JP7660899B2 - Method for inducing trophectoderm from naive pluripotent stem cells - Google Patents

Description

The present invention relates to a method for inducing trophectoderm from naive pluripotent stem cells. The present invention also relates to a culture medium used for inducing trophectoderm from naive pluripotent stem cells. The present invention also relates to a molecular marker for efficiently detecting or isolating trophectoderm. The present invention also relates to a molecular marker for efficiently detecting or isolating cytotrophoblast cells. The present invention also relates to a molecular marker for efficiently detecting or isolating amniotic cells. The present invention further relates to a method for inducing cytotrophoblast cells, cytotrophoblast stem cells, syncytiotrophoblast cells, and extravillous trophoblast cells from trophectoderm.

The fertilized egg develops by dividing into two major components: the fetal component and the extraembryonic component that supports the growth of the fetus. At the blastocyst stage, it can be divided into the epiblast, which develops into fetal tissue, and the hypoblast and trophectoderm, which become extraembryonic tissues. The hypoblast develops into the yolk sac, and the trophectoderm develops into the placenta.
Reproducing these developmental processes using pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is useful for analyzing developmental mechanisms and elucidating and treating diseases at the developmental stage. However, compared to mouse pluripotent stem cells, human pluripotent stem cells such as ES cells and iPS cells are classified as prime types that are more advanced in development, and it has been difficult to differentiate them into hypoblast or trophectoderm.

Takashima, one of the inventors of this study, succeeded in obtaining naive pluripotent stem cells by resetting human pluripotent stem cells to the pre-implantation epiblast stage by forcibly expressing two genes, NANOG and KLF2, in human ES cells and iPS cells (Non-Patent Document 1). Regarding early fetal development, the differentiation control mechanism is being elucidated by primed pluripotent stem cells. On the other hand, the differentiation control mechanism of early development of human extraembryonic tissues, especially the placenta, has not been elucidated. The placenta, which supplies nutrients and oxygen to the fetus, is mainly composed of trophoblast cells, which are roughly classified into three types: cytotrophoblast cells, syncytiotrophoblast cells, and extravillous trophoblast cells. The development of trophoblast cells in the body begins from the trophectoderm on the surface of the fertilized egg. The trophectoderm implants in the endometrium, and terminally differentiates into cytotrophoblast cells, syncytiotrophoblast cells, and extravillous trophoblast cells. 25% of all pregnancies develop pregnancy complications such as miscarriage, fetal growth restriction, and pregnancy-induced hypertension. The main causes of these complications are thought to be dysfunction and abnormal differentiation of trophoblast cells. Attention is being paid to elucidating the mechanism of differentiation control from trophectoderm to these trophoblast cells and to approaching the cause of placental dysfunction by molecular and cell biological approaches. In addition, the trophectoderm plays a very important role in all stages of the implantation process, including fixation, adhesion, and invasion, but studies using preimplantation embryos are not only limited by the number of preimplantation embryos that can be used, but are also ethically restricted. Therefore, induction of differentiation into trophectoderm in vitro is expected to overcome these challenges, elucidate the mechanism of implantation, and bring good news to reproductive medicine.

Attempts have been made to induce differentiation of pluripotent stem cells into trophectoderm or trophoblast cells in vitro. For example, it has been reported that primed ES cells were differentiated into trophoblast cells by inducing differentiation with BMP4 (Non-Patent Documents 2 and 3). However, the trophoblast cells obtained by these methods were completely different from trophoblast cells in vivo.
In addition, Non-Patent Document 4 reports that trophectoderm was induced to differentiate from expanded potential stem cells (EPSCs), but the trophectoderm obtained was completely different from the trophectoderm in vivo. On the other hand, Non-Patent Document 5 reports that trophoblast cells (trophoblasts) were induced from human placenta and human embryonic trophectoderm. However, access to such cells is limited and not practical.

Takashima Y. et al., Cell 2014 158(6):1254-1269. Xu et al., Nature Biotechnology 2002, Vol. 20 1261-1264 Amita et al., Proc Natl Acad Sci USA, 2013, E1212-E1221 Gao et al., Nature Cell Biology 2019, Vol 21, 687-699 Okae et al., Cell Stem Cell 2018, vol 22; 50-63 e56.

The present invention aims to provide a method for efficiently and simply inducing trophectoderm from pluripotent stem cells. A further objective of the present invention is to induce differentiation of trophectoderm into cytotrophoblast cells, cytotrophoblast stem cells, syncytiotrophoblast cells, and extravillous trophoblast cells, and to clarify the cell lineage of trophoblast cells.

The present inventors have conducted extensive research to solve the above problems. As a result, they have found that trophectoderm can be efficiently induced by using naive pluripotent stem cells and culturing them in a medium containing two or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor. They have also found that TACSTD2 (Tumor Associated Calcium Signal Transducer 2) and ENPEP (Glutamyl Aminopeptidase) can be used efficiently as trophectoderm and cytotrophoblast cells, and SIGLEC6 (Sialic Acid Binding Ig Like Lectin 6) can be used efficiently as a specific marker for cytotrophoblast cells to select and detect trophectoderm, cytotrophoblast cells and cytotrophoblast stem cells. In addition, it was found that ITGA6 (Integrin Subunit Alpha 6) can be used as a marker for trophectoderm, cytotrophoblast cells, and cytotrophoblast stem cells, HAVCR1 (Hepatitis A Virus Cellular Receptor 1) can be used as a marker for trophectoderm and cytotrophoblast stem cells, ITGB4 (Integrin Subunit Beta 4) can be used as a marker for cytotrophoblast cells and cytotrophoblast stem cells, and ITGA2 (Integrin Subunit Alpha 2) can be used as a marker for cytotrophoblast stem cells. In addition, it was found that TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, and/or HEY1 can be used as a marker for amniotic cells. Furthermore, it was found that the obtained trophectoderm can be further induced to differentiate into cytotrophoblast cells, cytotrophoblast stem cells, and even syncytiotrophoblast cells and extravillous trophoblast cells, thereby completing the present invention.

The present invention provides the following:
[1] A method for producing trophectoderm cells in vitro, comprising:
A method comprising the step of inducing differentiation of naive pluripotent stem cells into trophectoderm cells by culturing them in a medium containing two or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP inhibitor and a JAK inhibitor.
[2] The method for producing trophectoderm cells described in [1], wherein the medium contains three or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor.
[3] The method for producing trophectoderm cells according to [1] or [2], wherein the TGFβ inhibitor is A83-01, the MEK inhibitor is PD0325901, the BMP is BMP4, BMP2 or BMP6, and the JAK inhibitor is JAK inhibitor I.
[4] A method for producing trophectoderm cells according to any one of [1] to [3], further comprising a step of purifying the trophectoderm cells using the expression of TACSTD2 (Tumor Associated Calcium Signal Transducer 2), ENPEP (Glutamyl Aminopeptidase), ITGA6 (Integrin Subunit Alpha 6) and/or HAVCR1 (Hepatitis A Virus Cellular Receptor 1) as an indicator.
[5] The method for producing trophectoderm cells described in any one of [1] to [4], wherein the pluripotent stem cells are induced pluripotent stem cells.
[6] The method for producing trophectoderm cells described in any one of [1] to [5], wherein the pluripotent stem cells are human pluripotent stem cells.
[7] A method for producing cytotrophoblast cells, comprising the steps of producing trophectodermal cells by the method according to any one of [1] to [6], and culturing the obtained trophectodermal cells in a medium containing a TGFβ inhibitor, epidermal growth factor (EGF) and a Wnt signal activator to induce differentiation into cytotrophoblast cells.
[8] The method for producing cellular trophoblast cells according to [7], further comprising a step of purifying the cellular trophoblast cells using as an indicator the expression of TACSTD2, ENPEP, SIGLEC6 (Sialic Acid Binding Ig Like Lectin 6), Ki67 (MKI67 (Marker Of Proliferation Ki-67)), ITGA2 (Integrin Subunit Alpha 2), ITGB4 (Integrin Subunit Beta 4), PTGES (Prostaglandin E Synthase), HSD3B (Hydroxy-Delta-5-Steroid Dehydrogenase, 3 Beta-And Steroid Delta-Isomerase), S100P (S100 Calcium Binding Protein P), ITGA6 (Integrin Subunit Alpha 6) and/or HAVCR1.
[9] A method for producing syncytiotrophoblast cells and/or extravillous trophoblast cells, comprising the steps of preparing cytotrophoblast cells by the method described in [7] or [8], and culturing the obtained cytotrophoblast cells to induce differentiation into syncytiotrophoblast cells and/or extravillous trophoblast cells.
[10] A method for producing cytotrophoblast cells, comprising the steps of providing trophectoderm and inducing differentiation of the trophectoderm into cytotrophoblast cells.
[11] The method for producing cellular trophoblast cells described in [10], wherein the step of inducing differentiation of the trophectoderm into cellular trophoblast cells is a step of inducing differentiation of the trophectoderm into cellular trophoblast cells by culturing the trophectoderm in a medium containing a TGFβ inhibitor, epidermal growth factor (EGF) and a Wnt signal activator.
[12] A trophectoderm cell prepared by the method according to any one of [1] to [6].
[13] A method for separating trophectoderm cells from a cell population containing trophectoderm cells, the method comprising a step of selecting trophectoderm cells using the expression of TACSTD2, ENPEP, ITGA6 and/or HAVCR1 as an indicator.
[14] A method for detecting trophectoderm cells in a cell population containing trophectoderm cells, the method comprising a step of detecting trophectoderm cells using the expression of TACSTD2, ENPEP, ITGA6 and/or HAVCR1 as an indicator.
[15] A reagent for detecting or selecting trophectoderm cells, comprising a molecule that specifically binds to TACSTD2, a molecule that specifically binds to ENPEP, a molecule that specifically binds to ITGA6, and/or a molecule that specifically binds to HAVCR1.
[16] A naive pluripotent stem cell culture medium for inducing trophectoderm cell differentiation, comprising two or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP inhibitor, and a JAK inhibitor.
[17] The medium for naive pluripotent stem cells according to [16], wherein the TGFβ inhibitor is A83-01, the MEK inhibitor is PD0325901, the BMP is BMP4, BMP2 or BMP6, and the JAK inhibitor is JAK inhibitor I.
[18] A method for separating cytotrophoblast cells from a cell population containing cytotrophoblast cells, the method comprising a step of selecting cytotrophoblast cells using the expression of TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6 and/or HAVCR1 as an indicator.
[19] A method for detecting cellular trophoblast cells from a cell population containing cellular trophoblast cells, the method comprising a step of detecting cellular trophoblast cells using the expression of TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6 and/or HAVCR1 as an indicator.
[20] A reagent for detecting or selecting cytotrophoblast cells, comprising a molecule that specifically binds to TACSTD2, a molecule that specifically binds to ENPEP, a molecule that specifically binds to SIGLEC6, a molecule that specifically binds to ITGA2, a molecule that specifically binds to ITGB4, a molecule that specifically binds to PTGES, a molecule that specifically binds to HSD3B, a molecule that specifically binds to S100P, a molecule that specifically binds to ITGA6 and/or a molecule that specifically binds to HAVCR1.
[21] A method for separating amniotic cells from a cell population containing amniotic cells, comprising a step of selecting cellular trophoblast cells using the expression of TNC (Tenascin C), CDH10 (Cadherin 10), PKDCC (Protein Kinase Domain Containing, Cytoplasmic), GABRP (Gamma-Aminobutyric Acid Type A Receptor Subunit Pi), IGFBP5 (Insulin Like Growth Factor Binding Protein 5), POSTN (Periostin), VTCN1 (V-Set Domain Containing T Cell Activation Inhibitor 1), VIM (Vimentin), and/or HEY1 (Hes Related Family BHLH Transcription Factor With YRPW Motif 1) as an indicator.
[22] A method for detecting amniotic cells from a cell population containing amniotic cells, comprising a step of detecting cellular trophoblast cells using the expression of TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM and/or HEY1 as an indicator.
[23] A reagent for detecting or selecting amniotic cells, comprising a molecule that specifically binds to TNC, a molecule that specifically binds to CDH10, a molecule that specifically binds to PKDCC, a molecule that specifically binds to GABRP, a molecule that specifically binds to IGFBP5, a molecule that specifically binds to POSTN, a molecule that specifically binds to VTCN1, a molecule that specifically binds to VIM, and/or a molecule that specifically binds to HEY1.

According to the present invention, trophectoderm can be easily induced from pluripotent stem cells by culture alone, without forced expression of genes. Furthermore, trophectoderm can be induced to differentiate into cytotrophoblast cells, and cytotrophoblast stem cells having self-renewal and differentiation capabilities can be maintained and cultured. Furthermore, differentiation can be induced into syncytiotrophoblast cells and extravillous trophoblast cells, which are considered to be the terminal differentiation stage of trophoblast cells. The method of the present invention realizes the induction of differentiation into trophectoderm, which is an extraembryonic cell, which was difficult to achieve with conventional primed pluripotent stem cells, in a simple procedure, and is a groundbreaking method that can become a fundamental technology for inducing functional mature cells and tissues using pluripotent stem cells. The method of the present invention is useful for elucidating developmental mechanisms, regenerative medicine, elucidating mechanisms and treating diseases at the developmental stage, etc. In addition, the method of selecting and detecting trophectoderm using the molecular marker of the present invention is useful because it can selectively select and detect trophectoderm cells. In addition, the method of selecting and detecting cytotrophoblast cells using the molecular marker of the present invention is useful because it can selectively select and detect cytotrophoblast cells. Furthermore, the method for selecting and detecting amniotic cells using the molecular markers of the present invention is useful because it enables selective selection and detection of amniotic cells.

Graph 1 shows the percentage of TACSTD2- and ENPEP-positive cells when naive pluripotent stem cells were cultured under each condition. 1 shows the results of flow cytometry using TACSTD2 and ENPEP antibodies when naive pluripotent stem cells were cultured under various conditions, with the vertical axis representing TACSTD2 and the horizontal axis representing ENPEP. Microscopic image of cells obtained by culturing naive pluripotent stem cells under C1 conditions. Graph 2 shows the percentage of both TACSTD2- and ENPEP-positive cells when naive pluripotent stem cells were cultured under each condition. Figure (microscope photograph) showing the results of immunostaining to examine the expression of trophectoderm markers (KRT19, TFAP2C, GATA3, CDX2, TACSTD2) in cells obtained by culturing naive pluripotent stem cells under C1 conditions. Figure 1 shows the results of examining the expression of HLA-A and B in cells obtained by culturing naive ES cells or primed ES cells. "BMP-Treated Primed ESCs" are cells obtained by inducing the differentiation of primed ES cells using the method of Non-Patent Document 3, "Primed ESCs" are primed ES cells, "9-week Placenta" is an in vivo placenta, "Cytotrophoblast-like cells" are cytotrophoblast cells induced to differentiate using the method of the present invention, "Trophectoderm-lile cells" are trophectoderm cells induced to differentiate using the method of the present invention, "Naiive ESC cells" are naive ES cells, and Control is a negative control (unstained). This figure (microscope photograph) shows the results of immunostaining to examine the expression of trophoblast cell markers (GATA3, TFAP2C, KRT7) in cellular trophoblast cells obtained by further culturing cells obtained by culturing naive pluripotent stem cells under C1 conditions. Figure 1 shows the results of examining the expression of chromosome 19 microRNA in cells obtained by culturing naive ES cells. JAR is a choriocarcinoma cell line used as an indicator of the expression level of chromosome 19 microRNA. Figure showing the results of investigating the methylation status of the ELF5 promoter in cells obtained by culturing naive or primed ES cells. Results of bisulfite sequencing of the ELF5-2b promoter region. At least eight clones of CpG regions were analyzed for each donor. The positions from the ELF5-2b transcription start site are indicated by numbers. Demethylation is indicated by white circles, and methylation is indicated by black circles. The percentages indicate the methylation rates. A micrograph showing the results of immunostaining to examine the expression of Ki67 in human chorionic villi at 5 and 11 weeks of gestation. The scale bar indicates 100 μm. A micrograph showing the morphology of cytotrophoblast cells obtained by further culturing trophectoderm cells obtained by culturing naive ES cells, and a figure showing the results of investigating the expression of various markers in the cytotrophoblast cells. The figure shows the results of immunostaining to examine the expression of a cytotrophoblast marker (Ki67) in cytotrophoblast cells obtained by further culturing trophectoderm cells obtained by culturing naive ES cells. The scale bar indicates 100 μm. FIG. 1 shows micrographs showing the morphology of syncytiotrophoblast cells obtained by further culturing the cytotrophoblast cells, and the results of investigating cell fusion and human chorionic gonadotropin (hCG) expression in the syncytiotrophoblast cells. FIG. 1 shows micrographs illustrating the morphology of extravillous trophoblast cells obtained by further culturing the cytotrophoblast cells, and the results of examining the expression of HLA-G and LVRN (Laeverin) in the extravillous trophoblast cells. Figure 1 shows the results of flow cytometry to examine marker expression in TE-like cells and cytotrophoblast (CT) stem cells. Figure 2 shows the results of flow cytometry of marker expression in TE-like cells and cytotrophoblast (CT) stem cells. Figure (micrograph) showing the results of immunostaining to examine the expression of CT markers (PTGES, HSD3B) in cellular trophoblast (CT) stem cells and cells derived from primed pluripotent stem cells (pBAP). Scale bar indicates 100 μm. Graph showing the results of quantitative RT-PCR examination of gene expression of HAVCR1, S100P, ITGA6, ITGA2, and SIGLEC6 in TE, CT, and pBAP. Figure (microscope photograph) showing the results of immunostaining to examine the expression of TE and/or CT markers in human chorionic villi at 5 weeks of gestation. The scale bar indicates 100 μm. FIG. 1 shows the results of flow cytometry analysis of the expression of TE and/or CT markers in cells (pBAP) induced from primed pluripotent stem cells. FIG. 13 shows the results of comparing the expression levels of differentially expressed genes in cynomolgus monkey amniotic membrane between TE or CT obtained by the method of the present invention and cells (pBAP) induced from primed pluripotent stem cells. FIG. 1 is a diagram comparing the expression levels of genes that are significantly expressed in human amniotic membrane in TE and CT obtained by the method of the present invention and cells (pBAP) induced from primed pluripotent stem cells. FIG. 1 shows the results of quantitative RT-PCR of the expression levels of amniotic cell marker genes in TE and CT obtained by the method of the present invention, and cells (pBAP) induced from primed pluripotent stem cells. FIG. 1 shows the results of flow cytometry examination of VTCN1 expression in CT obtained by the method of the present invention and cells (pBAP) induced from primed pluripotent stem cells. FIG. 1 shows the results of comparing the expression of genes specific to each stage in TE, CT, ST and EVT obtained by the method of the present invention. FIG. 1 is a graph showing the cumulative cell number after long-term maintenance culture of CT obtained by the method of the present invention. FIG. 1 shows the results of quantitative RT-PCR to examine the expression levels of markers for ST and EVT differentiated by long-term maintenance culture of CT obtained by the method of the present invention. 1 is a micrograph showing the morphology of organoids formed using CT obtained by the method of the present invention, with the scale bar indicating 100 μm. FIG. 1 shows the results of quantitative RT-PCR to examine the expression levels of trophoblast cell markers and ST markers in organoids formed using CT obtained by the method of the present invention. 1 shows the results of immunostaining to examine the expression of trophoblast cell markers in organoids formed using the CT obtained by the method of the present invention (microscope photographs). The scale bar indicates 100 μm.

<Method of producing trophectoderm cells>
The method for producing trophectoderm cells in vitro of the present invention comprises:
The method includes a step of inducing differentiation of naive pluripotent stem cells into trophectoderm cells by culturing them in a medium containing two or more (preferably three, more preferably four) factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor.
When two are selected from a TGFβ inhibitor, a MEK inhibitor, and a BMP and JAK inhibitor, any two of these combinations may be used, but a TGFβ inhibitor and a MEK inhibitor are preferred.
When three types are selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor, any combination of these three types may be used, but a combination of a TGFβ inhibitor, a MEK inhibitor and a BMP is preferable.

<Pluripotent stem cells>
In the present invention, pluripotent stem cells are stem cells that have pluripotency and can differentiate into many cells present in the living body, and also have the ability to proliferate, and include any cell induced into trophectoderm. Pluripotent stem cells are not particularly limited, but include, for example, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES) cells, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells), and the like. Preferred pluripotent stem cells are iPS cells and ES cells. The origin of pluripotent stem cells is preferably mammalian, more preferably primate, and even more preferably human.

Methods for producing iPS cells are known in the art, and iPS cells 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 alone or in combination. Combinations of reprogramming factors include 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, and WO2009/126251. , WO2009/126655, WO2009/157593, WO2010/009015, WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010 /068955, 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. (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, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem 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, Maekawa M, et al. (2011), Nature. 474:225-9. are exemplified.

Somatic cells include, but are not limited to, fetal (baby) somatic cells, neonatal (baby) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines.Specific 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, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

<Naive pluripotent stem cells>
Naive pluripotent stem cells are pluripotent stem cells with properties similar to those of preimplantation embryos, and specifically have the following characteristics (Cytometry Research 27 (1): 19-24, 2017).
It exhibits a dome-shaped colony morphology and is smaller in size than the prime type.
As markers, they express one or more of CD75, KLF4, KLF17 and TFCP2L1.
The genome is demethylated.

Naive pluripotent stem cells can be prepared, for example, by the method described below.
A method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014)
Method using 5i/L/A 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, primed pluripotent stem cells can be cultured using commercially available media for preparing naive pluripotent stem cells, such as t2iLGo (Ndiff227 [Takara Bio, Cat. Y40002], NaiveCult Induction Kit [STEMCELL Technologies, Cat. ST-05580], or NaiveCult Expansion Medium [STEMCELL Technologies, Cat. ST-05590].

<Primed pluripotent stem cells>
Primed pluripotent stem cells are pluripotent stem cells with properties similar to the epiblast of post-implantation embryos, but they include common induced pluripotent stem cells and human ES cells obtained by introducing reprogramming factors into somatic cells, and have not been naïvely treated as described above.
Primed pluripotent stem cells have the following characteristics:
The colony exhibits a flat colony morphology and is larger in size than the naive type.
Markers include CD75, KLF4, KLF17, SUSD2, and TFCP2L1, and are negative, while CD57 and CD24 are positive.
The genome is methylated.

<TGFβ inhibitors>
TGFβ inhibitors are substances that inhibit the signal transduction that continues from the binding of TGFβ to the receptor to SMAD, and include substances that inhibit the binding to the receptor ALK (activin receptor-like kinase) family, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples of such inhibitors include Lefty-1 (NCBI Accession No.: mouse: NM#010094, human: NM#020997), SB431542, SB202190 (RKLindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), SB-525334, GW6604, NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408) and derivatives thereof. The TGFβ inhibitor is preferably A83-01.
The concentration of the TGFβ inhibitor contained in the culture medium can be appropriately selected so as to exert a TGFβ inhibitory effect depending on the type of TGFβ inhibitor. For example, when A83-01 is used, the concentration is usually within the range of 0.1 to 100 μM, preferably 0.5 to 20 μM, and more preferably 1 to 5 μM.

<MEK inhibitors>
MEK inhibitors are substances known to block the MAPK pathway by inhibiting the activity of the phosphorylation enzyme MEK (mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase) present in the Raf/MEK/ERK signaling pathway (MAPK pathway).
Examples of MEK inhibitors include trametinib, cobimetinib, selumetinib, refametinib, pimasertib, U0126, MEK162/ARRY-162, AZD8330/ARRY-424704, GDC-0973/RG7420, and GDC-0623/RG7 421/XL518, CIF/RG7167/RO4987655, CK127/RG7304/RO5126766, E6201, TAK-733, PD0325901, AS703988/MSC2015103B, WX-554, CI-1040/PD184352, AS703026, PD318088, PD98059, SL327, and pharma- ceutically acceptable salts and solvates thereof. The MEK inhibitor is preferably PD0325901.
The concentration of the MEK inhibitor used is not particularly limited, and the MEK inhibitory concentration can be appropriately set depending on the type of inhibitor. For example, when PD0325901 is used, the concentration is usually within the range of 0.1 to 100 μM, preferably 0.5 to 20 μM, and more preferably 1 to 5 μM.

＜BMP＞
The BMP may be at least one BMP selected from the group consisting of BMP2, BMP4, and BMP6, and is preferably BMP4. The BMP is preferably derived from a mammal, and more preferably from a human. An example of human BMP4 is a protein having an amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NP#001193. BMP includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. Commercially available BMPs may be used, or proteins purified from cells or proteins produced by genetic recombination may be used. The concentration of BMP contained in the culture medium is 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 200 ng/ml, and more preferably 5 ng/ml to 50 ng/ml.

<JAK inhibitors>
The JAK inhibitor is not particularly limited as long as it inhibits Janus kinase. Examples of the JAK inhibitor include JAK Inhibitor I (CAS-457081-03-7), tofacitinib (CP-690550), ruxolitinib, baricitinib, INCB039110, oclacitinib, AZD1480, federatinib (SAR302503, TG101348), AT9283, AG-490, momelotinib, WP1066, TG101209, gandotinib (LY2784544), NVP-BSK805, AZ960, CEP-33779, pacritinib (SB1518), WHI-P154, XL019, S-ruxolitinib, ZM39923, decernotinib (VX-509), cerduratinib (PRT062070, PRT2070), filgotinib (GLPG0634), FLLL32, FM-381, BMS-911543, itacitinib (INCB39110), peficitinib (ASP015K, JNJ-54781532), GLPG0634, GLPG0634 analogs, Go6976, curcumol, cucurbitacin, lestaurtinib, upadacitinib (ABT-494), WHI-P258, PF-06700841, PF-04965842, PF-06651600, FLLL32, WHI-P97, CHZ868, solcitinib (GSK2586184), NS-018, or derivatives thereof. In addition, the JAK inhibitors described in US Patent No. 2002/0019526 can also be used. The concentration of the JAK inhibitor used is not particularly limited, and the JAK inhibitory concentration can be set appropriately depending on the type of inhibitor. For example, when JAK Inhibitor I is used, the concentration is usually within the range of 0.05 to 50 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM.

The culture medium used in the trophectoderm induction step is not particularly limited, but can be prepared by adding two or more (preferably three or more, more preferably four) selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor to a naive pluripotent stem cell maintenance medium. These factors may be added at different times. For example, a JAK inhibitor may be added 24 hours after the start of induction. Also, BMP may be added only up to 24 hours after the start of induction.
In addition to two or more selected from the group consisting of a TGFβ inhibitor, a MEK inhibitor, and a BMP and a JAK inhibitor, a Wnt signal activator (such as CHIR99021) described below can also be added.

As a maintenance medium for naive pluripotent stem cells, for example, the following medium can be used.
・t2iLGo
N2B27 + PD0325901 (1μM) + CHIR99021 (1μM) + LIF (10 ng/ml) + Go6983 (2-3μM)
Takashima et al., Cell 158 : 1254-1269, 2014
・5i/L/A
N2B27 +PD0325901(1μM) +CHIR99021(1μM) +SB590885 (0.5μM) +WH-4-023 (1μM) +Y-27632(10μM) +LIF(10 ng/ml)+Activin A(20 ng/ml)
Theunissen, TW, et al. (2014). Cell Stem Cell 15(4): 471-487.
・tt2iLc +iWNT
N2B27 +PD0325901(1μM) +LIF(10 ng/ml) + Go6983(2μM) +XAV939(2μM)
Guo, G., et al. (2017). Development 144(15): 2748-2763.

The medium may contain serum, or may be serum-free or may use a serum substitute. If necessary, it may also contain one or more substances such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamic acid, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines. The medium may further contain a ROCK inhibitor. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), but for example, Y-27632 may be used.

In the trophectoderm induction step, naive pluripotent stem cells may be cultured in an adherent or floating state.
In the case of adhesion culture, the culture vessel may be coated and used, or the cells may be co-cultured with feeder cells, etc. Examples of the feeder cells to be co-cultured include mitomycin C-treated mouse embryo-derived primary fibroblasts (MEF), STO cells, SNL cells, OP9 cells, C3H10T1/2 cells, etc.

In the trophectoderm induction step, when adhesion culture is performed, it can be performed by culturing using a culture vessel coated with an extracellular matrix. The coating process can be performed by putting a solution containing the extracellular matrix into the culture vessel and then removing the solution as appropriate. Here, the extracellular matrix is a supramolecular structure that exists outside the cells, and may be naturally derived or artificial (recombinant). Examples include substances such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, and laminin, and fragments thereof. These extracellular matrices may be used in combination, and may be preparations from cells, such as BD Matrigel (trademark).

In the trophectoderm induction step, when the cells are cultured by suspension culture, it is desirable to culture the cells by forming aggregates (also called spheres) in a non-adherent state to the culture vessel. This type of culture can be performed, but is not limited to, by using a culture vessel that has not been artificially treated (e.g., coated with an extracellular matrix or the like) for the purpose of improving adhesion to the cells, or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA), nonionic surface-active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (e.g., a water-soluble polymer (Lipidure) whose constituent unit is 2-methacryloyloxyethyl phosphorylcholine).

The culture temperature conditions when culturing naive pluripotent stem cells in the trophectoderm induction step are not particularly limited, but are preferably, for example, about 37°C to about 42°C, and about 37°C to about 39°C. The culture oxygen conditions when culturing naive pluripotent stem cells in the trophectoderm induction step are not particularly limited and may be normal oxygen concentrations, but are preferably, for example, low oxygen conditions of about 2% to about 8%, and about 4% to about 6%. In addition, a person skilled in the art can appropriately determine the culture period while monitoring the cell count, etc. The number of days is not particularly limited as long as trophectoderm is obtained, but is, for example, at least one day or more, and preferably 2 to 5 days.

<Trophyectoderm>
The trophectoderm can be obtained by carrying out the above-mentioned culture process. The trophectoderm is characterized by the expression of one or more trophectoderm markers such as GATA2 (GATA Binding Protein 2), GATA3 (GATA Binding Protein 3), CDX2 (Caudal Type Homeobox 2), DAB2 (Disabled Homolog 2), PTGES (Prostaglandin E Synthase), TFAP2C (Transcription Factor AP-2 Gamma), KRT19 (Keratin 19), and ABCG2 (ATP Binding Cassette Subfamily G Member 2). In addition to these one or more markers, the cells are more preferably cells that express TACSTD2 and ENPEP, which will be described later. Furthermore, the cells are more preferably cells that express ITGA6 (Integrin Subunit Alpha 6) and HAVCR1 (Hepatitis A Virus Cellular Receptor 1). Trophectoderm cells are usually negative for OCT4 (Octamer-Binding Protein 4), NANOG (Nanog Homeobox), VIM (Vimentin), and SIGLEC6 (Sialic Acid Binding Ig Like Lectin 6). Trophectoderm cells are also characterized by being negative for HLA-A and HLA-B. Trophectoderm cells are also characterized by the same or higher expression of microRNAs from the chromosome 19 microRNA cluster compared to choriocarcinoma cell lines such as JAR, BeWo, and JEG. In addition, they are characterized by a high demethylation rate of the ELF5 promoter.

In order to enrich for trophectoderm cells, a step of selecting trophectoderm can be performed after the trophectoderm induction step. Selection can be performed using the expression of one or more types of trophectoderm-specific markers as described above as an indicator. When extracting trophectoderm cells using trophectoderm markers, the indicator may be the expression of each marker protein, or the indicator may be the expression of genes (expression of mRNA) encoding each of the marker proteins.

The reagent used to select (or extract or detect) trophectoderm cells from a cell population containing trophectoderm cells may be any molecule that specifically binds to the above-mentioned trophectoderm markers, such as an antibody, an aptamer, a peptide, or a compound that specifically recognizes the trophectoderm marker, and is preferably an antibody or a fragment thereof. When examining the gene expression of these markers, primers or probes that hybridize to these marker genes can be used.

The antibody may be a polyclonal or monoclonal antibody. These antibodies can be prepared using techniques well known to those skilled in the art. Specifically, when the antibody of the present invention is a polyclonal antibody, a marker protein expressed and purified in E. coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence can be synthesized, and a non-human animal such as a rabbit can be immunized with the antibody and obtained from the serum of the immunized animal according to a conventional method. On the other hand, when the antibody is a monoclonal antibody, the antibody can be obtained from hybridoma cells prepared by cell fusion of spleen cells obtained from the immunized non-human animal and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11). Examples of antibody fragments include a part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment "ScFv"). Antibody fragments such as Fab and F(ab) ₂ fragments can also be prepared by a well-known genetic engineering method. When the marker is a membrane protein, it is preferable to use an antibody against the extracellular domain. The antibody may be a commercially available antibody.

In order to distinguish and separate the bound cells, reagents such as antibodies having the relevant affinity may be bound or conjugated to detectable substances such as, for example, fluorescent labels, radioactive labels, chemiluminescent labels, enzymes, biotin, streptavidin, or substances that enable isolation and extraction, such as protein A, protein G, beads, magnetic beads, etc.

Methods for selecting (or extracting or detecting) trophectoderm cells include, for example, a method using a flow cytometer. Other examples include a method of precipitation using an antibody bound to a carrier, a method of magnetically selecting cells using magnetic beads (e.g., MACS), a method using a cell sorter with fluorescent labels, or a method using a carrier to which antibodies or the like are immobilized (e.g., a cell enrichment column).

<Method for selecting or detecting trophectoderm cells using expression of TACSTD2 and/or ENPEP as an indicator>
Since the present inventors have found that TACSTD2 and ENPEP can be suitably used as markers for naive pluripotent stem cell-derived trophectoderm, the present invention provides a method for selecting (separating) or detecting trophectoderm cells from a cell population containing trophectoderm cells, the method comprising a step of selecting (separating) or detecting trophectoderm cells using TACSTD2 and/or ENPEP. For example, as described above, trophectoderm cells can be selected (separated) or detected as cells positive for these markers using antibodies against TACSTD2 and/or ENPEP.
TACSTD2 is a protein that controls Wnt signaling to activate the proliferation of epithelial cells and the self-renewal of stem cells (reference: Stoyanova T et al., Genes Dev. 2012;Vol.26:2271-2285), and an example of this is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM#002353.
ENPEP (aminopeptidase A) is an endopeptidase that is a protein that has the function of decomposing angiotensin II of the renin-angiotensin system into angiotensin III (reference: Holmes RS et al., J Data Mining Genomics Proteomics. 2017; Vol.8:2). For example, there is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM#001977.

In the present invention, a "cell population containing trophectoderm cells" refers to a collection of cells containing trophectoderm cells, regardless of their origin, but is preferably a cell population containing trophectoderm cells obtained by inducing differentiation of naive pluripotent stem cells (preferably human naive pluripotent stem cells) into trophectoderm.

In the present invention, "selection (separation) of trophectoderm cells" means increasing the proportion of trophectoderm cells compared to before selection (separation), and preferably concentrating the cells to contain 50%, 60%, 70%, 80%, or 90% or more of trophectoderm cells. More preferably, it means obtaining cells that are 100% trophectoderm cells.

When trophectoderm cells are selected (separated) from a cell population containing trophectoderm cells using the expression of TACSTD2 and/or ENPEP as an indicator, they may be used in combination with the above-mentioned trophectoderm markers such as GATA2, GATA3, DAB2, CDX2, etc. Furthermore, they may be used in combination with ITGA6 and HAVCR1. This increases the enrichment rate of trophectoderm compared to when TACSTD2 and/or ENPEP are used as indicators.
ITGA2 is a protein that binds to collagen and laminin (reference: Rubel D et al., Matrix Biol. 2014; Vol. 34: 13-21), and an example of this is a protein having the amino acid sequence of NCBI accession number: NP#002194.
HAVCR1 is a membrane protein that is involved in the activation of T cells and acts as a receptor for Hepatitis A virus and Ebola virus (Reference: Moller-Tank S et al., J Virol. 2014;Vol.88:6702-6713). For example, there is a protein having the amino acid sequence of NCBI accession number: NP#036338.

Selectively selecting (separating) cells positive for a marker may mean selecting (separating) all cells positive for the marker, or it may mean selecting (separating) cells expressing a certain amount or more of the marker. For example, in a cell population containing trophectoderm, cells expressing the marker in the top 50%, top 40%, top 33%, top 30%, top 20%, or top 10% can be selectively collected.

<Reagents for selecting or detecting trophectoderm cells using expression of TACSTD2 and/or ENPEP as an indicator>
The present invention also provides a reagent for selecting or detecting trophectoderm cells, comprising a molecule that specifically binds to TACSTD2 and/or a molecule that specifically binds to ENPEP. The molecule that specifically binds to TACSTD2 or ENPEP is as described above, and includes an antibody against TACSTD2 or ENPEP, and a polynucleotide that hybridizes to the TACSTD2 gene or ENPEP gene. The reagent for selecting or detecting trophectoderm cells may also include an instruction manual that describes how to use the detection reagent, together with the molecule that specifically binds to TACSTD2 or ENPEP. The reagent for selecting or detecting trophectoderm cells may comprise a molecule that specifically binds to ITGA6 and/or HAVCR1.

<Culture medium for inducing differentiation into trophectoderm>
The present invention also provides a medium for naive pluripotent stem cells for inducing differentiation into trophectoderm, which contains two or more (preferably three or more, more preferably four) factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP, and a JAK inhibitor. These may be media prepared in advance so that each component is contained at an effective concentration for inducing differentiation into trophectoderm, or may be prepared and used by adding each component immediately before use. These factors may be added at different times. For example, the JAK inhibitor may be added 24 hours after the start of induction. In addition, the BMP may be added at the start of induction and the addition may be completed by 24 hours after induction. Therefore, some factors may be provided separately. The medium (kit) may be accompanied by an instruction manual describing the method of use and the method of preparation. The medium for inducing differentiation into trophectoderm may further contain other components necessary for the culture of naive pluripotent stem cells.

Method for preparing cytotrophoblast cells
The present invention also provides a method for preparing cytotrophoblast cells, which comprises a step of culturing trophectoderm in a medium containing a TGFβ inhibitor, epidermal growth factor (EGF) and a Wnt signal activator to differentiate into cytotrophoblast cells.
The trophectoderm may be obtained by the method described above or by other methods.

TGFβ inhibitors can be similar to those used in the production of trophectoderm (e.g., A83-01), and the preferred concentrations are also similar.

<Wnt signal activator>
The Wnt signal activator is not particularly limited, and examples thereof include Wnt proteins, GSK3β inhibitors, etc. Wnt signal activators are also disclosed in WO2007/126077.
GSK-3β inhibitors are defined as substances that inhibit the kinase activity of GSK-3β protein (e.g., the ability to phosphorylate β-catenin), and examples thereof include the indirubin derivative BIO (also known as GSK-3β inhibitor IX; 6-bromo indirubin 3'-oxime), the maleimide derivative SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), the phenyl α bromomethyl ketone compound GSK-3β inhibitor VII (4-dibromoacetophenone), and the cell membrane-permeable phosphorylated peptides L803-mts and CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino] ethylamino]pyridine-3-carbonitrile). The GSK-3β inhibitor as a Wnt signal activator used in the present invention may preferably be CHIR99021.
The concentration of the GSK3β inhibitor in the culture medium, for example, when CHIR99021 is used, is usually within the range of 0.05 to 50 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM.

＜EGF＞
EGF is preferably derived from a mammal, and more preferably from a human. An example of human EGF is a protein having an amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NP#001954. EGF includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. EGF may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of EGF contained in the culture medium is 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 500 ng/ml, more preferably 10 ng/ml to 200 ng/ml.

The medium used for inducing differentiation of cytotrophoblast cells can be a naive pluripotent stem cell maintenance medium as described above to which a TGFβ inhibitor, a Wnt signal activator, and EGF have been added, and further to which a serum substitute, a ROCK inhibitor, etc. have been added. Differentiation of cytotrophoblast cells can be induced by adhesion culture or suspension culture. In the case of adhesion culture, the cells can be cultured using a culture vessel coated with the above-mentioned extracellular matrix.

The culture temperature conditions for culturing trophectoderm cells in the cytotrophoblast differentiation induction step are not particularly limited, but for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, those skilled in the art can appropriately determine the culture period while monitoring the cell count, etc. The number of days is not particularly limited as long as cytotrophoblast cells can be obtained, but is, for example, at least one day or more, preferably 2 to 12 days.
In addition, the cytotrophoblast cells remain undifferentiated even after subculture, and can be maintained for a long period of time, for example, 6 months or more, while maintaining their self-renewal and differentiation abilities. The cells can be detached and subcultured every 2 to 4 days using Accutase (Sigma-Aldrich, Cat. A6964) or TrypLE Express (ThermoFisher Scientific, Cat. 12604021). It is desirable to seed the cells for new subculture in a ratio of 1:2 to 1:4 to the number of cells detached and recovered from the culture dish or the like by the above-mentioned method. When seeding the cells, it is preferable to add 10 μM Y-27632 to the maintenance medium for cytotrophoblast cells containing a TGFβ inhibitor, a Wnt signal activator, and EGF.
In this way, cytotrophoblast cells that maintain the ability to self-renew and the ability to differentiate are called cytotrophoblast stem cells, and in this specification, unless otherwise specified, the term cytotrophoblast cells also includes cytotrophoblast stem cells.

Cytotrophoblast cells are characterized by being positive for TACSTD2, ENPEP, SIGLEC6, ITGB4, and ITGA6, negative for CDX2, negative for OCT4, and negative for NANOG. Highly proliferative cytotrophoblast cells are additionally positive for ITGA2 and/or HAVCR1. Cytotrophoblast cells may be positive for one or more of Ki67, PTGES, HSD3B, and S100P. Thus, cytotrophoblast cells can be detected, separated, selected, or extracted using the expression of one or more markers, TACSTD2, ENPEP, SIGLEC6, Ki67, ITGB4, ITGA6, PTGES, HSD3B, and S100P. In addition, cellular trophoblast stem cells can be detected, separated or extracted using the expression of one or more markers, including TACSTD2, ENPEP, SIGLEC6, Ki67, ITGA2, ITGB4, HAVCR1, ITGA6, PTGES, HSD3B and S100P.

The present invention also provides a reagent for selecting or detecting cytotrophoblast cells, comprising one or more molecules that specifically bind to TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6, or HAVCR1. Examples of molecules that specifically bind to TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6, or HAVCR1 include antibodies against TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6, or HAVCR1 proteins, and polynucleotides that hybridize to TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6, or HAVCR1 genes. Reagents for selecting or detecting cellular trophoblast cells may also include a molecule that specifically binds to TACSTD2, ENPEP, SIGLEC6, ITGA2, ITGB4, PTGES, HSD3B, S100P, ITGA6 or HAVCR1, as well as instructions describing how to use the detection reagent.

SIGLEC6 is a protein that regulates cell proliferation and invasion in a leptin-dependent manner (reference: Rumer KK et al., Endocr Relat Cancer. 2012;Vol.19:827-840), and an example of this is a protein having the amino acid sequence of NCBI accession number: NP#001236.
ITGB4 is a structural adhesion molecule that forms a heterodimer with ITGA6 and other molecules. It is a protein involved in the activation of Rho GTPase, MAP kinase, and NK-κB signaling (reference: Han L et al., J Neuroinflammation. 2018;Vol.15:246), and an example of this is the protein with the amino acid sequence of NCBI accession number: NP#001005731.
ITGA6 is a structural adhesion molecule that forms a heterodimer with ITGB4 and other molecules. It is a protein involved in cell adhesion and migration (reference: Hu T et al., Sci Rep. 2016;Vol.6:33376), and one example is the protein with the amino acid sequence of NCBI accession number: NP#001073286.
Ki67 is a protein associated with the cell cycle (Reference: Brown DC et al., Histopathology. 2002; Vol. 40: 2-11), and an example thereof is a protein having the amino acid sequence of NCBI accession number: NP#001139438.
PTGES is a prostaglandin E synthesis enzyme and is a protein that increases with inflammation (Reference: Zhao FQ et al., Cell Stress Chaperones. 2013;Vol.18:773-783). For example, there is a protein having the amino acid sequence of NCBI accession number: NP#004869.
HSD3B is called 3beta hydroxysteroid dehydrogenase and is a protein involved in steroid production (reference: Majdic G et al., Biol Reprod. 1998; Vol. 58: 520-525). For example, there is a protein having the amino acid sequence of NCBI accession number: NP#000853.
S100P is a protein that has an EF-hand type calcium-binding domain and is involved in cell proliferation by activating Erk signaling and NK-κB signaling (reference: Arumugam T et al., J Biol Chem. 2004;Vol.279:5059-5065). For example, there is a protein having the amino acid sequence of NCBI accession number: NP#005971.

<Method for preparing syncytiotrophoblast cells>
The cytotrophoblast cells obtained as described above can be cultured to obtain syncytiotrophoblast cells.
Syncytiotrophoblast cells can be obtained by culturing cytotrophoblast cells in a medium containing a cAMP enhancer such as forskolin, preferably a medium further containing EGF. For example, when forskolin is used, its concentration is usually within the range of 0.05 to 50 μM, preferably 0.1 to 10 μM, more preferably 0.5 to 5 μM.
The medium preferably further contains a ROCK inhibitor and a serum substitute. Differentiation into syncytiotrophoblast cells may be induced by adhesion culture or suspension culture. In the case of adhesion culture, the cells can be cultured using a culture vessel coated with the above-mentioned extracellular matrix.

The culture temperature conditions for culturing the cytotrophoblast cells in the syncytiotrophoblast cell differentiation induction step are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, a person skilled in the art can appropriately determine the culture period while monitoring the cell count, etc. The number of days is not particularly limited as long as syncytiotrophoblast cells can be obtained, but is, for example, at least one day or more, preferably 2 to 10 days.

Syncytiotrophoblast cells can be identified by the expression of their marker molecules, such as CGA (Glycoprotein Hormones, Alpha Polypeptide), CGB5 (Chorionic Gonadotropin Subunit Beta 5), CSH1 (Chorionic Somatomammotropin Hormone 1), and SDC1 (Syndecan 1; CD138). Therefore, syncytiotrophoblast cells can be detected, separated, or extracted using the expression of one or more of these markers as an indicator.

<Method for preparing extravillous trophoblast cells>
The extravillous trophoblast cells can be obtained by culturing the above-mentioned cytotrophoblast cells in a medium containing a TGFβ inhibitor and Neuregulin 1.
The TGFβ inhibitor used can be the same as that used in the production of trophectoderm (e.g., A83-01), and the concentration is usually within the range of 0.1 to 100 μM, preferably 1 to 20 μM, and more preferably 5 to 10 μM.
Neuregulin 1 is preferably derived from a mammal, and more preferably from a human. An example of human Neuregulin 1 is a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NP#001153467. Neuregulin 1 includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. Neuregulin 1 may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of Neuregulin 1 contained in the culture medium is 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 500 ng/ml, and more preferably 10 ng/ml to 200 ng/ml.
The medium preferably further contains a ROCK inhibitor, a serum substitute, and an extracellular matrix such as Matrigel or Geltrex. When Matrigel is selected as the extracellular matrix to be added, the amount of Matrigel added to the culture medium is 0.1% to 10%, preferably 0.3% to 5%, and more preferably 0.5% to 3%. When Geltrex is selected as the extracellular matrix to be added, the amount of Geltrex added to the culture medium is 0.1% to 10%, preferably 0.3% to 5%, and more preferably 0.5% to 3%. Differentiation into extravillous trophoblast cells may be induced by adhesion culture or suspension culture. In the case of adhesion culture, the above-mentioned extracellular matrix may be coated on a culture vessel to perform the culture.

The culture temperature conditions for culturing the cellular trophoblast cells in the extravillous trophoblast cell differentiation induction step are not particularly limited, but are, for example, about 37°C to about 42°C, preferably about 37°C to about 39°C. Furthermore, those skilled in the art can appropriately determine the culture period while monitoring the cell count, etc. The number of days is not particularly limited as long as extravillous trophoblast cells can be obtained, but is, for example, at least one day or more, preferably 2 to 10 days.

They can also be identified by the expression of extravillous trophoblast cell marker molecules such as HLA-G and MCAM (Melanoma Cell Adhesion Molecule; CD146). Therefore, extravillous trophoblast cells can be detected, separated, or extracted using the expression of one or more of these markers as an indicator.

Methods and Reagents for Selecting or Detecting Amniotic Cells
Amniotic cells are characterized by being positive for one or more of TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, and HEY1. Thus, amniotic cells can be detected, separated, selected, or extracted using the expression of one or more markers, TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, and HEY1, as an index. Amniotic cells can also be detected, separated, or extracted using the expression of one or more markers, TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, and HEY1, as an index.
The present invention also provides a reagent for selecting or detecting amniotic cells, comprising one or more molecules that specifically bind to TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, or HEY1. Examples of molecules that specifically bind to TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, or HEY1 include antibodies against TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, or HEY1 proteins, and polynucleotides that hybridize to TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, or HEY1 genes. Reagents for selecting or detecting amniotic cells may also include a molecule that specifically binds to TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM or HEY1, as well as instructions describing how to use the detection reagent.

TNC is a large glycoprotein of the extracellular matrix involved in embryonic development and morphogenesis (reference: Chiquet-Ehrismann R et al., Cell. 1986; Vol. 47: 131-139), and an example of this is the protein having the amino acid sequence of NCBI accession number: NP#002151.
CDH10 is a protein that controls the balance between excitatory and inhibitory synapses in the cerebral neocortex (reference: Smith KR et al., J Neurosci. 2017;Vol.37:11127-11139), and an example of this is the protein having the amino acid sequence of NCBI accession number: NP#001304151.
PKDCC is a protein kinase involved in the control of Wnt signaling (Reference: Vitorino M et al., Plos One. 2015;Vol.10:e0135504), and an example of such a protein is the protein having the amino acid sequence of NCBI accession number: NP#612379.
GABRP is a protein involved in macrophage infiltration (Reference: Jiang SH et al., Gut. 2019;Vol.68:1994-2006), and an example of this is a protein having the amino acid sequence of NCBI accession number: NP#001278914.
IGFBP5 is a protein that controls cell proliferation and migration (reference: Akkiprik M et al., BMC Cancer. 2009; Vol. 9: 103), and an example of such a protein is the protein having the amino acid sequence of NCBI accession number: NP#000590.
POSTN is a protein that controls cell proliferation and migration (Reference: Tang Y et al., Cell Prolif. 2017;Vol.50:e12369), and an example of such a protein is the protein having the amino acid sequence of NCBI accession number: NP#006466.
VTCN1 is an immune checkpoint-related protein (Reference: Guo M et al., EMBO Mol Med. 2017;Vol.9:462-481), and an example of such a protein is the protein having the amino acid sequence of NCBI accession number: NP#078902.
VIM is a protein involved in dynamic regulation of cell function and migration (reference: Battaglia RA et al., F1000Res. 2018;Vol.7:F1000 Faculty Rev-1796), and an example of this is the protein with the amino acid sequence of NCBI accession number: NP#003371.
HEY1 is a protein that controls cell differentiation downstream of the Notch signal (Reference: Nakahara Y et al., Genes Cells. 2016; Vol. 21: 492-504), and an example of HEY1 is a protein having the amino acid sequence of NCBI accession number: NP#001035798.

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

Material and method
Cell culture
Human primed pluripotent stem cell (PSC) lines (H9ES (embryonic stem) cells, 409B2 cells, and 201B7 cells) were maintained on γ-irradiated MEFs in conventional condition (F12/KSR) (Dulbecco's modified Eagle medium [DMEM/F12; Nacalai Tesque, Cat. 08460-95], 20% [v/v] KSR [Thermo Fisher Scientific, Cat. 10828028], nonessential amino acids [NEAA; Thermo Fisher Scientific, Cat. 11140-050], 4 ng/ml recombinant human bFGF [bFGF; Oriental Yeast, Cat. NIB 47079000], and 0.1 mM 2-mercaptoethanol [Sigma-Aldrich, Cat. M3148]). Cells were passaged every 4 to 6 days by detaching them into small clumps using Dissociation Buffer (DB; 0.025% Trypsin [Thermo Fisher Scientific, Cat. 15090-046], 1 mg/ml Collagenase IV [Thermo Fisher Scientific, Cat. 17104-019], ₂₀ % KSR, 1 mM CaCl ).

Human naïve pluripotent stem cell lines (derived from H9ES, 409B2, and 201B7 cells) were maintained on MEFs using t2iLGo (Ndiff227 [Takara Bio, Cat. Y40002], 1 μM PD0325901 [PD03; Tocris, Cat.4192], 1 μM CHIR99021 [CH; Sigma-Aldrich, Cat.SML1046], 10 ng/ml Recombinant human LIF [human LIF; Peprotech, Cat.300-05], 3 μM Go6983 [Go; Tocris, Cat.2285]). Cells were detached and passaged every 3–5 days using Accutase (Sigma-Aldrich, Cat.A6964).

Naïve H9ES cells and naïve 409B2 cells were established using the 5i/L/A condition (Theunissen et al., Cell Stem Cell. 2014 Oct 2;15(4):471-487.). Primed H9 cells and/or 409B2 cells were detached into single cells with trypsin/EDTA, and 1× ¹⁰⁵ cells/ ^cm2 were seeded onto MEFs in F12/KSR medium supplemented with 10 μM Y-27632. The next day, the medium was changed to 5i/L/A (Ndiff, 1μM PD03, 1μM CH, 1μM WH-4-023 [A Chemtek H620061], 0.5μM SB590885 [R and D 2650], 10μM Y-27632, 10ng/ml human LIF, 20ng/ml Activin A [R&D, Cat.388-AC]) and cultured. When most of the cells formed colonies showing a dome-shaped morphology (4-6 passages), the medium was switched to t2iLGo and maintained for establishment.

Naive 201B7 cells were established by overexpressing NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014). A plasmid capable of inducing overexpression by doxycycline was electroporated and introduced into primed 201B7 cells (201B7 NK2). A drug resistance marker gene (neomycin resistance) was incorporated into the plasmid, and after introduction into the cells, cells with the plasmid were selected by drug selection using Geneticin (Thermo Fisher Scientific, Cat.10131035). Primed 201B7 NK2 were detached into single cells using trypsin/EDTA, and 1× ¹⁰⁵ cells/ ^cm2 were seeded onto MEFs in F12/KSR medium supplemented with 10μM Y-27632 (Wako, Cat.034-24024). The next day (day 1), 1 μg/ml doxycycline hyclate (Dox; Sigma-Aldrich, Cat. D9891) was added. From day 2, the medium was switched to 2iL (1 μM PD03, 1 μM CH, LIF) + Dox medium and cultured for about 1 week. After that, the medium was switched to t2iLGo and maintained for establishment.

Trophectoderm (TE) induction
Naïve pluripotent stem cells cultured on MEFs were detached and collected using Accutase, then seeded on gelatin-coated dishes and cultured in t2iLGo containing 10μM Y-27632 (ROCK inhibitor) at 37℃ for 2 hours to remove the MEFs. The cells were then resuspended in each induction medium, and induction was initiated immediately after seeding.

When induction was performed using a compound, a 12-well dish was coated with iMatrix-511 silk (Nippi, Cat. 892021) and 1.5 × 10 ⁵ cells/cm ² of cells were seeded.
Ndiff227 medium was used as the induction medium, and various compounds were added under the conditions described in Table 2 or 3. Culture was performed to screen for compounds that induce trophectoderm differentiation.
The TGFβ inhibitor used was A83-01, the MEK inhibitor used was PD0325901, the BMP inhibitor used was BMP4, and the JAK inhibitor used was JAK inhibitor I.

In addition, for evaluation of the obtained trophectoderm cells and induction into cytotrophoblast cells and beyond, trophectoderm cells obtained by culturing naive iPS cells under the following conditions were used.
The induction medium used was Ndiff227 medium supplemented with 2 μM A83-01 (Tocris, Cat. 2939), 2 μM PD0325901 (Tocris, Cat. 4192), and 10 ng recombinant human BMP-4 (BMP-4; R&D, Cat. 314-BP). After washing the dishes with saline 24 hours after the start of induction, Ndiff227 medium supplemented with 2 μM A83-01 (Tocris, Cat. 2939), 2 μM PD0325901, and 1 μM JAK inhibitor (MERCK) was used until 72 hours after the start of induction.

FACS analysis/sorting
Trophectoderm cells, cytotrophoblast cells, cytotrophoblast stem cells, and naive pluripotent stem cells were detached into single cells using Accutase and collected. Ndiff227 medium was added to the collected cell clusters and incubated at 37°C for 30 minutes. Blocking was then performed on ice for 30 minutes using HBSS (Thermo Fisher Scientific, Cat.14185052) containing 1% BSA (Sigma-Aldrich, Cat.A2153). Biotinylated anti-TACSTD2 antibody (Miltenyi biotec, Cat.130-115-054) and PE-conjugated anti-ENPEP antibody (Thermo Fisher Scientific, Cat.12-5891-82) were added and incubated on ice for 30 minutes. After washing, Streptavidin-APC (Biolegend, Cat.405207) was added and incubated on ice for 30 minutes. FACS analysis was performed using a BD LSR Fortessa (BD), sorting was performed using a FACS AriaII (BD), and data analysis was performed using Flow Jo V10.2 software.

Induction of trophectoderm into cytotrophoblast (CT) cells. On the third day after induction into trophectoderm, positive cells were purified by flow cytometry using anti-TACSTD2 and anti-ENPEP antibodies. After that, 1μM A83-01, 50 ng/ml EGF, and 2μM CHIR99021 were added to Ndiff227 medium, and the culture was continued on iMatrix511 silk. At the time of cell seeding, 10μM Y-27632 (ROCK inhibitor) was added to the medium. On the 12th day, cytotrophoblast cells were purified by flow cytometry. For purification by flow cytometry, Alexa-Fluor488-conjugated anti-TACSTD2 antibody (R&D, Cat# FAB650G), PE-conjugated anti-ENPEP antibody, and biotinylated anti-SIGLEC6 antibody (Miltenyi biotec, Cat.130-112-708) were added and incubated on ice for 30 minutes. After washing, Streptavidin-APC (Biolegend, Cat.405207) was added and incubated on ice for 30 minutes. BD LSR Fortessa (BD) was used for FACS analysis, and FACS AriaII (BD) was used for sorting. Data analysis was performed using Flow Jo V10.2 software.

On the 12th day, the cytotrophoblast cells were purified by flow cytometry and cultured on iMatrix511 silk in Ndiff227 medium supplemented with 1μM A83-01, 50 ng/ml EGF, and 2μM CHIR99021. When the cells were seeded, 10μM Y-27632 (ROCK inhibitor) was added to the medium. This made it possible to maintain and culture the cells as cytotrophoblast stem cells with self-renewal and differentiation capabilities for more than 6 months.

Induction of syncytiotrophoblast cells (ST) from cytotrophoblast stem cells The cytotrophoblast stem cells purified above were cultured on iMatrix511 silk for 6 days using a medium (DMEM/F12) containing 4% KSR (knockout serum replacement), 2 μM forskolin, and 2.5 μM Y-27632 (ROCK inhibitor).

Induction of extravillous trophoblast cells (EVT) from cytotrophoblast stem cells The cytotrophoblast stem cells purified above were cultured on iMatrix511 silk for 3 days (Day 1 to Day 3) using a medium (DMEM/F12) containing 4% KSR (knockout serum replacement), 7.5μM A83-01, 100ng/ml Neuregulin1, 2.5μM Y-27632 (ROCK inhibitor), and Matrigel. The medium was then replaced with 4% KSR (knockout serum replacement), 7.5μM A83-01, 2.5μM Y-27632 (ROCK inhibitor), and Matrigel (or Geltrex), and cultured for 3 days (Day 4 to Day 6). Furthermore, the medium was replaced with 7.5 μM A83-01, 2.5 μM Y-27632 (ROCK inhibitor), and Matrigel (or Geltrex), and the cells were cultured for 2 days (Day 7 to Day 8).

Reverse Transcription Quantitative Real-time PCR
Total RNA was extracted using the RNeasy kit (Qiagen, Cat. 74106), and cDNA was synthesized from 1000 ng of RNA using SuperScriptIV (Thermo Fisher Scientific, Cat. 18090050) and oligo-dT primers. Real-time PCR was performed using PowerUP Sybr Green Master Mix (Thermo Fisher Scientific, Cat. A25743), and PCR amplification was performed using QuantStudio3 (Thermo Fisher Scientific) or QuantStudio12K. Analysis of real-time RT-PCR reactions was performed using QuantStudio Design&Analysis Software v1.4.1.

Immunostaining
Cells were fixed with 4% paraformaldehyde (Nacalai Tesque, Cat. 09154-85) for 10 min at room temperature, and then permeabilized with PBS + 0.5% Triton X-100 for 1 h at room temperature. Cells were blocked with PBS + 1% BSA + 0.05% Tween-20 (PBS-BT) for 2 h. Primary antibodies were added after dilution in PBS-BT and incubated for 2 h at room temperature. After washing, secondary antibodies were added at 1:2000 dilution in PBS-BT and incubated for 2 h at room temperature. Nuclei were stained with DAPI (Sigma-Aldrich, Cat. D9542). The following antibodies were used:

Results <Selection of trophectoderm (TE) specific markers>
We reanalyzed single-cell RNA sequencing data of human preimplantation embryos previously reported (Petropoulos S et al. 2016 Cell, Stirparo GG et al. 2018 Development). The developmental stages of preimplantation embryos are the zygote immediately after fertilization, the 4 cell stage, the 8 cell stage, the morula stage, and the blastocyst stage. The blastocyst stage can be further subdivided according to cell type into three parts: the epiblast, which will become the components of the fetus, the hypoblast, which will form the yolk sac, and the trophectoderm, which will become the placenta.
TACSTD2 transcript expression was high in the trophectoderm at embryonic ages 5 and 7, whereas transcript expression was low in other preimplantation embryo developmental stages (zygote immediately after fertilization, 4-cell stage, 8-cell stage, morula stage, fetal age 5 epiblast, fetal age 7 epiblast, fetal age 5 hypoblast, fetal age 7 hypoblast).
While ENPEP transcript expression was high in the trophectoderm at embryonic age 7, transcript expression was low in other preimplantation embryo developmental stages (zygote immediately after fertilization, 4-cell stage, 8-cell stage, morula stage, fetal age 5 epiblast, fetal age 7 epiblast, fetal age 5 hypoblast, fetal age 7 hypoblast, and fetal age 5 trophectoderm).
Based on the expression patterns of TACSTD2 and ENPEP in preimplantation embryos, we found that TACSTD2 and ENPEP can be used as surface antigen proteins of the trophectoderm and selected them as indicators of trophectoderm differentiation.

<Trophyectoderm differentiation>
Naive iPS cells were cultured in a medium containing various differentiation-inducing factors, and the expression of TACSTD2 and ENPEP was used to screen for trophoblast differentiation-inducing factors. Candidate factors for trophoblast differentiation-inducing factors were predicted from the receptor expression patterns in trophoblasts and used for screening.
As a result of the screening, four factors (TGFβ inhibitor, MEK inhibitor, BMP4, JAK inhibitor) were identified. Table 2 shows the culture conditions in which these factors were combined, and Figure 1 shows the percentage of TACSTD2- and ENPEP-positive cells cultured under each condition. Figure 2 also shows the representative flow cytometry results, and Figure 3 shows the morphology of the obtained TACSTD2- and ENPEP-positive cells.

As a result, differentiation was most efficient under the C1 condition, but when two or more of the following were used: TGFβ inhibitor (A83-01), MEK inhibitor (PD0325901), BMP4, or JAK inhibitor, both TACSTD2- and ENPEP-positive cells were obtained. However, under the C7 condition, in which activin (a TGFβ signal activator) was added instead of A83-01, neither TACSTD2- nor ENPEP-positive cells were obtained.

Furthermore, we investigated the preferable combination of TGFβ inhibitor, MEK inhibitor, BMP4, and JAK inhibitor. Table 3 shows the culture conditions, and Figure 4 shows the ratio of TACSTD2- and ENPEP-positive cells when cultured under each condition.
As a result, it was found that it is preferable for TGFβ inhibitors and MEK inhibitors to be present throughout the entire differentiation induction process, while it is preferable for BMP4 to be present at least during the first half of the differentiation induction process (e.g., between the start of differentiation induction and 24 hours), and it is preferable for JAK inhibitors to be present at least during the second half of the differentiation induction process (e.g., more than 24 hours after the start of differentiation induction).

As a result, by using a combination of a TGFβ inhibitor, a MEK inhibitor, and a BMP4 and JAK inhibitor, we were able to obtain TACSTD2- and ENPEP-positive cells from naive iPS cells. These TACSTD2- and ENPEP-positive cells were evaluated as trophectoderm (TE)-like cells as follows.

<Evaluation of TE-like cells 1>
To analyze the properties of TE-like cells obtained by differentiation induction, the gene expression patterns of TE-like cells were compared with those of in vivo TE cells and in vivo epiblast cells (EPI: epiblast).
The genes analyzed were differential expression genes (DEGs) in in vivo TE and in vivo epiblast cells. Specifically, DEG01 was examined using 201 genes with a p-value of less than 0.001 that could distinguish epiblast from trophectoderm, as shown in Table S1 of Stirparo (2018) Development#Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.
A similar comparison was also performed for cells obtained by culturing primed iPS cells under similar conditions and for EPSCs from Non-Patent Document 4. Each experiment was performed twice, and gene expression patterns were observed on Day 0 (before the start of differentiation induction) or Day 3 (3 days after the start of differentiation induction, EPSCs 4 days later).

The results are shown in Table 4 (N#D3 and P#D3 indicate Day 3 of naive iPSCs and primed iPSCs, respectively). Here, a value closer to 1 indicates a high correlation.
When the correlation between in vivo trophectoderm (TE) and in vitro differentiation-induced cells was examined using DEG (EPI vs TE), naive-derived differentiation day 3 (trophectoderm-like cells) showed a higher correlation coefficient than EPSC Day 4 and Primed Day 3 ("trophoblast-like cells" in Non-Patent Document 3). (N#D3 was closer to TE than P#D3 and EPSC#Day 4.)
Thus, differentiated TE-like cells showed gene expression patterns similar to those of in vivo TEs, whereas prior art primed pluripotent stem cells and EPSCs did not.

Next, the expression of TE-specific markers in TE-like cells was examined by immunohistochemical staining using marker-specific antibodies. The results are shown in Figure 5. As a result, the formation of TE was also confirmed by the expression of protein markers.

<Evaluation of CT-like cells>
The criteria for early pregnancy trophoblast cells are: 1) no expression of HLA-A and HLA-B, 2) expression of GATA3, TFAP2C, and KRT7, 3) expression of chromosome 19 microRNA, and 4) demethylation of the ELF5 promoter.
We investigated whether the CT-like cells obtained by the method of the present invention fulfilled the above four criteria.

First, the expression levels of HLA-A and HLA-B were evaluated. As a result, as shown in Figure 6, it was found that the CT-like cells obtained by the method of the present invention barely expressed HLA-A or HLA-B, similar to in vivo CT in the placenta. On the other hand, primed ES cells were positive for HLA-A and HLA-B even after BMP treatment.

Next, we evaluated the expression levels of trophoblast cell markers GATA3, TFAP2C, and KRT7. As a result, as shown in Figure 7, it was found that the CT-like cells obtained by the method of the present invention expressed these markers.

Next, the expression levels of chromosome 19 microRNAs (miR518c-5p, miR520c-3p) were evaluated. As a result, as shown in Figure 8, it was found that the TE-like cells and CT-like cells obtained by the method of the present invention had high expression levels of chromosome 19 microRNAs.

Next, the methylation state of the ELF5 promoter was evaluated. The results are shown in Figure 9. The methylation rates of trophectoderm-like cells, cytotrophoblast-like cells, naive ES cells, primed ES cells, and BMP-treated cells (primed ES cells cultured for 3 days with BMP4 added) were 10.0%, 0.5%, 29.3%, 85.2%, and 22.7%, respectively, and strong demethylation was observed in the trophectoderm-like cells and cytotrophoblast-like cells. Thus, it was found that the TE-like cells and CT-like cells obtained by the method of the present invention had a high demethylation rate of the ELF5 promoter.
As described above, it was demonstrated that CT-like cells obtained by inducing differentiation from naive ES cells faithfully reproduce the properties of in vivo CT.

<Differentiation of TE into cytotrophoblast (CT)>
Single-cell RNAseq of human preimplantation embryos showed no expression of SIGLEC6. On the other hand, RNA sequence data of early pregnancy chorionic villi predicted that TACSTD2, ENPEP, and SIGLEC6 were expressed in CT. We confirmed protein expression by FACS using clinical samples from 9 and 11 weeks of pregnancy, and found that all of TACSTD2, ENPEP, and SIGLEC6 were expressed. In other words, SIGLEC6 is not expressed in TE, but is expressed in CT. Based on this finding, we investigated differentiation into CT based on the presence or absence of SIGLEC6 expression.
It is also known that Ki67 is expressed in CT (Chang and Parast, 2017; Muhlhauser et al., 1993). In fact, when immunostaining was performed using human chorionic villi from 5 weeks of pregnancy, it was confirmed that CT expressed Ki67, as shown in Figure 10. On the other hand, in chorionic villi from 11 weeks of pregnancy, cells expressing Ki67 disappeared. This indicates that Ki67 is a marker expressed in the early stage of CT.

Figures 11 and 12 show the results of examining the expression of markers in the cells obtained by culturing the TE-like cells obtained by the method of the present invention in a medium containing a TGFβ inhibitor, epidermal growth factor (EGF) and a Wnt signal activator. As a result, the TE-like cells were SIGLEC6 negative, but the expression of SIGLEC6 was confirmed in the obtained cells. Furthermore, the obtained cells expressed Ki67. These results indicate that it is possible to induce differentiation of TE into CT, and that the differentiated CT has high proliferation ability and is in the early stage of CT.

<Differentiation of CT into syncytiotrophoblast cells (ST)>
The CT obtained by the method of the present invention was further cultured in a medium containing Forskolin, etc., and the results of examining the expression of markers in the cells obtained are shown in Figure 13. As a result, the obtained cells were fused and multiple nuclei were visible, and the expression of CGA and CGB was confirmed. This shows that it is possible to induce differentiation from CT to ST.

<Differentiation of CT into extravillous trophoblast cells (EVT)>
The CT obtained by the method of the present invention was further cultured in a medium containing a TGFβ inhibitor and Neuregulin 1, and the results of examining the expression of markers in the cells obtained are shown in Figure 14. As a result, the expression of HLA-G and LVRN (Laeverin) was confirmed in the obtained cells. This indicates that it is possible to induce differentiation from CT to EVT.

Marker expression in TE-like cells and cytotrophoblast (CT) stem cells
The marker expression in the TE-like cells and cytotrophoblast (CT) stem cells obtained above was examined by flow cytometry using antibodies against each marker. As shown in Figures 15 and 16, in addition to TACSTD2 and ENPEP, ITGA6 (Integrin Subunit Alpha 6) and HAVCR1 (Hepatitis A Virus Cellular Receptor 1) were found to be positive in the TE-like cells. On the other hand, in addition to TACSTD2, ENPEP, and SIGLEC6, ITGA2 (Integrin Subunit Alpha 2), ITGB4 (Integrin Subunit Beta 4), ITGA6 (Integrin Subunit Alpha 6), and HAVCR1 (Hepatitis A Virus Cellular Receptor 1) were found to be positive in the CT stem cells.
Furthermore, immunostaining revealed that PTGES and HSD3B were expressed in CT stem cells, as shown in FIG. 17.
In addition, gene expression of HAVCR1, S100P, ITGA6, ITGA2 and SIGLEC6 was examined by quantitative RT-PCR. As a result, it was found that HAVCR1, S100P, and ITGA6 were expressed in CT and TE, and ITGA2 and SIGLEC6 were expressed in CT (FIG. 18).
Furthermore, in human chorionic villi at 5 weeks of gestation, the results of immunostaining shown in FIG. 19 revealed that in addition to GATA3 and KRT7, HAVCR1, ITGA6, ITGA2, S100P, HSD3B and PTGES were expressed.
From the above, ITGA2, ITGB4, PTGES, HSD3B, and SIGLEC6 can be CT markers, and ITGA6, HAVCR1, and S100P can be CT or TE markers.

<Cells derived from primed pluripotent stem cells>
The cells were generated by a known method of inducing differentiation into CT using primed iPSCs (Proc Natl Acad Sci USA 110, E1212-1221.2013; Proc Natl Acad Sci USA 112, E2337-2346. 2015), and designated as pBAP. When pBAP was examined by flow cytometry and immunostaining, it was found that CT markers and/or TE markers such as ITGA2, ITGB4, SIGLEC6, ITGA6, HAVCR1, PTGES, and HSD3B were not expressed, as shown in Figures 17 and 20.
Furthermore, the expression levels of differentially expressed genes in cynomolgus monkey amniotic membrane for TE and CT obtained by the method of the present invention, and pBAP, were analyzed from the results of RNA sequencing, and the scatter plots are shown in FIG.
As a result, genes expressed in trophoblast cells, such as GATA2, GATA3, TFAP2C, TACSTD2, ENPEP, and DAB2, were commonly expressed in TE, CT, and pBAP, whereas pBAP showed significantly higher expression levels of over 200 amnion-specific genes compared to TE and CT.
From the above, it was found that pBAP were not CTs but rather amniotic-like cells, although they were generated by a known method that is believed to induce differentiation into CTs.

<Amniotic cell marker genes>
Furthermore, the expression levels of genes significantly expressed in human amnion in TE and CT obtained by the method of the present invention were compared with those in pBAP using a heat map, as shown in Figure 22. As a result, TNC, CDH10, PKDCC, GABRP, IGFBP5, POSTN, VTCN1, VIM, and HEY1 were identified as genes that were hardly expressed in TE and CT but significantly expressed in pBAP. It was also confirmed by quantitative RT-PCR that these genes were more strongly expressed in pBAP than in TE and CT (Figure 23).
In addition, when the cell surface antigen VTCN1 was examined by flow cytometry, it was confirmed that it was expressed in pBAP but not in CT, as shown in Figure 24. Based on the above, the above nine genes can be used as markers for selecting and/or detecting amniotic cells.

<Gene expression patterns of trophectoderm (TE), cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT)>
Xiang et al. (Nature 577, 537-542.2020) report genes specific to the preimplantation TE (PreTE), postimplantation CT (PostCT), early EVT (EarlyEVT), EVT, early ST (EarlyST), or ST stages in human embryos.
Therefore, for TE, CT, ST and EVT obtained by the method of the present invention, the expression levels of genes specific to each of the above are shown in FIG.
As a result, the TE, CT, ST, and EVT obtained by the method of the present invention showed gene expression patterns in which the expression levels of genes specific to the corresponding cells were high.

<Maintenance of cytotrophoblast (CT)>
The CT obtained by the method of the present invention was passaged every 2 to 3 days by detaching the cells with TrypLE and reseeding at a density of 1/2 to 1/4. As shown in Figure 26, the CT could be passaged 35 times or more and maintained in culture for 70 days or more.
Furthermore, when CTs that had been passaged 30 times or more (90 days or more) and maintained in culture were differentiated into STs and EVTs by the method of the present invention, the expression of the respective markers was confirmed from the results of quantitative RT-PCR shown in Figure 27. That is, the CTs obtained by the method of the present invention maintained the differentiation ability into STs and EVTs even after passaged 30 times or more and maintained in culture for 90 days or more.

<Organoid formation>
Using the CT obtained by the method of the present invention, organoids were formed according to the method of Turco et al. (Nature 564, 263-267. 2018). The CT-derived organoids obtained by this method had a morphology very similar to that of trophoblast-derived organoids obtained from the placenta, as shown in Figure 28. The CT-derived organoids could be passaged more than 10 times and maintained in culture for more than 2 months.
Analysis by quantitative RT-PCR showed that the above CT-derived organoids expressed both CT and ST markers, as shown in Figure 29. This is consistent with the reports by Haider et al. (Stem Cell Reports 11, 537-551.2018) and Turco et al. (Nature 564, 263-267. 2018).
The results of immunostaining the CT-derived organoids are shown in FIG. 30. As a result, it was found that trophoblast cell markers, including KRT7, GATA3, and TFAP2A, were expressed. Furthermore, in the report by Haider et al. (Stem Cell Reports 11, 537-551.2018) and the report by Turco et al. (Nature 564, 263-267. 2018), it was reported that the outer layer of the trophoblast cell organoid was composed of CT-like cells, and the CT-derived organoids expressed ITGA6, a CT marker, in the surface layer, as shown in FIG. 30.
From the above, it is considered that the organoids derived from the CT were three-dimensional organoids similar to organoids derived from trophoblast cells obtained from the placenta. Therefore, the CT obtained by the method of the present invention was capable of forming three-dimensional organoids.

Claims (10)

1. A method for producing trophectoderm cells in vitro, comprising:
A method comprising the step of inducing differentiation of naive pluripotent stem cells into trophectoderm cells by culturing them in a medium containing three or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP and a JAK inhibitor. The method for producing trophectoderm cells according to claim 1 , wherein the TGFβ inhibitor is A83-01, the MEK inhibitor is PD0325901, the BMP is BMP4, BMP2 or BMP6, and the JAK inhibitor is JAK inhibitor I. Furthermore, trophectoderm cells were transduced with TACSTD2 (Tumor Associated Calcium Signal Transducer 2)
The method for producing trophectoderm cells according to claim 1 or 2 , comprising a purification step using the expression of ENPEP (Glutamyl Aminopeptidase), ITGA6 (Integrin Subunit Alpha 6) and/or HAVCR1 (Hepatitis A Virus Cellular Receptor 1) as an indicator. The method for producing trophectoderm cells according to any one of claims 1 to 3 , wherein the pluripotent stem cells are induced pluripotent stem cells. The method for producing trophectoderm cells according to any one of claims 1 to 4 , wherein the pluripotent stem cells are human pluripotent stem cells. A method for producing cytotrophoblast cells, comprising: a step of producing trophectodermal cells by the method according to any one of claims 1 to 5 ; and a step of inducing differentiation of the obtained trophectodermal cells into cytotrophoblast cells by culturing the cells in a medium containing a TGFβ inhibitor, an epidermal growth factor (EGF) and a Wnt signal activator. The method for producing cytotrophoblast cells according to claim 6, further comprising a step of purifying the cytotrophoblast cells using the expression of TACSTD2, ENPEP, SIGLEC6 (Sialic Acid Binding Ig Like Lectin 6), Ki67 (MKI67 (Marker Of Proliferation Ki-67)), ITGA2 (Integrin Subunit Alpha 2), ITGB4 (Integrin Subunit Beta 4), PTGES (Prostaglandin E Synthase), HSD3B (3 beta-hydroxysteroid dehydrogenase), S100P, ITGA6 (Integrin Subunit Alpha 6 ) and/or HAVCR1 as indicators. A method for producing syncytiotrophoblast cells and/or extravillous trophoblast cells, comprising the steps of preparing cytotrophoblast cells by the method described in claim 6 or 7 , and culturing the obtained cytotrophoblast cells and inducing their differentiation into syncytiotrophoblast cells and/or extravillous trophoblast cells. A naive pluripotent stem cell culture medium for inducing differentiation of trophectoderm cells, comprising three or more factors selected from a TGFβ inhibitor, a MEK inhibitor, a BMP inhibitor, and a JAK inhibitor. The medium for naive pluripotent stem cells according to claim 9 , wherein the TGFβ inhibitor is A83-01, the MEK inhibitor is PD0325901, the BMP is BMP4, BMP2 or BMP6, and the JAK inhibitor is JAK inhibitor I.