Granted Patent
B2
US 12,618,830 · App. 18/065,480 · Granted May 5, 2026
Methods, culture medias and devices for generating embryos in vitro from stem cells
Inventors:
Magdalena D. Zernicka-Goetz (Pasadena, CA); Gianluca Amadei (Cambridge, GB); Charlotte Handford (Cambridge, GB)
Assignee:
California Institute of Technology
G01N33/5088C12N5/0604C12Q1/6841C12N2500/02C12N2501/119C12N2501/33C12N2501/392C12N2503/02C12N2506/025C12N2506/45C12N2513/00C12Q1/6869C12Q2600/158
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Abstract
Disclosed herein include methods and compositions for culture medias for in vitro culture of synthetic embryos from mammalian pluripotent stem cells and extra-embryonic stem cells. The methods and compositions described herein can generate synthetic embryos at different developmental stage reaching early organogenesis and beyond. Disclosed herein also include an embryo culturing system and methods of using same.
Claims (34)
1 . A method of generating a synthetic embryo in vitro, the method comprising:
(a) co-culturing at least one mammalian pluripotent stem cell and an extra-embryonic stem cell in a first culture media under a static condition allowing the mammalian pluripotent stem cell and the extra-embryonic stem cell to self-assemble into a post-implantation embryo structure;
(b) culturing the post-implantation embryo structure in a second culture media under a static condition allowing the post-implantation embryo structure embryo structure to develop into a neurulating embryo structure, wherein the second culture media comprises a basal culture medium, human cord serum, and bicarbonate or HEPES; and
(c) culturing the neurulating embryo structure for at least one day in the second culture media under a dynamic condition in a culture chamber allowing the neurulating embryo structure to develop into a synthetic embryo of at least early organogenesis stage.
2 . The method of claim 1 , wherein the at least one mammalian pluripotent stem cell comprises a wild type mammalian embryonic stem cell and a mammalian embryonic stem cells modified to express an inducible GATA transcription factor upon induction.
3 . The method of claim 1 , wherein the at least one extra-embryonic stem cell comprises a trophoblast stem cell.
4 . The method of claim 2 , wherein the GATA transcription factor is GATA4.
5 . The method of claim 1 , wherein the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in the first culture media for up to 4 days.
6 . The method of claim 1 , wherein step (a) is from embryonic day E0-E5.5 of a mouse embryo structure, step (b) is from embryonic day E5.5 to E8.0 of a mouse embryo structure, and/or step (c) is from embryonic day E8.0 to at least E8.5 of a mouse embryo structure.
7 . The method of claim 1 , wherein the post-implantation embryo structure is a post-implantation pre-gastrulation embryo structure.
8 . The method of claim 1 , wherein the post-implantation embryo structure resembles an E5.5 natural mouse embryo structure, the neurulating embryo structure resembles an E8.0 natural mouse embryo structure, and/or the generated synthetic embryo resembles an E8.5 natural mouse embryo structure, an E9.0 natural mouse embryo structure or beyond.
9 . The method of claim 1 , wherein the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in a substrate, and wherein the substrate comprises a dish, a U-plate, a flask, a microwell plate, or inverted pyramidal microwells.
10 . The method of claim 1 , wherein step (a) comprises culturing the mammalian pluripotent stem cell and the extra-embryonic stem cell in a feeder cell (FC) media and culturing the mammalian pluripotent stem cell and the extra-embryonic stem cell in an in vitro culture (IVC) media following culturing the mammalian pluripotent stem cell and the extra-embryonic stem cell in the FC media,
wherein the FC media and the IVC media comprise a basal culture medium and wherein the basal culture medium comprises Dulbecco's Modified Eagle Media (DMEM), DMEM Nutrient Mixture 12 (DMEM/F12), a non-human serum or serum substitute thereof, a reducing agent, an antibiotic, L-glutamine or an analogue thereof, or any combination thereof.
11 . The method of claim 10 , wherein the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in the FC media for about 2 days and in the IVC media for about 2 days.
12 . The method of claim 10 , wherein the FC media comprises DMEM, fetal bovine serum, sodium pyruvate, L-glutamine or an analogue thereof, MEM non-essential amino acids, 2-mercaptoethanol, penicillin and/or streptomycin, or any combination thereof.
13 . The method of claim 10 , wherein the IVC media comprises:
a) insulin, an insulin analogue, or an insulin receptor agonist;
b) estrogen, an estrogen analogue, or an estrogen receptor agonist; and
c) progesterone, a progesterone analogue, or a progesterone receptor agonist.
14 . The method of claim 10 , wherein the IVC media comprises DMEM/F12, fetal bovine serum, L-glutamine or an analogue thereof, ITS-X, β-estradiol, progesterone, N-acetyl-L-cysteine, penicillin and/or streptomycin, or any combination thereof.
15 . The method of claim 1 , wherein the post-implantation embryo structure is cultured in the second culture media for up to 3 days.
16 . The method of claim 1 , wherein the second culture media comprises DMEM, rat serum, human cord serum, L-glutamine or an analogue thereof, penicillin and/or streptomycin, HEPES, or any combination thereof.
17 . The method of claim 1 , wherein step (b) comprising supplying the second culture media with at least 3 mg/ml glucose.
18 . The method of claim 1 , wherein step (b) comprises culturing the post-implantation embryo structure in a media comprising about 1 mg/ml glucose for two days and culturing the post-implantation embryo structure in a media comprising about 3 mg/ml glucose for one day.
19 . The method of claim 1 , wherein the second culture media in step (c) comprises at least 30% non-human serum.
20 . The method of claim 1 , wherein the dynamic condition comprises suspension agitation.
21 . The method of claim 1 , wherein the culturing the neurulating embryo comprises providing a plurality of gases to the culture chamber at a gas pressure of about 0.5 to 1 psi.
22 . The method of claim 1 , wherein the culture chamber has an atmosphere comprising an incremental increase of oxygen concentration from about 5% to about 25%.
23 . The method of claim 1 , wherein the synthetic embryo has established brain regions, a neural tube, a beating heart, a gut tube, developing somites, and/or primordial germ cells.
24 . The method of claim 1 , wherein the method does not comprise any in vivo step.
25 . The method of claim 1 , wherein the synthetic embryo is a mouse embryo.
26 . A synthetic embryo obtained by the method of claim 1 .
27 . The method of claim 1 , wherein oxygen is supplied to the culture chamber at a constant concentration.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Feb 15, 2023
From: ZERNICKA-GOETZ, MAGDALENA D.
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 062712/0715 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 11, 2023
From: AMADEI, GIANLUCA; HANDFORD, CHARLOTTE
To: CAMBRIDGE ENTERPRISE LIMITED
Reel/Frame 062349/0637 →
Continuity (2)
Provisional Application
63289587
· Dec 14, 2021
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