Differentiation of trophectoderm lineage cells from pluripotent stem cells
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsIlyas Singec, Jaroslav Slamecka, Anton Simeonov, Tao Deng
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Abstract
Methods for generating in culture of cells resembling mammalian trophectoderm-lineage cells from mammalian pluripotent stem cells are provided, along with the related compositions.
Claims14
A method of producing a cytotrophoblast-like cell, the method comprising:
- plating a mammalian pluripotent stem cell onto a substrate-coated surface;
- culturing the plated mammalian pluripotent stem cell for at least 12 hours in a first culture medium, thereby producing a plurality of adherent cells;
- and
- culturing the plurality of adherent cells in a second culture medium for approximately 24-96 hours,
- wherein the plurality of adherent cells is approximately 25-50% confluent before the culturing in the second culture medium;
- and
- wherein the second culture medium comprises:
- i) an effective concentration of at least one inhibitor of transforming growth factor beta (TGF-beta) signaling;
- ii) an effective concentration of at least one activator of Wnt signaling;
- iii) an effective concentration of at least one inhibitor of fibroblast growth factor (FGF) receptor signaling;
- iv) an effective concentration of bone morphogenic protein 4 (BMP4);
- and
- v) an effective concentration of bone morphogenic protein 10 (BMP10).
A method of producing a syncytiotrophoblast-like structure, the method comprising culturing a cytotrophoblast-like cell in a culture medium for approximately 168-312 hours, wherein the culture medium comprises an effective concentration of at least one inhibitor of transforming growth factor beta (TGF-beta) signaling and an effective concentration of at least one inhibitor of Wnt signaling.
A method of producing a syncytiotrophoblast-like structure, the method comprising:
- plating a mammalian pluripotent stem cell onto a substrate-coated surface;
- culturing the plated mammalian pluripotent stem cell for at least 12 hours in a first culture medium, thereby producing a plurality of adherent cells;
- and
- culturing the plurality of adherent cells in a second culture medium for approximately 24-96 hours, thereby producing a cytotrophoblast-like cell;
- and
- culturing the cytotrophoblast-like cell in a third culture medium,
- wherein:
- the plurality of adherent cells is approximately 25-50% confluent before the culturing in the second culture medium;
- the second culture medium comprises:
- i) an effective concentration of at least one first inhibitor of transforming growth factor beta (TGF-beta) signaling;
- ii) an effective concentration of at least one first activator of Wnt signaling;
- iii) an effective concentration of at least one inhibitor of fibroblast growth factor (FGF) receptor signaling;
- iv) an effective concentration of bone morphogenic protein 4 (BMP4);
- and
- V) an effective concentration of bone morphogenic protein 10 (BMP 10);
- and
- the third culture medium comprises an effective concentration of at least one second inhibitor of TGF-beta signaling and an effective concentration of at least one second inhibitor of Wnt signaling.
Dependencies are inferred from the claim text.