Cell-based treatment and drug discovery in Hirschsprung's disease enabled by pluripotent stem cell-derived human enteric neural crest lineages
Inventors
Studer, Lorenz • Fattahi, Faranak
Assignees
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Publication Number
US-12371662-B2
Publication Date
2025-07-29
Expiration Date
Abstract
The presently disclosed subject matter provides for in vitro methods of inducing differentiation of stem cells into enteric neural crest lineage cells, and enteric neural crest lineage cells by such methods. The presently disclosed subject matter also provides for uses of such enteric neural crest lineage cells for preventing and/or treating enteric nervous system disorders (e.g, Hirschsprung's disease), and for screening compounds suitable for preventing and/or treating enteric nervous system disorders (e.g., Hirschsprung's disease).
Core Innovation
The invention is an in vitro method inducing differentiation of human stem cells to vagal neural crest lineage cells expressing PAX3 and at least one enteric neural crest lineage marker selected from HOXB3 and HOXB5. The method contacts the human stem cells with at least one inhibitor of TGFβ/Activin-Nodal signaling and at least one activator of Wnt signaling.
The cells are further contacted with at least one molecule that induces vagal neural crest patterning. The initial contact of the patterning molecule is between about 2 days and about 6 days from the initial contact of the Wnt activator, and the patterning molecule is selected from retinoic acid, retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling, and combinations thereof.
Claims Coverage
The independent claims cover 2 inventive features directed to in vitro differentiation of human stem cells into vagal neural crest lineage cells expressing PAX3 and HOXB3 and/or HOXB5.
Inducing vagal neural crest lineage cells via coordinated TGFβ/Activin-Nodal inhibition, Wnt activation, and vagal neural crest patterning
An in vitro method inducing differentiation of human stem cells to vagal neural crest lineage cells expressing PAX3 and at least one enteric neural crest lineage marker selected from HOXB3 and HOXB5 by contacting the human stem cells with at least one inhibitor of TGFβ/Activin-Nodal signaling and at least one activator of Wnt signaling, and further contacting the cells with at least one molecule that induces vagal neural crest patterning, where the initial contact of the vagal neural crest patterning molecule is between about 2 days and about 6 days from the initial contact of the Wnt activator, and where the patterning molecule is selected from retinoic acid, retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling, and combinations thereof.
Differentiation protocol with defined vagal patterning timing windows tied to Wnt activation
An in vitro method inducing differentiation of human stem cells to vagal neural crest lineage cells expressing PAX3 and at least one enteric neural crest lineage marker selected from HOXB3 and HOXB5 by contacting the stem cells with at least one inhibitor of TGFβ/Activin-Nodal signaling and at least one activator of Wnt signaling, and further contacting the cells with at least one molecule that induces vagal neural crest patterning, where the initial contact of the patterning molecule is between about 2 days and about 6 days from the initial contact of the Wnt activator, and where the patterning molecule is selected from retinoic acid, retinol, retinal, tretinoin, isotretinoin, alitretinoin, etretinate, acitretin, tazarotene, bexarotene, adapalene, activators of FGF signaling, and combinations thereof.
The claims combine TGFβ/Activin-Nodal signaling inhibition, Wnt signaling activation, and a vagal neural crest patterning-inducing molecule, with specified timing windows relative to Wnt activation.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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