Patent Application
20230115640
Current methods for generating mouse blastocyst-like structures, termed blastoids, require the sequential aggregation of embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) in microwells. However, these blastoids are unable to model post-implantation development since they poorly develop into post-implantation embryo-like structures in vitro and only generate trophoblast cell types in vivo. These blastoids also are unable to model the pre-implantation because of the nature of the assembly method. Thus, there remains an unmet need for blastoids that are derived from one cell type and which develop to include all three founder tissues of a blastocysts: pluripotent epiblast (EPI) cells, trophectoderm (TE), and primitive endoderm (PE).
SUMMARY
In an aspect, there are provided methods of producing a blastoid, the method comprising: (a) obtaining or providing an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632, the FGF is FGF4, the Wnt agonist is Wnt-3a or CHIR99021, the BMP is BMP4, and/or the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, after about 24 hours, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the culturing is conducted for about 5 days. In some cases, the method further comprises at step (b) culturing the EPS cell with a trophectoderm (TE) cell.
In another aspect, there are provided methods of assisted reproduction of an individual, the method comprising: (a) obtaining or providing an extended pluripotent stem (EPS) cell derived from the individual; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor; (c) isolating a resulting blastoid; (d) transferring the resulting blastoid to a uterus. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632, the FGF is FGF4, the Wnt agonist is Wnt-3a or CHIR99021, the BMP is BMP4, and/or the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, after about 24 hours, the medium is replaced with a medium without Y-27632. In some cases, the culturing is conducted for about 5 days. In some cases, the individual is a mammal selected from a mouse, a rat, a rabbit, a horse, a sheep, a cow, a dog, a cat, an elephant, a whale, a rhinoceros, a non-human primate, or a human. In some cases, the EPS cell is an induced EPS cell derived from a somatic cell. In some cases, the method further comprises at step (b) culturing the EPS cell with a trophectoderm (TE) cell. In some cases, the uterus is receptive to implantation.
In another aspect, there are provided methods of determining a drug toxicity, the method comprising: (a) obtaining or providing a blastoid produced by a method according to any method provided herein; (b) contacting the blastoid to the drug; and (c) detecting signs of toxicity. In some cases, the signs of toxicity comprise cell death, loss of blastoid cell organization, arrest in blastoid growth or development.
In various embodiments of methods herein, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium.
In another aspect, there are provided blastoids, produced or producible by a method comprising: (a) obtaining an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632, the FGF is FGF4, the Wnt agonist is Wnt-3a or CHIR99021, the BMP is BMP4, and/or the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, after about 24 hours, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the culturing is conducted for about 5 days. In some cases, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium. In some cases, the Wnt agonist is CHIR99021. In some cases, the TGF-β signaling inhibitor comprises A83-01, SB431543, OR REPSOX. In some cases, the EPS cell is an induced EPS cell derived from a somatic cell. In some cases, the blastoid is produced by a method where at step (b) culturing the EPS cell with a trophectoderm (TE) cell. In some cases, the EPS is derived from a mammal selected from a mouse, a rat, a rabbit, a horse, a sheep, a cow, a dog, a cat, an elephant, a whale, a rhinoceros, a non-human primate, or a human.
Provided herein are 3D differentiation systems that, in some embodiments, enable generation of blastocyst-like structures (EPS-blastoids) which are derived from a single stem cell type, the extended pluripotent stem (EPS) cell. When cultured, the EPS-blastoids generate structures characteristic of an E5.0-E5.5 post-implantation embryo. EPS-blastoids are transcriptionally similar to natural E3.5 blastocysts and contain all three blastocyst cell lineages. In utero EPS-blastoids are capable of implantation, triggering decidualization, and give rise to structures containing live tissues of EPI, TE, and PE origins. Also, EPS-blastoids can be generated from mouse fibroblasts; thus, embryo-like structures are produced from somatic cells. Accordingly, the herein disclosed EPS-blastoids serve as a model of early embryogenesis (both preimplantation and postimplantation) for testing candidate gene mutations, drug screening, and understanding the basic principles of embryo development. Further, the 3D differentiation system also serves as a framework for advancing the development of fully functional synthetic blastocysts in mice or other mammalian species.
In certain aspects, there are provided methods of producing a blastoid. The method comprising steps of (a) obtaining or providing an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid.
In embodiments, the medium comprises two or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises three or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor.
In embodiments, the medium comprises four or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises five or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor.
In embodiments, the EPS cell is cultured in a v-bottomed microwell plate. In embodiments, the v-bottomed microwell plate is an AggreWell plate. In embodiments, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate.
In embodiments, after about 24 hours, the medium is replaced with a medium without a ROCK inhibitor, such as Y-27632.
In embodiments, the culturing is conducted for about 5 days.
In additional aspects, there are provided, methods of assisted reproduction of an individual. The method comprising steps of: (a) obtaining or providing an extended pluripotent stem (EPS) cell derived from the individual; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; (c) isolating a resulting blastoid; (d) transferring the resulting blastoid to a uterus.
In embodiments, the medium comprises two or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises three or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises four or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises five or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor.
In embodiments, the EPS cell is cultured in a v-bottomed microwell plate. In embodiments, the v-bottomed microwell plate is an AggreWell plate. In embodiments, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate.
In embodiments, after about 24 hours, the medium is replaced with a medium without a ROCK inhibitor, such as Y-27632.
In embodiments, the culturing is conducted for about 5 days.
In embodiments, the individual is a mammal. In embodiments, the individual is a mouse, a rat, a rabbit, a horse, a sheep, a cow, a dog, a cat, an elephant, a whale, a rhinoceros, a non-human primate, or a human. In embodiments, the uterus is receptive to implantation.
In embodiments, the EPS cell is an induced EPS cell derived (e.g., reprogrammed) from a somatic cell.
In embodiments, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium.
In embodiments, the Wnt agonist comprises CHIR99021 or Wnt-3a.
In embodiments, the TGF-β signaling inhibitor comprises A83-01, SB431543, OR REPSOX.
In additional aspects, there are provided methods of determining a drug toxicity. The method comprising steps of: (a) obtaining or providing a blastoid produced by a method according to any herein-described method (b) contacting the blastoid to the drug; and (c) detecting signs of toxicity.
In embodiments, the signs of toxicity comprise cell death, loss of blastoid cell organization, arrest in blastoid growth or development.
In embodiments, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium.
In embodiments, the Wnt agonist comprises CHIR99021 or Wnt-3a.
In embodiments, the TGF-β signaling inhibitor comprises A83-01, SB431543, OR REPSOX.
In certain aspects, there are provided, blastoids, e.g., produced or producible by a method comprising steps of: (a) obtaining an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid.
In embodiments, the medium comprises two or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises three or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises four or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises five or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor. In embodiments, the medium comprises Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor.
In embodiments, the EPS cell is cultured in a v-bottomed microwell plate. In embodiments, the v-bottomed microwell plate is an AggreWell plate. In embodiments, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate.
In embodiments, after about 24 hours, the medium is replaced with a medium without a ROCK inhibitor, such as Y-27632.
In embodiments, the culturing is conducted for about 5 days.
In embodiments, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium.
In embodiments, the Wnt agonist comprises CHIR99021 or Wnt-3a.
In embodiments, the TGF-β signaling inhibitor comprises A83-01, SB431543, OR REPSOX.
In embodiments, the EPS cell is an induced EPS cell derived (e.g., reprogrammed) from a somatic cell.
In additional aspects, there are provided blastoids in which at least ⅛ of its cells are derived from a common progenitor. As examples, at least ¼, at least ½, or at least ¾ of the cells in a blastoid are derived from a common progenitor.
Any herein described aspect or embodiment may be combined with any other herein described aspect or embodiment.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Data above are represented as mean±SEM. Scale bar, 500 μm (
Provided herein are 3D differentiation systems that enable generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization and which are derived from a single stem cell type, extended pluripotent stem (EPS) cell.
Additionally provided herein are EPS-blastoids that resemble blastocysts in morphology and cell lineage allocation and recapitulate key morphogenetic events during preimplantation and early postimplantation development in vitro. Upon transfer, EPS-blastoids undergo implantation, induce decidualization, and generate live tissues in utero. EPS-blastoids contain all three blastocyst cell lineages and share transcriptional similarity with natural blastocysts. EPS-blastoids can be generated from adult cells which have acquired stem-cell like characteristics via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to create viable synthetic embryos using cultured cells.
Indeed, since the EPS-blastoids undergo morphogenetic events characteristic of post-implantation development upon further culturing, the EPS-blastoid model provided herein allows a unique platform for studying the peri- and post-implantation in a high throughput manner.
Moreover, the EPS-blastoid model provided herein offers a unique platform for studying the effects of genetic variants on early embryogenesis. Compared to the use of genetically modified mouse, the herein-described EPS-blastoid model bypasses the time-consuming process of establishing mouse model and serves as a screening process at the beginning of an animal study. Similarly, the EPS-blastoid model offers a unique platform for studying the effects of de novo mutations (DNMs) on early embryogenesis.
Also, the EPS-blastoid model serves as a platform to test drug toxicity on early embryo development, and in a high throughput manner. Compared to normal mouse embryos, the herein-disclosed model has an advantage of integrating genetic variants and drug toxicology, hence providing a chance to look into how genetic variants affect the response to drugs. Further, since the EPS-blastoids may be derived from a single cell, or at least one cell type, each or a majority of cells in the resulting blastoid may similarly respond to a drug and/or express the same genetic variant. In other words, the present disclosure enables more straightforward interpretation of the readouts after introducing genetic and/or epigenetic changes.
The EPS-blastoids as disclosed herein are useful to produce specific lineage progenitors in a 3D setting, which has the advantages of mimicking the natural environment; this is a significant advantage over methods employing 2D cultures.
Additionally, the EPS-blastoid model is useful for the development of ways to preserve the endangered species, by creating adult animals that are derived from somatic cells, e.g., from a male or from an infertile female.
Provided herein are methods of producing a blastoid, such as a mammalian blastoid. In some cases, the method comprises obtaining or providing an extended pluripotent stem (EPS) cell. In some cases, the method comprises culturing the EPS cell in a medium comprising one or more factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor. In some cases, the method comprises isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632. In some cases, the FGF is FGF4. In some cases, the Wnt agonist is Wnt-3a or CHIR99021. In some cases, the BMP is BMP4. In some cases, the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX.
In additional aspects, there are provided methods of producing a blastoid. The method comprising steps of (a) obtaining or providing an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid.
Methods herein culture EPS cells in methods of producing a blastoid in any suitable culture vessel. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate.
In additional aspects, methods of producing a blastoid herein comprise centrifuging a culture vessel comprising the EPS cells and the culture media. In some cases, the v-bottomed plate is centrifuged at about 50×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 100×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 150×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 200×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 250×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 350×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 400×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 450×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 500×g after the cell and medium is added to the plate.
In further aspects of methods of producing a blastoid provided herein, the contents of the culture medium are changed after culturing the EPS cell for a period of time. In some cases, the contents of the culture medium are changed after about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours after culturing the EPS cell. In some cases, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the medium is replaced with a medium without the FGF. In some cases, the medium is replaced with a medium without heparin. In some cases the medium is replaced with a medium without the Wnt agonist. In some cases, the medium is replaced with a medium without the BMP. In some cases, the medium is replaced with a medium without the TGF-β signaling inhibitor.
In further aspects of methods of producing a blastoid provided herein, the EPS cell is cultured for an appropriate period of time sufficient to form the blastoid. In some cases, the culturing is conducted for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more as needed. In some cases, the culturing is conducted for about 1-5 days, about 2-6 days, about 3-7 days, about 4-8 days, about 5-9 days, or about 6-10 days.
In additional aspects of methods of producing a blastoid provided herein, the method comprises culturing the EPS cell with additional cell types that facilitate production of the blastoid. For example, in some cases the method comprises culturing the EPS cell with a trophectoderm (TE) cell.
In further aspects provided herein, in some cases, blastoids are derived from a mammalian EPS cell. In some cases, the mammalian EPS cell is an EPS cell from a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.
Further provided herein are methods of assisted reproduction of an individual. In some cases, the method comprises obtaining or providing an extended pluripotent stem (EPS) cell derived from the individual. In some cases, the method comprises culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor. In some cases, the method comprises isolating a resulting blastoid. In some cases, the method comprises transferring the resulting blastoid to a uterus. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632. In some cases, the FGF is FGF4. In some cases, the Wnt agonist is Wnt-3a or CHIR99021. In some cases, the BMP is BMP4. In some cases, the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX.
In further aspects, there are provided methods of assisted reproduction of an individual. The method comprising steps of: (a) obtaining or providing an extended pluripotent stem (EPS) cell derived from the individual; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; (c) isolating a resulting blastoid; (d) transferring the resulting blastoid to a uterus.
In further aspects of methods of assisted reproduction provided herein, a blastoid is produced in any suitable culture vessel. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate.
In additional aspects, methods of assisted reproduction provided herein comprise centrifuging a culture vessel comprising the EPS cells and the culture media. In some cases, the v-bottomed plate is centrifuged at about 50×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 100×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 150×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 200×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 250×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 350×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 400×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 450×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 500×g after the cell and medium is added to the plate.
In further aspects of methods of assisted reproduction provided herein, the contents of the culture medium are changed after culturing the EPS cell for a period of time. In some cases, the contents of the culture medium are changed after about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours after culturing the EPS cell. In some cases, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the medium is replaced with a medium without the FGF. In some cases, the medium is replaced with a medium without heparin. In some cases the medium is replaced with a medium without the Wnt agonist. In some cases, the medium is replaced with a medium without the BMP. In some cases, the medium is replaced with a medium without the TGF-β signaling inhibitor.
In further aspects of methods of assisted reproduction provided herein, the EPS cell is cultured for an appropriate period of time sufficient to form the blastoid. In some cases, the culturing is conducted for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more as needed. In some cases, the culturing is conducted for about 1-5 days, about 2-6 days, about 3-7 days, about 4-8 days, about 5-9 days, or about 6-10 days.
In further aspects of assisted reproduction provided herein, in some cases, the individual is a mammal. In some cases, the mammal is a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.
In additional aspects of methods of assisted reproduction provided herein, the EPS cell is an induced EPS cell derived from a somatic cell. In some cases, the somatic cell is any cell derived from an individual that is not a germ cell. Any suitable somatic cell is contemplated to be used in methods herein. A non-limiting list of somatic cells for use in methods herein include an endothelial cell, an epithelial cell, a blood cell, an adipocyte, a neuron, an osteoclast, a chondrocyte, a myocyte, or other cell type.
In additional aspects of methods of assisted reproduction provided herein, the method comprises culturing the EPS cell with additional cell types that facilitate production of the blastoid. For example, in some cases the method comprises culturing the EPS cell with a trophectoderm (TE) cell.
In additional aspects of methods of assisted reproduction provided herein the uterus of the individual is receptive to implantation. In some cases, the individual is treated with a medication in order to prepare the uterus for implantation. In some cases, the menstrual cycle and endometrial thickness of the individual is monitored for receptivity to implantation.
In an aspect there provided, compositions comprising an extended pluripotent stem cell and at least one factor selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor. In some cases, isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632. In some cases, the FGF is FGF4. In some cases, the Wnt agonist is Wnt-3a or CHIR99021. In some cases, the BMP is BMP4. In some cases, the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX. In some cases, the composition further comprises a trophectoderm cell. In some cases, the composition comprises a blastoid.
In some aspects there are provided blastoids that are produced or producible by a method herein. In some cases, the method comprises obtaining an extended pluripotent stem (EPS) cell. In some cases, the method comprises culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor. In some cases, the method comprises isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632. In some cases, the FGF is FGF4. In some cases, the Wnt agonist is Wnt-3a or CHIR99021. In some cases, the BMP is BMP4. In some cases, the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX.
In additional aspects, there are provided blastoids, e.g., produced or producible by a method comprising steps of: (a) obtaining an extended pluripotent stem (EPS) cell; (b) culturing the EPS cell in a medium comprising one or more of factors selected from the group consisting of Y-27632, FGF4, Heparin, a Wnt agonist, BMP4, and a TGF-β signaling inhibitor; and (c) isolating the resulting blastoid.
In additional aspects, blastoids herein are produced or producible by a method comprising centrifuging a culture vessel comprising the EPS cells and the culture media. In some cases, the v-bottomed plate is centrifuged at about 50×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 100×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 150×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 200×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 250×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 350×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 400×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 450×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 500×g after the cell and medium is added to the plate.
In further aspects, blastoids herein are produced or producible by a method wherein the contents of the culture medium are changed after culturing the EPS cell for a period of time. In some cases, the contents of the culture medium are changed after about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours after culturing the EPS cell. In some cases, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the medium is replaced with a medium without the FGF. In some cases, the medium is replaced with a medium without heparin. In some cases the medium is replaced with a medium without the Wnt agonist. In some cases, the medium is replaced with a medium without the BMP. In some cases, the medium is replaced with a medium without the TGF-β signaling inhibitor.
In further aspects, blastoids herein are produced or producible by a method wherein the EPS cell is cultured for an appropriate period of time sufficient to form the blastoid. In some cases, the culturing is conducted for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more as needed. In some cases, the culturing is conducted for about 1-5 days, about 2-6 days, about 3-7 days, about 4-8 days, about 5-9 days, or about 6-10 days.
In further aspects, blastoids herein are produced or producible by a method wherein the EPS cell is an induced EPS cell derived from a somatic cell. In some cases, the somatic cell is any cell derived from an individual that is not a germ cell. Any suitable somatic cell is contemplated to be used in methods herein. A non-limiting list of somatic cells for use in methods herein include an endothelial cell, an epithelial cell, a blood cell, an adipocyte, a neuron, an osteoclast, a chondrocyte, a myocyte, or other cell type.
In additional aspects, blastoids herein are produced or producible by a method wherein the EPS cell is cultured with additional cell types that facilitate production of the blastoid. For example, in some cases the method comprises culturing the EPS cell with a trophectoderm (TE) cell.
In additional aspects provided herein, in some cases, blastoids are derived from a mammalian EPS cell. In some cases, the mammalian EPS cell is an EPS cell from a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.
Mammalian blastoids created using methods disclosed herein are contemplated for a variety of uses including drug screening, reproductive medicine, and other research uses and methods. In some cases, uses of mammalian blastoids provided herein is determining a drug toxicity.
In further aspects, there are provided methods of determining a drug toxicity. The method comprising steps of: (a) obtaining or providing a blastoid produced by a method according to any herein-described method (b) contacting the blastoid to the drug; and (c) detecting signs of toxicity. In some cases, the method comprises obtaining or providing an extended pluripotent stem (EPS) cell. In some cases, the method comprises culturing the EPS cell in a medium comprising one or more factors selected from the group consisting of a ROCK inhibitor, a FGF, Heparin, a Wnt agonist, a BMP, and a TGF-β signaling inhibitor. In some cases, the method comprises isolating the resulting blastoid. In some cases, the medium comprises two or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises three or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises four or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the medium comprises five or more factors selected from the group consisting of the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and a TGF-β signaling inhibitor. In some cases, the medium comprises the ROCK inhibitor, the FGF, Heparin, the Wnt agonist, the BMP, and the TGF-β signaling inhibitor. In some cases, the ROCK inhibitor is Y-27632. In some cases, the FGF is FGF4. In some cases, the Wnt agonist is Wnt-3a or CHIR99021. In some cases, the BMP is BMP4. In some cases, the TGF-β signaling inhibitor is A83-01, SB431543, OR REPSOX.
Methods herein culture EPS cells in methods of producing a blastoid for testing drug toxicity in any suitable culture vessel. In some cases, the EPS cell is cultured in a v-bottomed microwell plate. In some cases, the v-bottomed microwell plate is an AggreWell plate.
In additional aspects, methods of producing a blastoid for testing drug toxicity herein comprise centrifuging a culture vessel comprising the EPS cells and the culture media. In some cases, the v-bottomed plate is centrifuged at about 50×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 100×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 150×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 200×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 250×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 300×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 350×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 400×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 450×g after the cell and medium is added to the plate. In some cases, the v-bottomed plate is centrifuged at about 500×g after the cell and medium is added to the plate.
In further aspects of methods of producing a blastoid for testing drug toxicity provided herein, the contents of the culture medium are changed after culturing the EPS cell for a period of time. In some cases, the contents of the culture medium are changed after about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours after culturing the EPS cell. In some cases, the medium is replaced with a medium without the ROCK inhibitor. In some cases, the medium is replaced with a medium without the FGF. In some cases, the medium is replaced with a medium without heparin. In some cases the medium is replaced with a medium without the Wnt agonist. In some cases, the medium is replaced with a medium without the BMP. In some cases, the medium is replaced with a medium without the TGF-β signaling inhibitor.
In further aspects of methods of producing a blastoid for testing drug toxicity provided herein, the EPS cell is cultured for an appropriate period of time sufficient to form the blastoid. In some cases, the culturing is conducted for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or more as needed. In some cases, the culturing is conducted for about 1-5 days, about 2-6 days, about 3-7 days, about 4-8 days, about 5-9 days, or about 6-10 days.
In additional aspects of methods of producing a blastoid for testing drug toxicity provided herein, the method comprises culturing the EPS cell with additional cell types that facilitate production of the blastoid. For example, in some cases the method comprises culturing the EPS cell with a trophectoderm (TE) cell.
In further aspects provided herein, in some cases, blastoids are derived from a mammalian EPS cell. In some cases, the mammalian EPS cell is an EPS cell from a human, a mouse, a rat, a rabbit, a cat, a dog, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, a goat, an elephant, a rhinoceros, an orangutan, a gorilla, a bonobo, a chimpanzee, a monkey, a panda, a tiger, a whale, a dolphin, a sea lion, a narwhal, a beluga, a fox, a wolf, a pronghorn, a kangaroo, a sloth, a koala, a hippopotamus, a bear, or a leopard.
In embodiments, the EPS cell is cultured in a medium comprising a KSOM or comprising an M16 medium.
In embodiments, the EPS cell is cultured in a medium comprising a Wnt agonist, e.g., CHIR99021 or Wnt-3a.
In embodiments, the EPS cell is cultured in a medium comprising a TGF-β signaling inhibitor, e.g., A83-01, SB431543, OR REPSOX.
Indeed, the present disclosure provides for use of single EPS which differentiates and self-organize into blastocyst-like structures which comprises all three blastocyst lineages.
In embodiments, differentiation conditions are modified by addition of, as examples, different growth factors, cytokines, and small molecules. These additions enable the generation of structures mimicking blastocysts (EPS-blastoids), which contain a cavity and an inner cell mass. As described herein, combinations of FGF4, Heparin, BMP4, CHIR99021, and/or A83-01 provide high efficiency EPS-blastoid formation. Each of these additions may be added alone or in any combination thereof. EPS cells treated with Y-27632, a Rho kinase (ROCK) inhibitor, on the day of seeding enhances cell survival. In embodiments, combinations of the additions provide emergence of EPS-blastoids around three days after cell seeding.
In embodiments, EPS-blastoids enlarge and acquire an early blastocyst-like size at around day five or day six.
. The average diameter of EPS-blastoids produced according to embodiments provided herein are comparable to that of E3.5 blastocysts.
WNT/β-catenin signaling pathway is dispensable for blastocyst formation and , the WNT antagonists, XAV939 or IWR-1-endo, significantly inhibited EPS-blastoids formation.
In embodiments, a single EPS cell gives rise to an entire EPS-blastoid.
The extended pluripotency of EPS cells are helpful for generating blastoids, when compared to use of ES cells.
Key cellular and molecular events characteristic of early preimplantation development are recapitulated during EPS-blastoid formation.
Like blastocysts, EPS cell aggregates undergo polarization and recapitulate the process of polarization characteristic of early preimplantation development.
Like blastocysts, EPS-blastoid formation also requires an intact Hippo/YAP signaling pathway.
EPS-blastoid formation recapitulates key molecular and cellular processes characteristic of early preimplantation development. For example, cells of the EPS-blastoid gradually reactivate inactivated paternal X chromosome.
The cellular composition of EPS-blastoids resembles early blastocysts. For example, EPS-blastoids possess the three lineages of blastocysts
In particular, like E3.5 mouse blastocysts, EPS blastoids have two cell lineages, the external trophectoderm (TE) layer and the internal inner cell mass (ICM).
Like blastocysts, the TE and ICM lineages segregate during EPS-blastoid formation.
Similar to how early blastocysts further develop, the ICM of EPS-blastoids segregates into two lineages: epiblast (EPI) cells and primitive endoderm (PE) cells.
EPS-blastoids exhibit blastocyst-like allocation of cell lineages. For example, they exhibit CDX2, SOX2/NANOG, and GATA4 staining consistent with blastocysts.
RNA expression of EPS-blastoids more resembled blastocysts than morulae.
EPS-blastoids contain all three blastocyst cell lineages.
Like blastocysts, ESCs, TSCs, and XEN cells, which are considered the in vitro counterparts of EPI, TE, and PE lineages are derivable from EPS-blastoids. Also, cells of the EPS-blastoids express genes consistent with natural blastocysts.
Upon further cultivation, EPS-blastoids give develop into postimplantation embryo-like structures.
Data above are represented as mean±SEM. Scale bar, 500 μm (
EPS-blastoids implant, trigger decidualization, and continue to grow inside the uterus.
Scale bar, 1 mm (
Induced EPS (iEPS) cells can be used to generate iEPS-blastoids. iEPS-blastoids generated from adult somatic cells are similar to those from embryo-derived stem cells. Similar to EPS-blastoids, iEPS-blastoids also morphologically resemble natural blastocysts and are of similar size as E3.5 blastocysts.
The process of the induction of iEPS-blastoids recapitulates the compaction, polarization, and changes in subcellular YAP localization
iEPS-blastoids displayed the correct spatial expression of markers for both embryonic and extraembryonic lineages.
Further culture of iEPS-blastoids generates egg-cylinder structures containing ExE-, EPI-, and VE-like compartments (marked by TFAP2C, SOX2/OCT4, and GATA4, respectively).
iEPS-blastoids implant into the uterus and induce the formation of decidua.
Data are represented as mean±SEM. Scale bar, 1 mm (
In the herein-disclosed data, it is shown that EPS cells alone differentiate and self-organize to generate blastocyst-like structures that share several cellular, molecular, and functional features with natural blastocysts (
In addition, the herein disclosed EPS-only blastoid approach has several additional advantages. First, unlike ETS-blastoids, which are generated by sequential aggregation using multiple cells from two stem cell types (ESCs and TSCs), all cells within EPS-blastoids come from a single cell type (even from a single cell). This enables a more straightforward interpretation of the readouts after introducing genetic or epigenetic changes. Second, EPS-blastoids further cultured in vitro recapitulate several key morphogenetic processes of early postimplantation development, forming an egg cylinder embryo-like structure. Without wishing to be bound by theory, an advantage provided herein is due to EPS cells' superior ability to generate PE cells than ESCs.
Bulk RNA-Seq analysis of individual EPS-blastoids revealed that they were more similar to blastocysts than morulae. Single-cell RNA-Seq analysis confirmed that EPS-blastoids contained all three cell lineages of blastocysts. Several DEGs for each lineage (EPFICM, TE, and PE) were determined between EPS-blastoids and blastocysts. For PE, DEGs seem to be enriched in terms related to vesicle transport and endocytosis. For ICM/EPI, a group of DNA methylation- and genomic imprinting-related genes, namely Tet1, Dnmt3L, Zfp42, Atrx, and Tdrd12, were expressed at lower levels in EPS-blastoids than blastocysts. These data suggest that epigenetic abnormalities, well-known defects for embryonic development (Barton et al., 1991; Surani et al., 1990), likely play a negative role in EPS-blastoids development in utero. Another notable observation from single-cell RNA-Seq analysis is that there are several subpopulations between EPI/ICM and TE. These cells likely represent uncommitted or improperly differentiated cells, likely as a result of suboptimal differentiation condition.
Although mouse EPS cells exhibit bi-directional developmental potency (in vivo chimera formation (Yang et al., 2017b) and in vitro blastoid generation) as well as some molecular features of early preimplantation embryos, they are clearly not equivalent to totipotent blastomeres. Nonetheless, and despite that, the EPS-blastoids provided herein followed evolutionary conserved developmental processes which faithfully recapitulated highlights of the plastic yet remarkably regulatory nature of early mammalian embryos. The transcriptional differences between laboratory created and naturally evolved blastocysts uncovered in the herein disclosed data, reflect distinct molecular trajectories that, nonetheless, lead to the generation of similarly patterned structures.
In summary, the present disclosure provides a 3D differentiation system for generating blastoids from cultured EPS cells derived from embryonic or adult sources. The herein disclosed data serves as a framework for advancing the development of fully functional synthetic blastocysts, not only in mice but also in other mammalian species, including humans. As such this system could be used as an in vitro model for studying fundamental questions in both preimplantation and early postimplantation mammalian embryogenesis, modeling diseases related to early pregnancy, high-throughput pharmacological and toxicological screens, and possibly bioengineered embryogenesis.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
The ability of a single EPS cell to contribute to all three blastocyst lineages suggests that, under certain condition, EPS cells differentiate and self-organize into blastocyst-like structures (
To further improve the differentiation conditions, different growth factors, cytokines, and small molecules were included to identify condition(s) that enable the generation of structures mimicking blastocysts (EPS-blastoids), which contain a cavity and an inner cell mass. A combination of FGF4, Heparin, BMP4, CHIR99021, and A83-01 resulted in the highest efficiency of EPS-blastoid formation. EPS cells treated with Y-27632, a Rho kinase (ROCK) inhibitor, on the day of seeding enhanced cell survival. Using this optimized condition, EPS-blastoids consistently emerged three days after cell seeding (
WNT/β-catenin signaling pathway is dispensable for blastocyst formation (
Next, it was determined that a single EPS cell could give rise to an entire EPS-blastoid. Surprisingly, individual EPS cells did not survive in the herein-disclosed 3D differentiation system. To overcome this problem, puromycin resistant and mCherry+ EPS cells were mixed with helper wild type EPS cells at the ratio of 1:10 for the initial plating. Low concentration of puromycin (0.25 μg/ml) was added to the differentiation medium 24 hours later to eliminate helper cells gradually (
Extended pluripotency was then shown to be helpful for generating blastoids. To this end, blastoid formation efficiencies were compared between an ES cell line (Tanimoto et al., 2008) and EPS cells converted from the same ES cell line. While approximately 8% of ES-converted EPS cell aggregates formed EPS-blastoids, little to no (˜0.2%) ES cell aggregates generated blastocyst-like structures (ES-blastoids) (
Besides EPS cells cultured in LCDM condition (Yang et al., 2017b), EPS cells generated and cultured under a different culture condition developed by Pengtao Liu's group (referred to as Liu-EPS; Yang et al., 2017a) also generated EPS-blastoids using the herein-disclosed 3D differentiation system in approximately 11% of cell aggregates (
Key cellular and molecular events characteristic of early preimplantation development were recapitulated during EPS-blastoid formation. Beginning at the 8-cell stage, blastomeres undergo compaction, which is characterized by the formation of intercellular junctions (Rossant and Tam, 2009; Wang et al., 2008). A similar process occurs during EPS-blastoid formation. To assay this, the dynamics of EPS cell aggregation during the first 18 hours was monitored. Four hours after seeding, cells were found loosely connected. At approximately 18 hours, cells started to form compact aggregates (
Following blastomere compaction, polarization begins. Apical domain proteins, such as atypical protein kinase C (aPKC) and the Par complex proteins (PAR3 and PAR6), start to accumulate at the apical side of the blastomeres (Chazaud and Yamanaka, 2016; Rossant and Tam, 2009). EPS cell aggregates also underwent polarization. At day 1, PAR6 protein was absent from all cell aggregates examined, which homogenously expressed the pluripotency factor SOX2 (
In an early female blastocyst, while both X-chromosomes are active in the ICM, the paternally-inherited X chromosome is preferentially inactivated in TE (Takagi and Sasaki, 1975; West et al., 1977). The X-chromosome status in different lineages of EPS-blastoids were assayed. Here, an epiblast stem cell (EpiSC) line, which contained a green fluorescent protein (GFP) transgene in the paternal X-chromosome (X-GFP). X-GFP EpiSCs were FACS-sorted to obtain a pure GFP negative population (Paternally X inactivated, or Xpi-GFP) (Bao et al., 2009). Xpi-GFP EpiSCs were converted to EPS cells by culture adaptation. Over passaging, the percentage of GFP+ cells was increased, indicating gradual reactivation of the inactivated paternal X chromosome during conversion. Next, GFP+ EPS cells were FACS-sorted out and used to generate EPS-blastoids. These blastoids were stained with NANOG and CDX2, which showed that a large fraction of them (79%, 11/14) contained GFP+ cells only in the ICM-like compartment (
E3.5 mouse blastocysts have two cell lineages, the external trophectoderm (TE) layer and the internal inner cell mass (ICM). Whether EPS-blastoids also had these two early blastocyst lineages was tested. Immunofluorescence analysis revealed that cells in the outer layer of EPS-blastoids expressed the TE transcription factors CDX2 and EOMES (
The segregation of TE and ICM lineage occurred during EPS-blastoid formation and how the cells of the two lineages were spatially distributed in the aggregates. To this end expression of CDX2 and SOX2 in EPS cell aggregates collected from day 1 to day 5 was analyzed. At day 1, —55% of the aggregates exhibited a composition of mixed CDX2+ and SOX2+ cell. This percentage continued to increase from day 1 to day 3 (
As early blastocysts further develop, ICM segregates into two lineages: epiblast (EPI) cells and primitive endoderm (PE) cells. Therefore, whether EPS-blastoids could develop into a late blastocyst-like structure comprised of all three blastocyst lineages: TE, EPI, and PE, was examined. Indeed, in some EPS-blastoids, GATA4+ PE-like cells enclosing the NANOG+ EPI-like compartment was detected, reminiscent of a peri-implantation E4.5 blastocyst (
The expression of the lineage markers in EPS-blastoids generated from ES-converted EPS, Liu-EPS, as well as a single EPS cell was assayed. Consistently, all EPS-blastoids exhibited blastocyst-like allocation of cell lineages, as revealed by CDX2, SOX2/NANOG, and GATA4 staining (
RNA-Seq analysis using individual EPS-blastoids was performed. Their transcriptomes were compared to E3.5 early blastocysts and E3.0 morulae from published datasets (Sampath Kumar et al., 2017; Wang et al., 2018). Principle component analysis revealed that EPS-blastoids were closer to blastocysts than morulae on both PC1 and PC2 axis (
928 DEGs (including novel genes from a de novo transcriptome assembly; 1.8% of all genes) were identified between EPS-blastoids and blastocysts (
To uncover differences within each lineage between EPS-blastoids and blastocysts, functional annotation of DEGs was performed. 53 DEGs were identified for the ICM/EPI lineage between the two samples (
For the PE lineage, 67 DEGs were identified between EPS-blastoids and blastocysts, which were mostly enriched in terms related to vesicle transport and endocytosis (
For the TE lineage, only two DEGs (Gjb2 and Arhgel6) were identified to be significantly different between the two samples (
ESCs, TSCs, and XEN cells, which are considered the in vitro counterparts of EPI, TE, and PE lineages, respectively, could all be derived directly from blastocysts. That EPS-blastoids could also give rise to these three stem cell lines was determined. Using the ground state culture condition (Ying et al., 2008), ESC lines were successfully established from four out of five EPS-blastoids. These ESCs formed colonies with similar morphologies to those generated from natural blastocysts and expressed the pluripotency factors OCT4, NANOG, and SOX2, but not the trophoblast marker CDX2 (
A recently developed in vitro culture (IVC) system enabled the development of mouse and human blastocysts beyond the implantation stages in vitro, and has also been successfully used to assemble postimplantation embryo-like structures (Bedzhov and Zernicka-Goetz, 2014; Bedzhov et al., 2014a; Harrison et al., 2017; Sozen et al., 2018). It was tested whether the IVC condition could support the development of EPS-blastoids beyond the implantation stage. In agreement with the reports from the Zernicka-Goetz group (Bedzhov and Zernicka-Goetz, 2014), in vitro culture of blastocysts generated an egg cylinder structure with extraembryonic ectoderm (ExE, marked by TFAP2C) and EPI (marked by SOX2) as two hemispheres enclosed by the visceral endoderm (VE, marked by GATA6) (
The cellular and molecular events that contributed to the generation of the egg cylinder structures from EPS-blastoids were examined. During the peri-implantation stage, EPI cells become polarized and form a rosette-like structure with apical domains clustered in the center. In cultured EPS-blastoids, F-actin was enriched in the center of the EPI-like compartment and the cells adopted a rosette-like configuration, reminiscent of a pen-implantation embryo at ˜E4.5-E4.75 stage (
In sum, EPS-blastoids could give rise to functional ESCs, TSCs, and XENs, and upon further cultivation, could develop into postimplantation embryo-like structures.
A more stringent functional test for blastoids is to determine whether they develop into fetuses in utero. To this end, EPS-blastoids were transferred into pseudopregnant mice at 2.5 days post coitum (dpc) and their in vivo developmental potential was analyzed. At 7.5 dpc, decidua formed in the uteri of both control mice and surrogates that had been transferred with EPS-blastoids (
To show that EPS-blastoids form without using any cells of embryonic origin, EPS-blastoids were generated from somatic cells. Through somatic cell reprogramming, EPS cells were established from mouse ear fibroblasts (induced EPS cells, or iEPS cells), which were subsequently used for blastoid formation. Blastoids were successfully generated from iEPS cells (referred to as iEPS-blastoids) in ˜15% of aggregates (
All procedures related to animals were performed following the ethical guidelines of the Salk Institute for Biological Studies. Animal protocols were reviewed and approved by the Salk Institute Institutional Animal Care and Use Committee (IACUC) before any experiments were performed. C57BL/6J (Stock No: 000664 Black 6), ICR mice (Stock No: 009122), and C57BL/6-Tg(CAG-EGFP)1Osb/J (Stock No: 003291) were obtained from The Jackson Laboratory. To prepare pseudopregnant surrogates, ICR female mice (8-12 weeks old) in the estrus were mated with vasectomized ICR male mice. Mice were housed in a 12 hr light/12 hr dark cycle in a temperature-controlled facility with free access to water and food.
Mouse 2-cell embryo or blastocysts were flushed out of the uterus of pregnant female C57BL/6J or ICR and cultured in drops of KSOM medium (homemade or Millipore, MR-020P-5D; see also Summers 2013) covered by a layer of mineral oil (Sigma-Aldrich, M8410) in a humidified incubator under 5% CO2 at 37° C. Homemade KSOM was prepared according to a previously published recipe (Wu et al., 2017). The KSOM medium contains: NaCl (95 mM), KCl (2.5 mM), KH2PO4 (0.35 mM), MgSO4 (0.2 mM), NaHCO3 (25 mM), CaCl2 (1.71 mM), Naz-EDTA (0.01 mM), L-glutamine (1.0 mM), Na lactate (10 mM), Na pyruvate (0.2 mM), glucose (5.56 mM), essential amino acid (EAA; 10.0 m/l), non-essential amino acid (NEAA; 5.0 ml/l), and BSA (4 g/l). In some experiments, KSOM-HEPES medium was used. KSOM-HEPES medium was prepared using the same amount of chemicals as KSOM with the following changes: using lower amount of NaHCO3 (5 mM), the addition of HEPES-Na (20 mM), without EAA or NEAA, and substitution of BSA by PVA (0.1 g/l). All reagents were from Sigma-Aldrich except for NEAA and EAA, which were from Thermo Fisher Scientific. The sex of the mouse embryos was not determined. Both male and female embryos were used in all experiments.
To culture blastocysts beyond the implantation stage, a protocol developed by the Zernicka-Goetz group (Bedzhov and Zernicka-Goetz, 2014; Bedzhov et al., 2014b) was used. Blastocysts were first treated in drops of Tyrode's Solution, Acidic (Sigma-Aldrich) for one to two minutes to digest the zona pellucida. The zona-free blastocysts were washed in drops of KSOM medium and transferred into u-Slide 8 well (ibidi, 80826) containing pre-equilibrated IVC-1 medium (Cell Guidance Systems, M11-25). 20-25 blastocysts were plated into each well of u-Slide. Within 2 to 3 days, blastocysts attached to the plate and medium was replaced with pre-equilibrated IVC-2 (Cell Guidance Systems, M12-25). The culture continued for an additional 4-6 days and was fixed with 4% PFA for 15 min at room temperature for immunofluorescence analysis.
All stem cell lines were cultured on a layer of irradiated CF1 mouse embryonic fibroblasts (MEF) under 20% O2 and 5% CO2 at 37° C. The chimera-competent naïve ES cell line (B6N-22; male) was a gift from Fumihiro Sugiyama (Tanimoto et al., 2008). ES cells were cultured in N2B27-based medium. N2B27 basal medium was composed of 1:1 mixture of DMEM/F-12 (11330-032) and Neurobasal (21103-049) supplemented with 0.5X N2 (17502-048), 0.5X B27 (17504-044), 1X NEAA (11140-050), 1X GlutaMAX (35050-061), 0.1 mM 2-mercaptoethanol (21985-023), and 0.1% BSA (15260-037, optional) or 5% KnockOut Serum Replacement (10828-028, optional) (all from Thermo Fisher Scientific). Mouse ESCs were maintained in N2B27 medium supplemented with 10 ng/ml hLIF (Peprotech, 300-05), 3 μM CHIR99021 (Reagents Direct, 27-H76), and 1 μM PD0325901 (Selleck Chemicals, S1036) (hereinafter referred to as N2B272iL) (Ying et al., 2008) on a layer of irradiated MEF and passaged every two to three days using TrypLE (Thermo Fisher Scientific, 12604-013). The B6 GFP+ naïve ES cell line was derived from C57BL/6-Tg(CAG-EGFP)10sb/J blastocyst using the 2iL protocol. Blastocysts were collected from timed-pregnant mice and transferred onto a MEF feeder layer in a 96-well plate and cultured in N2B272iL medium. The cell outgrowth was dissociated and replated on new MEF feeder cells. Cell lines were established by dissociating individual colony using 0.05% trypsin-EDTA and replating into a new well.
The two EPS cell lines (EPS 1 and EPS 2, tdTomato+; both were male) derived from 8-cell embryos were obtained from the Hongkui Deng's lab. These two cell lines EPS cells were cultured on irradiated MEF cells in N2B27 basal medium supplemented with 10 ng/ml LIF (Peprotech, 300-05), 3 μM CHIR99021 (Reagents Direct, 27-H76), 2 μM (S)-(+)-Dimethindene maleate (Tocris, 1425), and 2 μM minocycline hydrochloride (Santa Cruz Biotechnology, sc-203339) (hereinafter referred to as N2B27LCDM). In some experiments, the EPSC culture protocol developed by Pentao Liu's lab was used (Yang et al., 2017b). The Liu-EPS culture medium was CDF12 basal medium supplemented with 10 ng/ml hLIF (Peprotech, 300-05), 3 μM CHIR99021 (Reagents Direct, 27-H76), 1 μM PD0325901 (Selleck Chemicals, S1036), 4 μM JNK Inhibitor VIII (Millipore, 420135), 10 μM SB203580 (Tocris, 1402), 0.3 μM A-419259 (Tocris, 3914), and 5 μM XAV939 (Sigma-Aldrich, X3004). EPS cells were routinely passaged every two days at a ratio of 1:10 to 1:20. CDF12 basal medium was composed of DMEM/F-12 (11330-032) supplemented with 20% KnockOut Serum Replacement (10828-028), 1X NEAA (11140050), 1X GlutaMAX (35050-061), and 0.1 mM 2-mercaptoethanol (21985-023) (all from Thermo Fisher Scientific). The female X-GFP mEpiSC (female) was a gift from Dr. Azim Surani (Bao et al., 2009). EpiSCs were cultured in CDF12 medium supplemented with 12.5 ng/ml bFGF (Peprotech, 100-18B). To convert naïve ESCs or EpiSCs into EPS cells, naïve ESC or EpiSCs were first seeded on MEF feeder cells with ESC or EpiSC medium, respectively. After 24 hr, the medium was removed and replaced with EPS medium. The conversion process usually completed after five passages in the EPS conditions.
TSCs were maintained in basal TSC medium supplemented with 25 ng/ml rhFGF4 (R&D, 235F4025) and 1 μg/ml Heparin (Sigma-Aldrich, H3149) on a layer of irradiated MEF (Tanaka et al., 1998). TSC basal medium was composed of RPMI 1640 (11875-093) supplemented with 20% Fetal Bovine Serum (FBS) (16000-044), 1X GlutaMAX (35050061), 1X Sodium pyruvate (11360-070), and 0.1 mM 2-mercaptoethanol (21985-023) (all from Thermo Fisher Scientific). TSCs were passaged every five to seven days at 1:5-1:10 using 0.05% trypsin (Thermo Fisher Scientific, 25300-054). In addition, XEN cells were cultured in the TSC basal medium and passaged every 4 to 5 days using TrypLE.
Mouse ear fibroblasts were derived by plating minced ear tissue in DMEM (11950-040) supplemented with 10% FBS (16000-044), 1X NEAA (11140-050), and 1X GlutaMAX (35050-061) (all from Thermo Fisher Scientific) under 20% O2 and 5% CO2 at 37° C. When confluent, ear fibroblasts were split using TrypLE (Thermo Fisher Scientific, 12604-013) at 1:5.
Retrovirus containing the four Yamanaka factors (Takahashi and Yamanaka, 2006) were packaged in 293 cells by transfection of pMXs-c-Myc, pMXs-Klf4, pMXs-Sox2, pMXs-Oct3/4 (all from Addgene). Mouse ear fibroblasts at passage 1 to 3 were used for reprogramming into iPS cells by incubation with the mixed retrovirus for two days. Then medium was replaced with CDF12 supplemented with 10 ng/ml LIF (CDF12LIF). iPS colonies were picked up, dissociated with TrypLE, and replated into a new MEF well for establishing individual iPS cell line. iPS cells were routinely cultured in either CDF12LIF or N2B272iL. iPS cells were converted into EPS cells by culturing in N2B27LCDM for at least five passages.
Lentiviral particle encoding the puromycin resistant gene and the mCherry gene (Lenti-EFla-puromycin-mcherry) (Liao et al., 2015) were packaged in 293 cells via transfection. Medium supernatant containing the lentiviral particles was collected 48 hr after transfection and concentrated by ultracentrifugation at 25,000 r.p.m (82,700g) at 4° C. for 2 hr using a Beckman ultracentrifuge (Beckman Coulter) (Kutner et al., 2009). The lentivirus pellet was resuspended with cold DPBS. EPS cells were transduced by incubating with lentivirus-containing N2B27LCDM for 48 hr. Upon passaging, puromycin (1 μg/ml; InvivoGen, ant-pr-1) was supplemented in the medium to eliminate untransduced cells.
EPS colonies were dissociated into single cells by incubation with TrypLE (Thermo Fisher Scientific, 12604-013). Cell resuspension was transferred into a 0.1% gelatin-coated plate and incubated at 37° C. for 30 min to allow irradiated MEF cells attach to the plate. The supernatant containing the EPS cells were collected, filtered through a 40 μm cell strainer, and counted using the TC-10 counter (Bio-Rad, 1450001). AggreWell 400 (STEMCELL Technologies, 34415) was prepared following the manufacturer's instructions. EPS-blastoid basal medium is composed of 25% TSC basal medium (see above), 25% N2B27 basal medium (see above), and 50% KSOM (see above). In some experiments, M16 (Sigma-Aldrich, M7292) was used to replace KSOM. Approximately 6,000 cells (five cells per microwell for 1200 microwells) were resuspended in EPS-blastoid basal medium supplemented with 2 μM ROCK inhibitor Y-27632 (Reagents Direct, 53-B80-50), 12.5 ng/ml rhFGF4 (R&D, 235F4025), 0.5 μg/ml Heparin (Sigma-Aldrich, H3149), 3 μM GSK3 inhibitor CHIR99021 (Reagents Direct, 27-H76), 5 ng/ml BMP4 (Proteintech, HZ-1040), and 0.5 μM A83-01 (Axon Medchem, 1421) and seeded into one well of the 24-well AggreWell plate. The plate was centrifuged at 300g for one minute and transferred into an incubator. The day of cell seeding was counted as day 0 of the process. Medium was removed 24 h later (day 1) and replaced with fresh medium without Y-27632. Additional medium change is optional for the rest of the EPS-blastoid formation process. Starting from day 4, blastoids were manually picked up using a mouth pipette (Sigma-Aldrich, A5177) under a stereomicroscope for analysis or downstream experiments. For testing of the effect of antagonists or inhibitors on EPS-blastoid induction, chemicals were added to the medium at day 1. XAV939 (5 μM; Tocris, 3748) or IWR-1-endo (10 μM; STEMCELL Technologies, 72562) was used to inhibit Wnt signaling. Verteporfin (2 μM; Tocris, 5305) was used to inhibit YAP/TEAD interaction. For testing whether a single EPS cell forms into a blastoid, WT cells and Puromycin+/mcherry+ cells were mixed at a ratio of 10:1 and seeded onto Aggrewell 400 as stated above. Puromycin (0.25 μg/ml; InvivoGen, ant-pr-1) was added to the medium 24 hours later to eliminate helper cells gradually.
Derivation of Three Types of Stem Cells from EPS-Blastoids
To derive ES and TS cells, individual EPS-blastoid was transferred onto a MEF feeder layer in a 96-well plate and cultured with ES culture medium (N2B272iL) or TS culture medium, respectively. Within 2-3 days, EPS-blastoids attached to the plate and outgrowth was observed. Outgrowth was dissociated with 0.05% trypsin and plated into a new plate with MEF feeders. Individual colony was manually picked, dissociated, and seeded onto a new 96-cell MEF feeders for cell line derivation. The derivation of XEN cell line was performed following an established protocol (Rugg-Gunn, 2017) with modifications. EPS-blastoids were plated individually in a 24-well plate pre-coated with MEF feeders in XEN derivation medium (TSC basal medium supplemented with 25 ng/ml rhFGF4 and 1 μg/ml Heparin). The outgrowth was formed around day 3. And medium was changed every 3 days until the XEN cells are around 80% confluent. The XEN cells were dissociated into single cells using TrypLE Express for 5 min at 37° C. with gentle pipetting. Dissociated cells were transferred into a 15 ml falcon tube containing 5× volume of digestion mixture and collected by centrifugation at 300 g for 5 min. After removal of the supernatant, the XEN cells were resuspended and seeded into a 6-well plate pre-coated with MEF feeders. After 2-3 passages, the medium was switched to XEN culture medium. Chimeric assays of ES and TS cells from EPS-blastoids
ICR female mice were superovulated by intraperitoneal (i.p.) injection of 7.5 Unit of pregnant mares' serum gonadotrophin (PMSG; Prospec-Tany Technogene, HOR-272) and 46-48 hr later 7.5 Unit of human chorionic gonadotrophin (HCG; Sigma-Aldrich, CG10-1VL), and then mated with ICR male mice immediately. The blastocysts were flushed from female mice 3.5 days after detection of the vaginal plug, and cultured in KSOM under 37° C. with an atmosphere of 5% CO2 in the air. The blastoid-derived ES or TS cells were dissociated into single cells and placed into the working medium before blastocyst injection. For each chimeric blastocyst, 12-15 ES or TS cells were injected into the cavity of blastocyst assisted with a PIEZO impact drive (Primetech, Ibaraki, Japan). After injection, the chimeric blastocysts were rinsed three times and cultured in KSOM at 37° C. with an atmosphere of 5% CO2 in the air. Fifteen to twenty chimeric blastocysts, which re-expanded after injection, were transferred into the uterine horn of 2.5 dpc pseudopregnant ICR female mice. For the ES chimeric group, full-term pups were delivery naturally from the pregnant mice at 17.5 days after embryo transfer; for TS chimeric group, the E14.5 fetus was dissected from uterine of pregnant mice 12.5 days after embryo transfer. Placenta was fixed with 4% PFA overnight and embedded in OCT. Frozen sections (10 μm thick) were cut using a microtome cryostat (Leica, model #CM1900-3-1).
Chimeric Assay of XEN Cells Derived from EPS-Blastoids
ICR female mice in natural estrous cycles were mated with same strain males, and blastocysts were collected from uterine at 3.5 dpc in KSOM-HEPES. They were cultured in the KSOM under 37° C. and 5% CO2 until microinjection of XEN cells. The EPS-blastoid derived XEN cells were dissociated into single cells and placed into KSOM on ice before blastocyst injection. Fifteen cells were introduced into the blastocoel assisted with a PIEZO impact drive. Microinjected blastocysts were cultured in KSOM until embryo transfer to the surrogates. Microinjected blastocysts were surgically transferred to the uterine horn of 2.5 dpc pseudopregnant ICR females. 15-18 blastocysts were transferred to each surrogate. E11.5 fetuses were dissected from uterine for analysis.
EPS-blastoids were cultured beyond the implantation stage using a protocol as for blastocyst (see above). EPS-blastoids were manually picked up using a mouth pipette, washed twice with pre-equilibrated IVC-1 medium (Cell Guidance Systems, M11), and transferred into a μ-Slide 8-well (ibidi, 80826) containing the IVC-1 medium. Around 20-30 EPS-blastoids were plated in one well of the μ-Slide. Within one or two days, EPS-blastoids attached to the plate. Once the EPS-blastoids attached, the medium was switched to IVC-2 medium (Cell Guidance Systems, M12). In two to four days, postimplantation embryo-like structures emerged and were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature for immunofluorescence staining analysis.
EPS-blastoids were manually picked up under a stereomicroscope and transferred into KSOM droplets using a mouth pipette. The surrogate at 2.5 days post coitum (dpc) was anesthetized with ketamine (Putney) and xylazine (Akorn) and the uterine horn was exposed surgically. After three washes in KSOM, EPS-blastoids were loaded to the pipette with air bubble and transferred to the uterine horn, which was previously punctured with a 27 G needle. Around 20 EPS-blastoids were transferred into each uterine horn. The process of transfer was typically performed within 20-30 min per surrogate. A C-section was performed at 6.5, 7.5, or 8.5 dpc, and the uterus was dissected out. For staining with Evans blue, the surrogate mice received a tail vein injection of 0.5% Evans Blue (MP Biomedicals, 151108) 15 min before the C-section. Deciduae were dissected out of the uterus, and embryo-like structures were dissected out of the deciduae. Tissue samples were fixed with 4% PFA overnight and embedded in OCT. Frozen sections (10 μm thick) were cut using a microtome cryostat (Leica, model #CM1900-3-1).
Immunofluorescence for 2D cell culture, 3D cell aggregates, EPS-blastoid, early mouse embryos, and postimplantation embryo-like structures was performed following a previously established protocol (Gu et al., 2018) with small modifications. The samples were fixed with freshly prepared 4% PFA in PBS for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 15 min. Samples were then blocked with blocking buffer (PBS containing 5% normal donkey serum (NDS), 2% BSA, and 0.1% Tween 20) at room temperature for two hours or overnight (O/N) at 4° C. Primary antibodies diluted in blocking buffer were applied to samples and incubated for two hours at room temperature or O/N at 4° C. Samples were washed for three times with PBS containing 0.1% Tween 20 followed by the incubation with fluorescence-conjugated secondary antibodies diluted in blocking buffer (2-5 μg/ml) for 1 hr (2D culture) or 2 hr (3D structures and postimplantation embryo-like structures) at room temperature. Samples were washed for three times with PBS containing 0 1% Tween 20. Nuclei were counterstained with Hoechst 33342 at 1 μg/ml. In some experiments for staining with membrane-associated protein (E-cadherin, ZO1, and PARE), Saponin (0.1%; MP Biomedicals, 102855) was used for permeabilization and wash to replace Triton X-100 and Tween20. For staining of tissue cryosections, an additional step of antigen retrieval between permeabilization and blocking was performed to incubate the sections in 1X HistoVT One (Nacalai Tesque, 06380-05) at 70° C. for 20 min. When using mouse antibody on mouse tissue, Mouse on Mouse Basic Kit (Vector Laboratories, Cat# BMK-2202) was used after blocking with normal serum and BSA. Also, to reduce background, 1X TruBlack Lipofuscin Autofluorescence Quencher (Biotium, 23007) was applied to sections as the last step of the staining process. Image acquisition was performed using a Zeiss LSM 710 or 880 confocal microscope. Images were processed using Fiji (ImageJ, v2.0.0) (Rueden et al., 2017)(Schindelin et al., 2012) or Zen (Zeiss). 3D cell counting was performed using the Imaris software (Oxford Instruments). The primary antibodies and dilutions used were: anti-CDX2 (1:100; Biogenex, MU392A), anti-KRT8 (1:5; Developmental Studies Hybridoma Bank, TROMA-1), anti-EOMES (1:200; Abcam, Ab23345), anti-ECAD (1:200; Dako, M3612), anti-ZO1 (1:150, Invitrogen, 61-7300), Rabbit anti-OCT4 (1:200; Abcam, ab19857), Rabbit anti-GATA6 (1:100; Cell Signaling Technology, 5851), Mouse anti-laminin gamma 1 (1:5; DSHB, 2E8), Goat anti-GATA6 (1:200; R and D Systems, AF1700), anti-PAR6 (1:50, Santa Cruz Biotechnology, sc-166405), anti-YAP (1:100, Santa Cruz Biotechnology, sc101199), anti-active YAP (1:100, Abcam, ab205270), anti-GFP (1:1000, MBL, M048-3), anti-TFAP2C (1:50; Santa Cruz Biotechnology, sc-12762), anti-OCT4 (1:100; Santa Cruz Biotechnology, sc-5279), anti-NANOG (1:100; Invitrogen, 14-5761-80), anti-SOX2 (1:100; R&D, AF2018), anti-GATA4 (1:100; Santa Cruz Biotechnology, sc-1237), anti-aPKC (1:50; Santa Cruz Biotechnology, sc-17781), and anti-Podocalyxin (PCX) (1:200; R&D, MAB1556). Secondary antibodies used were: Alexa Fluor 488 Donkey anti-Rat IgG (H+L) (Thermo Fisher Scientific, A-21208), Alexa Fluor 488-AffiniPure Donkey Anti-Goat IgG (H+L) (Jackson ImmunoResearch Labs, 705545-147), Alexa Fluor 488-AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 715-545-151), Alexa Fluor 488-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 711-545-152), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L) (Thermo Fisher Scientific, A-31570), Alexa Fluor 555 Donkey anti-Rat IgG (H+L) (Abcam, ab150154), Alexa Fluor 647 Donkey anti-Rat IgG (H+L) (Abcam, ab150155), Alexa Fluor 647-AffiniPure Donkey Anti-Goat IgG (H+L) (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647-AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, 715-605-151), Alexa Fluor 647-AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, 711-605-152), DyLight 550 Donkey anti-Goat IgG (H+L) (Thermo Fisher Scientific, SA5-10087), DyLight 550 Donkey anti-Rabbit IgG (H+L) (Thermo Fisher Scientific, SA5-10039). F-actin was directly stained with Phalloidin CruzFluor 488 Conjugated antibody (1:1000; Santa Cruz Biotechnology, sc-363791) along with other secondary antibodies in blocking buffer.
Immunohistochemistry (IHC) was performed using the ImmPRESSTM HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection Kit (Vector laboratories, MP-7401) according to the manufacturer's instructions with minor modifications. The frozen sections were firstly treated with citrate-based (Vector laboratories, H-3300) for antigen unmasking. Blocking was done with 2.5% normal horse blocking serum included in the kit for 1 hr at room temperature. The primary antibody for tdTomato (1:200; Rockland, 600-401-379) was applied to the sections and incubated overnight at 4° C. All washes were performed with 0.1% Tween-20 (PBS) for 5 min. The color was developed using ImmPACT DAB Peroxidase (HRP) Substrate (Vector laboratories, SK-4105) according to the manufacturer's instructions. Lastly, the sections were counterstained with hematoxylin and went through a series of ethanol-based dehydration and xylene-based clearing and mounted with mounting medium.
Decidua tissue sample was minced and resuspended in TE buffer. Tissue was digested by treating with 0.3 mg/ml proteinase K (Thermo Fisher Scientific, AM2546) at 55° C. overnight. Genomic DNA preparations were incubated at 95° C. for 5 min to inactivate proteinase K before used for PCR. An ultraconserved noncoding element (UNCE) overlapping with the Tfap2a locus was used as an internal control (Cohen et al., 2016). The tdTomato gene was amplified by a nested PCR. The first round of PCR was done with an external primer set: 5′-GGC GAG GAG GTC ATC AAA GAG T-3′, 5′-ATG GTG TAG TCC TCG TTG TGG G-3′. PCR product of the first PCR was diluted at 1:200 and 1 μl of the diluted sample was used as the template for the second round nested PCR with the following primers: 5′-ACA TCC CCG ATT ACA AGA AGC-3′, 5′-TTG TAG ATC AGC GTG CCG TC-3′. All PCR reactions were performed with PrimeSTAR GXL DNA Polymerase (Clontech, R050B). PCR products were resolved in a 2% agarose gel with TBE buffer. Images were acquired using a Bio-Rad Gel Doc XR+ system with Image Lab software.
Total RNA was isolated from eight individual EPS-blastoids collected at day five using the TRIzol (Thermo Fisher Scientific, 15596026) method. RNA-Seq libraries were constructed using the Illumina Smart-Seq2 (Picelli et al., 2013) using Nextera XT DNA sample preparation kit (Illumina, FC-131-1096) and Nextera XT 24-index kit (Illumina, FC-131-1001), and 2×150 bp pair-end sequencing was performed on an Illumina HiSeq Xten. Sequencing reads were filtered and mapped to the mouse genome build mm10 using the HISAT2 alignment program (Kim et al., 2019). De novo transcriptome assembly and transcript and gene abundance calculations were performed using the StringTie assembler (Pertea et al., 2015). The expression values of each gene were normalized using FPKM. RNA-Seq data of morula stage and E3.5 early blastocyst stage embryos were obtained from published datasets (GSE98150 and GSE87504, respectively) (Sampath Kumar et al., 2017; Wang et al., 2018). Raw read data were downloaded and processed using the same pipeline as that used for EPS-blastoid data. Differentially expressed genes (DEGs) were calculated using the R package ballgown (Frazee et al., 2015). DEGs were deemed significant if they passed the following cutoff parameters: FPKM>1, absolute value of log 2 ratio >1, and Q-value (adjusted p-value) <0.05. Gene ontology (GO) and KEGG pathway analyses were performed using Fisher's exact test, and the false discovery rate (FDR) was controlled by the BH method. Principle components analysis was performed using the R package ade4 (Dray and Dufour, 2007). Cluster analysis was performed using the R package pvclust (Suzuki and Shimodaira, 2006). Heatmaps were generated using the R package pheatmap (Kolde, 2012).
EPS-blastoids were manually picked up using mouth pipette and washed three times in PBS containing 0.04% BSA. Around 500 EPS-blastoids were harvested and dissociated with a homemade enzyme mix composed of 0.5X versene (Lonza, 17711E), 0.5X Acumax (Innovative Cell Tech, AM105), and 0.05X Dnase (STEMCELL Technologies, 07900) at 37° C. for 30min with agitation. Dissociated cells were spun down and wash with PBS+0.04% BSA for three times and resuspended in the same buffer. Cell density was determined by a TC10 cell counter (Bio-Rad, 1450001). Blastocysts were dissociated using the same protocol. Dissociated cells (˜4800 cells for EPS-blastoids and 1000 cells for blastocysts) were loaded into the Chromium Single Cell B Chip (10X Genomics, PN-120262) and processed in the Chromium single cell controller (10X Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. The library was generated using the Chromium Single Cell 3′ Reagent Kits v3 (10X Genomics, PN-1000092) and Chromium i7 Multiplex Kit (10X Genomics, PN-120262) according to the manufacturer's manual. The two libraries were pooled and sequenced using Nextseq 500 (150 cycles, high output).
STAR v2.5.1b1 (Dobin et al., 2013) was used to align reads to the 10x Genomics pre-built mm10 reference genome and utilized the CellRanger v3.0.2 (10X Genomics) software for blastocysts (288 cells) and EPS-blastoids (3528 cells) datasets with the default setting for de-multiplexing to generate feature-barcode matrix. The R package Seurat v3.0.12 (Stuart et al., 2019) was used to read and analyze feature-barcode matrix following the steps: First, cells were filtered that have unique feature counts over 5000 according to quality control matrix plots (184 and 2518 cells in the blastocysts and EPS-blastoids group passed the filter, respectively); Then UMI counts were normalized with NormalizeData function using the default settings. Seurat's RunUMAP function was used to perform a non-linear dimension reduction and clustering with resolution setting at 0.2. Differentially expressed genes within the clusters between blastocysts and EPS-blastoids were determined by the FindMarkers function using a bimodal likelihood ratio test. For the differentially expressed genes, whether each had enriched GO terms in biological process and molecular functions was tested using the ToppGene Suite3 (Chen et al., 2009). Unsupervised clustering analysis (UCA) was performed using the R package ComplexHeatmap v2.1.0 (Gu et al., 2016) with clustering_distance_columns=“manhattan”.
The sample size was not predetermined using any statistical methods or packages before experimentation. Quantification details on the number of biological replicates (n value) and data presentation were included in figure legends. Values were shown as the mean and error bars represented SEM unless otherwise indicated. Statistical analysis details were described in figure legends or method details. No method was used to determine whether the data met assumptions of the statistical approach. Differences were considered to be significant when the P (or adjusted P) values were smaller than 0.05. Graphs were generated using Prism or R package ggplot2 (Wickham, 2016) or other R packages described in the method details.
R scripts used for the single-cell RNA-Seq analysis are available upon request. The sequencing data have been deposited at the NCBI Gene Expression Omnibus under the following accession number: GSE135289 (bulk RNA-Seq) and GSE135701 (single-cell RNA-Seq).
All stem cell lines were cultured on a layer of irradiated CF1 mouse embryonic fibroblasts (MEF) under 20% O2 and 5% CO2 at 37° C.
Human primed embryonic stem cells were cultured in CDF12 medium, which was composed of DMEM/F-12 (11330-032) supplemented with 20% KnockOut Serum Replacement (10828-028), 1X NEAA (11140-050), 1X GlutaMAX (35050-061), 0.1 mM 2-mercaptoethanol (21985-023) (all from Thermo Fisher Scientific), and 10 ng/mL FGF2 (Peprotech). To convert human primed ESCs into EPS or Liu-EPSC cells, human primed ESCs were first seeded on MEF feeder cells with CDF12 medium. After 24 h, the medium was removed and replaced with EPS or Liu-EPSC medium. After 3-4 days, cell colonies were dissociated into single cells with Accumax (Stemcell Technology, 07921), and passaged into new CF1 MEF plate at 1:5-1:10 in EPS or Liu-EPSC medium with 10 μM Rock inhibitor Y-27632 (Reagents Direct, 53-B80-50). Change medium without Rock inhibitor Y-27632 next day. The conversion process usually completed after three to five passages in the EPS conditions according to the references (Yang et al, Cell, 2017; Gao et al, Nature Cell Biology).
EPS medium is composed of N2B27 basal medium supplemented with 10 ng/mL LIF (Peprotech, 300-05), 1.5 μM CHIR99021 (Reagents Direct, 27-H76), 2 μM (S)-(+)-Dimethindene maleate (Tocris, 1425), 2 μM minocycline hydrochloride (Santa Cruz Biotechnology, sc-203339) (hereinafter referred to as N2B27-LCDM), and 2 μM IWR endo-1 (Selleck, S7086). EPSC medium is composed of N2B27 basal medium supplemented 1.0 μM CHIR99021, 0.1 μM A419259 (Tocris, cat. no. 3914), 2.5 μM XAV939 or 2.0 μM IWR-1, 65 μg/ml vitamin C, 10 ng/ml LIF (SCI), 0.25 μM SB590885 and 2.0 μM SP600125. N2B27 basal medium was composed of 1:1 mixture of DMEM/F-12 (11330-032) and Neurobasal (21103-049) supplemented with 0.5X N2 (17502-048), 0.5X B27 (17504-044), 1X NEAA (11140-050), 1X GlutaMAX (35050-061), 0.1 mM 2-mercaptoethanol (21985-023), and 0.1% BSA (15260-037, optional) or 5% KnockOut Serum Replacement (10828-028, optional) (all from Thermo Fisher Scientific).
EPS or EPSC colonies were dissociated into single cells by incubation with Accumax (Stemcell Technology, 07921). Cell resuspension was transferred into a 0.1% gelatin-coated plate and incubated at 37° C. for 30 min to allow irradiated MEF cells attach to the plate. The supernatant containing the EPS or EPSC cells were collected, filtered through a 40 μm cell strainer, and counted using the TC-10 counter (Bio-Rad, 1450001). AggreWell 400 (STEMCELL Technologies, 34415) was prepared following the manufacturer's instructions. EPS-blastoid basal medium is composed of 25% TSC basal medium, 25% N2B27 basal medium (see above), and 50% KSOM. In some experiments, M16 (Sigma-Aldrich, M7292) was used to replace KSOM. Approximately 24,000 cells (20 cells per microwell for 1200 microwells) were resuspended in EPS-blastoid basal medium supplemented with 2 μM ROCK inhibitor Y-27632 (Reagents Direct, 53-B80-50), 12.5 ng/mL rhFGF4 (R&D, 235F4025), 0.5 μg/mL Heparin (Sigma-Aldrich, H3149), 3 μM GSK3 inhibitor CHIR99021 (Reagents Direct, 27-H76), 5 ng/mL BMP4 (Proteintech, HZ-1040), and 0.5 μM A83-01 (Axon Medchem, 1421) and seeded into one well of the 24-well AggreWell plate. The plate was centrifuged at 300 g for one minute and transferred into an incubator. The day of cell seeding was counted as day 0 of the process. Medium was removed 24 h later (day 1) and replaced with fresh medium without Y-27632. Additional medium change is optional for the rest of the EPS-blastoid formation process. Starting from day 5 or day 6, blastoids were manually picked up using a mouth pipette (Sigma-Aldrich, A5177) under a stereomicroscope for analysis or downstream experiments. TSC basal medium was composed of RPMI 1640 (11875-093) supplemented with 20% Fetal Bovine Serum (FBS) (16000-044), 1X GlutaMAX (35050-061), 1X Sodium pyruvate (11360-070), and 0.1 mM 2-mercaptoethanol (21985-023) (all from Thermo Fisher Scientific). Homemade KSOM was prepared according to a previously published recipe (Wu et al., 2017). The KSOM medium contains: NaCl (95 mM), KC1 (2.5 mM), KH2PO4 (0.35 mM), MgSO4 (0.2 mM), NaHCO3 (25 mM), CaCl2 (1.71 mM), Na2-EDTA (0.01 mM), L-glutamine (1.0 mM), Na lactate (10 mM), Na pyruvate (0.2 mM), glucose (5.56 mM), essential amino acid (EAA; 10.0 m/l), non-essential amino acid (NEAA; 5.0 m/l), and BSA (4 g/l).
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Various explicit examples of compositions of matter and processes/methods are described herein, the components or steps of which are optionally utilized in any composition of matter and/or process/method described herein, as applicable. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 62/910,335, filed Oct. 3, 2019, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/054128 | 10/2/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62910335 | Oct 2019 | US |
