Bilayered embryonic disc model and method for manufacturing the same

Abstract

A method for producing a mammalian blastocystis bilaminaris model, comprising a step for forming a cell mass containing a hypoblast-like cell derived from a naive mammalian pluripotent stem cell and a mammalian pluripotent stem cell corresponding to an epiblast-like cell.

Description

[Technical Field]

[0001] The present invention relates to a bilayered blastocyst model and a method for producing the same. [Background technology]

[0002] During mammalian development, an inner cell mass forms inside the blastocyst, from which the epiblast and hypoblast (primitive endoderm) emerge. The epiblast differentiates into the embryonic mesoendoderm through the formation of the primitive intestinal tract, while the primitive endoderm differentiates into extraembryonic tissues, particularly the yolk sac. Reproducing these developmental processes with pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is useful for analyzing developmental mechanisms, elucidating the mechanisms of diseases at the developmental stage, and developing treatments. However, human ES cells and iPS cells are classified as primed cells, which are more advanced in development than mouse pluripotent stem cells, making differentiation into epiblasts and primitive endoderm difficult. Therefore, Takashima et al. succeeded in obtaining naive pluripotent stem cells by expressing two genes, NANOG and KLF2, in human pluripotent stem cells, thereby resetting the human pluripotent stem cells to the same state as the basal state (Non-Patent Document 1), and reported on the induction of primitive endoderm (Patent Document 1) and trophectoderm (Patent Document 2) using these cells. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] WO 2019 / 093340 [Patent Document 2] WO2021 / 106765 [Non-patent literature]

[0004] [Non-Patent Document 1] Cell 158: 1254-1269, 2014 [Overview of the project] [Problems that the invention aims to solve]

[0005] As mentioned above, while the induction of primitive endoderm and trophectoderm from pluripotent stem cells has been reported, no reports have been made regarding blastocyst models that mimic the developmental stages of mammals. Therefore, the object of the present invention is to provide a bilayered blastodisc model derived from pluripotent stem cells and a method for producing the same, in order to understand early development, to obtain differentiated cells using a model that mimics early development, and to enable the induction of tissues with physiological functions. [Means for solving the problem]

[0006] The inventors diligently conducted research to solve the above problems. As a result, they found that by co-culturing hypoblast (primordial endoderm)-like cells (nHyC) derived from naive pluripotent stem cells with pluripotent stem cells, a cell aggregate (bilayered blastodisc-like structure / Bilaminoid) containing a layer of hypoblast-like cells and a layer of epiblast (epiblast)-like cells (EpiC) is formed, thereby enabling the production of a bilayered blastodisc model. Further culturing of the obtained bilayered blastodisc model revealed the generation of gastrulation cells, amnion-like cells, and even primordial germ cells, thus completing the present invention.

[0007] The present invention provides the following: [1] A method for producing a bilayered embryonic model of a mammal, A method comprising the step of forming a cell aggregate containing naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) that resemble the subblast layer and mammalian pluripotent stem cells (second mammalian pluripotent stem cells) that resemble the epiblast layer. [2] The hypoblastoid-like cells are positive for at least one of PDGFRA, CEACAM1, and ANPEP. The method according to [1], wherein the blastocyst-like cells are PDFGRA-negative, OCT3 / 4-positive, and NANOG-positive. [3]The method according to [1] or [2], comprising the following step (a) or (b): (a) A step of co-culturing the epiblast-like cells induced from naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) and the mammalian pluripotent stem cells (second mammalian pluripotent stem cells) by suspension culture; (b) A step of inducing the epiblast-like cells from the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) while co-culturing the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) and the mammalian pluripotent stem cells (second mammalian pluripotent stem cells) by suspension culture. [4] The method according to any one of [1] to [3], wherein the epiblast-like cells are induced from the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) by forcibly expressing a GATA family transcription factor in the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells). [5] The method according to any one of [1] to [4], wherein the epiblast-like cells are induced from the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) by activating a signal via a BMP type 1 receptor and an FGFR2 signal in the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells). [6] The method according to any one of [1] to [5], wherein the cell mass forms a bilayered blastoid structure including a layer of mammalian pluripotent stem cells (second mammalian pluripotent stem cells) corresponding to the epiblast-like cells and a layer of the epiblast-like cells underlying the layer of the mammalian pluripotent stem cells (second mammalian pluripotent stem cells). [7] The method according to [6], wherein the bilayered blastoid structure has an anterior-posterior axis. [8] The method according to [6] or [7], wherein the bilayered blastoid structure contains cells of the gastrulation stage. [9] The method according to any one of [1] to [8], wherein the bilayered blastoid structure forms an amniotic cavity-like cavity structure.

[10] The method according to any one of [1] to [9], further comprising a step of co-culturing the cell mass and / or the bilayered blastoid structure with IL-6 secreting cells.

[11] The method according to

[10] , wherein the IL-6 secreting cells are trophoectoderm cells.

[12] The method according to any one of [1] to [9], further comprising a step of culturing the cell mass and / or the bilaminar blastoid structure in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF.

[13] A layer of mammalian induced pluripotent stem cells (iPS cells) and / or mammalian embryonic stem cells (ES cells) corresponding to epiblast-like cells (second mammalian iPS cells and / or mammalian ES cells), A bilaminar blastoid structure comprising a layer of the iPS cells and / or ES cells (second mammalian iPS cells and / or mammalian ES cells) lining the layer of hypoblast-like cells derived from mammalian iPS cells and / or mammalian ES cells (first mammalian iPS cells and / or mammalian ES cells).

[14] The bilaminar blastoid structure according to

[13] , comprising two or more cell types of different genotypes.

[15] A method for producing primordial germ cells, comprising: forming a cell mass comprising hypoblast-like cells derived from naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) and mammalian pluripotent stem cells corresponding to epiblast-like cells (second mammalian pluripotent stem cells); the method comprising culturing the cell mass for 10 days or more.

Advantages of the Invention

[0008] According to the present invention, a bilaminar blastoid model can be easily produced from pluripotent stem cells. Thereby, a model of an embryo at the pre-implantation stage, which has not been reported so far, can be provided, and important interactions that stimulate embryonic development before and after implantation between the primitive endoderm and the epiblast can be analyzed, which can be a useful tool for elucidating early development. Furthermore, the bilaminar blastoid model of the present invention may be able to differentiate into all the cells constituting a fetus by continuous culture, and it can be expected that various tissues and cells can be obtained from the model of the present invention. Furthermore, it can be expected to be a disease model by using disease iPS cells and the like. [Brief explanation of the drawing]

[0009] [Figure 1] Figure showing the culture scheme. Naive (WT), Naive-GFP (GATA6), or Naive (WT) and Naive-GFP (GATA6) were cultured in t2iLGo medium (Takara Bio) on a microwell array. After 24-36 hours (h), the medium was replaced with N2B27. DOX was added for the first two days. IL6 was added from day 0 to day 4, where mentioned. Naive (WT) represents wild-type naive pluripotent stem cells; Naive-GFP (GATA6) represents GFP-expressing naive pluripotent stem cells that express GATA6 under DOX treatment. [Figure 2] Representative images (photographs) of cell aggregates consisting of Naive (WT) (50 cells / well), Naive-GFP (GATA6) (50 cells / well), or Naive (WT) (10 cells / well) + Naive-GFP (GATA6) (40 cells / well), and a figure showing the ratio of the long axis to the short axis of the cell aggregates. The aggregates were cultured in t2iLGo medium on a microwell array. On day 4, the axes of aggregates generated by Naive (WT) (50 cells / well) or Naive (WT) (10 cells / well) + Naive-GFP (GATA6) (40 cells / well) were measured. Mixed aggregates of Naive (WT) + Naive-GFP (GATA6) are more spherical than those of Naive (WT). At least 100 aggregates were measured in each group. [Figure 3] Immunofluorescence images (photographs) of cell aggregates generated by Naive (WT) (10 cells / well) + Naive-GFP (GATA6) (40 cells / well) or Naive (WT) only (50 cells / well). Cells aggregated in N2B27 without Matrigel. On day 4, PAR6 accumulation begins to appear in the center of mixed aggregates of Naive (WT) and Naive-GFP (GATA6). Scale bar, 20 μm. [Figure 4]The graph shows the proportion of structures formed on day 2 (D2) and day 4 (D4) in cell aggregates generated by Naive(WT) + Naive-GFP or Naive(WT) alone, along with representative images (photographs) of the aggregates. [Figure 5] Time course images (photographs) of aggregates from 0 to 48 hours after induction. After DOX introduction, Naive-GFP (GATA6) (nHyC) and Naive-DsRed (nEpiC) separated, and after 48 hours, nHyC (G6) was located around the aggregates. Scale bar: 40 μm. [Figure 6] Relative distance from the center of the aggregate. The distance of nHyC(G6) and nEpiC nuclei from the center of the aggregate was measured at the level of the widest cross-section of each aggregate. Mixtures of GFP(nHyC) and DsRed(nEpiC) were cultured for 48 hours under conditions of no (-) or with (+) DOX. Ten aggregates were counted under each condition. The number of nuclei counted is shown above. [Figure 7] Cell counts for each aggregate on days 0, 2, 4, and 6. The nHyC(G6) and nEpiC aggregates proliferated from day 0 to day 6. [Figure 8] From day 0 to day 6, the maximum cross-sectional area of ​​each aggregate increases. From day 0 to day 6, the size of each aggregate increases. [Figure 9] After dissociating aggregates (Naive (WT) and Naive (WT) + Naive-GFP (GATA6)) into single cells, GFP+ or GFP- cells were sorted by flow cytometry, and genes related to pluripotency and hypodermia were evaluated. [Figure 10]Gene expression in cells sorted from aggregates generated by Naive (WT) or Naive (WT) + Naive-GFP (GATA6) on day 2 (D2) and day 4 (D4). After dissociating aggregates (Naive (WT) and Naive (WT) + Naive-GFP (GATA6)) into single cells, GFP+ or GFP- cells were sorted by flow cytometry, and genes related to pluripotency and the subblast layer were evaluated. In nHyC (G6 cells) of WT+G6 aggregates, GATA6 (Total) and OTX2 were upregulated. G(+): G(+): GFP+ cells, G(-): GFP- cells. G(+): GFP+ cells, G(-): GFP- cells, WT+G6: mixed aggregates of Naive (WT) + Naive-GFP (GATA6), WT: aggregates of Naive (WT), N: naive pluripotent stem cells, P: primed pluripotent stem cells. [Figure 11] GATA6 expression in human preimplantation embryos. The dot plot images were obtained from Genome-wide Rodent and Primate Preimplantation Atlas 17 (GRAPPA; https: / / app.stemcells.cam.ac.uk / GRAPPA / ). Expression in the hypoblast is approximately 20 times higher than in the epiblast (EPI). [Figure 12] GATA6 expression in nHyC of aggregates. Aggregates generated with Naive (WT) + Naive-GFP(GATA6) were sorted on days 2, 4, and 6. GATA6 (Total) and GATA6 (Endogenous) expression were measured by qPCR. [Figure 13] Gene expression patterns in Naive (WT) and Naive-GFP (GATA6) or Naive (WT) only on day 2 (D2) and day 4 (D4). Cell aggregates were sorted by GFP on D2 and D4. G(+): GFP+, G(-): GFP-, WT+G6: mixed aggregates of Naive (WT) and Naive-GFP (GATA6), WT: aggregates of Naive (WT) only; N: naive pluripotent stem cells, P: primed pluripotent stem cells. [Figure 14]Immunofluorescence images of polarity markers in aggregates on day 4 (photograph). Aggregates were generated using either Naive (WT) alone or Naive (WT) and Naive (GATA6) (naive pluripotent stem cells expressing GATA6 under DOX treatment). PAR6, PODXL, and aPKC proteins accumulated in the embryonic coel (center) of aggregates generated by Naive (WT) and Naive (GATA6). Aggregates generated by Naive (WT) alone did not have a coel. Scale bar, 20 μm; all PAR6-accumulating aggregates co-expressed either PODXL or aPKC (5 aggregates each; N=2). [Figure 15] Quantification of the efficiency of generating bilaminids (bilayered blastodisc-like structures). Aggregates were created from naive or primed pluripotent stem cells (PSCs) and naive-derived PDGFRA+ cells (G6- / 7F- / 4F-nHyC) induced with GATA6, 7F, or 4F, or ECAD+CXCR4+ endoderm cells. All sorted cells expressed GFP. Aggregates were fixed on day 4 of culture and stained with F-actin. Epiblastoid cells were surrounded by all types of sorted cells, but aggregates with preamniotic spaces were formed only by G6- / 7F- / 4F-nHyC. Aggregates made from a mixture of naive (WT) and naive-GFP (GATA6) most efficiently formed preamniotic spaces. Naive-derived PDGFRA+ cells were sorted with PDGFRA on day 3. The numbers above indicate the aggregates we counted. Representative images (photographs) of the aggregates are shown below. Scale bar: 50 μm. [Figure 16]Image of Bilaminoid on day 6. The epiblast layer of aggregates of Naive(WT) and Naive(GATA6) mixtures expresses T-positive cells, but aggregates of Naive(WT) alone do not. Scale bar, 20 μm. Only Naive(WT) aggregates did not express T (75 aggregates, N=2). For Naive(WT) and Naive(GATA6), aggregates surrounded by nHyC(G6) under N2B27, IL6, and PDGF-AA were collected and counted (66, 62, and 64, respectively; N=2). 47 aggregates under N2B27, 47 aggregates under IL6, and 43 aggregates under PDGF-AA expressed T. [Figure 17] Image of Bilaminoid on day 6. Aggregates were generated by primed pluripotent stem cells and 7F-, 4F-, or G6-nHyC. [Figure 18] Gene expression patterns in naive (WT) only or in naive (WT) and naive-GFP (GATA6). Aggregates were sorted by GFP on day 2 (D2), day 4 (D4), and day 6 (D6). G(-): GFP-, WT+G6: mixed aggregates of naive (WT) and naive-GFP (GATA6), WT: aggregates of naive (WT) only; N: naive pluripotent stem cells, P: primed pluripotent stem cells. [Figure 19] Gene expression patterns in bilaminoid cells on day 6. Aggregates generated by Naive (WT) and Naive-GFP (GATA6) were analyzed by scRNA-seq (10× genomics) on day 6 (n=1642 cells). UMAP divided the cells into two distinct clusters: UMAP1-High and UMAP1-Low. Gene expression levels of hypoblast and epiblast markers are plotted on UMAP. Hypoblast marker genes are expressed in UMAP1-Low, and epiblast marker genes are expressed in UMAP1-High. Hypoblast marker genes are expressed in UMAP1-Low. [Figure 20]Visualization of UMAP1-High (nEpiC) and UMAP1-Low (nHyC) clusters. nEpiC was grouped into clusters (CL) 0-7, and nHyC was grouped into clusters 0-2. [Figure 21] Relative expression of DEG between the epiblast and amnion, as revealed by cultured human embryos. The heatmap shows the expression of individual cells CL0-7 in nEpiC. CL5, 0, and 3 primarily express epiblast genes, while CL1 and 4 express amnion genes. [Figure 22] Expression levels in the epiblast and amnion of each cluster of nEpiC. Expression levels are shown in violin plots. [Figure 23] The lineage separation pathway constructed by Monocle2. The single-cell projection pathway reveals the pseudotemporal differentiation of the epiblast (CL5, 0) into the amnion (CL2, 1, 4) and the primitive streak and gastrulation cells (CL3, 6). [Figure 24] Expression of primitive streak and cystogenesis-related genes in each cluster of nEpiC. Expression levels are shown as violin plots. [Figure 25] Associated expression of stage-specific genes in the epiblast layer, as revealed by cultured human embryos. CL5 and CL0 express early and late epiblast layer genes, CL3 mainly expresses late epiblast layer genes, and CL6 expresses primitive streak genes. [Figure 26] The correlation between nEpiC CL5, 0, 3, and 6 and cynomolgus monkey embryos was determined based on the development of cynomolgus monkeys. CL6 showed a high correlation with Gast2b. [Figure 27] Photograph showing the spontaneous differentiation of KDR+PDGFRA+ mesodermal cells on 7F-nHyC. CD11b+CD14+ bone marrow / macrophage cells were detected in StemPro-34 without growth factors or inhibitors. CD71+CD235a+ erythrocytes were detected in StemPro-34 containing EPO. N=2. [Figure 28] Integrin expression levels in each cluster of nEpiC. Expression levels are shown in the violin plot. [Figure 29] Laminin expression in nHyC. Expression levels are shown in the violin plot. [Figure 30] Expression of visceral endoderm genes in nHyC of Bilaminoid on day 6. Gene expression levels are plotted on UMAP-Low and shown as a feature plot. [Figure 31] Anterior-posterior axis images of bilaminoid on day 6. Bilaminoid produced by naive (WT) and naive (GATA6) on day 6 was stained for GATA6 / PAR6, OTX2, and T (white). Scale bar, 20 μm. 24 aggregates surrounded by nHyC(G6) were counted, and 10 aggregates expressed T and OTX2 (N=2). [Figure 32] Images (photographs) (top) of bilaminoid co-cultured with trophectoderm (TE) cultured on a Transwell, and a graph (bottom) showing the amniotic cavity formation rate. AP: TE induced by the method described in Guo et al., Cell Stem Cell 2021. APB-APJ: TE induced by the method described in Io et al., Cell Stem Cell 2021. [Figure 33] A diagram (photograph) showing the morphology of bilaminoid co-cultured with TE cultured on a transwell. [Figure 34] Graph showing the amniotic cavity formation rate of bilaminoid cultured with either a JAK inhibitor (JAKi) (left) or IL6 (right), which are IL6 inhibitors. Bilaminoid + TE: Bilaminoid co-cultured with TE cultured on a Transwell. [Figure 35] Figure showing FACS analysis of bilaminoids derived from primordial germ cell reporter lines (BLIMP1-tdTomato, TFAP2C-EGFP) on day 12. Bilaminoid + TE: Bilaminoids co-cultured with TE cultured on a transwell. [Figure 36]This graph shows the expression of PGC-related genes analyzed by qPCR after collecting BILaminoid cells positive for both BLIMP1 and TFAP2C from primordial germ cell reporter lineage cells on day 12. Naive: Naive pluripotent stem cells, Primed: Primed pluripotent stem cells, BT+AG+ Bilaminoid: BLIMP1 and TFAP2C positive cells collected from Bilaminoid, BT+AG+ Bilaminoid+TE: BLIMP1 and TFAP2C positive cells collected from Bilaminoid co-cultured with TE cells cultured on Transwell. [Modes for carrying out the invention]

[0010] The present invention will be described below. In this specification, marker-positive cells refer to cells that express the marker to a degree that can be detected by immunohistochemistry or quantitative PCR using an antibody against the marker, and marker-negative cells refer to cells that do not express the marker to a degree that can be detected by immunohistochemistry or quantitative PCR using an antibody against the marker.

[0011] The method for producing the bilayered embryonic disc model of the present invention is as follows: The process includes forming a cell aggregate (cell aggregate) containing hypoblast-like cells (primitive endoderm) derived from naive mammalian pluripotent stem cells and mammalian pluripotent stem cells corresponding to epiblast-like cells.

[0012] <Bilayered embryonic disc model> The bilayered blastodisc model of the present invention is a cell mass containing two cell layers: a layer of hypoblastodisc-like cells derived from naive mammalian pluripotent stem cells, and a layer of mammalian pluripotent stem cells, with the layer of mammalian pluripotent stem cells corresponding to the layer of epiblastodisc-like cells. This cell mass containing these two cell layers is sometimes called a bilayered blastodisc-like structure.

[0013] The bilayered blastodisc model of the present invention is preferably a bilayered blastodisc-like structure comprising a layer of mammalian pluripotent stem cells corresponding to epiblast-like cells (epiblast-like layer) and a layer of hypoblast-like cells derived from naive mammalian pluripotent stem cells that underpins the epiblast-like layer (hypoblast-like layer). The bilayered blastodisc model of the present invention is more preferably a bilayered blastodisc-like structure comprising a layer of induced pluripotent stem cells (iPS cells) and / or embryonic stem cells (ES cells) corresponding to epiblast lobe-like cells, and a layer of hypoblast lobe-like cells derived from iPS cells and / or ES cells backing said layer.

[0014] The size of the bilayered blastocyst model is not particularly limited, but for example, the size of the cell aggregate is preferably 20 μm to 500 μm, more preferably 50 μm to 300 μm. In a bilayered blastocyst model, there are no particular restrictions on the ratio of hypoblast-like cells to epiblast-like cells, as long as there are enough of each to form a layer, but for example, it is 1:1 to 10:1.

[0015] The cell mass that is a bilayered blastodisc model of the present invention is preferably a bilayered blastodisc-like structure having a spherical (including substantially spherical) two-layer structure, containing an inner layer of mammalian pluripotent stem cells corresponding to epiblast-like cells and an outer layer of hypoblast-like cells surrounding it. Furthermore, the bilayered blastodisc-like structure may be a cell mass that forms an amniotic cavity-like cavity structure in its center, surrounded by a layer of epiblast-like cells.

[0016] Furthermore, in a bilayered blastocyst-like structure, it is preferable that the layer resembling the epiblast and the layer resembling the hypoblast are separated by a basement membrane. The basement membrane is preferably an extracellular matrix containing laminin. The laminin is preferably β1.

[0017] Furthermore, it is preferable that the bilayered blastocyst-like structure has an anterior-posterior axis. Having an anterior-posterior axis means that the polarity of the cells is from the head side to the tail side. The presence of an anterior-posterior axis can be confirmed, for example, by the expression of one or more anterior markers such as CER1 (Cerberus 1), DKK1 (Dickkopf-1), LEFTY1, 2 (Left-Right Determination Factor 1, 2), and OTX2 (Orthodenticle Homeobox 2) in a subblast-like layer, and the expression of posterior markers such as T in an epiblast-like layer. Furthermore, it is preferable that the bilayered blastocyst-like structure has apical-baseal polarity, indicated by PAR6 expression.

[0018] Furthermore, the bilayered blastodisc-like structure may also contain gastrulation-stage cells in the layer of blastodisc-like cells. Gastrulation-stage cells are characterized by the expression of one or more of the following: T, MIXL1 (Mix Paired-Like Homeobox), MESP1 (Mesoderm posterior protein 1), EVX1 (Even-Skipped Homeobox 1), SNAI2, and EOMES (Eomesodermin).

[0019] Furthermore, the bilayered blastodisc-like structure may include amnion-like cells in the layer of blastodisc-like cells. Amnion-like cells are characterized by the expression of one or more of GATA3, TFAP2A (Transcription Factor AP-2 Alpha), and ISL1 (islet 1).

[0020] The bilayered blastocyst-like structure of the present invention is more preferably composed of two or more cells of different genotypes. For example, the genotypes of the hypoblastoblast-like cells and the epiblastoblast-like cells may be different, and naive pluripotent stem cells from which the hypoblastoblast-like cells originate and pluripotent stem cells corresponding to the epiblastoblast-like cells can be of different genotypes.

[0021] <Euper layer-like cells of blastula> Epiblastoid-like cells are characterized by pluripotency, allowing differentiation into endoderm, ectoderm, and mesoderm, and are negative for hypoblastoid markers, as well as being positive for OCT3 / 4 and NANOG. Preferably, they are characterized by PDGFRA (PDGF receptor α) negativity, and positive for OCT3 / 4 and NANOG.

[0022] In the bilayered blastodisc model of the present invention, pluripotent stem cells having the above-described characteristics can be used as epiblast-like cells. Therefore, pluripotent stem cells correspond to epiblast-like cells in the bilayered blastodisc model. The pluripotent stem cells corresponding to epiblast-like cells may be naive pluripotent stem cells or primed pluripotent stem cells, and if mammalian pluripotent stem cells are allowed to form layers, these will become layers of epiblast-like cells.

[0023] The following explains pluripotent stem cells.

[0024] <Pluripotent stem cells> In this invention, pluripotent stem cells are stem cells that possess pluripotency, enabling differentiation into many cells present in the body, and also possess proliferative capacity. This includes any cells that can be induced into blastocyst-like cells. Furthermore, in this invention, the term "pluripotent stem cells" includes naive pluripotent stem cells and primed pluripotent stem cells. Pluripotent stem cells are not particularly limited, but include, for example, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), cultured fibroblasts, and pluripotent cells (Muse cells) derived from bone marrow stem cells. Preferred pluripotent stem cells are iPS cells and ES cells. The origin of pluripotent stem cells is preferably mammalian, including primates and rodents, more preferably primate, and even more preferably human.

[0025] Methods for producing iPS cells are well known in the field and can be produced by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D,et al.(2008),Nat.Biotechnol.,26:795-797,Shi Y,et al.(2008),Cell Stem Cell,2:525-528,Eminli S,et al.(2008),Stem Cells.26:2467-2474,Huangfu D,et al. al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al.(2008),Cell Stem Cell,3,568-574,Zhao Y,et al.(2008),Cell Stem Cell,3:475-479,Marson A,(2008),Cell Stem Cell,3,132-135,Feng B,et al.(2009),Nat.Cell Biol.11:197-203, RLJudson et al.,(2009), Nat.Biotechnol.,27:459-461, Lyssiotis CA,et al.(2009),Proc Natl Acad Sci US A.106:8912-8917, Kim JB,et al. (2009), Nature.461:649-643, Ichida JK, et al. (2009), Cell Stem Examples of combinations are given in Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, and Maekawa M, et al. (2011), Nature. 474:225-9.

[0026] Somatic cells include, without limitation, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells, passaged cells, and established cell lines. Specifically, examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0027] <Naive Pluripotent Stem Cells> Naive pluripotent stem cells are pluripotent stem cells that have properties similar to preimplantation embryos, and specifically possess the following characteristics (Cytometry Research 27(1):19 ~ 24, 2017). It exhibits a dome-shaped colony form, and the colony size is smaller than that of the prime type. The organism expresses one or more of the following markers: CD75, KLF4, and TFCP2L1. The genome is demethylated.

[0028] Naive pluripotent stem cells can be created, for example, by the following methods. A method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014) Method using the 5iLFA condition (Theunissen et al., Cell Stem Cell. 2016 Oct 6; 19(4):502-515.) Method using HDAC (histone deacetylase) inhibitors (Guo, G. et al. (2017). Development 144(15): 2748-2763.) Alternatively, it can also be obtained by culturing primed pluripotent stem cells using commercially available culture media for preparing naive pluripotent stem cells, such as t2iLGo(Ndiff227 [Takara Bio, Cat. Y40002]).

[0029] <Prime-type pluripotent stem cells> Primed pluripotent stem cells are pluripotent stem cells that possess properties similar to those of the epiblastocyte cells of a post-implantation embryo. These include typical induced pluripotent stem cells obtained by introducing reprogramming factors into somatic cells, and human ES cells, which have not undergone the naiveté treatment described above. Prime-type pluripotent stem cells have the following characteristics: They exhibit a flat colony morphology, and their colony size is larger than that of the naive type. The markers CD75, KLF4, and TFCP2L1 were negative. The genome is methylated.

[0030] Furthermore, the epiblastoid-like cells can differentiate into cell populations including cells positive for primitive streak-related genes (e.g., T, MIXL1, MESP1, EVX1, SNAI2, EOMES, etc.) (gastrulation-stage cells) and / or cells positive for amniotic membrane-related genes (e.g., GATA3, TFAP2A, ISL1, etc.). Therefore, the epiblastoid-like cell layer in the bilayered blastocyst model of the present invention may include gastrulation-stage cells and / or amniotic membrane-like cells.

[0031] <Hyperblast layer> Hypoblastic layer-like cells are classified as extraembryonic cells that express the extracellular matrix and support the epiblastic layer cells. They are characterized by the expression of one or more hypoblastic layer markers such as GATA3, GATA4, GATA6, SOX17, FOXA2 (Forkhead Box A2), HNF4A (Hepatocyte Nuclear Factor 4 Alpha), CER1 (Cerberus 1), OTX2 (Orthodenticle Homeobox 2), PDGFRA (Platelet Derived Growth Factor Receptor Alpha), COL4A1 (alpha-1 subunit of collagen type IV), SPARC (Secreted protein acidic and rich in cysteine), CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1), and ANPEP (alanyl aminopeptidase, membrane), and are preferably positive for at least one of PDGFRA, CEACAM1, and ANPEP.

[0032] Hypoblastic cells are derived from naive pluripotent stem cells; that is, they are cells differentiated from naive pluripotent stem cells. The following describes a method for differentiating naive pluripotent stem cells into hypoblastic cells.

[0033] <Process for inducing differentiation of naive pluripotent stem cells into blastoblast-like cells> The method for inducing differentiation of naive pluripotent stem cells into hypoblastoid-like cells is not particularly limited, and any method, including known methods, can be used. However, methods such as the forced expression of GATA family transcription factors (GATA6 or GATA4) in naive pluripotent stem cells, as disclosed in WO 2019 / 093340 and WO 2021 / 107117, and the method of induction by activating the signaling pathway via BMPR1A (BMP receptor 1A) / ACVR (activin receptor) and FGFR2 (FGF receptor 2) as type 1 receptors for BMP signaling (e.g., WO 2019 / 093340) are preferably used.

[0034] 1) Induction by forced expression of GATA family transcription factors By forcing the expression of GATA family transcription factors in naive pluripotent stem cells, hypoblastoid-like cells can be induced. The GATA family transcription factor to be forcibly expressed is preferably GATA6 or GATA4.

[0035] The method for forcing the expression of GATA family transcription factors in naive pluripotent stem cells is any method that increases the amount of GATA family transcription factors expressed in naive pluripotent stem cells, and is not particularly limited. Preferably, the method involves introducing a vector capable of forcing the expression of GATA family transcription factors into naive pluripotent stem cells. More preferably, the method involves introducing a vector capable of inducibly forcing the expression of GATA family transcription factors by stimulation such as a drug into naive pluripotent stem cells, and then forcing expression by adding stimulation such as a drug. Examples of such drugs include doxycycline (DOX), and examples of vectors capable of inducing forced expression by such drugs include, but are not limited to, vectors containing rtTA expression constructs.

[0036] 2) Induction by activating the BMPR1A / ACVR signal and the FGFR2 signal. In naive pluripotent stem cells, activating the signaling pathway via BMPR1A / ACVR, a type 1 receptor for BMP signaling, and the FGFR2 signaling pathway can induce hypoblastoid-like cells.

[0037] The signal via BMPR1A / ACVR is a signal from BMPR1A / ACVR activation to the phosphorylation of SMAD1 / 5 / 9, and is a signal cascade activated by BMP2, BMP4, BMP6, or BMP7. This signal cascade may be activated, for example, by using one or more cytokines from a group consisting of BMP2, BMP4, BMP6, and BMP7, or by using any compound that acts on any of the signal cascade, or a combination thereof, but is not limited to these, and any method that can activate the signal via BMPR1A / ACVR (the signal that phosphorylates SMAD1 / 5 / 9) can be used.

[0038] The FGFR2 signal is a signal cascade that begins with the activation of FGFR2 and is activated by FGF4. This signal cascade may be activated by FGF4, by any compound that acts on any part of the signal cascade, or by a combination thereof, but is not limited to these, and any method that can activate the FGFR2 signal can be used.

[0039] To efficiently induce hypoblastoid-like cells, it is preferable to further suppress the WNT / β-catenin signaling and / or the ALK4 / 5 / 7 signaling (a signal that phosphates SMAD2) in naive pluripotent stem cells, and more preferably to suppress the WNT / β-catenin signaling, suppress the ALK4 / 5 / 7 signaling (a signal that phosphates SMAD2), activate the STAT3 signaling, activate the PDGF-AA signaling and / or activate the retinoic acid signaling. Suppression of the WNT / β-catenin signaling pathway can be achieved, for example, by using compounds that act on and suppress the WNT / β-catenin signaling cascade. Examples of such compounds include XAV939, IWP-1, IWP-2, IWP-3, IWP-4, IWR-1, 53AH, and KY02111 (available from Abcam, Sigma-aldrich, etc.), but are not limited to these; any method capable of suppressing the WNT / β-catenin signaling pathway can be used.

[0040] Suppression of the ALK4 / 5 / 7 signaling pathway (the signaling pathway that phosphates SMAD2) can be achieved, for example, by using compounds that act on and suppress the ALK4 / 5 / 7 signaling pathway. Examples of such compounds include A83-01 (WO 2009146408), SB431542, SB202190 (RKLindemann et al., Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, and LY580276 (Lilly Research Laboratories), but are not limited to these; any method that can suppress the ALK4 / 5 / 7 signaling pathway can be used.

[0041] Activation of the STAT3 signal may be achieved, for example, by IL-6, or by using a compound that acts on and activates the STAT3 signal, but is not limited to these methods; any method capable of activating the STAT3 signal can be used.

[0042] Activation of the PDGF-AA signaling pathway may be achieved, for example, by PDGF-AA itself, or by using a compound that acts on and activates the PDGF-AA signaling pathway, but is not limited to these methods; any method capable of activating the PDGF-AA signaling pathway can be used.

[0043] Activation of the retinoic acid signaling pathway may be achieved, for example, by retinoic acid (RA), a retinoic acid receptor (RAR) agonist, or a retinoid X receptor (RXR), or by using a compound that acts on and activates the retinoic acid signaling pathway, but is not limited to these methods; any method capable of activating the retinoic acid signaling pathway can be used.

[0044] The culture medium used to induce differentiation of hypoblastoid-like cells can be a naive pluripotent stem cell maintenance medium such as t2iLGo, 5iL / AF, tt2iLGo, or Ndiff227, to which factors necessary for differentiation induction have been added. It is preferable to use serum-free medium, but serum medium may be used in the case of induction by GATA6 gene overexpression. Culture can be performed in suspension or adherent culture, but adherent culture can be performed by using culture vessels coated with extracellular matrix such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, enterin, elastin, fibrillin, or laminin.

[0045] The culture temperature conditions for culturing naive pluripotent stem cells in the subblast layer-like cell differentiation induction process are not particularly limited, but are preferably around 37°C to 42°C, or 37°C to 39°C. The culture period is also not particularly limited as long as subblast layer-like cells are obtained, but is preferably at least 1 day, and preferably 2 to 5 days.

[0046] <Method for manufacturing a bilayered embryonic disc model> In the method for producing the bilayered blastodisc model of the present invention, (a) hypoblastodisc-like cells may be induced from naive mammalian pluripotent stem cells, and the obtained hypoblastodisc-like cells may be co-cultured with mammalian pluripotent stem cells by suspension culture, or (b) hypoblastodisc-like cells may be induced from naive mammalian pluripotent stem cells while co-culturing them with mammalian pluripotent stem cells by suspension culture.

[0047] In step (a), hypoblastoid-like cells differentiated from naive pluripotent stem cells and pluripotent stem cells corresponding to epiblastoid-like cells are mixed and co-cultured in suspension. The mixing ratio of hypoblastoid-like cells to pluripotent stem cells is, for example, 1:1 to 1:10. Preferably, when hypoblastoid-like cells differentiated from naive pluripotent stem cells and pluripotent stem cells are mixed and co-cultured in suspension, aggregates are formed within 24 hours, and after about 4 days, each cell type forms layers, resulting in a bilayer aggregate containing an epiblastoid-like layer from the pluripotent stem cells and a hypoblastoid-like layer backing it, derived from the hypoblastoid-like cells.

[0048] In step (b), naive pluripotent stem cells for differentiation into subblast layer-like cells and pluripotent stem cells corresponding to elastomer-like cells are mixed and co-cultured, and subblast layer-like cells are specifically induced from the naive pluripotent stem cells. The mixing ratio of naive pluripotent stem cells for differentiation into subblast layer-like cells and pluripotent stem cells is, for example, 1:1 to 1:10. Specifically, for example, a step is to mix naive pluripotent stem cells into which a vector capable of inducibly forcing the expression of GATA family transcription factors by stimulation such as a drug is introduced, and pluripotent stem cells corresponding to elastomer-like cells are mixed and co-cultured, and then forcing the expression of GATA family transcription factors by adding the aforementioned drug or other stimulation, thereby induced to induce subblast layer-like cells from the naive pluripotent stem cells. Preferably, when naive pluripotent stem cells expressing inducible GATA family transcription factors and pluripotent stem cells corresponding to epiblast layer-like cells are mixed and co-cultured, aggregates are formed within 24 hours, and approximately 4 days after initiating induction of hypoblast layer-like cells, each cell type forms layers, resulting in the formation of a bilayer aggregate containing an epiblast layer-like layer formed by the pluripotent stem cells and a layer of hypoblast layer-like cells (cells differentiated from naive pluripotent stem cells) that underpins it. Co-culture is performed in suspension culture to efficiently form aggregates, but after the aggregates have formed, the culture may be continued by adhering the cells together.

[0049] The culture medium used to produce the bilayer blastodisc model is preferably a serum-free medium, but is not particularly limited, and known media such as media suitable for the differentiation of pluripotent stem cells may be used. The culture medium used to produce the bilayer blastodisc model preferably does not contain undifferentiated maintenance factors such as bFGF that maintain the undifferentiated state of pluripotent stem cells.

[0050] The culture temperature conditions in the method for producing the bilayered embryonic disc model of the present invention are not particularly limited, but for example, about 37°C to about 42°C, and about 37°C to about 39°C are preferred. The culture period for the bilayer blastodisc model is not particularly limited and may be changed depending on the purpose, but preferably, the culture of a mixture of hypoblastodisc-like cells and pluripotent stem cells is continued for 2 days or more, 4 days or more, or 6 days or more. There is no particular upper limit, but 30 days or less, 20 days or less, or 14 days or less are preferred.

[0051] <Method for accelerating maturation in a bilayered blastocyst model> By continuing step (a) or step (b) described above, the bilayered blastodisc model matures, forming an amniotic cavity-like structure in the inner blastodisc-like layer, and further, gastrulation cells and amniotic cells begin to appear. However, in order to more efficiently mature the bilayered blastocyst model of the present invention, it is more preferable to include the following steps (maturation acceleration steps).

[0052] In other words, the method for producing the bilayered blastodisc model of the present invention may include the step of co-culturing the above-mentioned cell aggregate and / or the above-mentioned bilayered blastodisc-like structure with IL-6 secreting cells, or the step of culturing it in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF.

[0053] The IL-6 secreting cells used in the co-culture process are not particularly limited as long as they are cells that secrete IL-6 extracellularly, but are preferably trophectderm cells. Tropicectoderm cells may be cells derived from the trophectoderm of the embryo, and may, for example, be trophectoderm cells differentiated by the method described in WO2021 / 106765, but are not particularly limited. It is more preferable for trophectoderm cells to secrete Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF in addition to IL-6.

[0054] When co-culturing the cell aggregate and / or the bilayered blastocyst-like structure with IL-6 secreting cells, it is preferable to use a partition that inhibits contact between the two types of cells but does not hinder the movement of secreted substances from one cell mass to the other. Such a partition may include, for example, a permeable membrane. An example of a culture method using a partition is a transwell, in which a cell culture vessel is divided into upper and lower sections with a permeable membrane in between. Other culture conditions can be adapted from the culture conditions used in the method for producing the bilayered embryonic disc model described above.

[0055] On the other hand, the step of culturing in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF may specifically be the step of culturing the above-mentioned cell aggregate and / or the above-mentioned bilayer blastodisc-like structure in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF to form an amniotic cavity-like cavity structure in the cell aggregate and / or the bilayer blastodisc-like structure.

[0056] Culturing in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF can be achieved, for example, by adding at least one cytokine selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF to the culture medium, or by co-culturing with cells that secrete at least one factor selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF extracellularly. When co-culturing, it is preferable to use the partitions described above. The concentrations of these cytokines are, for example, 5 ng / ml to 100 ng / ml.

[0057] Other culture conditions can be adapted from the culture conditions used in the method for producing the bilayered embryonic disc model described above.

[0058] <Method for producing primordial germ cells> A third aspect of the present invention relates to a method for producing primordial germ cells, comprising the steps of forming a cell aggregate containing subblast layer-like cells derived from naive mammalian pluripotent stem cells and mammalian pluripotent stem cells corresponding to epiblast layer-like cells, and culturing the cell aggregate for 10 days or more, preferably 12 days or more. There is no upper limit to the culture period, but for example, it may be 30, 40, or 50 days. In other words, primordial germ cells can be obtained by continuing to culture the cell aggregate obtained as described above. For example, primordial germ cells can be obtained by continuing to culture for a long period of time after performing steps (a) and (b) above, as well as the maturation acceleration step above.

[0059] Primordial germ cells possess the ability to differentiate into germ cells in the future and are characterized by the expression of primordial germ cell markers such as BLIMP1 and TFAP2C.

[0060] By culturing the aforementioned cell aggregate for more than 10 days, cells differentiated into primordial germ cells can be obtained. Other culture conditions can be derived from the culture conditions described above in the bilayered blastocyst model and its manufacturing method. The method for recovering primordial germ cells is not particularly limited as long as it can recover primordial germ cells from a cell population. For example, cells expressing BLIMP1 (B-lymphocyte-induced maturation protein 1) and TFAP2C are fluorescently labeled and then recovered by fluorescence-activated cell sorting (FACS). [Examples]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following embodiments.

[0062] cell culture Unless otherwise specified, the cells in this example were cultured under 5% O2 and 5% CO2 conditions. Human ES cell lines H1 and H9 (WiCell Laboratory, Wyoming, USA) and human iPS cells generated from adult adipose-derived stem cells (AdiPSCs) (Takashima et al., Cell 158, 1254-1269, 2014) were cultured on mouse embryonic fibroblasts (MEFs) (1 × 10⁶ cells per 6-well plate). 6 cell). Primed human pluripotent stem cells were maintained in DMEM / F12 (Nacalai Tesque) containing 20% ​​knockout serum replacement (KSR; Thermo Fisher Scientific), 1% non-essential amino acids (Thermo Fisher Scientific), 4 ng / mL recombinant human basic fibroblast growth factor 2 (bFGF; Oriental Yeast Co., Ltd.), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich). These cells were then passaged every 5–7 days as small aggregates using a separation buffer containing 0.025% trypsin (Thermo Fisher Scientific), 1 mg / mL collagenase IV (Thermo Fisher Scientific), 20% KSR, and 1 μM CaCl2.

[0063] Naive human pluripotent stem cells were maintained in t2iLGo medium consisting of N2B27 (Ndiff227; Takara Bio Inc.) containing 1 μM PD0325901 (PD03; Tocris), 1 μM CHIR99021 (CH; Sigma-Aldrich), 10 ng / mL recombinant human LIF (hLIF; Peprotech), and 3 μM Go6983 (Go; Tocris). Naive pluripotent stem cells were passaged every 3-5 days using Accutase (Sigma-Aldrich).

[0064] The reset of primed pluripotent stem cells to naive pluripotent stem cells by overexpression of NANOG and KLF2 was performed as briefly described below. PiggyBac (PB) vectors (2 μg) and PB-M2rtTA expression vectors (2 μg) containing doxycycline (DOX)-inducible KLF2 or NANOG were cotransfected with pBase helper plasmids (4 μg) using the Neon transfection system (program 14; Invitrogen). The culture medium was replaced with t2iL + DOX (1 μM) for reset. After separation using Accutase, cells were divided every 5-7 days. After 2 weeks, DOX was removed, PKC inhibitor Go6983 (3 μM) was added (t2iLGo), and the cells were maintained on an MEF feeder.

[0065] Chemical conversion to naive pluripotent stem cells was performed as described in Guo et al., 2017. Prime pluripotent stem cells (1 × 10⁻¹⁶) 4 cells / cm 2 The cells were seeded on MEF feeder cells in primed pluripotent stem cell medium containing 10 μM Y-27632. The following day, the medium was replaced with cRM-1 (N2B27, 1 μM PD03, 10 ng / mL hLIF, and 1 mM sodium valproate (VPA; Sigma-Aldrich)). On the third day, the medium was replaced with cRM-2 (N2B27, 1 μM PD03, 10 ng / mL hLIF, 2 μM Go, and 2 μM XAV939 (Sigma-Aldrich)). Dome-shaped naive colonies were observed approximately two weeks after seeding. The reset cells were passaged and maintained on MEF feeders in t2iLGo medium. Chemical conversion to naive pluripotent stem cells using 5iLA was performed as follows: Primed pluripotent stem cells (2 × 10⁶) 5 cells / cm 2)The cells were seeded onto MEF feeder cells in primed pluripotent stem cell medium containing 10 μM Y-27632. The following day, the medium was replaced with 5iLA medium (N2B27 + 1 μM PD03, 1 μM CH, 1 μM WH-4-023 (H620061, Morrisville, North Carolina, USA), 0.5 μM SB590885 (R&D Systems), 10 μM Y-27632, 10 ng / mL hLIF, and 20 ng / mL activin A (R&D Systems)). After conversion to naive pluripotent stem cells, the cells were maintained on MEF feeder cells in t2iLGo medium.

[0066] Mouse ES cells were cultured in 2 iL (N2B27, 1 μM PD03, 3 μM CH, and 10 ng / mL hLIF) on gelatin-coated dishes, and the cells were passaged every 2-3 days using Accutase.

[0067] Induction of differentiation of hypoblastoid-like cells GATA6, GATA4, and SOX17 were cloned into DOX-inducible PB vectors conjugated with an rtTA expression construct (KW110). PB-GATA6 vector (2 μg), PB-GATA4 vector (2 μg), or PB-SOX17 vector (2 μg) and pBase helper plasmid (2 μg) were transfected into naive or primed pluripotent stem cells using the Neon transfection system (Program 20 for naive pluripotent stem cells; Program 14 for primed pluripotent stem cells). After 2 days, G418 was added for approximately 2 weeks (200 μg / mL). Naive or primed pluripotent stem cells containing inducible GATA6, GATA4, or SOX17 were maintained in naive or primed medium. For transgene induction, MEF feeder cells were isolated at the single-cell level and then incubated on gelatin-coated dishes. Next, 1 × 10 5 cells / cm 2They were seeded onto dishes coated with fibronectin (Millipore) or iMatrix-511 silk (laminin 511-E8) (Matrixome). The serum medium consisted of GMEM (Sigma-Aldrich), FBS (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), NEAA, and 0.1 mM 2-ME. Except for these experiments, all other analyses were performed under serum-free conditions. As the serum-free basal medium, N2B27 medium (Ndiff227; catalog number Y40002, Takara Bio) was used. The components of the N2B27 medium were DMEM / F12, Neurobasal, N2, and B27. BSA was included in N2 and B27. 25 ng / ml recombinant human FGF4 (FGF4) and 1 μg / mL heparin sodium (Wako Pure Chemical Industries, Ltd.) were added to the basal medium. The medium was changed daily.

[0068] Induction of differentiation of hypoblast-like cells using chemical components is briefly shown below. 5×10 4 / cm 2 Naïve pluripotent stem cells were seeded onto laminin 511-E8 in N2B27 medium. On day 0, 6 factors, 25 ng / mL FGF4 (+1 μg / mL heparin sodium), 10 ng / mL recombinant human BMP4 (BMP4; R&D), 10 ng / mL recombinant human PDGF-AA (PDGF AA, Peprotech), 1 μM XAV939, 3 μM A83-01 (Tocris), and 0.1 μM RA (Sigma-Aldrich) were added. On day 2, the medium was changed to 7 factors (6 factors and 10 ng / mL recombinant human IL-6) (IL-6; Oriental Yeast Co., Ltd.). In some experiments, 500 ng / mL recombinant human BMP2 (BMP2; Oriental Yeast Co., Ltd.) or 50 ng / mL recombinant human BMP6 (BMP6; Peprotech) was used instead of BMP4. N2B27 medium without VitA was prepared in-house.

[0069] Two protocols were used to induce the subblast layer from mouse ESCs. Mouse ESCs were maintained under 2iL conditions. In the first protocol, 5 × 10 4 / cm 2 Mouse ESCs were seeded on gelatin under RPMI1640 (Thermo Fisher Scientific) containing 2 mM L-glutamine, B27-negative insulin (Gibco), 20 ng / mL activin A, 3 μM CH10 ng / mL, and hLIF. In the second protocol, 5 × 10 4 / cm 2 Mouse ESCs were seeded on gelatin under 10 nM RA and 20 ng / mL activin A. The culture medium was changed daily under both conditions.

[0070] Marmoset embryo culture All animal experiments were approved by the Animal Experiment Committees of CiRA and Kyoto University (Approval No. 16-75-6) and the Animal Experiment Committee of the Central Institute for Experimental Animals (CIEA: Approval Nos. 17029A and 18031A). Spontaneously fertilized embryos were recovered from the uterus by non-invasive flushing. Embryos (morula or blastocyst) were cultured under Sequential Blast (Origio 83050010). When the embryos reached the blastocyst stage, the zona pellucida was removed using acidic Tyrode's solution (Sigma), and the embryos were subjected to immunosurgery using a custom rabbit polyclonal anti-marmoset antibody. ICMs were seeded on laminin 511-E8 for 3 days under N2B27+ factor 7 (7F), factor 4 (4F) (FGF4, BMP4, A83, XAV), or control (PD03, LDN, A83, XAV), fixed, and analyzed with SOX17 antibody.

[0071] Creation of a bilayered embryonic disc model Ten naive pluripotent stem cells (naive, WT) and 40 naive pluripotent stem cells expressing GATA6 under DOX treatment or GFP-expressing naive pluripotent stem cells (naive (GATA6) or naive GFP (GATA6)) were seeded in each well of a microarray or Elplasia plate (Corning, Inc.) under t2iLGo + 10 μM Y27632 without Matrigel or Geltrex. 24–36 hours after aggregation (day 0), the medium was replaced with N2B27 containing 0.1 μM DOX. On day 2, the DOX was removed. To screen signaling pathways, on day 0, 25 ng / mL FGF4 and 1 μg / mL heparin sodium, 1 μM PD03, 0.1 μM RA, 10 μM BMS493 (BMS; catalog number 17418, Cayman, Ann Arbor, Michigan, USA), 10 ng / mL IL6, 1 μM JAK inhibitor 1 (JAKi, catalog number 420099, Sigma), 10 ng / mL PDGF-AA, 10 ng / mL BMP4, 300 nM LDN193189 (LDN, SML0559, Sigma), 3 μM A83-01, 10 ng / mL activin, 1 μM XAV, and 1 μM CHIR were added. The medium was changed daily.

[0072] Aggregates generated from human pluripotent stem cells and selected cells 100 naive or primed human pluripotent stem cells and 100 selected cells expressing GFP (naive 7F-PDFRA) + Cells, 4F-PDFRA + cells, GATA6(G6)-PDFRA + Cells, Prime G6-PDGFRA + cells, PDGFRA + RACL cells and CXCR4 + / CDH1 + Embryonic endoderm cells were seeded in each well of an Elplasia plate under N2B27 + 10 μM Y27632. The culture medium was changed every other day. Aggregates were evaluated on day 4.

[0073] Co-culture of a bilayered blastocyst model and trophectoderm To create a bilayered blastocyst model, wild-type naive human pluripotent stem cells (10 cells / microwell) and naive human pluripotent stem cells (40 cells / microwell) capable of inducing GATA6 overexpression by DOX addition were seeded on Elplasia plates (Corning) using t2iLGo (Ndiff227 (Takara Bio), 1 μM PD0325901 (PD03; Tocris), 1 μM CHIR99021 (CH; Sigma-Aldrich), 10 ng / ml Recombinant human LIF (hLIF; Peprotech), 3 μM Go6983 (Go; Tocris)) + 10 μM Y-27632 (Wako) medium. Cell aggregates were formed 24-36 hours after seeding. After cell aggregate formation, the culture medium was changed to N2B27 + DOX (0.1 μg / ml), and co-culture with trophectoderm cultured on a Transwell (Corning) was initiated. 48 hours after the start of co-culture, the DOX was removed, and culture was continued in N2B27. The culture medium was changed daily.

[0074] Tropicectoderm was differentiated from naive human pluripotent stem cells (Io et al., Cell stem cell 3;28(6):1023-1039.e13. 2021). After removing MEF, naive human pluripotent stem cells were differentiated into 0.5 × 10⁶ 4 pieces / cm 2 Seeds were seeded onto iMatrix-511 silk (MAX) coated Transwells and cultured in N2B27 + 2μM A83-01 (Tocris) + 2μM PD03 + 10ng / ml recombinant human BMP-4 (BMP-4; R&D) medium. After 24 hours, the medium was changed to N2B27 + 2μM A83-01 + 2μM PD03 + 1μM JAK inhibitor 1 (JAKi; MERCK) and cultured for another 48 hours. At 72 hours of differentiation induction, the embryos were washed with PBS and used in co-culture with a bilayered blastocyst model.

[0075] Flow cytometry and cell sorting Cells were isolated into single cells using Accutase or trypsin, washed, and blocked on ice for 30 minutes in HBSS (catalog no. 14185052, Thermo Fisher Scientific) containing 1% BSA (catalog no. A2153, Sigma-Aldrich). Staining was performed on ice with the following: biotinylated PDGFRA antibody (catalog no. BAF322, R&D), CEACAM1+CEACAM5 antibody (catalog no. ab91213, Abcam, Cambridge, UK), and PE-conjugated anti-CD13 (catalog no. 301703, Biolegend, San Diego, California, USA) in HBSS containing 1% BSA for 30 minutes. After washing, streptavidin-APC (catalog number 405207, Biolegend), streptavidin-PE (catalog number 12-4317-87, eBioscience, San Diego, California, USA), and streptavidin-BV421 (catalog number 405226, Biolegend) were used as secondary antibodies against PDGFRA-biotin. Alexa Flour 488 was used as the CEACAM1 antibody. Flow cytometry and cell sorting were performed using BD LSR Fortessa (BD) or FACS Aria II (BD). Data were analyzed using FlowJo.

[0076] Reverse transcription and real-time PCR quantification Total RNA was extracted using the RNeasy kit (catalog number 74106, Qiagen, Hilden, Germany). Total RNA (0.5 μg) was reverse transcribed into cDNA containing oligo-dT primers using SuperScript IV (catalog number 18090050, Thermo Fisher Scientific). Real-time PCR was performed using QuantStudio3 (Thermo Fisher Scientific) and QuantStudio12K (Thermo Fisher Scientific) equipped with TaqMan Fast Universal Master Mix (catalog number 4364103, Thermo Fisher Scientific) and either a TaqMan probe or PowerUP SYBR Green Master Mix (catalog number A25743, Thermo Fisher Scientific), according to the manufacturer's instructions. The results were analyzed using QuantStudio Design & Analysis v1.4.1 (Thermo Fisher Scientific).

[0077] immunostaining Cells were fixed in 4% paraformaldehyde (catalog no. 09154-85, Nacalai Tesque) at room temperature for 10 minutes. After fixation, cells were washed with PBS, permeabilized in PBS + 0.5% Triton X-100 for 1 hour, and blocked in PBS + 1% BSA and 0.05% Tween-20 (PBS-BT) for 2 hours. The primary antibody was diluted in PBS-BT and incubated overnight at 4°C. After washing, the secondary antibody was diluted 1:2000 and incubated at room temperature for 2 hours or overnight at 4°C. Nuclei were stained with DAPI. Fluorescence images were obtained using a confocal laser scanning microscope TCS SP8 (Leica, Wetzlar, Germany) or LSM710 (Zeiss, Oberkochen, Germany).

[0078] RNA-seq analysis For RNA-seq, the sample was collected after removing MEFs by gelatin treatment. RNA was purified using the miRNeasy mini-kit (catalog number 217004, Qiagen), and 200 ng of RNA was used for library construction using the TruSeq Stranded mRNA LT Sample Prep kit (RS-122-2101, Illumina, San Diego, California, USA). The RNA-seq library was sequenced using the NextSeq 500 High Output v2 kit (75 cycles, FC-404-2005) (Illumina). The sequenced reads were trimmed using cutadapt-1.15 to remove low-quality bases and adapter sequences. The trimmed reads were then processed using GENCODE v27. 60 TopHat2, equipped with [specific feature / tool], was used to map the genome to the human reference genome (hg38). Uniquely mapped reads (MAPQ≧20) were used for further analysis. Each gene expression level was calculated as reads per kilobase (FPKM) per million mapped reads using cufflink-2.2.1. Genes expressed at low levels across all samples in each dataset (FPKM<5: UHC, PCA; FPKM<1: correlation coefficient) were excluded from subsequent analysis. Expression values ​​were normalized to the median, mean, or specific conditions of the entire dataset. Heatmap creation, correlation analysis, hierarchical clustering analysis, and PCA were performed using R (v3.3.2).

[0079] scRNA-seq and data analysis Pyraminoids were generated by co-culturing naive (WT) and naive GFP (GATA6). +Pyraminoids surrounded by cells were manually picked up and isolated using Accutase. Single cells were recovered by flow cytometry and loaded into a 10× Genomics Chromium system. A 10× Genomics v.3.0 library was prepared according to the manufacturer's instructions. The library was then sequenced using an Illumina HiSeq 4000 with paired-end sequencing. scRNA-seq data were mapped and quantified against the hg38 human reference genome + reporter sequence (EGFP) using the Cell Ranger pipeline (v.4.0.0, 10× Genomics). Raw count data were imported into the Seurat package (v.3.1.5). Cells with nfeature > 4,000, nCount between 5,000 and 200,000, and low mitochondrial gene expression (<15%) were further analyzed. Raw counts were normalized using logarithmic normalization in the Seurat package. UMAP analysis and clustering were performed using the Seurat RunUMAP function, which includes the "FindClusters" function, with initial parameters (dims=1:50) and resolution sets of 0.8 for the hypoblast and 1.2 for the epiblast, respectively. Cell type was determined by the expression of selected markers. Flight trajectory analysis was performed using Monocle (v.2.410.1).

[0080] result The hypoblast blast controls the development of the epiblast blast before and during implantation. Naive human pluripotent stem cells (Naive(WT)) and a mixture of naive human pluripotent stem cells expressing GATA6 and GFP under DOX treatment (Naive-GFP(GATA6)) were cultured on a microwell array (Figure 1). To label aggregated cells, GFP or DsRed was introduced into naive PSCs (naive GFP and naive DsRed, respectively). Naive-GFP(GATA6) alone was unable to generate cell aggregates. However, both Naive(WT) and a mixture of Naive(WT) and Naive-GFP(GATA6) generated aggregates. The mixture of Naive(WT) and Naive-GFP(GATA6) formed spherical aggregates, while the aggregates from Naive(WT) alone were large but not spherical (Figure 2). On day 0 (D0), the aggregates showed a mixture of Naive (WT) and Naive-GFP (GATA6). On day 2 (D2), GATA6 was present, as is normally observed during maturation in late blastocysts. +Cells (naive pluripotent stem cell-induced subblastome-like cells (nHyC)(G6)) were located at the periphery of the aggregates (Figure 3). Approximately 40% of the aggregates were surrounded by nHyC(G6) (Figure 4). Time-course images of the aggregates also showed the dynamics of nHyC(G6) and naive pluripotent stem cell-derived subblastome-like cells (nEpiC) from 0 to 48 hours (Figure 5). A mixture of GFP(nHyC(G6)) and DsRed(nEpiC) was separated after DOX induction. nHyC(G6) was located at the periphery of the aggregates after 48 hours. The distance of nHyC(G6) and nEpiC from the center of the aggregate was measured (Figure 6). In DOX-treated aggregates, the distance of nHyC(G6) gradually increased, while the distance of nEpiC decreased. Previous reports on human embryos have suggested that the number of cells in the epiblast layer increases from approximately 20–40 at embryonic days 7–8 to approximately 80–100 at embryonic day 10, and the number of cells in the hypoblast layer increases from approximately 20–50 at embryonic day 8 to approximately 60–90 at embryonic day 10 (Deglincerti, A. et al. Nature 533, 251-254 (2016), Roode, M. et al. Developmental Biology 361, 358-363 (2012), Di Stefano, B. et al. Nat Methods 15, 732-740, (2018)). The number of nHyC (G6) and nEpiC cells in the aggregates also proliferated during differentiation, corresponding to human embryo data (Figure 7). The size of the aggregates also increased during culture (Figure 8).

[0081] Next, nHyC(G6)(GFP) + ) and nEpiC(GFP -Cells were sorted by flow cytometry on days 2 and 4 to evaluate gene expression patterns (Figure 9). First, the authors checked GATA6 expression after DOX treatment. Total GATA6 expression in nHyC(G6) on day 2 after DOX treatment was approximately 30-fold higher than in nEpiC on day 2 (Figure 10). Published scRNA-seq data showed that GATA6 expression in the hypoblastoblast was approximately 20-fold higher than in the epiblastoblast at the blastocyst stage (Figure 11). In aggregates on day 2, nHyC(G6) showed increased expression of hypoblastoblast genes such as SOX17, HNF4A, and FOXA2, as well as decreased expression of pluripotency-related genes, while nEpiC showed expression of naive or epiblastoblast genes such as NANOG, KLF17, and OCT3 / 4 (Figures 10, 12). Therefore, we concluded that the two cell types were sorted by the marker and expressed the marker, similar to the phenomenon reported in late human blastocysts. Furthermore, GATA6 continued to be expressed in nHyC even after DOX was removed on day 2 (Figure 12).

[0082] During the period before and after implantation, the epiblast layer acquires apical-basal polarity, which is labeled by PAR6 expression. On day 2, no accumulation of PAR6 was observed in aggregates, consistent with the blastocyst-like stage. However, by day 4, approximately 20% of aggregates surrounded by nHyC(G6) accumulated PAR6 protein in their center, and simultaneously, nEpiC formed rosette structures (Figures 3 and 4). Similar to the pre-implantation to post-implantation transition, nEpiC showed decreased expression of the naive pluripotency gene KLF17 and increased expression of the early post-implantation epiblast layer genes THY1, DNMT3B, and SFRP2 (Figures 10 and 13). The polarized nEpiC gradually formed rosette-like structures on day 4, which we call bilaminoids. PODXL and aPKC co-existed with PAR6 in these bilaminoids (Figure 14).

[0083] Furthermore, naive pluripotent stem cells and naive-derived PDGFRA induced and selected on laminin 511-E8 by GATA6, 7F, or 4F. +Aggregates produced by cells (G6-nHyC, 7F-nHyC, or 4F-nHyC, respectively) were also analyzed (Figure 15). Although the efficacy was lower than that of the Naive (WT) and Naive-GFP (GATA6) mixture, the mixture of naive pluripotent stem cells and 7F-nHyC, 4F-nHyC, or G6-nHyC was able to generate aggregates with rosette-like structures (Figure 15). Furthermore, the mixture of primed pluripotent stem cells and 7F-nHyC, 4F-nHyC, or G6-nHyC was also able to generate aggregates with rosette structures (Figure 15).

[0084] Once the bilayer disc is formed, a subset of epiblastocyte cells participates in a process called gastrulation, which determines the basic axis of the body. Indeed, when Bilaminoid was cultured for an additional two days, T-positive cells were observed (Figure 16). Aggregates generated by prime PSCs and 7F-nHyC, 4F-nHyC, or G6-nHyC also showed T expression in a portion of the epiblastocyte population (Figure 17). Interestingly, only epiblastocyte cells surrounded by nHyC expressed primitive streak-related genes (T, MIXL1, MESP1, EVX1, SNAI2, EOMES), while aggregates without nHyC did not (Figure 18). Bilaminoid was further analyzed on day 6 using scRNA-seq. Characteristic plots of hypoblast and epiblast gene clusters showed that nHyC and nEpiC clustered differentially via homogeneous manifold approximation and projection (UMAP) (Figure 19). GATA6, SOX17, PDGFRA, GATA4, FOXA2, ANPEP, and CEACAM1 were strongly expressed in the lower UMAP1 cluster (UMAP1-Low), while OCT3 / 4, NANOG, and SOX2 were expressed in the higher UMAP1 cluster (UMAP1-High). Subpopulation analysis revealed that the epiblast population could be divided into 7 clusters and the hypoblast population into 3 clusters (Figure 20). When the primitive amniotic cavity is formed, the epiblast differentiates into the post-implantation epiblast and amnion in both in vivo and in vitro models. Because bilaminoid may contain cells expressing amniotic genes, we analyzed the gene expression patterns of bilaminoid using differential gene expression (DEG) data from the epiblast layer and amniotic membrane established from in vitro cultured human embryos. Heatmaps revealed that cells in clusters 5, 0, and 3 tended to express epiblast layer genes, while cells in clusters 4 and 1 expressed amniotic genes (Figure 21). Violin plots also showed the expression of epiblast layer-related genes in clusters 5 and 0, and amniotic membrane-related genes in clusters 1 and 4 (Figure 22).

[0085] Next, nEpiCs were arranged in a pseudo-time using Monocle2. Flight trajectory analysis suggested that nEpiCs from clusters 5 and 0 differentiated into clusters 2, 1, and 4, and clusters 3 and 6, respectively (Figure 23). Clusters 6 and 3 expressed genes related to the primitive streak and gastrulation (Figure 24). Heatmaps of marker genes for epiblast development stages (ICM, pre-epic epiblast, late epiblast, and primitive streak) in in vitro cultured human embryos confirmed the development of nEpiCs from pre-epic epiblast to the primitive streak (Figure 25). In addition, correlation analysis of epiblast development in cynomolgus monkeys suggested that cluster 6 had a strong correlation with Gast2b (Figure 26). In summary, these data suggest that amnionoid cells in Bilaminoid may be neoamnionoid bud-like cells, but the epiblast layer of Bilaminoid showed the appearance of amnionoid and primitive streaky cells.

[0086] Next, we analyzed the role of nHyC and the interaction between nHyC and nEpiC during pre- and post-implantation development. Laminin formed a boundary between nHyC(G6) and nEpiC in Bilaminoid (Figure 27), reflecting the formation of the basement membrane between the hypoblastoblast and epiblastoblast in humans as well as in mice. To analyze the adhesion of nEpiC to the extracellular matrix (ECM), we studied integrin expression in nEpiC in Bilaminoid (Figure 28). Two pairs of heterodimers, ITGA6 and ITGB1(α6β1) and ITGAV and ITGB5(α v β5) was expressed in nEpiC. Integrin α6β1 and α v The ligands for β5 are known as laminin and vitronectin, respectively. We checked the expression of laminin and vitronectin in nHyC(G6). VTN expression was weak, but LAMA1, LAMB1, and LAMC1 were strongly expressed in nHyC(G6) (Figure 29). Since Lamb1 is required for the formation of the basement membrane between the epiblast and hypoblast layers in early mouse embryos, we hypothesized that LAMB1 may be a major ECM component for interaction in humans.

[0087] Interactions between the hypoblastoblast and epiblastoblast in cynomolgus monkey embryo data have revealed that the anterior-posterior axis in the visceral endoderm is determined during gastrulation. ScRNA-seq data showed that NODAL was expressed in nHyC(G6) (Figure 30), consistent with previous reports in mice. Several nHyC(G6) of Bilaminoid expressed the anterior markers CER1, DKK1, LEFTY1, 2, and OTX2 (Figure 30). This finding suggests the presence of an anterior-posterior axis in Bilaminoid nHyC(G6). Co-staining of OTX2 and T in Bilaminoid on day 6 showed that T-positive cells of nEpiC and OTX2-positive cells of nHyC(G6) did not come into contact with each other (Figure 31). This result suggests that the anterior-posterior axis may be formed and play a role in initiating the primitive streak.

[0088] These results demonstrate that the hypoblast and epiblast develop synergistically. More specifically, the authors show that the hypoblast is essential for primitive amniotic cavity formation in vitro, induces apical-base polarity formation in the epiblast, and initiates the primitive streak.

[0089] Furthermore, co-culture was performed with a bilayered blastodisc model using trophectoderm (TE) differentiated from naive human pluripotent stem cells. Wild-type naive human pluripotent stem cells and naive human pluripotent stem cells that can induce GATA6 overexpression by adding DOX were seeded on Elplasia plates. After cell aggregates were formed, the medium was changed to N2B27 + DOX (0.1 μg / ml), and co-culture with TE cultured on a Transwell was started. Co-culture with TE increased the amniotic cavity formation rate (Figure 32), and cells exhibiting amniotic cell-like morphology appeared (Figure 33). Therefore, it was found that co-culture with TE is preferable for the bilayered blastodisc model. Furthermore, since this effect was obtained through co-culture on a Transwell, it is presumed that it is due to factors secreted from TE. Therefore, we investigated the factors secreted by TE. As a result, IL-6 added to the culture medium promoted amniotic cavity formation in a bilayered blastocyst model, and JAK inhibitors (JAKi) inhibited the increase in amniotic cavity formation rate by co-culturing with TE (Figure 34). However, the addition of IL-6 did not promote the appearance of cells exhibiting amniotic cell-like morphology. From these results, it was found that the effect of co-culturing with TE is partly due to the secretion of IL-6.

[0090] A bilayered blastodisc model cultured for 12 days on N2B27 was analyzed by the expression of primordial germ cell (PGC) markers. PGC reporter lines (BLIMP1-tdTomato, TFAP2C-EGFP) were used for the analysis. FACS analysis of the PGC reporter line-derived bilayered blastodisc model after 12 days of culture revealed cells positive for both BLIMP1-tdTomato and TFAP2C-EGFP (Figure 35). These cells were collected, and the expression of the genes shown in Figure 36 was analyzed. The results showed that cells collected from the bilayered blastodisc model after 12 days of culture exhibited PGC-related gene positivity. This strongly suggests that differentiation into PGCs can occur in some cells within the bilayered blastodisc model.

Claims

1. A method for producing a bilayered embryonic dissection model of a mammal, The following steps (a) or (b) (a) A step of co-culturing naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) with mammalian pluripotent stem cells (second mammalian pluripotent stem cells) in suspension culture; (b) A step of co-culturing naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells) with mammalian pluripotent stem cells (second mammalian pluripotent stem cells) in suspension culture, while inducing subblast layer-like cells from the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells). by The process includes forming a bilayered cell mass comprising a first mammalian pluripotent stem cell-like cell and a second mammalian pluripotent stem cell, The first mammalian pluripotent stem cell and the second mammalian pluripotent stem cell are, respectively, induced mammalian pluripotent stem cells and / or mammalian embryonic stem cells. The method involves a bilayered cell mass forming a bilayered blastocyst-like structure, the latter containing a layer of second mammalian pluripotent stem cells on the inside and a layer of hypoblast-like cells surrounding it on the outside.

2. The aforementioned hypoblastoid-like cells are positive for at least one of PDGFRA, CEACAM1, and ANPEP. The second mammalian pluripotent stem cell is PDFGRA-negative, OCT3 / 4-positive, and NANOG-positive. The method according to claim 1.

3. The aforementioned hypoblastoid-like cells contain GATA family transcription factors in the aforementioned naive mammalian pluripotent stem cells. The offspring is induced from the naive mammalian pluripotent stem cells by forcing their expression, claim The method described in item 1 or 2.

4. The aforementioned subblast lobe-like cells activate the BMP signaling receptor type 1 and FGFR2 signaling pathways in the aforementioned naive mammalian pluripotent stem cells, thereby enhancing the naive type The method according to any one of claims 1 to 3, derived from mammalian pluripotent stem cells.

5. The method according to any one of claims 1 to 4, wherein the bilayered blastocyst-like structure has an anterior-posterior axis. 。

6. The method according to any one of claims 1 to 5, wherein the bilayered blastocyst-like structure includes cells in the gastrulation stage.

7. The method according to any one of claims 1 to 6, wherein the bilayered blastocyst-like structure forms an amniotic cavity-like cavity structure.

8. The process involves co-culturing the cell aggregate and / or the bilayered blastocyst-like structure with IL-6 secreting cells. The method according to any one of claims 1 to 7, further comprising the step.

9. The method according to claim 8, wherein the IL-6 secreting cells are trophectoderm cells.

10. The method according to any one of claims 1 to 9, further comprising the step of culturing the cell aggregate and / or the bilayered blastodisc-like structure in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11, and VEGF.

11. A first mammalian induced pluripotent stem cell (iPS cell) and / or mammalian embryonic stem cell (ES cell) A layer of hypoblast-like cells derived from ) Second mammalian induced pluripotent stem cells (iPS cells) and / or mammalian embryonic stem cells (ES cells) A bilayered blastocyst-like structure consisting of a bilayered cell mass including a layer of ), The aforementioned bilayered cell mass is a bilayered blastocyst-like structure, containing an inner layer of second mammalian pluripotent stem cells and an outer layer of hypoblast-like cells surrounding it.

12. A bilayered blastocyst-like structure according to claim 11, comprising two or more cells of different genotype origins.

13. A method for producing primordial germ cells, A bilayered blastocyst-like structure is produced by the method described in any one of claims 1 to 10. A method comprising the step of culturing the bilayered blastocyst-like structure for 10 days or more.

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