Efficient and universal method for neural differentiation of human pluripotent stem cells
Inventors
Kim, Dong-Wook • Kim, Dae-sung
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Publication Number
US-8551783-B2
Publication Date
2013-10-08
Expiration Date
Abstract
The present invention relates to a method for inducing neural differentiation of stem cells. In more detail, the present invention relates to a method for inducing neural differentiation of stem cells by inhibiting both BMP (bone morphogenetic protein) and Activin/Nodal signaling pathways in the stem cells. The present invention allows all types of stem cells to effectively differentiate into neural precursor cells regardless of conventional methods for stem cell differentiation including floating culture and attachment culture. In addition, since the neural precursor cells induced by the present invention may be differentiated into specific cells (e.g., dopaminergic neurons) or oligodendrocytes in higher efficient manner, they may be applied to treatment of incurable nerve diseases (e.g., Parkinson's disease or spinal cordinjury) and further provide fundamental data on new drug development.
Core Innovation
The invention relates to a universal neural differentiation method for stem cells selected from the group consisting of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Neural differentiation is induced by inhibiting BMP (bone morphogenetic protein) signaling and Activin/Nodal signaling during differentiation using dorsomorphin and 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide.
The method provides neural precursors with reported reductions in unwanted germ-layer and undifferentiated markers. The generated neural precursor cells differentiate efficiently into dopamine neurons and also oligodendrocytes, and the improvements occur across multiple hPSC lines regardless of innate differentiation propensity.
The generated neural precursors are further differentiated toward dopamine lineage and maturation. Dopamine neuron induction is described using bFGF, Sonic hedgehog, FGF8, GDNF, BDNF, and ascorbic acid, with marker expression and marker-reduction rationale using neuroectoderm markers and suppression of undifferentiation, germ layer, and trophoblast markers.
Claims Coverage
The independent claim family includes one independent claim directed to inducing neural differentiation of hESCs and hiPSCs, based on dual inhibition of BMP and Activin/Nodal signaling with specific small molecules (2 inventive features). A related dependent claim adds sequential culturing steps to progress from neural precursor cells to dopamine precursor cells and then to dopaminergic neurons.
Dual inhibition of BMP and Activin/Nodal signaling
A method for inducing neural differentiation of stem cells selected from hESCs and hiPSCs by inhibiting BMP and Activin/Nodal signaling pathway in the stem cells.
Use of dorsomorphin and 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide
In the inhibition, BMP and Activin/Nodal signaling pathway are inhibited using dorsomorphin and 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide.
Culturing to induce neural differentiation
After the inhibition, culturing the stem cells induces neural differentiation of the stem cells.
Sequential culturing to dopamine lineage
The method further includes sequential culturing steps that progress from neural precursor cells to dopamine precursor cells and then to dopaminergic neurons using bFGF, Sonic hedgehog, FGF 8, GDNF, BDNF, and ascorbic acid.
The claims coverage centers on a neural differentiation method for hESCs and hiPSCs that combines dual pathway inhibition (BMP and Activin/Nodal) using dorsomorphin and a specific benzo-dioxole pyridinyl imidazole benzamide, followed by culturing to induce neural differentiation. A dependent claim further specifies sequential factor-driven progression toward dopamine precursor cells and dopaminergic neurons.
Stated Advantages
Reduces unwanted germ-layer and undifferentiated markers.
Generates neural precursor cells that differentiate efficiently into dopamine neurons.
Reported improvements occur across multiple hPSC lines.
Improvements occur regardless of innate differentiation propensity.
Generated neural precursors can also differentiate into oligodendrocytes.
Documented Applications
Neurodegenerative disease contexts are described, including Parkinson's disease.
Spinal cord injury is described as an application context.
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