METHODS, CULTURE MEDIAS AND DEVICES FOR GENERATING EMBRYOS IN VITRO FROM STEM CELLS
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.
1 . A method of generating a synthetic embryo in vitro, the method comprising:
culturing a post-implantation embryo structure in a post-implantation culture media under a static condition allowing the post-implantation embryo structure to develop into a neurulating embryo structure, wherein the post-implantation culture media comprises a basal culture medium, human cord serum, and bicarbonate or HEPES; and
culturing the neurulating embryo structure for at least one day in the post-implantation 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 post-implantation embryo structure is generated by co-culturing at least one mammalian pluripotent stem cell and an extra-embryonic stem cell in a pre-implantation culture media under a static condition.
3 . The method of claim 2 , wherein the at least one mammalian pluripotent stem cell comprises a wild type mammalian embryonic stem cell and a mammalian embryonic stem cell modified to express an inducible GATA transcription factor upon induction, and the extra-embryonic stem cell comprises a trophoblast stem cell.
4 . The method of claim 3 , wherein the GATA transcription factor is GATA4.
5 . The method of claim 1 , wherein
the post-implantation embryo structure (a) is a post-implantation pre-gastrulation embryo structure or (b) 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.
6 . The method of claim 1 , wherein the post-implantation embryo structure is cultured in a substrate, and wherein the substrate comprises a dish, a U-plate, a flask, a microwell plate, or inverted pyramidal microwells.
7 . The method of claim 1 , wherein the post-implantation embryo structure is cultured in the post-implantation culture media under the static condition for up to 3 days.
8 . The method of claim 1 , wherein the post-implantation culture media comprises DMEM, rat serum, human cord serum, L-glutamine or an analogue thereof, penicillin and/or streptomycin, HEPES, or any combination thereof.
9 . The method of claim 1 , wherein culturing the post-implantation embryo structure in the post-implantation culture media under the static condition comprises
(a) supplying the post-implantation culture media with at least 3 mg/ml glucose; or
(b) 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.
10 . The method of claim 1 , wherein the post-implantation culture media comprises at least 30% non-human serum.
11 . The method of claim 1 , wherein the dynamic condition comprises suspension agitation.
12 . The method of claim 1 , wherein culturing the neurulating embryo structure comprises providing a plurality of gases to the culture chamber at a gas pressure of about 0.5 to 1 psi.
13 . The method of claim 1 , wherein the culture chamber has an atmosphere comprising (a) an incremental increase of oxygen concentration from about 5% to about 25% or (b) a constant concentration of oxygen.
14 . The method of claim 2 , wherein the at least one mammalian pluripotent stem cell and the extra-embryonic stem cell are co-cultured in the pre-implantation culture media for up to 4 days.
15 . The method of claim 2 , wherein co-culturing the at least one mammalian pluripotent stem cell and the extra-embryonic stem cell comprises culturing the at least one mammalian pluripotent stem cell and the extra-embryonic stem cell in a feeder cell (FC) media and culturing the at least one mammalian pluripotent stem cell and the extra-embryonic stem cell in an in vitro culture (IVC) media following culturing the at least one 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.
16 . The method of claim 15 , 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, and
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.
17 . 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.
18 . The method of claim 1 , wherein the method does not comprise any in vivo step.
19 . A synthetic embryo obtained by the method of claim 1 .
20 . A method of investigating the effect of a test agent on embryonic development, comprising
contacting the synthetic embryo of claim 19 with a test agent; and
determining the effect of the test agent on the synthetic embryo.
21 . A method of diagnosing or treating a disease or disorder in a subject, comprising:
transplanting the synthetic embryo of claim 19 into the subject, wherein the synthetic embryo is generated using at least one mammalian pluripotent stem cell and an extraembryonic stem cell obtained from the subject.