US-12618830-B2 - Methods, culture medias and devices for generating embryos in vitro from stem cells

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.

Inventors

• Magdalena D. Zernicka-Goetz
• Gianluca Amadei
• Charlotte Handford

Assignees

• CALIFORNIA INSTITUTE OF TECHNOLOGY
• CAMBRIDGE ENTERPRISE LIMITED

Dates

Publication Date

20260505

Application Date

20221213

Claims (20)

1. 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. 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. 3 . The method of claim 1 , wherein the at least one extra-embryonic stem cell comprises a trophoblast stem cell.
4. 4 . The method of claim 2 , wherein the GATA transcription factor is GATA4.
5. 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. 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. 7 . The method of claim 1 , wherein the post-implantation embryo structure is a post-implantation pre-gastrulation embryo structure.
8. 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. 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. 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. 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. 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. 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. 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. 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. 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. 17 . The method of claim 1 , wherein step (b) comprising supplying the second culture media with at least 3 mg/ml glucose.
18. 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. 19 . The method of claim 1 , wherein the second culture media in step (c) comprises at least 30% non-human serum.
20. 20 . The method of claim 1 , wherein the dynamic condition comprises suspension agitation.

Description

RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/289,587, filed Dec. 14, 2021, the content of this related application is incorporated herein by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY SPONSORED R&D This invention was made with government support under Grant No. HD104575 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 30KJ-365851-WO_Sequence_Listing, created Dec. 13, 2022, which is 9 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. BACKGROUND Field The present disclosure relates generally to the field of cell culture, in particular, culturing embryos and stem cells. Description of the Related Art In natural development, the zygote develops into the epiblast, which will form the organism; the extraembryonic visceral endoderm (VE), which contributes to the yolk sac; and the extraembryonic ectoderm (ExE), which contributes to the placenta. Stem cells corresponding to these three lineages offer the possibility to completely regenerate the mammalian organism from multiple components, instead of from a single totipotent zygote. Embryonic stem (ES) cells, which are derived from the epiblast, show a remarkable ability to form embryo-like structures upon aggregation and, when embedded in Matrigel, can be induced to form trunk-like structures with somites, a neural tube and a gut. Although neural development can be promoted in such ‘gastruloids’ by inhibiting the initial burst of Wnt activity, they do not accurately replicate gastrulation movements, nor do they represent the complete anatomy of natural embryos. Other model embryoids generated from ES cells aggregated with an ectopic morphogen signaling center can develop the posterior midbrain, neural tube, cardiac tissue and gut tube only. Thus, these models do not recapitulate the entirety of development to neurulation. There is a need for stem-cell-based embryonic models that can capture natural stages of mammalian development in vitro through and beyond gastrulation and neurulation. SUMMARY Disclosed herein includes a method of generating a synthetic embryo in vitro. The method, in some embodiments, comprises: (a) co-culturing a mammalian pluripotent stem cell and at least one extra-embryonic stem cell in a first culture media under a first 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 second static condition allowing the post-implantation embryo structure embryo structure to develop into a neurulating embryo structure; and (c) culturing the neurulating embryo structure for at least one day 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. The mammalian pluripotent stem cell can comprise a mammalian embryonic stem cell. In some embodiments, the at least one extra-embryonic stem cell comprises a trophoblast stem cell, an inducible extra-embryonic endoderm stem cell, or both. In some embodiments, the inducible extra-embryonic endoderm stem cell is capable of expressing a GATA transcription factor upon induction. The GATA transcription factor can be, for example, GATA4. In some embodiments, the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in the first culture media for up to 4 days. In some embodiments, step (a) is from embryonic day E0-E5.5. In some embodiments, the post-implantation embryo structure is a post-implantation pre-gastrulation embryo structure. In some embodiments, the post-implantation pre-gastrulation embryo structure resembles an E5.5 natural embryo structure. In some embodiments, the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in a substrate, for example a substrate comprising a dish, a U-plate, a flask or a microwell plate. In some embodiments, the mammalian pluripotent stem cell and the extra-embryonic stem cell are cultured in inverted pyramidal microwells. In some embodiments, each of the inverted-pyramidal microwells is about 400 μm or about 800 μm in size, optionally about 400 μm or about 800 μm diameter. In some embodiments, step (a) comprises culturing the mammalian pluripotent stem cell and the extra-embryonic stem cell in a feeder cell (FC) media, optionally passaging the mammalian pluripotent stem cell and the extra-embryonic stem cell in the feeder cell media at least two times. In some embodiments, the mammalian pluripotent stem cell and the extra-