WO2022114188A1 - Modèle de blastocystis bilaminaris et procédé de production de celui-ci - Google Patents

Modèle de blastocystis bilaminaris et procédé de production de celui-ci Download PDF

Info

Publication number
WO2022114188A1
WO2022114188A1 PCT/JP2021/043655 JP2021043655W WO2022114188A1 WO 2022114188 A1 WO2022114188 A1 WO 2022114188A1 JP 2021043655 W JP2021043655 W JP 2021043655W WO 2022114188 A1 WO2022114188 A1 WO 2022114188A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
pluripotent stem
naive
stem cells
mammalian
Prior art date
Application number
PCT/JP2021/043655
Other languages
English (en)
Japanese (ja)
Inventor
康弘 ▲高▼島
巧 大久保
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2022565490A priority Critical patent/JPWO2022114188A1/ja
Publication of WO2022114188A1 publication Critical patent/WO2022114188A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a bilayer scutellum model and a method for producing the same.
  • blastocyst During the developmental stage of mammals, an inner cell mass is formed inside the blastocyst, from which the upper blastocyst (epiblast) and the lower blastocyst (hypoblast) emerge, and the epiblastocyst is formed. Blast undergoes primordial intestinal formation and differentiates into embryonic mesoderm, while primordial endoderm differentiates into epiblast tissue, especially the epiblast. Recreating these developmental processes with pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is the analysis of developmental mechanisms, elucidation and treatment of disease mechanisms at the developmental stage, etc. It is useful for.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • Non-Patent Document 1 Korean ES cells and iPS cells were classified into advanced developmental prime type compared to mouse pluripotent stem cells, and it was difficult to differentiate into epiblasts and primitive endoderms. Therefore, Takashima et al. Decided to obtain naive pluripotent stem cells in which human pluripotent stem cells were reset to the same state as the basal state by expressing two genes, NANOG and KLF2, in human pluripotent stem cells.
  • Non-Patent Document 1 reported on the induction of primitive endoderm (Patent Document 1) and the induction of trophorid ectoderm (Patent Document 2) using the same.
  • the present invention is a pluripotent stem cell for understanding early development, for obtaining differentiated cells using a model that mimics early development, and for enabling the induction of tissues having physiological functions. It is an object of the present invention to provide a bilayer scutellum model derived from the above and a method for producing the same.
  • hypoblast primary endoderm
  • nHyC hypoblast-like cells
  • nHyC naive pluripotent stem cells
  • the present invention provides: [1] A method for producing a two-layered scutellum model of mammals.
  • Subepithelial lobe-like cells derived from naive-type mammalian pluripotent stem cells (first mammalian pluripotent stem cells) and mammalian pluripotent stem cells corresponding to pelvic lobe upper layer-like cells (second mammalian pluripotency) A method comprising the step of forming a cell mass comprising (stem cells).
  • the subblastocyst-like cells are positive for at least one of PDGFRA, CEACAM1 and ANPEP.
  • Step of co-culturing by suspension culture (B) The naive-type mammal while co-culturing the naive-type mammalian pluripotent stem cell (first mammalian pluripotent stem cell) with the mammalian pluripotent stem cell (second mammalian pluripotent stem cell) by suspension culture.
  • the naive mammalian pluripotent stem cells are forced to express the GATA family transcription factor in the naive mucosal pluripotent stem cells (the naive mammalian pluripotent stem cells).
  • the method according to any one of [1] to [3], which is derived from a first mammalian pluripotent stem cell).
  • the sublamellar layer-like cells activate signals via the BMP signal type 1 receptor and FGFR2 signals in the naive mammalian pluripotent stem cells (first mammalian pluripotent stem cells).
  • the cell mass consists of a layer of mammalian pluripotent stem cells (second mammalian pluripotent stem cells) corresponding to the upper lamellar layer-like cells and the mammalian pluripotent stem cells (second mammalian pluripotent stem cells).
  • IL-6 secreting cells are vegetative ectoderm cells.
  • iPS cells mammalian induced pluripotent stem cells
  • ES cells mammalian embryonic stem cells
  • second mammalian iPS cells and / or mammalian ES cells second mammalian iPS cells and / or mammalian ES cells
  • first mammalian iPS cells and / or mammalian ES cells first mammalian iPS cells and / or mammalian ES cells.
  • [15] A method for producing primordial germ cells. Subepithelial lobe-like cells derived from naive-type mammalian pluripotent stem cells (first mammalian pluripotent stem cells) and mammalian pluripotent stem cells corresponding to pelvic lobe upper layer-like cells (second mammalian pluripotency) Including the step of forming a cell mass containing stem cells) A method comprising culturing the cell mass for 10 days or longer.
  • a bilayer scutellum model can be easily produced from pluripotent stem cells. This provides a previously unreported model of pre-implantation and pre-implantation stage embryos, analyzing important pre- and post-implantation stimulating interactions between primitive endoderm and epiblast. It can be a useful tool for elucidating early development. Furthermore, the bilayer scutellum model of the present invention may be able to differentiate into all the cells constituting the fetus by continuing the culture, and it is expected that various tissues and cells can be obtained from the model of the present invention. can. Furthermore, it can be expected to become a model of disease by using diseased iPS cells and the like.
  • Naive (WT), Naive-GFP (GATA6), or Naive (WT) and Naive-GFP (GATA6) were cultured on a microwell array in t2iLGo medium (TakaraBio). After 24-36 hours (h), the medium was replaced with N2B27. DOX was added for the first 2 days. If mentioned, IL6 was added from day 0 to day 4.
  • 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.
  • Nive (50 cells / well), Nive-GFP (GATA6) (50 cells / well), or Nive (WT) (10 cells / well) + Nive-GFP (GATA6) (40 cells / well)
  • WT Nive
  • GATA6 Nive-GFP
  • GATA6 Nive-GFP
  • the mixed aggregate of Naive (WT) + Naive-GFP (GATA6) is more spherical than Naive (WT). At least 100 aggregates were measured in each group. Immunofluorescent images of cell aggregates produced 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 the mixed aggregate of Naive (WT) and Naive-GFP (GATA6). Scale bar, 20 ⁇ m.
  • nHyC (G6) and nEpiC nuclei were from the center of the aggregate was measured at the level of the maximum cross section of each aggregate.
  • a mixture of GFP (nHyC) and DsRed (nEpiC) was cultured for 48 hours under the conditions of no (-) or (+) DOX.
  • Ten aggregates under each condition were counted. The number of nuclei counted is shown above.
  • Aggregates of nHyC (G6) and nEpiC grew from day 0 to day 6. From the 0th day to the 6th day, the maximum cross-sectional area of each aggregate increases.
  • GFP + or GFP-cells are sorted by flow cytometry to identify genes associated with pluripotency and low germ layer. evaluated. Gene expression in cells selected from aggregates produced by Naive (WT) or Naive (WT) + Naive-GFP (GATA6) on days 2 (D2) and 4 (D4).
  • GFP + or GFP-cells are sorted by flow cytometry and are associated with pluripotency and subembryonic lobe. The gene was evaluated.
  • GATA6 Total
  • OTX2 are upregulated.
  • GATA6 GATA6 in human pre-implantation embryos. The dot plot image was acquired from Genome-wide Association and Primate Preimplantation Atlas17 (GRAPPA; https: //app.stemcells.cam.ac.uk/GRAPPA/). Expression in the hypoblast layer is about 20 times higher than that in the upper blastoderm layer (EPI). Expression of GATA6 in nHyC of aggregates.
  • GATA6 Total Aggregates produced by Naive (WT) + Naive-GFP (GATA6) were sorted on days 2, 4, and 6. The expression of GATA6 (Total) and GATA6 (endogenous: Endo) was measured by qPCR. Gene expression pattern in Naive (WT) and Naive-GFP (GATA6) or Naive (WT) only on day 2 (D2) and day 4 (D4). Cell aggregates were sorted into D2 and D4 by GFP.
  • Aggregates are naive pluripotent stem cells (Nive PSCs) or prime pluripotent stem cells (Primed PSCs) and gATA6, 7F or 4F-induced naive-derived PDGFRA + cells (G6- / 7F- / 4F-nHyC). ), Or ECAD + CXCR4 + germ layer cells in the embryo. All selected cells expressed GFP.
  • the aggregate On the 4th day after culturing the aggregate, the aggregate was fixed and stained with F-actin. The blastoderm cells were surrounded by all types of sorted cells, but aggregates with preamniotic cavities were formed only by G6- / 7F- / 4F-nHyC.
  • GFP GFP-
  • WT + G6 mixed aggregate of Naive (WT) and Naive-GFP (GATA6)
  • WT aggregate of only Naive (WT)
  • N naive pluripotent stem cell
  • P prime type Pluripotent stem cells.
  • GATA6 GFP-, WT + G6: mixed aggregate of Naive (WT) and Naive-GFP (GATA6)
  • WT aggregate of only Naive (WT)
  • N naive pluripotent stem cell
  • P prime type Pluripotent stem cells.
  • Gene expression pattern in Bilaminoid on day 6.
  • UMAP divided the cells into two different clusters: UMAP1-High and UMAP1-Low.
  • the gene expression levels of the subblastocyst and upper blastoderm markers are plotted on the UMAP.
  • the lower scutellum marker gene is expressed in UMAP1-Low
  • the upper scutellum marker gene is expressed in UMAP1-High.
  • the subblastocyst marker gene is expressed.
  • nEpiC was grouped as clusters (CL) 0-7 and nHyC was grouped as CL0-2. Relative expression of DEG between the upper blastoderm and amniotic membrane revealed by cultured human embryos.
  • Heatmaps show the expression of individual cells of CL0-7 in nEpiC.
  • CL5, 0, and 3 predominantly express the blastoderm upper layer gene, while CL1 and 4 express the amniotic membrane gene. Expression of upper blastoderm and amniotic membrane in each nEpiC cluster.
  • the expression level is shown by a violin plot. Series separation path constructed by Monocle2. The single cell projection pathway reveals pseudo-temporal differentiation of the upper blastoderm (CL5, 0) into the amniotic membrane (CL2, 1, 4) and primitive streak and gastrulatory cells (CL3, 6). Expression of primitive streak and cyst formation-related genes in each nEpiC cluster. The expression level is shown as a violin plot.
  • CD71 + CD235a + erythrocyte cells were detected in StemPro-34 containing EPO.
  • N 2. Expression level of integrin in each cluster of nEpiC. The expression level is shown by a violin plot. Expression of laminin in nHyC. The expression level is shown by a violin plot. Expression of the visceral endoderm gene in nHyC of Bilaminoid on day 6. Gene expression levels are plotted on UMAP-Low and shown as feature plots. Photograph of the front-back axis of Bilaminoid on the 6th day. Day 6 Bilaminoid produced by Naive (WT) and Naive (GATA6) was stained against GATA6 / PAR6, OTX2, and T (white). Scale bar, 20 ⁇ m.
  • Bilaminoid + TE Bilaminoid co-cultured with TE cultivated on the transwell.
  • Bilaminoid + TE Bilaminoid co-cultured with TE cultivated on the transwell.
  • Nive naive pluripotent stem cells
  • Primed prime pluripotent stem cells
  • BT + AG + Bilaminoid both BLIMP1 and TFAP2C positive cells recovered from Bilaminoid
  • BT + AG + Bilaminoid + TE co-cultured with TE cultured on transwell. Both BLIMP1 and TFAP2C positive cells recovered from Bilaminoid.
  • marker-positive cells refer to cells that express the marker to the extent that it can be detected by immune tissue staining using an antibody against the marker or quantitative PCR
  • marker-negative cells refer to the marker.
  • the method for producing the bilayer scutellum model of the present invention is: Cell clusters (cell aggregation) containing sublamellar (hypoblast) -like cells derived from naive mammalian pluripotent stem cells and mammalian pluripotent stem cells corresponding to epiblast-like cells. Includes the step of forming a mass).
  • the bilayered scutellum model of the present invention is a cell mass containing two cell layers, including a layer of subepithelial lobe-like cells derived from naive mammalian pluripotent stem cells and a layer of mammalian pluripotent stem cells.
  • the layer of mammalian pluripotent stem cells corresponds to the layer of upper scutellum-like cells.
  • a cell mass containing these two cell layers may be referred to as a bilayer scutellum-like structure.
  • the two-layered scutellum model of the present invention preferably comprises a layer of mammalian pluripotent stem cells (upper scutellum-like layer) corresponding to the upper scutellum-like cells and the upper scutellum-like layer. It is a bilayered scutellum-like structure containing a layer of subblastocyst-like cells derived from the lining naive mammalian pluripotent stem cells (subseroblastal sap-like layer).
  • the bilayered scutellum model of the present invention more preferably comprises a layer of induced pluripotent stem cells (iPS cells) and / or embryonic stem cells (ES cells) corresponding to the upper lamellar layer-like cells and the layer. It is a bilayered scutellum-like structure that contains a layer of subseroblast-like cells derived from iPS cells and / or ES cells, which is lined.
  • the size of the bilayer scutellum model is not particularly limited, but for example, the size of the cell mass is preferably 20 ⁇ m to 500 ⁇ m, and more preferably 50 ⁇ m to 300 ⁇ m.
  • the ratio of the subblastocyst-like cells and the upper scutellum-like cells is not particularly limited as long as each cell has a sufficient number to form a layer, but for example, It is 1: 1 to 10: 1.
  • the cell mass which is the bilayer scutellum model of the present invention, contains a layer of mammalian pluripotent stem cells corresponding to blastocyst upper layer-like cells on the inside, and a layer of subblastocyst-like cells surrounding the cell on the outside. It is preferably a bilayer scutellum-like structure having a spherical (including substantially spherical) bilayer structure. Further, the bilayer scutellum-like structure may be a cell mass that forms an amnion cavity-like cavity structure surrounded by a layer of scutellum lobule-like cells in the center thereof.
  • the upper scutellum-like layer and the lower scutellum-like layer are separated by a basement membrane.
  • the basement membrane is preferably an extracellular matrix containing laminin.
  • Laminin is preferably ⁇ 1.
  • the bilayer scutellum-like structure preferably has an anterior-posterior axis.
  • Having an anterior-posterior axis means the polarity of cells from the head side to the tail side, and having an anterior-posterior axis means, for example, anterior markers CER1 (Cerberus 1), DKK1 in a layer like the subblastocyst layer. (Dickkopf-1), LEFTY1, 2 (Left-Right Determination Factor 1, 2), OTX2 (Orthodenticle Homeobox 2) or more are expressed, and T, which is a posterior marker, is expressed in the upper blastocyst layer. It can be confirmed by expressing it. Further, it is preferable that the bilayer scutellum-like structure has an apical polarity indexed by the expression of PAR6.
  • the bilayer blastoderm-like structure may contain cells in the invagination stage of the archenteron in the layer of blastoderm-like cells.
  • Cells in the invagination stage are expressed by one or more of T, MIXL1 (Mix Paired-Like Homeobox), MESS1 (Mesoderm posterior protein 1), EVX1 (Even-Skipped Homeobox 1), SNAI2, and EOMEs (Eomesodermin). Be characterized.
  • the bilayer scutellum-like structure may contain amnion-like cells in the layer of scutellum lobe upper layer-like cells.
  • Amniotic membrane-like cells are characterized by the expression of one or more of GATA3, TFAP2A (Transcription Factor AP-2 Alpha), ISL1 (islet 1).
  • the bilayer scutellum-like structure of the present invention contains two or more types of cells having different genotype origins.
  • the genotypes of the sub-embryonic lobe-like cells and the supra-embryonic lobe-like cells may be different, and the naive pluripotent stem cells from which the sub-embryonic lobe-like cells are derived and the supra-embryonic lobe-like cells. It is possible to use pluripotent stem cells corresponding to those having different genotypes.
  • Upper germ layer-like cells have pluripotency capable of differentiating into endoderm, ectoderm and mesoderm, characterized by negative subgerm marker and OCT3 / 4 positive and NANOG positive. Be done. Preferably, it is characterized by PDGFRA (PDGF receptor ⁇ ) negative and OCT3 / 4 positive and NANOG positive.
  • PDGFRA PDGF receptor ⁇
  • pluripotent stem cells having the above-mentioned characteristics can be used as they are. Therefore, pluripotent stem cells correspond to blastoderm-like cells in the bilayer scutellum model.
  • the pluripotent stem cells corresponding to the upper lamellar layer-like cells may be naive-type pluripotent stem cells or prime-type pluripotent stem cells. It becomes a layer of upper layer-like cells.
  • the pluripotent stem cells will be described below.
  • the pluripotent stem cell is a stem cell having pluripotency capable of differentiating into many cells existing in a living body and also having a proliferative ability, and is induced into erythrogram upper layer-like cells. Any cell is included.
  • the term "pluripotent stem cell” in the present invention includes naive pluripotent stem cells and prime pluripotent stem cells.
  • the pluripotent stem cells are not particularly limited, and are, for example, embryonic stem (ES) cells, artificial pluripotent stem (iPS) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transplantation, and sperm.
  • GS cells stem cells
  • EG cells embryonic germ cells
  • fibroblasts pluripotent cells derived from bone marrow stem cells (Muse cells) and the like.
  • Preferred pluripotent stem cells are iPS cells and ES cells.
  • the pluripotent stem cells are preferably derived from mammals including primates and rodents, more preferably from primates, and even more preferably from humans.
  • the method for producing iPS cells is known in the art, and can be produced by introducing a reprogramming factor into any somatic cell or the like.
  • the reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15.
  • Genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, All4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. Is also good.
  • the combinations of initialization factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 /.
  • Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passed cells, and established cells are all included.
  • the somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) 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, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.) Etc.
  • differentiated cells such as brain cells, lung cells, kidney cells and fat cells are exemplified.
  • Naive-type pluripotent stem cells are pluripotent stem cells with properties similar to those of preimplantation embryos, but specifically, they have the following characteristics (Cytometry Research 27 (1): 19). ⁇ 24, 2017). It shows a dome-shaped colony morphology, and the size of the colony is smaller than that of the prime type. Express one or more of CD75, KLF4 and TFCP2L1 as markers. The genome is demethylated.
  • Naive pluripotent stem cells can be produced, for example, by the following methods. Method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014) 5 Using iLFA conditions (The unissen et al., Cell Stem Cell. 2016 Oct 6; 19 (4): 502-515.) Method using HDAC (histone deacetylase) inhibitor (Guo, G. et al. (2017).
  • Primed pluripotent stem cells are pluripotent stem cells with properties similar to those of post-embryo embryonic pelvic lobe superior cells, but are commonly artificial obtained by introducing reprogramming factors into somatic cells. Pluripotent stem cells and human ES cells fall under this category and have not been subjected to the above-mentioned naive treatment. Prime-type pluripotent stem cells have the following characteristics. It shows a flat colony morphology, and the size of the colony is larger than that of the naive type. As markers, CD75, KLF4 and TFCP2L1 are negative. The genome is methylated.
  • Primitive streak-related genes eg, T, MIXL1, MESS1, EVX1, SNAI2, EOMES, etc.
  • T, MIXL1, MESS1, EVX1, SNAI2, EOMES, etc. eg., T, MIXL1, MESS1, EVX1, SNAI2, EOMES, etc.
  • the upper blastoderm-like cell layer in the bilayered scutellum model of the present invention may contain cells in the invagination stage of the archenteron and / or amniotic membrane-like cells.
  • Subepithelial layer-like cells are classified as extraembryonic cells, express extracellular matrix, and support cells in the upper lamellar layer, 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) It is characterized by the expression of one or more subplatelet markers such as cysteine), CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1), ANPEP (alanyl aminopeptidase, membrane), preferably at least PDGFRA, CEACAM1 and ANPEP. One is positive.
  • Subblastocyst cells are derived from naive pluripotent stem cells, that is, cells induced to differentiate from naive pluripotent stem cells.
  • naive pluripotent stem cells that is, cells induced to differentiate from naive pluripotent stem cells.
  • the method for inducing the differentiation of naive pluripotent stem cells into subseroblast-like cells is not particularly limited, and any method including known methods can be used.
  • any method including known methods can be used.
  • Disclosed methods for forcibly expressing GATA family transcription factors (GATA6 and GATA4) in naive pluripotent stem cells, and BMPR1A (BMP receptor 1A) / ACVR (Actibin) as type 1 receptors for BMP signals A method of inducing by activating a signal via the receptor) and an FGFR2 (FGF receptor 2) signal (eg, WO 2019/093340) is preferably used.
  • GATA family transcription factor induction by forced expression of GATA family transcription factor
  • the method for forcibly expressing the GATA family transcription factor in naive pluripotent stem cells may be any method that increases the amount of GATA family transcription factor expressed in naive pluripotent stem cells, and is not particularly limited, but is preferable.
  • This is a method in which a possible vector is introduced into naive pluripotent stem cells and forcibly expressed by adding a stimulus such as a drug.
  • agents include doxycycline (DOX), and vectors capable of inducing forced expression by such agents include, for example, vectors containing rtTA expression constructs, but are limited thereto. It's not a thing.
  • the signal via BMPR1A / ACVR is a signal from activation of BMPR1A / ACVR to phosphorylation of SMAD1 / 5/9, and is a signal cascade activated by BMP2, BMP4, BMP6 or BMP7.
  • the signal cascade may be activated by using, for example, one or more of a group of cytokines composed of BMP2, BMP4, BMP6 and BMP7, and any compound acting on any of the signal cascades. It may be activated by use, or a combination thereof, but is not limited to these, and any method capable of activating a signal via BMPR1A / ACVR (a signal for phosphorylating SMAD1 / 5/9) is used. Be done.
  • the FGFR2 signal is a signal cascade that begins with the activation of FGFR2 and is a signal cascade that is activated by FGF4.
  • the signal cascade may be activated, for example, by FGF4, may be activated by using any compound that acts on any of the signal cascades, or may be a combination thereof, but is not limited thereto. , Any technique capable of activating the FGFR2 signal is used.
  • the WNT / ⁇ -catenin signal and / or the ALK4 / 5/7 signal (the signal that phosphates SMAD2) is further suppressed in naive pluripotent stem cells. Suppresses WNT / ⁇ -catenin signal, suppresses ALK4 / 5/7 signal (signal that phosphates SMAD2), activates STAT3 signal, activates PDGF-AA signal and / or retinoic acid It is more preferable to activate the signal.
  • Suppression of the WNT / ⁇ -catenin signal may be achieved, for example, by using a compound that acts on and suppresses the WNT / ⁇ -catenin signal cascade, such as XAV939, IWP-1, Examples include, but are not limited to, IWP-2, IWP-3, IWP-4, IWR-1, 53AH, KY02111 (available from Abcam, Sigma-aldrich, etc.), but suppress WNT / ⁇ -catenin signaling. Any method that can be used is used.
  • Suppression of the ALK4 / 5/7 signal may be achieved, for example, by using a compound that acts on and suppresses the ALK4 / 5/7 signal, as such a compound.
  • a compound that acts on and suppresses the ALK4 / 5/7 signal may be achieved, for example, by using a compound that acts on and suppresses the ALK4 / 5/7 signal, as such a compound.
  • A83-01 WO2009146408)
  • SB431542 SB202190 (R.K.Lindemann et al., Mol. Cancer 2:20 (2003)), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761
  • Examples include, but are not limited to, LY364947, LY580276 (Lilly Research Laboratories), and any method that can suppress the ALK4 / 5/7 signal is used.
  • Activation of the STAT3 signal may be achieved, for example, by IL-6 or by using a compound that acts and activates the STAT3 signal, but is not limited to activating the STAT3 signal. Any method that can be converted is used.
  • Activation of the PDGF-AA signal may be achieved, for example, by PDGF-AA or by using a compound that acts and activates the PDGF-AA signal, but is not limited to this. Any technique that can activate the PDGF-AA signal is used.
  • Activation of the retinoic acid signal may be achieved, for example, by a retinoic acid (RA), a retinoic acid receptor (RAR) agonist, or a retinoid X receptor (RXR), which acts on and activates the retinoic acid signal. It may be achieved by using a compound, but is not limited to these, and any method capable of activating the retinoic acid signal is used.
  • RA retinoic acid
  • RAR retinoic acid receptor
  • RXR retinoid X receptor
  • a naive pluripotent stem cell maintenance medium such as t2iLGo, 5iL / AF, tt2iLGo, Ndiff227, etc., to which factors necessary for inducing differentiation can be added can be used. It is preferable to use a serum-free medium, but in the case of induction by overexpression of the GATA6 gene, a serum medium may be used.
  • the culture may be suspension culture or adhesive culture, but the adhesive culture is a culture vessel coated with extracellular matrix such as polylysine, polyornitine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, and laminin. It can be done by culturing using.
  • extracellular matrix such as polylysine, polyornitine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, and laminin. It can be done by culturing using.
  • the culture temperature conditions for culturing naive pluripotent stem cells in the step of inducing subblastocyst-like cell differentiation are not particularly limited, but are, for example, about 37 ° C to about 42 ° C and about 37 to about 39 ° C. Is preferable.
  • the number of culture days is not particularly limited as long as subblastocyst-like cells can be obtained, but is, for example, at least 1 day or more, preferably 2 to 5 days.
  • ⁇ Manufacturing method of bilayer blastocyst model of the present invention (a) subserosal lobe-like cells are induced from naive mammalian pluripotent stem cells, and the obtained subserosmal lamellae-like cells are used as mammalian pluripotent cells. It may be co-cultured with sexual stem cells by suspension culture, or (b) co-cultured naive mammalian pluripotent stem cells with mammalian pluripotent stem cells by suspension culture, and scutellate from the naive mammalian pluripotent stem cells. Subleaflet-like cells may be induced.
  • step (a) the sub-embryonic lobe-like cells induced to differentiate from naive pluripotent stem cells and the pluripotent stem cells corresponding to the supra-embryonic lobe-like cells are mixed and co-cultured by suspension culture.
  • the mixing ratio of subblastocyst-like cells and pluripotent stem cells is, for example, 1: 1 to 1:10.
  • each cell forms a layer, and includes a layer like the upper lamellar layer due to the pluripotent stem cells and a layer like the lower lamellar layer derived from the sublayer-like cells that support it. Layered aggregates are formed.
  • the naive-type pluripotent stem cells for differentiating into sub-embryonic lobe-like cells and the pluripotent stem cells corresponding to the supra-embryonic lobe-like cells are mixed and co-cultured, and the naive-type polypluripotent cells are co-cultured. It specifically induces subembryonic lobe-like cells from pluripotent stem cells.
  • the mixing ratio of naive pluripotent stem cells and pluripotent stem cells for differentiating into subblastocyst-like cells is, for example, 1: 1 to 1:10.
  • naive pluripotent stem cells into which a vector capable of inducibly forcibly expressing the GATA family transcription factor by stimulation with a drug or the like and pluripotent stem cells corresponding to the upper lamellar layer-like cells are introduced.
  • Examples thereof include a step of forcibly expressing the GATA family transcription factor by mixing and co-culturing the cells and adding a stimulus such as the above-mentioned drug to induce the naive pluripotent stem cells to induce sublamellar lobe-like cells.
  • inducible GATA family transcription factor-expressing naive pluripotent stem cells and pluripotent stem cells corresponding to the upper lamellar lobe-like cells are mixed and co-cultured, aggregates are formed within 24 hours. Approximately 4 days after the start of induction of sublayer-like cells, each cell forms a layer, and the pluripotent stem cells form an upper layer-like layer and the sublayer-like cells that support it. A bilayer aggregate containing a layer of (cells induced to differentiate from inducible GATA family transcription factor-expressing naive pluripotent stem cells) is formed. The co-culture is performed by suspension culture in order to efficiently form aggregates, but after the aggregates are formed, they may be adhered and the culture may be continued.
  • the medium used for producing the bilayer scutellum model is preferably a serum-free medium, but is not particularly limited, and a known medium such as a medium preferably used for the differentiation of pluripotent stem cells may be used. It is preferable that the medium used for producing the bilayer scutellum model does not contain undifferentiated maintenance factors such as bFGF that maintain the undifferentiated state of pluripotent stem cells.
  • the culture temperature conditions in the method for producing the bilayer scutellum model of the present invention are not particularly limited, but are preferably about 37 ° C. to about 42 ° C. and about 37 ° C. to about 39 ° C., for example.
  • the number of culture days of the bilayer scutellum model is not particularly limited and may be changed depending on the purpose, but preferably, the culture in which the subblastocyst-like cells and the pluripotent stem cells are mixed is performed for 2 days or more, 4 days or more. Continue for more than a day, or more than 6 days.
  • the upper limit is not particularly limited, but is preferably 30 days or less, 20 days or less, or 14 days or less.
  • step (a) or step (b) above the bilayer scutellum model matures, forming an amniotic cavity-like cavity structure in the inner blastoderm-like layer, and further, the archenteron. Invaginated cells and amnion-like cells will appear.
  • steps (maturation promoting steps) it is more preferable to include the following steps (maturation promoting steps).
  • the method for producing the bilayer blastocyst model of the present invention is a step of co-culturing the above-mentioned cell mass and / or the above-mentioned bilayer blastocyst-like structure with IL-6 secreting cells, or IL-. 6.
  • the step of culturing in the presence of at least one selected from the group consisting of Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF may be included.
  • the IL-6 secreting cells used in the step of co-culturing with IL-6 secreting cells are not particularly limited as long as they are cells that secrete IL-6 extracellularly, but are preferably trophectderm cells.
  • the vegetative ectoderm cell may be a cell derived from the vegetative ectoderm of the embryo, and may be, for example, a vegetative ectoderm cell whose differentiation is induced by the method described in WO2021 / 106765, but is not particularly limited. It is more preferred that the vegetative ectoderm cells further secrete Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF in addition to IL-6.
  • the cell mass and / or the bilayer blastocyst-like structure When the cell mass and / or the bilayer blastocyst-like structure is co-cultured with IL-6 secreting cells, it inhibits contact between the two cells and secretes from one cell tumor to the other. It is preferable to use a partition that does not hinder the movement of substances, and examples of such a partition include a permeable film. Examples of the culture method using a partition include a transwell in which a cell culture container is divided into upper and lower parts and a permeable membrane is inserted between them. For other culture conditions, the culture conditions in the above-mentioned method for producing a bilayer scutellum model can be used.
  • 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 specifically comprises the above cell mass and / or the above bilayered structure.
  • the amniotic structure is cultured in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF to form the cell mass and / or the bilayered amniotic structure. It may be a step of forming an amniotic cavity-like cavity structure.
  • Culturing in the presence of at least one selected from the group consisting of IL-6, Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF can be carried out, for example, from the group consisting of IL6, Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF in the medium. It may be achieved by adding at least one selected cytokine, and co-cultured with cells secreting at least one factor selected from the group consisting of IL6, Wnt6, Wnt7A, Wnt7B, Wnt11 and VEGF. It may be achieved by doing. In the case of co-culturing, it is preferable to use the above-mentioned partition. Concentrations of these cytokines are, for example, 5 ng / ml to 100 ng / ml.
  • the culture conditions in the above-mentioned method for producing a bilayer scutellum model can be used.
  • a third aspect of the present invention is a method for producing primordial germ cells, which is a mammalian pluripotency corresponding to sub-embryonic lobe-like cells derived from naive mammalian pluripotent stem cells and supra-embryonic lobe-like cells.
  • the present invention relates to a method comprising a step of forming a cell mass containing sexual stem cells, and comprising a step of culturing the cell mass for 10 days or longer, preferably 12 days or longer. There is no upper limit to the culture period, but it is, for example, 30, 40, or 50 days. That is, primordial germ cells can be obtained by further culturing the cell mass obtained as described above. For example, primordial germ cells can be obtained by performing the above-mentioned steps (a) and (b) and further the above-mentioned maturation promoting step and continuing the culture for a long time.
  • Primordial germ cells have 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.
  • primordial germ cells By culturing the cell mass for 10 days or more, cells differentiated into primordial germ cells can be obtained.
  • the culture conditions in the above-mentioned bilayer scutellum model and the method for producing the same can be used.
  • Recovery of primordial germ cells is not particularly limited as long as it is a method capable of recovering primordial germ cells from a cell population, but for example, cells expressing BLIMP1 (B-lymphocyte-induced maturation protein 1) and TFAP2C are fluorescently labeled and then labeled. Examples thereof include a method of recovery by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Human iPS cells (Takashima et al., Cell 158, 1254-1269, 2014) generated from human ES cell lines H1 and H9 (WiCell Research Institute, Wyoming, USA) and adult fat-induced stem cells (AdiPSC) are mouse embryonic fibers. Cultured on blast cells (MEF) (1 x 106 cells per 6-well plate). Prime-type human pluripotent stem cells are 20% knockout serum replacement (KSR; Thermo Fisher Scientific), 1% non-essential amino acid (Thermo Fisher Scientific), 4 ng / mL recombinant human basic fibroblast growth factor 2 (bFGF;).
  • KSR knockout serum replacement
  • Thermo Fisher Scientific Thermo Fisher Scientific
  • bFGF basic fibroblast growth factor 2
  • Naive human pluripotent stem cells include 1 ⁇ M PD0325901 (PD03; Tocris), 1 ⁇ M CHIR99021 (CH; Sigma-Aldrich), 10 ng / mL recombinant human LIF (hLIF; Peprotech), and 3 ⁇ M Go6983 (Go; Tocris). It was maintained in t2iLGo medium consisting of N2B27 (Ndiff227; Takara Bio Inc.). Naive pluripotent stem cells were passaged every 3-5 days using Accutase (Sigma-Aldrich).
  • naive pluripotent stem cells Chemical conversion to naive pluripotent stem cells is described in Guo et al. , 2017. Prime-type pluripotent stem cells (1 ⁇ 10 4 cells / cm 2 ) were seeded on MEF feeder cells under prime-type pluripotent stem cell medium containing 10 ⁇ M Y-27632. The next day, the medium was replaced with cRM-1 (N2B27, 1 ⁇ M PD03, 10 ng / mL hLIF, and 1 mM sodium valproate salt (VPA; Sigma-Aldrich)).
  • cRM-1 N2B27, 1 ⁇ M PD03, 10 ng / mL hLIF, and 1 mM sodium valproate salt (VPA; Sigma-Aldrich)
  • naive pluripotent stem cells were passaged and maintained on a MEF feeder under t2iLGo medium.
  • the chemical conversion to naive pluripotent stem cells using 5iLA was performed as follows. Prime-type pluripotent stem cells ( 2 ⁇ 105 cells / cm 2) were seeded on MEF feeder cells under prime-type pluripotent stem cell medium containing 10 ⁇ M Y-27632.
  • 5iLA medium N2B27 + 1 ⁇ M PD03, 1 ⁇ M CH, 1 ⁇ M WH-4-023 (H620061, Morrisville, NC, USA), 0.5 ⁇ M SB590885 (R & D Systems), 10 ⁇ M Y-27632, 10ng / mL hLIF, and 20ng / mL.
  • the medium was replaced with A (R & D Systems).
  • the cells were maintained on MEF feeder cells and under t2iLGo medium.
  • Mouse ES cells were cultured on gelatin-coated dishes in 2iL (N2B27, 1 ⁇ M PD03, 3 ⁇ M CH, and 10 ng / mL hLIF), and cells were subcultured every 2 to 3 days using Accutase.
  • GATA6, GATA4, and SOX17 were cloned into DOX-inducible PB vectors bound to the rtTA expression construct (KW110).
  • the PB-GATA6 vector (2 ⁇ g), PB-GATA4 vector (2 ⁇ g), or PB-SOX17 vector (2 ⁇ g) and pBase helper plasmid (2 ⁇ g) were programmed into the Neon transfection system (naive pluripotent stem cells 20; Prime-type pluripotent stem cells were transfected into naive or prime-type pluripotent stem cells using program 14).
  • G418 was added for about 2 weeks (200 ⁇ g / mL).
  • Naive or prime-type pluripotent stem cells containing inducible GATA6, GATA4, or SOX17 were maintained in naive or prime medium.
  • MEF feeder cells were isolated at the single cell level and then removed by incubation on a gelatin-coated dish. Then, 1 ⁇ 10 5 cells / cm 2 was seeded in a dish coated with fibronectin (Millipore) or iMatrix-511 silk (laminin 511-E8) (Matrixome).
  • Serum media were GMEM (Sigma-Aldrich), FBS (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific, 0.1 from Thermo Fisher Scientific). .. Except for these experiments, all other analyzes were performed under serum-free conditions.
  • N2B27 medium Ndiff227; Catalog No. Y40002, Takara Bio
  • the components of N2B27 medium were DMEM / F12, Neurobasal, N2, and B27.
  • BSA is included in N2 and B27. 25 ng / ml recombinant human FGF4 (FGF4) and 1 ⁇ g / mL heparin sodium (Wako Pure Chemical Industries, Ltd.) were added to the basal medium. The medium was changed daily.
  • naive pluripotent stem cells were seeded on laminin 511-E8 in N2B27 medium.
  • Day 0 6 factors, 25 ng / mL FGF4 (+ 1 ⁇ g / mL heparin sodium), 10 ng / mL recombinant human BMP4 (BMP4; R & D), 10 ng / mL recombinant human PDGF-AA (PDGF AA, Peprotech), 1 ⁇ M XAV939 , 3 ⁇ M A83-01 (Tocris), and 0.1 ⁇ M RA (Sigma-Aldrich) were added.
  • the medium was replaced with 7 factors (6 factors and 10 ng / mL recombinant human IL-6) (IL-6; Oriental Yeast Co., Ltd.).
  • 7 factors and 10 ng / mL recombinant human IL-6) IL-6; Oriental Yeast Co., Ltd.
  • 500 ng / mL recombinant human BMP2 (BMP2; Oriental Yeast Co., Ltd.) or 50 ng / mL recombinant human BMP6 (BMP6; Peprotech) was used in place of BMP4.
  • N2B27 medium containing no VitA was prepared in-house.
  • mice ESC was maintained under 2iL conditions.
  • RPMI1640 Thermo Fisher Scientific
  • 20 ng / mL activin A 3 ⁇ M CH 10 ng / mL
  • hLIF hLIF.
  • 5 ⁇ 10 4 / cm 2 mouse ESCs were seeded on gelatin under 10 nM RA and 20 ng / mL activin A.
  • Medium was changed daily under both conditions.
  • the zona pellucida was removed with acidic tie load solution (Sigma) and the embryos were subjected to immunosurgery with a custom rabbit polyclonal anti-marmoset antibody.
  • ICM was seeded and fixed on laminin 511-E8 under N2B27 + 7 factor (7F), 4 factor (4F) (FGF4, BMP4, A83, XAV) or control (PD03, LDN, A83, XAV) for 3 days. And analyzed with SOX17 antibody.
  • naive pluripotent stem cells naive, WT 10 cells and naive pluripotent stem cells expressing GATA6 under DOX treatment or GFP-expressing naive pluripotent stem cells (naive (GATA6) or 40 cells of Naive GFP (GATA6)) were seeded in each well of a microarray or Elplasia plate (Corning, Inc.) under t2iLGo + 10 ⁇ M Y27632 containing neither Matrix nor Geltrex. After 24-36 hours of aggregation (day 0), the medium was replaced with N2B27 containing 0.1 ⁇ M DOX. On the second day, DOX was removed.
  • Aggregates produced by human pluripotent stem cells and sorted cells 100 cells of naive or prime human pluripotent stem cells and 100 sorted 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 embryonic follicle cells) were seeded in each well of the Elplasia plate under N2B27 + 10 ⁇ M Y27632. The medium was changed every other day. Aggregates were evaluated on day 4.
  • GFP naive 7F-PDFRA + cells, 4F-PDFRA + cells, GATA6 ( G6) -PDFRA + cells, prime G6-PDGFRA + cells, PDGFRA + RACL cells, and CXCR4 + / CDH1 + embryonic embryonic follicle cells
  • bilayer scutellum model Excess of GATA6 by wild-type naive human pluripotent stem cells (10 cells / microwell) and DOX addition for formation.
  • Naive human pluripotent stem cells 40 cells / microwell capable of inducing expression on Elplasia plate (Corning) t2iLGo (Ndiff227 (Takara Bio), 1 ⁇ M PD0325901 (PD03; Tocris), 1 ⁇ M CHIR99021 (CH; Sig) ), 10 ng / ml Recombinant human LIF (hLIF; Proprotech), 3 ⁇ M Go6983 (Go; Tocris,)) + 10 ⁇ M Y-27632 (Wako) medium. Cell masses were formed 24-36 hours after seeding.
  • the medium was exchanged to N2B27 + DOX (0.1 ⁇ g / ml), and co-culture with vegetative ectoderm cultivated on Corning was started. DOX was removed 48 hours after the start of co-culture, and culture was continued with N2B27. The medium was changed daily.
  • naive human pluripotent stem cells The vegetative ectoderm was induced to differentiate from naive human pluripotent stem cells (Io et al., Cell stem cell 3; 28 (6): 1023-1039.e13.2021). After removing the MEF, naive human pluripotent stem cells were seeded at 0.5 ⁇ 10 4 cells / cm 2 onto an iMatrix-511 silk (MAX) -coated transwell and N2B27 + 2 ⁇ M A83-01 (Tocris) + 2 ⁇ M. Culture was started in PD03 + 10 ng / ml recombinant human BMP-4 (BMP-4; R & D) medium.
  • BMP-4 human BMP-4
  • the medium is replaced with N2B27 + 2 ⁇ M A83-01 + 2 ⁇ M PD03 + 1 ⁇ M JAK inhibitor1 (JAKi; MERCK) and cultured for another 48 hours.
  • JAKi 1 ⁇ M JAK inhibitor1
  • Flow Cytometry and Cell Sorting Cells are separated into single cells by Accutase or trypsin, washed and HBSS containing 1% BSA (Catalog No. A2153, Sigma-Aldrich) on ice for 30 minutes (Catalog No. 14185052, Thermo Fisher Scientific). Shut off inside. Staining on ice with the following: biotinylated PDGFRA antibody (catalog number BAF322, R & D) in HBSS containing 1% BSA for 30 minutes, CEACAM1 + CEACAM5 antibody (catalog number ab91213, Abcam, Cambridge, UK), PE binding. Anti-CD13 (Catalog No.
  • Immunostained cells were fixed in 4% paraformaldehyde (catalog number 09154-85, Nacalai Tesque) for 10 minutes at room temperature. 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 at 4 ° C. overnight. The nuclei were stained with DAPI. Fluorescent images were obtained using a confocal laser scanning microscope TCS SP8 (Leica, Wetzlar, Germany) or LSM710 (Zeiss, Oberkochen, Germany).
  • RNA-seq analysis For RNA-seq, the MEF was removed by gelatin treatment and then samples were collected. RNA was purified using the miRNeasy minikit (Catalog No. 217004, Qiagen) and 200 ng of RNA was lived using the TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-2101, Illumina, San Diego, CA, USA). Used for rally construction. The RNA-seq library was sequenced with the NextSeq 500 HighUput v2 kit (75 cycles, FC-404-2005) (Illumina). The sequenced readings were trimmed with cutapt-1.15 to remove low quality bases and adapter sequences.
  • Trimmed readings were mapped to the human reference genome (hg38) using TopHat2 with GENCODE v2760. Uniquely mapped readings (MAPQ ⁇ 20) were used for further analysis. Each gene expression level was calculated using cufflink-2.2.1 as a reading per kilobase per million mapped readings (FPKM). 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 median or mean or specific conditions for all datasets. Heat map creation, correlation analysis, hierarchical clustering analysis, and PCA were performed using R (v3.3.2).
  • scRNA-seq and data analysis were produced by co-culture with naive (WT) and naive GFP (GATA6). Vilaminoids surrounded by GFP + cells were manually picked up and isolated by Accutase. Single cells were harvested by flow cytometry and loaded into a 10 ⁇ Genomics Chromium system. 10 ⁇ Genomics v. A 3.0 library was made according to the manufacturer's instructions. The library was then sequenced on Illumina HiSeq 4000 with paired end sequencing. The scRNA-seq data was mapped and quantified using the Cell Ranger pipeline (v. 4.0.0, 10 ⁇ Genomics) against the hg38 human reference genome + reporter sequence (EGFP).
  • WT naive
  • GATA6 naive GFP
  • the subblastocyst is a naive type that expresses GATA6 and GFP under DOX treatment and Naive human pluripotent stem cells (Naive (WT)) that control the development of the upper blastocyst before and after implantation.
  • a mixture of human pluripotent stem cells (Nive-GFP (GATA6)) was cultured on a microwell array (Fig. 1).
  • GFP or DsRed was introduced into naive PSCs (naive GFP and naive DsRed, respectively) to label aggregated cells.
  • Naive-GFP (GATA6) alone was unable to produce cellular aggregates.
  • nHyC nHyC
  • nEpiC naive pluripotent stem cell-derived blastoderm suprastric-like cells
  • nHyC G6
  • nEpiC decreased.
  • Previous reports of human embryos indicate that the number of cells in the upper lamellar foliage increases from about 20-40 on the 7th to 8th day of embryonic period to about 80-100 on the 10th day of the embryonic period, and the number of cells in the lower lamellar layer is embryonic. It has been suggested to increase from about 20-50 on the 8th day of the embryo to about 60-90 on the 10th day of the embryonic period (Deglincerti, A. et al. Nature 533, 251-254 (2016), Road, M. et. al. Developmental Biologic 361, 358-363 (2012), Di Stephano, B.
  • nHyC (G6) and nEpiC cells in the aggregate also proliferated during differentiation and corresponded to human embryo data (FIG. 7).
  • the size of the aggregates also increased during culture (Fig. 8).
  • nHyC (G6) (GFP + ) and nEpiC (GFP-) cells were screened by flow cytometry to assess gene expression patterns on days 2 and 4 (FIG. 9).
  • the authors checked GATA6 expression after DOX treatment.
  • the expression of GATA6 (total) in nHyC (G6) on day 2 after DOX treatment was increased about 30-fold higher than that in nEpiC on day 2 (FIG. 10).
  • Published scRNA-seq data showed that GATA6 expression in the subblastocyst layer during the blastocyst stage was approximately 20-fold higher than that in the upper blastocyst layer (FIG. 11).
  • nHyC G6
  • nEpiC showed a decrease in NANOG, KLF17
  • naive or upper scutellum gene such as OCT3 / 4 (FIGS. 10 and 12). Therefore, it was concluded that the two cell types were sorted by the marker and expressed the marker, similar to the phenomenon reported in late human blastocysts.
  • GATA6 continued to be expressed in nHyC even after DOX was removed on day 2 (FIG. 12).
  • nEpiC showed decreased expression of the naive pluripotent gene KLF17 and increased expression of the early post-implantation blastocyst upper layer genes THY1, DNMT3B, and SFRP2 ( Figure).
  • nEpiC The acquired polarity of nEpiC gradually forms a rosette-like structure on the 4th day and is called Bilaminoid.
  • PODXL and aPKC co-existed with PAR6 in the Bilaminoid (FIG. 14).
  • naive pluripotent stem cells and naive-derived PDGFRA + cells (G6-nHyC, 7F-nHyC or 4F-nHyC, respectively) induced and sorted by GATA6, 7F, or 4F on laminin 511-E8.
  • the produced aggregates were also analyzed (Fig. 15).
  • WT Naive
  • GATA6 Naive-GFP
  • the mixture of naive pluripotent stem cells and 7F-nHyC, 4F-nHyC, or G6-nHyC has a rosette-like structure.
  • a mixture of prime-type pluripotent stem cells and 7F-nHyC, 4F-nHyC, or G6-nHyC was also able to produce aggregates with rosette structures (FIG. 15).
  • Bilaminoid on day 6 was further analyzed using scRNA-seq. Characteristic plots of subblastocyst and upper blastoderm genes showed that nHyC and nEpiC were differentially clustered by uniform manifold approximation and projection (UMAP) (FIG. 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). did.
  • UMAP uniform manifold approximation and projection
  • the blastocyst superior layer differentiates into the post-implantation blastoderm upper layer and amniotic membrane in both in vivo and in vitro models.
  • Bilaminoid may contain cells expressing the amniotic membrane gene
  • the gene expression pattern of Bilaminoid using the expression variation gene (DEG) between the upper layer of the pelvis and the amniotic membrane established from human embryos cultured in vitro. was analyzed.
  • nEpiC was arranged in pseudo time using Monocle2. Flight orbit analysis suggested that nEpiC in clusters 5 and 0 differentiated into clusters 2, 1, and 4, and clusters 3 and 6, respectively (FIG. 23). Clusters 6 and 3 expressed primitive streak and gastulation-related genes (FIG. 24).
  • the heat map of the marker genes at the developmental stage of the blastocyst upper layer (ICM, early blastocyst upper layer, late blastocyst upper layer, and primitive streak) in human embryos cultured in vitro is from the early blastocyst upper layer to the primitive streak.
  • ICM early blastocyst upper layer
  • late blastocyst upper layer and primitive streak
  • nEpiC Two pairs of heterodimers, ITGA6 and ITGB1 ( ⁇ 6 ⁇ 1 ) and ITGAV and ITGB 5 ( ⁇ v ⁇ 5 ), were expressed in nEpiC.
  • Ligands for integrin ⁇ 6 ⁇ 1 and ⁇ v ⁇ 5 are known as laminin and vitronectin, respectively.
  • the expression of laminin and vitronectin in nHyC (G6) was checked.
  • VTN was weakly expressed, but LAMA1, LAMB1, and LAMC1 were strongly expressed in nHyC (G6) (FIG. 29). Since Lamb1 is required for the formation of the basement membrane between the upper and subblastocyst layers of early mouse embryos, it was hypothesized that LAMB1 could be the major ECM component for human interaction.
  • TE trophic ectoderm
  • the bilayer scutellum model is preferably co-cultured with TE. Furthermore, since this effect was obtained by co-culture with Transwell, it is speculated that it may be due to the factor secreted from TE. Therefore, the factors secreted from TE were examined. As a result, IL-6 added to the medium promoted amnion cavity formation in the bilayer scutellum model, and the JAK inhibitor (JAKi) inhibited the increase in amnion cavity formation rate by co-culturing with TE. (Fig. 34). However, the addition of IL-6 did not promote the emergence of cells exhibiting amniotic epithelial-like morphology. From these results, it was found that the effect of co-culture with TE was partly due to the secretion of IL-6.
  • PGC primordial germ cell
  • the PGC reporter series (BLIMP1-tdTomato, TFAP2C-EGFP) was used for the analysis.
  • FACS FACS-activated cell sorting

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de production d'un modèle de blastocystis bilaminaris mammifère, comprenant une étape de formation d'une masse cellulaire contenant une cellule de type hypoblaste dérivée d'une cellule souche pluripotente de mammifère naïve et une cellule souche pluripotente de mammifère correspondant à une cellule de type épiblaste.
PCT/JP2021/043655 2020-11-30 2021-11-29 Modèle de blastocystis bilaminaris et procédé de production de celui-ci WO2022114188A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022565490A JPWO2022114188A1 (fr) 2020-11-30 2021-11-29

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063119482P 2020-11-30 2020-11-30
US63/119,482 2020-11-30

Publications (1)

Publication Number Publication Date
WO2022114188A1 true WO2022114188A1 (fr) 2022-06-02

Family

ID=81754501

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/043655 WO2022114188A1 (fr) 2020-11-30 2021-11-29 Modèle de blastocystis bilaminaris et procédé de production de celui-ci

Country Status (2)

Country Link
JP (1) JPWO2022114188A1 (fr)
WO (1) WO2022114188A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016508365A (ja) * 2013-03-01 2016-03-22 国立大学法人京都大学 多能性幹細胞から生殖細胞への分化誘導方法
JP2016518831A (ja) * 2013-04-16 2016-06-30 ユニフェルシテイト マーストリヒトＵｎｉｖｅｒｓｉｔｅｉｔ Ｍａａｓｔｒｉｃｈｔ ブラストイド、細胞株に基づく人工胚盤胞
JP2016521971A (ja) * 2013-04-23 2016-07-28 イェダ リサーチ アンド ディベロップメント カンパニー リミテッド 単離ナイーブ型多能性幹細胞およびそれを発生させる方法関連出願本出願は、米国特許法１１９条第（ｅ）項に基づき、２０１４年１月２９日出願の米国特許仮出願第６１／９３２，９３５号、２０１３年９月１７日出願の米国特許仮出願第６１／８７８，７６９号、および２０１３年４月２３日出願の米国特許仮出願第６１／８１４，９２０号の優先権を主張する。また、本出願は、同時に提出された同出願人による同時係属出願である、ＹａｑｕｂＨＡＮＮＡ、ＮｏａＮＯＶＥＲＳＨＴＥＲＮ、およびＹｏａｃｈＲＡＩＳによる米国特許出願（発明の名称「単離ナイーブ型多能性幹細胞およびそれを発生させる方法（ＩＳＯＬＡＴＥＤＮＡＩＶＥＰＬＵＲＩＰＯＴＥＮＴＳＴＥＭＣＥＬＬＳＡＮＤＭＥＴＨＯＤＳＯＦＧＥＮＥＲＡＴＩＮＧＳＡＭＥ）」）（代理人事件記録簿第５８８７０号）にも関する。上記出願の内容はその全体を参考として本明細書に組み込む。
WO2019093340A1 (fr) * 2017-11-07 2019-05-16 国立大学法人京都大学 Procédé d'induction d'endoderme primitif à partir de cellules souches pluripotentes naïves
WO2021106765A1 (fr) * 2019-11-25 2021-06-03 国立大学法人京都大学 Procédé d'induction de trophectoderme à partir de cellules souches pluripotentes naïves

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016508365A (ja) * 2013-03-01 2016-03-22 国立大学法人京都大学 多能性幹細胞から生殖細胞への分化誘導方法
JP2016518831A (ja) * 2013-04-16 2016-06-30 ユニフェルシテイト マーストリヒトＵｎｉｖｅｒｓｉｔｅｉｔ Ｍａａｓｔｒｉｃｈｔ ブラストイド、細胞株に基づく人工胚盤胞
JP2016521971A (ja) * 2013-04-23 2016-07-28 イェダ リサーチ アンド ディベロップメント カンパニー リミテッド 単離ナイーブ型多能性幹細胞およびそれを発生させる方法関連出願本出願は、米国特許法１１９条第（ｅ）項に基づき、２０１４年１月２９日出願の米国特許仮出願第６１／９３２，９３５号、２０１３年９月１７日出願の米国特許仮出願第６１／８７８，７６９号、および２０１３年４月２３日出願の米国特許仮出願第６１／８１４，９２０号の優先権を主張する。また、本出願は、同時に提出された同出願人による同時係属出願である、ＹａｑｕｂＨＡＮＮＡ、ＮｏａＮＯＶＥＲＳＨＴＥＲＮ、およびＹｏａｃｈＲＡＩＳによる米国特許出願（発明の名称「単離ナイーブ型多能性幹細胞およびそれを発生させる方法（ＩＳＯＬＡＴＥＤＮＡＩＶＥＰＬＵＲＩＰＯＴＥＮＴＳＴＥＭＣＥＬＬＳＡＮＤＭＥＴＨＯＤＳＯＦＧＥＮＥＲＡＴＩＮＧＳＡＭＥ）」）（代理人事件記録簿第５８８７０号）にも関する。上記出願の内容はその全体を参考として本明細書に組み込む。
WO2019093340A1 (fr) * 2017-11-07 2019-05-16 国立大学法人京都大学 Procédé d'induction d'endoderme primitif à partir de cellules souches pluripotentes naïves
WO2021106765A1 (fr) * 2019-11-25 2021-06-03 国立大学法人京都大学 Procédé d'induction de trophectoderme à partir de cellules souches pluripotentes naïves

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DEGLINCERTI ALESSIA; CROFT GIST F.; PIETILA LAUREN N.; ZERNICKA-GOETZ MAGDALENA; SIGGIA ERIC D.; BRIVANLOU ALI H.: "Self-organization of the in vitro attached human embryo", NATURE, vol. 533, no. 7602, 4 May 2016 (2016-05-04), London, pages 251 - 254, XP037445781, ISSN: 0028-0836, DOI: 10.1038/nature17948 *
MA HUAIXIAO, ZHAI JINGLEI, WAN HAIFENG, JIANG XIANGXIANG, WANG XIAOXIAO, WANG LIN, XIANG YUNLONG, HE XIECHAO, ZHAO ZHEN-AO, ZHAO B: "In vitro culture of cynomolgus monkey embryos beyond early gastrulation", SCIENCE, vol. 366, no. 6467, 15 November 2019 (2019-11-15), US , XP055933698, ISSN: 0036-8075, DOI: 10.1126/science.aax7890 *
MASAHITO TACHIBANA, MICHELLE SPARMAN, CATHY RAMSEY, HONG MA, HYO-SANG LEE, MARIA CECILIA T. PENEDO, SHOUKHRAT MITALIPOV: "Generation of Chimeric Rhesus Monkeys", CELL, vol. 148, no. 1-2, 1 January 2012 (2012-01-01), Amsterdam NL , pages 285 - 295, XP055459089, ISSN: 0092-8674, DOI: 10.1016/j.cell.2011.12.007 *
MASCETTI VICTORIA L.; PEDERSEN ROGER A.: "Contributions of Mammalian Chimeras to Pluripotent Stem Cell Research", CELL STEM CELL, vol. 19, no. 2, 10 August 2016 (2016-08-10), AMSTERDAM, NL , pages 163 - 175, XP029675887, ISSN: 1934-5909, DOI: 10.1016/j.stem.2016.07.018 *
OKUBO, TAKUMI : "Construction of Implantation-Phase Human Embryo Model Using Pluripotent Stem Cells", EXPERIMENTAL MEDICINE, vol. 39, no. 18, 1 November 2021 (2021-11-01), JP , pages 2891 - 2976, XP009536873, ISSN: 0288-5514 *
TAKASHIMA, YASUHIRO: "Induction of Pre-Implantation Human Embryo Extracorporeal Tissue Using Initialization Technology, Analysis of Early Human Embryogenesis", GRANT-IN-AID FOR SCIENTIFIC RESEARCH DATABASE; PROJECT / AREA NUMBER 15H06329, KAKEN, JP, 6 January 2017 (2017-01-06), JP, XP009537292 *

Also Published As

Publication number Publication date
JPWO2022114188A1 (fr) 2022-06-02

Similar Documents

Publication Publication Date Title
JP7088496B2 (ja) 網膜組織の製造方法
JP7176764B2 (ja) ナイーブ型多能性幹細胞からの原始内胚葉誘導方法
US10100283B2 (en) Efficient chondrocyte induction method
US10557121B2 (en) Method for chondrogenic induction
KR102318706B1 (ko) 모양체 주연부-유사 구조체의 제조 방법
KR20140050514A (ko) 세포 배양 기재, 및 그것을 사용한 세포 배양 방법 및 만능 줄기 세포의 분화 유도 방법
WO2021106765A1 (fr) Procédé d'induction de trophectoderme à partir de cellules souches pluripotentes naïves
WO2018138281A1 (fr) Cellules souches pluripotentes
JP2014506447A (ja) 多能性幹細胞から樹状細胞を製造する方法
JP6646311B2 (ja) 多能性幹細胞から中胚葉前駆細胞および血液血管前駆細胞への分化誘導法
JP7274683B2 (ja) 多能性幹細胞から樹状分岐した集合管を伴う腎臓構造を作製する方法
Blin et al. Human embryonic and induced pluripotent stem cells in basic and clinical research in cardiology
JP7410518B2 (ja) 脳オルガノイドの製造方法
WO2022114188A1 (fr) Modèle de blastocystis bilaminaris et procédé de production de celui-ci
US20240174985A1 (en) Method for producing ovarian somatic cell-like cells, and method for inducing differentiation of primate pluripotent stem cells into ovarian somatic cell-like cells
JP7072756B2 (ja) 多能性幹細胞から中胚葉前駆細胞および血液血管前駆細胞への分化誘導法
US20220162550A1 (en) Induced stem cells
US20240084265A1 (en) Methods to convert somatic human cell to a totipotency-like state with engineered sox17
Wu et al. Chi-Hun Park,* Young-Hee Jeoung, Kyung-Jun Uh, 3 Ki-Eun Park, Jessica Bridge, Anne Powell, 2, 4
Parris Isolation and characterisation of muscle satellite cells from differentiating human embryonic stem cells.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21898160

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022565490

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21898160

Country of ref document: EP

Kind code of ref document: A1