with feeder co-culture, producing primitive monocytes from the blood progenitor stage in the presence of IL-3 and/or GM-CSF, and generating microglia by using astrocytes or astrocyte supernatant with M-CSF.
Feeder co-culture to generate blood progenitor cells from a pluripotent stem cell
Co-culturing a pluripotent stem cell with a feeder cell for 7 days or more to obtain a blood progenitor cell.
Cytokine-supported feeder co-culture to obtain primitive monocytes
Co-culturing the blood progenitor cell obtained by step (a) with a feeder cell in the presence of IL-3 and/or GM-CSF to obtain a primitive monocyte.
Astrocyte-dependent M-CSF step to obtain microglia
Co-culturing the primitive monocyte obtained by step (b) with an astrocyte in the presence of M-CSF, or culturing the primitive monocyte obtained by step (b) using an astrocyte supernatant in the presence of M-CSF.
Overall, the claim coverage centers on sequential feeder co-culturing that produces a blood progenitor cell, then a primitive monocyte under IL-3 and/or GM-CSF, and then microglia generation via an astrocyte or astrocyte supernatant with M-CSF.
Stated Advantages
Efficient production of microglia from pluripotent cells via sequential feeder co-culture.
Documented Applications
Downstream screening uses for microglia-involved diseases.
Interested in licensing this patent?