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US 2026/0298908 A1Published application
Reconstructing human early embryogenesis in vitro with pluripotent stem cells
AssigneeCalifornia Institute of Technology
InventorsMagdalena D. Zernicka-Goetz, Berna Sozen, Victoria Jorgensen
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Abstract
Disclosed herein include methods and compositions for in vitro culture of three-dimensional expanded pluripotency (EP) structures from pluripotent stem cells. In some embodiments, the method can include generating expanded pluripotent stem cells (EPSCs) and culturing the EPSCs in a composition capable of supporting generation of the EP structure.
Claims21
-92. (canceled)
An in vitro method of generating an expanded pluripotency (EP) structure in three dimensions, the method comprising:
- a) contacting pluripotent stem cells (PSCs) with EP media to generate expanded pluripotent stem cells (EPSCs);
- b) culturing at least 5 EPSCs with
- a first substrate,
- one or more of a TGFβ ligand, a WNT agonist and a ROCK inhibitor, and
- a fibroblast growth factor (FGF) protein,
- and a composition capable of supporting generation of an EP structure, wherein the composition comprises 20% to 30% EP media, 20% to 30% trophoblast stem cell (TSC) media, 45% to 55% in vitro fertilization (IVF) media, for at least about 4 days;
- wherein step b) comprises reducing the concentration of the FGF protein after at least 48 hours of culturing in the composition,
- whereby the EPSCs self-organize to generate an EP structure.
5 more elements
A composition comprising:
- a culture media comprising 20% to 30% EP media, 20% to 30% trophoblast stem cell (TSC) media, 45% to 55% in vitro fertilization (IVF) media and one or more of a TGFβ ligand, a WNT agonist, a fibroblast growth factor (FGF) protein, and a ROCK inhibitor, and
- an EP structure comprising a single outside layer, an enlarged cavity, and an internal acentric compartment,
- wherein the composition does not comprise an ALK5 kinase inhibitor.
Dependencies are inferred from the claim text.