Method for inducing differentiation of stem cell into dopaminergic neural precursor cell
Inventors
Cho, Myung Soo • EOM, Jang Hyeon • NAM, Seung Taek
Assignees
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Publication Number
US-11655450-B2
Publication Date
2023-05-23
Expiration Date
Abstract
Disclosed herein are a method for inducing differentiation of stem cells into dopaminergic neural precursor cells and a method for mass production of dopaminergic neural precursor cells. Having ability to effectively differentiate stem cells into neural precursor cells, the methods can find advantageous applications in research and development and commercialization associated therewith.
Core Innovation
The invention describes inducing differentiation of stem cells into dopaminergic neural precursor cells through a staged differentiation workflow that includes monolayer stem-cell culture, embryoid body formation and maintenance, neural rosette generation, and differentiation of the neural rosette into dopaminergic neural precursor cells. In the monolayer stage, the method involves adding a BMP signaling inhibitor and an activin/nodal signaling inhibitor to the stem cells.
For embryoid body formation and maintenance, the method further includes adding a sonic hedgehog (SHH) signaling activator and a GSK-3 inhibitor as a Wnt signaling activator in addition to the BMP signaling inhibitor and the activin/nodal signaling inhibitor. The document specifies that the BMP signaling inhibitor and the activin/nodal signaling inhibitor are added daily from the starting day of the embryoid body step, and that the SHH signaling activator and GSK-3 inhibitor are added daily from days 2-6 after the starting day.
After generating the neural rosette, the method differentiates the neural rosette into dopaminergic neural precursor cells, and the differentiation workflow is extended for mass production by proliferating the dopaminergic neural precursor cells through passage. The document also reports marker expression such as FOXA2, LMX1A, and En1, together with high yield and mass production capacity using passage-based proliferation.
Claims Coverage
The partial content provides two independent claims: a differentiation method and a mass-production method. Across both independent claims, the inventive features are organized around a staged workflow and a day-by-day regimen for adding signaling modulators, with an additional proliferating step via passage in the mass-production claim.
Staged differentiation from stem cells via monolayer culture
A method for inducing differentiation of stem cells into dopaminergic neural precursor cells by culturing stem cells in a monolayer format with a BMP signaling inhibitor and an activin/nodal signaling inhibitor.
Embryoid body formation with SHH activation and Wnt modulation
Forming and maintaining an embryoid body by adding a BMP signaling inhibitor, an activin/nodal signaling inhibitor, a sonic hedgehog (SHH) signaling activator, and a GSK-3 inhibitor as a Wnt signaling activator.
Neural rosette generation and differentiation to dopaminergic neural precursor cells
Generating a neural rosette and differentiating the neural rosette into dopaminergic neural precursor cells.
Daily timing schedule for signaling modulator additions
In the embryoid body step, adding the BMP signaling inhibitor and the activin/nodal signaling inhibitor daily from the starting day of the step and adding the SHH signaling activator and GSK-3 inhibitor daily from days 2-6 after the starting day.
Mass production by passaging dopaminergic neural precursor cells
Proliferating the dopaminergic neural precursor cells through passage as part of a mass production method for dopaminergic neural precursor cells.
The independent claims cover a staged differentiation workflow that combines monolayer culture with BMP and activin/nodal inhibition, embryoid body formation with SHH activation and GSK-3 modulation, neural rosette generation, differentiation into dopaminergic neural precursor cells, and passaging for mass production, with a defined daily timing schedule during the embryoid body step.
Stated Advantages
Increases marker expression (FOXA2, LMX1A, En1).
Provides high yield.
Enables mass production with reported capacity of approximately 130 billion cells from one MCB vial.
Stated in vivo efficacy in a 6-OHDA rat Parkinson’s model.
Documented Applications
Use in a 6-OHDA rat Parkinson’s disease model, with stated efficacy and outcomes described as alleviating Parkinson’s symptoms.
Use of intermediate cells stored as a working cell bank (WCB) for clinical entry and transplant preparation.
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