JP7176764B2 - Method for inducing primitive endoderm from naive pluripotent stem cells - Google Patents

Description

The present invention relates to a method for inducing primitive endoderm from naive pluripotent stem cells. The present invention also relates to media suitably used for inducing primitive endoderm from naive pluripotent stem cells. The present invention also relates to molecular markers for efficient detection and isolation of primitive endoderm. The present invention further relates to a method of mimicking gastrulation and inducing mesendoderm differentiation by co-culturing primitive endoderm and epiblast.

During mammalian development, the inner cell mass is formed inside the blastocyst, from which the epiblast and primitive endoderm emerge, and the epiblast undergoes primitive gut formation and develops into the embryonic mesendoderm. While differentiating, the primitive endoderm differentiates into extraembryonic tissue, particularly the yolk sac. Reproducing these developmental processes with pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is useful for analysis of developmental mechanisms, elucidation and treatment of disease mechanisms at the developmental stage, etc. useful for
However, compared to mouse pluripotent stem cells, human ES cells and iPS cells are classified as the prime type, which is more advanced in development and difficult to differentiate into epiblasts and primitive endoderm.
Therefore, Takashima, one of the present inventors, reset human pluripotent stem cells to the same state as the basal state by expressing two genes, NANOG and KLF2, in human pluripotent stem cells. have succeeded in obtaining sex stem cells (Non-Patent Document 1), and using them to induce primitive endoderm and analyze early development (Non-Patent Document 2).

Cell 158: 1254-1269, 2014 Induction of human pre-implantation extra-embryonic tissue using reprogramming technology and analysis of early human development-Grants-in-Aid for Scientific Research Report https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-15H06329/

An object of the present invention is to provide a method for efficiently and conveniently inducing primitive endoderm from pluripotent stem cells. Another object of the present invention is to provide a method for efficiently inducing mesendoderm differentiation using primitive endoderm and epiblasts (primed pluripotent stem cells). Another object of the present invention is to provide a molecular marker for efficiently detecting and isolating primitive endoderm.

The present inventors have made intensive studies to solve the above problems. As a result, we found that primitive endoderm can be efficiently induced by culturing naive pluripotent stem cells in a medium containing BMP (Bone morphogenetic protein) and FGF4 (Fibroblast growth factor 4). Furthermore, they found that by co-culturing the obtained primitive endoderm with prime-type pluripotent stem cells, it was possible to mimic gastrulation and induce mesendoderm differentiation. Furthermore, we found that CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1) and ANPEP (alanyl aminopeptidase, membrane) can be efficiently used as specific markers of primitive endoderm for screening and detection of primitive endoderm, and completed the present invention. .

The present invention provides the following.
[1] A method for preparing primitive endoderm in vitro from pluripotent stem cells, comprising:
A method comprising the step of culturing naive pluripotent stem cells in a medium containing BMP and FGF4 to induce primitive endoderm differentiation.
[2] The medium further contains one or more selected from PDGF (Platelet-Derived Growth Factor), IL-6 (Interleukine-6), TGF (Transforming Growth Factor) β inhibitor, Wnt signal inhibitor and retinoic acid. The method for preparing primitive endoderm according to [1], comprising:
[3] The method for preparing primitive endoderm according to [1], wherein the medium contains BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor.
[4] any of [1] to [3], wherein the BMP is BMP4, BMP2 or BMP6, the PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939 A method for preparing primitive endoderm as described in .
[5] The method for preparing primitive endoderm according to any one of [1] to [4], further comprising the step of purifying primitive endoderm cells using CEACAM1 or ANPEP.
[6] The method for preparing primitive endoderm according to any one of [1] to [5], wherein the pluripotent stem cells are induced pluripotent stem cells.
[7] The method for preparing primitive endoderm according to any one of [1] to [6], wherein the pluripotent stem cells are human pluripotent stem cells.
[8] A step of preparing primitive endoderm by the method according to any one of [1] to [7], and coculturing the obtained primitive endoderm with prime pluripotent stem cells to obtain prime pluripotent A method for preparing mesendoderm, comprising a step of differentiating sex stem cells to mesendoderm.
[9] Myocardium, comprising the step of preparing mesendoderm by the method of [8], and further culturing the obtained mesendoderm to differentiate into myocardial progenitor cells and/or pancreatic progenitor cells. A method for preparing progenitor cells and/or pancreatic progenitor cells.
[10] providing primitive endoderm; Preparation method.
[11] Primitive endoderm cells prepared by the method according to any one of [1] to [7].
[12] A method for isolating primitive endoderm cells from a cell population containing primitive endoderm cells, comprising the step of selecting primitive endoderm cells using CEACAM1 or ANPEP.
[13] A method for detecting primitive endoderm cells in a cell population comprising primitive endoderm cells, comprising the step of detecting primitive endoderm cells using CEACAM1 or ANPEP.
[14] A reagent for detecting primitive endoderm cells, comprising a molecule that specifically binds to CEACAM1 or ANPEP.
[15] A medium for naive pluripotent stem cells containing BMP and FGF4.
[16] The medium for naive pluripotent stem cells of [15], further comprising one or more selected from PDGF, IL-6, TGFβ inhibitors, Wnt signal inhibitors and retinoic acid.
[17] The medium for naive pluripotent stem cells of [15], containing BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor.
[18] any of [15] to [17], wherein the BMP is BMP4, BMP2 or BMP6, the PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939 3. The medium for naive pluripotent stem cells according to .

According to the present invention, primitive endoderm can be easily induced from pluripotent stem cells only by culturing without forced gene expression. Furthermore, by co-culturing primitive endoderm with primed pluripotent stem cells, mesendoderm differentiation mimicking gastrulation in the physiological developmental process, and furthermore, mesendoderm differentiation can be achieved. It can be induced to differentiate into myocardial progenitor cells and pancreatic progenitor cells. The method of the present invention realizes differentiation induction into primitive endoderm, which is extra-embryonic cells, which was difficult with conventional primed pluripotent stem cells, in a simple procedure. This is an epoch-making method that can serve as a basic technology for inducing mature cells and tissues. INDUSTRIAL APPLICABILITY The method of the present invention is useful for elucidation of developmental mechanisms, regenerative medicine, and elucidation and treatment of disease mechanisms at the developmental stage. In addition, the method for selecting and detecting primitive endoderm using the molecular marker of the present invention is useful because it can selectively select and detect primitive endoderm cells.

The figure which shows the procedure of the expression induction of the GATA gene or the SOX gene in pluripotent stem cells (PSC). Photomicrographs showing colony morphology of naïve or primed PSCs overexpressing the GATA gene or SOX gene. Graphs showing temporal changes in expression levels of endogenous GATA4, GATA6, and SOX17 genes in naive-type PSCs or primed-type PSCs overexpressing the GATA gene or SOX gene. Graph showing time-dependent changes in the expression level of the endogenous PDGFRA gene in naive-type PSCs or primed-type PSCs overexpressing the GATA gene or the SOX gene. FIG. 10 shows the results of analysis by flow cytometry of changes over time in the expression level of the endogenous PDGFRA gene in naïve-type PSCs or primed-type PSCs overexpressing the GATA gene or SOX gene. Graph showing changes over time in the expression levels of endogenous genes in naive PSCs in which the GATA6 or GATA4 gene was overexpressed, divided into PDGFRA-positive cells and PDGFRA-negative cells, respectively. A graph comparing the expression levels of endogenous genes on Day 0 and Day 3 (PDGFRA-positive cells) in naive-type or prime-type PSCs overexpressing the GATA6 gene. A graph comparing the expression levels of endogenous genes on Day 0 and Day 3 (PDGFRA-positive cells) in naïve PSCs or primed PSCs overexpressing the GATA4 gene. Graph showing the results of analysis by flow cytometry of changes over time in the expression level of the endogenous PDGFRA gene in naive PSCs (H9) cultured by overexpressing the GATA6 gene and adding FGF4. Graph showing expression levels of endogenous genes on Day 0 and Day 3 (PDGFRA-positive cells) in naïve PSCs (H9) overexpressing the GATA6 gene and cultured in serum-free medium (SFO3) or serum medium (GMEM). Naive PSCs (H9) overexpressing the GATA6 gene and cultured in serum-free medium (SFO3) were immunostained to analyze the expression of GATA6, GATA4, SOX17, or NANOG on Days 0 and 3 (PDGFRA-positive cells). Photo shown. Blue indicates DAPI (nuclear) staining. A diagram showing the results of PCA analysis using all genes in PDGFRA-positive cells (Day 1, Day 3) induced by overexpressing GATA6 in naïve PSCs or primed PSCs. The results for Day 0 are also shown. FIG. 10 shows the results of analyzing the expression levels of primitive streak-related genes or endoderm-related genes in PDGFRA-positive cells (Day 1, Day 3) induced by overexpressing GATA6 in naive or primed PSCs (N=2). The results for Day 0 are also shown. Shows the results of comparing the expression of Top100 genes related to epiblast and primitive endoderm (PrE) in human embryos in cells (H9-naive-GATA6) induced by expressing GATA6 in naive PSCs (Day 0, Day 1, Day 3). figure. Results of ChIP-seq (GATA6, GATA4, SOX17, HNF4A or PDGFRA) in cells (H9-naive-GATA6 and H9-primed-GATA6) induced by expressing GATA6 in naïve or primed PSCs. illustration. Figure showing the results of ChIP-seq (BMP2, BMP6, IL6ST or FRZB) in cells induced by expressing GATA6 in naive or primed PSCs (H9-naive-GATA6 and H9-primed-GATA6). . A diagram showing the results of analyzing the expression levels of BMP2, BMP6, PDGFRA, LEFTY1, IL6ST, or FRZB genes in PDGFRA-positive cells (Day 1, Day 3) induced by overexpressing GATA6 in naive PSCs (N=2). The results for Day 0 are also shown. Photographs showing the results of Western blot analysis of phosphorylation of SMAD1/5/8, MAPK, STAT3 and SMAD2. Fig. 2 shows the results of analysis by flow cytometry of changes over time in the expression level of the endogenous PDGFRA gene in naive PSCs differentiated in a medium containing 7 factors (BMP is BMP2). Fig. 2 shows the results of analysis by flow cytometry of changes over time in the expression level of the endogenous PDGFRA gene in naive PSCs differentiated in a medium containing 7 factors (BMP is BMP6). FIG. 10 shows the results of analysis by flow cytometry of changes over time in the expression level of the endogenous PDGFRA gene in naive PSCs differentiated in a medium containing 7 factors (BMP is BMP4). Graph comparing expression levels of endogenous genes on Day 3 (PDGFRA-positive cells) in naïve PSCs differentiated by GATA6 forced expression or 7-factor (BMP is BMP4) culture. The results for Day 0 are also shown. A diagram (photograph) showing the results of immunostaining analysis of GATA6 and GATA4 or SOX17 expression on Day 3 (PDGFRA-positive cells) in naive PSCs differentiated by GATA6 forced expression or seven-factor (BMP is BMP4) culture. FIG. 3 shows the results of flow cytometry analysis of the expression levels of CD57 and CD75 in naïve PSCs differentiated in a medium containing 7 factors or in primed PSCs forced to express GATA6. FIG. 4 shows the results of comparing the expression level of the CEACAM1 gene between naive PSC-derived PDGFRA-positive cells and primed PSC-derived PDGFRA-positive cells. CEACAM1 expression levels in naïve PSCs (before differentiation induction), GATA6-forced expression naïve PSCs, 7-factor cultured naïve PSCs, primed PSCs (before differentiation induction), or GATA6-forced primed PSCs were analyzed by flow cytometry. The figure which shows a result. A diagram showing the results of flow cytometry analysis of the expression levels of ANPEP and CEACAM1 in naive PSCs (before induction of differentiation), GATA6 forced expression naive PSCs, and 7-factor cultured naive PSCs. Shows the results of comparing the expression levels of visceral endoderm genes, yolk sac genes, and extraembryonic mesoderm genes in naïve PSC-derived PDGFRA-positive cells and primed PSC-derived PDGFRA-positive cells (Day 0, Day 3, reculture 13 days). figure. A diagram (photograph) showing the results of immunostaining analysis of T and OCT3/4 expression in co-cultures of naive PSC-derived PDGFRA-positive cells (primitive endoderm cells) and primed PSCs. When naïve PSCs are subtracted from 7 factors or 1 factor from 7 factors and induced to differentiate with 6 factors, PD03 (MEC inhibitor), LDN193189 (BMP inhibitor), JaKi (JaK inhibitor), A diagram showing the results of analysis by flow cytometry of the expression level of PDGFRA when differentiation was induced by adding Activin and CH (Wnt activator). GP130Y118F chimeric receptor-introduced naïve PSCs were activated with G-CSF, and PDGFRA expression levels were analyzed by flow cytometry when differentiation was induced by adding 6 factors (excluding IL-6) from the 7 factors. figure. FIG. 10 shows the results of flow cytometry analysis of the expression level of CEACAM1 in naive PSCs in which GATA6 was knocked down using shGATA6 and differentiation was induced using 7 factors.

<Method for preparing primitive endoderm>
The method of preparing primitive endoderm in vitro from pluripotent stem cells of the present invention comprises:
naive pluripotent stem cells,
BMP (Bone morphogenetic protein) and FGF4 (Fibroblast growth factor 4)
Inducing primitive endoderm differentiation by culturing in a medium comprising
In addition to BMP and FGF4, the medium contains one or more (preferably two or more, more preferably three or more, more preferably three or more types selected from PDGF, IL-6, TGFβ inhibitors, Wnt signal inhibitors and retinoic acid. preferably contains 4 or more types, particularly preferably all 5 types).
In a more preferred embodiment, the medium contains a TGFβ inhibitor and a Wnt signal inhibitor in addition to BMP and FGF4.

<Pluripotent stem cells>
In the present invention, the pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into many cells existing in the body and that also has the ability to proliferate. Cells are included. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, and spermatozoa. Included are stem cells (“GS cells”), embryonic germ cells (“EG cells”), cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), and the like. Preferred pluripotent stem cells are iPS cells and ES cells. Pluripotent stem cells are preferably derived from mammals, more preferably from primates, and even more preferably from humans.

Methods for producing iPS cells are known in the art, and can be produced by introducing reprogramming factors into arbitrary somatic cells. Here, the initialization factor is, for example, 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 genes or gene products are exemplified, and these reprogramming factors may be used alone or in combination Also good.初期化因子の組み合わせとしては、ＷＯ２００７／０６９６６６、ＷＯ２００８／１１８８２０、ＷＯ２００９／００７８５２、ＷＯ２００９／０３２１９４、ＷＯ２００９／０５８４１３、ＷＯ２００９／０５７８３１、ＷＯ２００９／０７５１１９、ＷＯ２００９／０７９００７、ＷＯ２００９／０９１６５９、ＷＯ２００９／１０１０８４、ＷＯ２００９／ １０１４０７、ＷＯ２００９／１０２９８３、ＷＯ２００９／１１４９４９、ＷＯ２００９／１１７４３９、ＷＯ２００９／１２６２５０、ＷＯ２００９／１２６２５１、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 USA. 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. The combination described in is exemplified.

Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , passaged cells, and cell lines are all included. Specifically, somatic cells are, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue progenitor cells, (3) blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosa cells, enterocytes, splenocytes, pancreatic cells (pancreatic exocrine cells) etc.), differentiated cells such as brain cells, lung cells, kidney cells and adipocytes.

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

Naive pluripotent stem cells can be produced, for example, by the method described below.
A method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014)
5iLFA condition method (Theunissen et al., Cell Stem Cell. 2016 Oct 6; 19(4):502-515.)
A method using an HDAC (histone deacetylase) inhibitor (Guo, G. et al. (2017). Development 144(15): 2748-2763.)
They can also be obtained by culturing primed pluripotent stem cells using a commercially available naive pluripotent stem cell preparation medium such as t2iLGo (Ndiff227 [Takara Bio, Cat. Y40002]).

<Prime type pluripotent stem cells>
Primed-type pluripotent stem cells are pluripotent stem cells with properties similar to epiblasts of post-implantation embryos, but they are general induced pluripotent cells obtained by introducing reprogramming factors into somatic cells. This includes stem cells and human ES cells, which have not been naiveized as described above.
Primed pluripotent stem cells have the following characteristics.
It shows a flat colony morphology and the colony size is larger than the naive type.
As markers, CD75, KLF4 and TFCP2L1 are negative.
The genome is methylated.

<TGFβ inhibitor>
A TGFβ inhibitor is a substance that inhibits the signal transduction from binding of TGFβ to a receptor to SMAD. Substances that inhibit phosphorylation of SMAD by the family include, for example, Lefty-1 (NCBI Accession No. includes 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 20089 ) and derivatives thereof.
The TGFβ inhibitor used in the present invention is preferably A83-01.
As the concentration of the TGFβ inhibitor contained in the culture medium, the concentration that exerts the TGFβ inhibitory effect can be appropriately selected according to the type of TGFβ inhibitor. It is within the range of 100 μM, preferably 0.075-50 μM, more preferably 0.25-10 μM.

<Wnt signal inhibitor>
The Wnt signal inhibitor is not particularly limited as long as it can suppress the Wnt signal pathway involved in the regulation of gene expression and cytoskeleton. 2, IWP-3, IWP-4, IWR-1, 53AH (above porcupine inhibitor), low molecular weight compounds such as KY02111 and their derivatives, proteins such as IGFBP4, DKK1, Wnt-C59, Wnt and Wnt signals Antisense nucleic acids, RNA interference-inducing nucleic acids (e.g., siRNA), competitive peptides, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant-negative mutants, and expression vectors thereof that suppress the expression or function of pathway-constituting proteins Wnt signal inhibitors, which are low-molecular-weight compounds, are preferable, and XAV939 and IWP-4 can be preferably exemplified.
As the concentration of the Wnt signal inhibitor contained in the culture medium, the concentration that exerts the Wnt signal inhibitory effect can be appropriately selected according to the type of Wnt signal inhibitor. It is within the range of 100 μM, preferably 0.075-50 μM, more preferably 0.25-10 μM.

<PDGF>
The PDGF used in step (i) is preferably a protein that takes a dimer structure, such as a dimer by A-type monomer (PDGF-AA), a dimer by B-type monomer (PDGF-BB), A A dimer of a type monomer and a type B monomer (PDGF-AB) is exemplified, but PDGF-AA is preferred.
PDGF is preferably of mammalian origin, preferably of human origin. Human PDGF-A includes, for example, a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM_002607. PDGF includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. Commercially available PDGF may be used, or proteins purified from cells or proteins produced by genetic recombination may be used. The concentration of the PDGF dimer contained in the culture medium is 0.1 ng/ml to 100 ng/ml, preferably 0.5 ng/ml to 50 ng/ml, more preferably 5 ng/ml to 20 ng/ml.

<BMP>
BMP includes at least one BMP selected from the group consisting of BMP2, BMP4 and BMP6, preferably BMP4. BMPs are preferably of mammalian origin, preferably of human origin. Human BMP4 includes, for example, a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: AAH20546.1. BMPs include fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. A commercially available BMP may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. The concentration of BMP contained in the culture solution 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.

<Retinoic acid>
Retinoic acid may be retinoic acid itself or a retinoic acid derivative that retains the differentiation-inducing function of natural retinoic acid. Examples of retinoic acid derivatives include 3-dehydroretinoic acid, 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoic acid ( AM580) (Tamura K, et al., Cell Differ. Dev. 32:17-26 (1990)), 4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8 , 8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoic acid (TTNPB) (Strickland S, et al., Cancer Res. 43:5268-5272 (1983)), and Tanenaga, K.; et al. , Cancer Res. 40:914-919 (1980), retinol palmitate, retinol, retinal, 3-dehydroretinol, 3-dehydroretinal and the like.
The concentration of retinoic acid or its derivative contained in the culture medium is, for example, 1 nM to 1000 nM, preferably 5 nM to 500 nM, more preferably 10 nM to 250 nM.

<FGF4>
FGF4 is preferably of mammalian origin, preferably of human origin. Examples of human FGF4 include proteins having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM_002007. FGF4 includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. Commercially available FGF4 may be used, or proteins purified from cells or proteins produced by genetic recombination may be used. The concentration of FGF4 contained in the culture solution is 0.1 ng/ml to 1000 ng/ml, preferably 1 ng/ml to 500 ng/ml, more preferably 10 ng/ml to 100 ng/ml.

<IL-6>
IL-6 is preferably of mammalian origin, preferably of human origin. Human IL-6 includes, for example, a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: M18403. IL-6 includes fragments and functional variants thereof as long as they have the desired differentiation-inducing activity. Commercially available IL-6 may be used, or proteins purified from cells or proteins produced by genetic recombination may be used. The concentration of IL-6 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 100 ng/ml.

The culture medium used in the primitive endoderm induction step is not particularly limited, but a naive pluripotent stem cell maintenance medium containing BMP and FGF4, preferably further PDGF, IL-6, a TGFβ inhibitor, a Wnt signal inhibitor and retinoin It can be prepared by adding one or more (preferably two or more, more preferably three or more, still more preferably four or more, particularly preferably all five) selected from acids. These factors may be added at different times. For example, IL-6 can be added 48 hours after initiation of induction. As the naive pluripotent stem cell maintenance medium, for example, the following medium can be used.
・t2iLGo
N2B27 + PD0325901 (1 µM) + CHIR99021 (1 µM) + LIF + Go6983 (2-3 µM)
Takashima et al., Cell 158: 1254-1269, 2014
・5iL/AF
N2B27 +PD0325901 (1 μM) +CHIR99021 (1 μM) +SB590885 (0.5 μM) +WH-4-023 (1 μM) +Y-27632 (10 μM) +LIF +Activin A
Theunissen, TW, et al. (2014). Cell Stem Cell 15(4): 471-487.
・tt2iLGo
N2B27 + PD0325901 (1 μM) + LIF + Go6983 (2 μM) + XAV939 (2 μM)
Guo, G., et al. (2017). Development 144(15): 2748-2763.
The medium may contain serum or may be serum-free. If necessary, for example albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics It may also contain one or more substances such as substances, antioxidants, pyruvate, buffers, inorganic salts, cytokines, and the like.

In the primitive endoderm induction step, the naive pluripotent stem cells may be adherently cultured or suspended cultured. In the case of adherent cultured, the culture vessel may be coated and used, or co-cultured with feeder cells or the like. good. Feeder cells to be co-cultured include primary fibroblasts (MEF) derived from mouse embryos treated with mitomycin C, STO cells, SNL cells, OP9 cells, C3H10T1/2 cells, and the like.

In the primitive endoderm induction step, when culture is performed by suspension culture, it is desirable to culture the cells by forming aggregates (also referred to as spheres) in a non-adherent state to the culture vessel, and such culture is particularly Although not limited, a culture vessel that has not been artificially treated (e.g., coating treatment with extracellular matrix or the like) for the purpose of improving adhesion to cells, or a treatment that artificially suppresses adhesion (e.g., poly Hydroxyethyl methacrylate (poly-HEMA), nonionic surfactant polyol (Pluronic F-127, etc.) or phospholipid-like structure (e.g., water-soluble polymer (Lipidure) with 2-methacryloyloxyethylphosphorylcholine as a structural unit ) by using a culture vessel coated with

When adherent culture is performed in the primitive endoderm induction step, it can be performed by culturing using a culture vessel coated with an extracellular matrix. The coating treatment can be performed by adding a solution containing an extracellular matrix to the culture vessel and then removing the solution as appropriate. Here, the extracellular matrix is a supramolecular structure existing outside the cell, and may be naturally derived or artificial (recombinant). Examples include substances such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, and fragments thereof. These extracellular matrices may be used in combination, eg, preparations from cells such as BD Matrigel™.

The culture temperature conditions for culturing naive pluripotent stem cells in the primitive endoderm induction step are not particularly limited, but are preferably about 37°C to about 42°C, and about 37°C to about 39°C, for example. In addition, the culture period can be appropriately determined by a person skilled in the art while monitoring the number of cells and the like. The number of days is not particularly limited as long as primitive endoderm can be obtained, but is, for example, at least 1 day or longer, preferably 2 to 5 days.

<Primitive endoderm>
Primitive endoderm can be obtained by performing the above-described culture steps.
The primitive endoderm contains GATA3, GATA4, GATA6, SOX17, FOXA2 (Forkhead Box A2), HNF4A (Hepatocyte Nuclear Factor 4 Alpha), CER1 (Cerberus 1), OTX2 (Orthodenticle Homeobox 2), PDGFRA (Platelet Derived Growth Factor Receptor Alpha). ), COL4A1 (alpha-1 subunit of collagen type IV), SPARC (Secret protein acidic and rich in cysteine), and the expression of one or more primitive endoderm markers. More preferably, the cell expresses CEACAM1 or ANPEP, which will be described later, in addition to one or more of these markers.

In order to enrich primitive endoderm cells, the primitive endoderm inducing step may be followed by a primitive endoderm selection step. The selected primitive endoderm can be further differentiated into visceral endoderm and yolk sac by further culturing.
Selection can be performed using the expression of one or more types of primitive endoderm-specific markers as described above as an index.
When extracting primitive endoderm cells using primitive endoderm markers, the expression of each marker protein may be used as an index, or the expression of the gene encoding each marker protein (mRNA is expressed) You can use what you are doing as an indicator.

Reagents used for sorting (extracting or detecting) primitive endoderm cells from a cell population containing primitive endoderm cells include reagents having specific affinity for the above primitive endoderm markers. Antibodies, aptamers, peptides, compounds that specifically recognize, and the like can be used, preferably antibodies or fragments thereof. In addition, when examining the gene expression of these markers, primers and probes that hybridize to these marker genes can be used.

Antibodies may be polyclonal or monoclonal antibodies. These antibodies can be generated using techniques well known to those skilled in the art (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Section 11.12-11.13). Specifically, when the antibody of the present invention is a polyclonal antibody, a marker protein expressed in Escherichia coli or the like and purified according to a conventional method, or an oligopeptide having a partial amino acid sequence is synthesized, and then used in non-human animals such as rabbits. can be obtained from the sera of the immunized animals according to a conventional method. On the other hand, monoclonal antibodies can be obtained from hybridoma cells prepared by fusing spleen cells obtained from the above-mentioned 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). Antibody fragments include antibody portions (eg, Fab fragments) or synthetic antibody fragments (eg, single-chain Fv fragments “ScFv”). Antibody fragments such as Fab and F(ab) ₂ fragments can also be produced by methods well known in genetic engineering. If the marker is a membrane protein, it is preferably an antibody against the extracellular domain. A commercially available antibody may be used as the antibody.

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

Methods for sorting (also for extracting or detecting) primitive endoderm cells include, for example, methods using a flow cytometer. In addition, a method of sedimentation using an antibody bound to a carrier, a method of magnetically sorting cells using magnetic beads (e.g., MACS), a method of using a cell sorter using fluorescent labeling, or immobilizing an antibody or the like. exemplified is a method using a carrier (eg, a cell concentration column).

<Method for Extracting or Detecting Primitive Endoderm Cells Using CEACAM1 or ANPEP as an Indicator>
Since the present inventors found that CEACAM1 or ANPEP can be suitably used as a marker for naive pluripotent stem cell-derived primitive endoderm, the present invention aims to identify primitive endoderm cells from a cell population containing primitive endoderm cells. A method of sorting (separating) or detecting is provided, comprising sorting (separating) or detecting primitive endoderm cells using CEACAM1 or ANPEP. Both CEACAM1 and ANPEP may be used.

CEACAM1, also called CD66, is a type I transmembrane glycoprotein expressed in epithelial cells and T cells, and is classified as an intercellular adhesion factor. For example, the amino acid sequence of human CEACAM1 includes the amino acid sequence registered as P13688 (CEAM1_HUMAN) in UniProt Knowledgebase (UniProtKB).

ANPEP, also called Aminopeptidase N (APN), CD13, or PEPN, is a membrane peptidase. For example, the amino acid sequence of human ANPEP is the amino acid sequence registered as P15144 (AMPN_HUMAN) in UniProtKB.

In the present invention, the term "cell population containing primitive endoderm cells" refers to any aggregate of cells containing primitive endoderm cells of any origin, but naive pluripotent stem cells ( A cell population containing primitive endoderm cells obtained by inducing differentiation from human naive pluripotent stem cells (preferably human naive pluripotent stem cells) to primitive endoderm is preferred. The method for inducing differentiation from naïve pluripotent stem cells to primitive endoderm is not limited to the above, and may be obtained by a method of inducing differentiation by introducing a gene such as GATA6.

In the present invention, “sorting (separating) primitive endoderm cells” means increasing the ratio of primitive endoderm cells compared to before sorting (separating). %, 60%, 70%, 80% or 90% or more. More preferably, cells consisting of 100% primitive endoderm cells are obtained.

When using CEACAM1 or ANPEP as an index to select (separate) primitive endoderm cells from a cell population containing primitive endoderm cells, it may be used in combination with the above-mentioned primitive endoderm markers such as GATA4, GATA6, SOX17, and FOXA2. good. This results in increased enrichment of primitive endoderm compared to using CEACAM1 or ANPEP alone.

The selective selection (separation) of marker-positive cells may be the selection (separation) of all marker-positive cells. (Separate) may be used. For example, in a cell population containing primitive endoderm, the expression level of the marker is within the top 50% cells, the top 40% cells, the top 33% cells, the top 30% cells, the top 20% Cells, or cells within the top 10% can be selectively collected.

<Kit for selecting or detecting primitive endoderm cells using CEACAM1 or ANPEP as an indicator>
The present invention also provides kits for sorting or detecting primitive endoderm cells containing reagents that specifically bind to CEACAM1 or ANPEP. The detection reagents contained in this extraction kit are as described above, and include antibodies against CEACAM1 or ANPEP, polynucleotides that hybridize to the CEACAM1 gene or ANPEP gene, and the like. The extraction kit of the present invention can also contain a reagent that specifically binds to CEACAM1 or ANPEP and instructions describing how to use the detection reagent.

<Method for Preparing Mesendoderm and Cardiac Progenitor Cells and/or Pancreatic Progenitor Cells>
The present invention also provides a method for preparing mesendoderm, comprising co-culturing primitive endoderm with primed pluripotent stem cells to differentiate the primed pluripotent stem cells to mesendoderm. Furthermore, the present invention provides a method for preparing myocardial progenitor cells and/or pancreatic progenitor cells, which includes the step of culturing the obtained mesendoderm to differentiate into myocardial progenitor cells and/or pancreatic progenitor cells. Primitive endoderm may be obtained by the above method, or may be obtained by other methods such as forced expression of the GATA6 gene or the like in naive pluripotent stem cells.

In ontogeny, the epiblast induces gastrulation, forms mesoderm and endoderm, and develops and differentiates. Primitive endoderm is present in contact with the epiblast and plays an essential role in gastrulation. Therefore, by co-culturing primitive endoderm and primed pluripotent stem cells (iPS cells and ES cells), it is possible to create a gastrulation state in vitro that is close to physiological early-implantation-stage development. .

By co-culturing the primitive endoderm with primed pluripotent stem cells, the primed pluripotent stem cells differentiate into mesendoderm as an epiblast, and the primitive endoderm differentiates into the yolk sac. Mesendoderm can be identified by expression of the marker molecule T (Brachyury).

For example, by inoculating primed pluripotent stem cells to cultured primitive endoderm cells, primitive endoderm and primed pluripotent stem cells can be co-cultured. As the medium, it is preferable to use a medium for iPS/ES cells such as AK03, mTeSR1, F12/KSR, etc., and extracellular matrix components such as Matrigel may be added for culturing. The number ratio of primitive endoderm and primed pluripotent stem cells to be co-cultured is preferably 1:3 to 3:1, more preferably 1:2 to 2:1, and particularly preferably 1:1. The culture temperature conditions for cocultivation are not particularly limited, but are preferably about 37°C to about 42°C, and about 37°C to about 39°C, for example. The culture period is not particularly limited, but is, for example, at least 12 hours or more, preferably 24 to 48 hours.

Cardiac progenitor cells and/or pancreatic progenitor cells can be obtained by further culturing the mesendoderm. In order to induce differentiation into myocardial progenitor cells, a TGFβ inhibitor, BMP, Wnt signal inhibitor, activin or the like may be added to the medium and cultured. On the other hand, in order to induce differentiation into pancreatic progenitor cells, KGF (keratinocyte growth factor), sonic hedgehog inhibitor, retinoic acid, BMP inhibitor, protein kinase C activator (such as phorbol ester), etc. are added to the culture medium. It is recommended to culture as follows.
Cardiac progenitor cells can be identified by expression of the marker molecule Troponin T (TnT). It can also be identified by the expression of PDX1, a pancreatic progenitor cell marker molecule.

<Medium for inducing differentiation into primitive endoderm>
The present invention also provides a medium for naive pluripotent stem cells for inducing differentiation to primitive endoderm containing BMP and FGF4, preferably BMP and FGF4 and PDGF, IL-6, TGFβ inhibitor, Wnt signal inhibitor and For induction of differentiation into primitive endoderm, including one or more (preferably two or more, more preferably three or more, still more preferably four or more, particularly preferably all five) selected from retinoic acid A culture medium for naive pluripotent stem cells is provided. These may be media prepared in advance so that each component is contained at concentrations effective for inducing differentiation into primitive endoderm, or media prepared by adding each component immediately before use. good. These factors may be added at different times. For example, IL-6 can be added 48 hours after initiation of induction. Therefore, some factors may be provided separately. The medium (kit) may be accompanied by an instruction manual describing usage and preparation methods. The medium for inducing differentiation into primitive endoderm can further contain other components necessary for culturing naive pluripotent stem cells.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following embodiments.

Materials and methods
Cell culture
Human primed pluripotent stem cell (PSC) lines (H9ES (embryonic stem) cells, H1ES cells, AdiPS cells) were prepared under Conventional conditions (referred to as 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], 0.1 mM 2-mercaptoethanol [Sigma-Aldrich, Cat. Cells were washed every 5-7 days with Dissociation Buffer (DB; 0.025% Trypsin [Thermo Fisher Scientific, Cat. 15090-046], 1mg/ml Collagenase IV [Thermo Fisher Scientific, Cat. 17104-019], 20% KSR, 1mM CaCl ₂ ) was used to detach like a small clump and passage.

Human naive pluripotent stem cell lines (H9ES, H1ES, AdiPS cell-derived) are 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 [hLIF; Peprotech, Cat.300-05], 3 μM Go6983 [Go; Tocris, Cat.2285]) was used to maintain on MEFs. Cells were detached and passaged every 3-5 days using Accutase (Sigma-Aldrich, Cat.A6964).

Naive H9PSCs were established by a method using an HDAC inhibitor (Guo, G. et al. (2017). Development 144(15): 2748-2763.). Upon establishment, the EOS plasmid was introduced into primed H9PSCs by electroporation (primed H9 EOS). A drug resistance marker gene (puromycin resistance) is incorporated into the plasmid, and after introduction into the cells, drug selection is performed using Puromycin Dihydrochloride (Thermo Fisher Scientific, Cat.A1113802). was selected. Primed H9 EOS was detached into single cells by trypsin/EDTA (Nacalai Tesque, Cat.32777-15), and 1×10 ⁵ cells/cm ² of cells were added to 10 μM Y-27632 (Wako, Cat.034-15). 24024) on MEFs in F12/KSR medium. From the next day, cRM1 (Ndiff, 1μM PD03, 10ng/ml hLIF, 1mM Valproic acid sodium salt [VPA; Sigma-Aldrich, Cat.P4543]) was incubated for 48 hours, and then cRM2 (Ndiff, 1μM PD03, 10ng/ml hLIF, Cells were maintained with 2 μM Go, 2 μM XAV939 [Sigma-Aldrich, Cat.X3004]). From about passage 3 to 5, most of the cells form dome-shaped colonies. At this point, it was established by switching to t2iLGo medium and culturing.

In some experiments, 5iLFA conditions were also used to establish naive H9 (Theunissen et al., Cell Stem Cell. 2016 Oct 6;19(4):502-515.). Primed H9 were detached into single cells by trypsin/EDTA and 1×10 ⁵ cells/cm ² were seeded onto MEFs in F12/KSR medium supplemented with 10 μM Y-27632. 5iLFA medium (Ndiff, 1μM PD03, 1μM CH, 1μM WH-4-023[A Chemtek H620061], 0.5μM SB590885[R and D 2650], 10μM Y-27632, 10ng/ml hLIF, 20ng/ml Activin A [R&D, Cat.388-AC], 8ng/ml bFGF) and culture was continued. When most of the cells formed dome-shaped colonies (passage 3-5), they were established by switching to t2iLGo and maintaining it.

Naive H1 PSCs and AdiPS were established by a method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014). Plasmids capable of inducing overexpression by DOX were electroporated and introduced into H1 and AdiPS (H1 NK2, AdiPS NK2). A drug resistance marker gene (neomycin resistance) is incorporated into the plasmid, and after introduction into the cells, drug selection is performed using Geneticin (Thermo Fisher Scientific, Cat.10131035) to select cells into which the plasmid has been introduced. did. Primed H1 NK2, AdiPS NK2 were detached into single cells by trypsin/EDTA, and 1×10 ⁵ cells/cm ² cells were added to F12/F12 with 10 μM Y-27632 (Wako, Cat.034-24024). KSR medium was plated onto MEFs. On the following day (day 1), 1 μg/ml doxycycline hyclate (Dox; Sigma-Aldrich, Cat.D9891) was added. Switch to 2iL (1 μM PD03, 1 μM CH, LIF)+Dox medium from Day2 and culture for about 1 week. After that, it was established by switching to t2iLGo and maintaining it.

primitive endoderm induction
Naive PSCs cultured on MEFs were detached with Accutase and harvested, seeded on gelatin-coated dishes, and cultured in t2iLGo containing 10 μM Y-27632 (ROCK inhibitor) at 37°C for 1-2 hours. removed. They are then resuspended in their respective induction medium and induction initiated upon seeding.

When induced by gene overexpression, cells were plated at 1×10 ⁵ /cm ² on Fibronectin (Millipore, Cat.FC010). Serum medium (Glasgow Minimum Essential Medium [GMEM; Sigma-Aldrich, Cat.G5154], 15% FBS [Thermo Fisher Scientific, Cat. 10437028], 2mM L-Glutamine [Thermo Fisher Scientific, Cat. 25030081], 0.1% bovine serum albumin (BSA; Wako, Cat.012-23881), 50μM 2-mercaptoethanol, 25ng/ml recombinant human FGF4 (FGF4; Peprotech, Cat.100-31), 1μg/ml heparin sodium (Wako, Cat. 081-00131) added did. 0.1 μg/ml Dox was added until 48 hours after initiation of induction.

When induced with compounds, cells were seeded at 5×10 ⁴ /cm ² on iMatrix-511 silk (MAX, Cat.892021). Induction medium contains 25ng/ml FGF4, 1μg/ml heparin sodium, 10-200ng/ml recombinant human BMP-4 (BMP-4; R&D, Cat.314-BP), 10ng/ml recombinant human PDGF-AA in Ndiff227 medium. (PDGF-AA, Peprotech, Cat.100-13A), 10ng/ml recombinant human IL-6 (IL-6; Oriental Yeast, Cat.47066000), 1μM XAV939, 3μM A83-01 (Tocris, Cat.2939), 0.1 μM retinoic acid (RA; Sigma-Aldrich, Cat.R2625) was used. IL-6 was added 48 hours after initiation of induction. In some experiments, BMP-4 was replaced with 10-500ng/ml recombinant human BMP-2 (BMP-2; Oriental Yeast, Cat.47304000), or 50ng/ml recombinant human BMP-6 (BMP-6). 6; Peprotech, Cat.120-06) was used.

FACS analysis/sorting
Primitive endoderm-like cells and primed PSCs were detached into single cells by trypsin/EDTA, and naive PSCs were detached by Accutase. Thereafter, blocking was performed on ice for 30 minutes using HBSS (Thermo Fisher Scientific, Cat.14185052) containing 1% BSA (Sigma-Aldrich, Cat.A2153). Biotinylated anti-PDGFRA antibody (R&D, Cat.BAF322), anti-CEACAM1+5 antibody (abcam, Cat.ab91213), anti-ANPEP antibody (Biolegend, Cat.301703), Dylight650-anti-CD75 antibody (novusbio, Cat.NBP2-47890) , BV421-anti-CD57 antibody (BD, Cat.563896) was added to each combination and incubated on ice for 30 minutes. After washing, Streptavidin-APC (Biolegend, Cat.405207), Streptavidin-PE (eBioscience, Cat.12-4317-87), and Streptavidin-BV421 (Biolegend, Cat.405226) were added and incubated on ice for 20 minutes. BD LSR Fortessa (BD) was used for FACS analysis, and FACS AriaII (BD) was used for sorting. Flow Jo V10.2 software was used for data analysis.

Induction from primitive endoderm to visceral endoderm and yolk sac cells Three day old cells were induced to primitive endoderm and positive cells were purified by flow cytometry using PDGFRA or CEACAM1 antibody. After that, culture was continued on MEFs using DMEM+10% FBS medium. RNA was extracted on the 13th day, and differentiation into visceral endoderm and yolk sac cells was confirmed.

Reverse Transcription Quantitative Real-time PCR
Total RNA was extracted with RNeasy kit (Qiagen, Cat.74106), and cDNA was synthesized from 1000 ng of RNA using SuperScriptIV (Thermo Fisher Scientific, Cat.18090050) and oligo-dT primer. Real-time PCR uses TaqMan Fast Universal Master Mix (Thermo Fisher Scientific, Cat.4364103) and TaqMan probe, or PowerUP Sybr Green Master Mix (Thermo Fisher Scientific, Cat.A25743), and QuantStudio3 ( Thermo Fisher Scientific) was used. Analysis after real-time RT-PCR reaction was performed using QuantStudio Design&Analysis Software v1.4.1.

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

Co-culture of primitive endoderm cells and primed PSCs
PDGFRA-positive cells were collected by FACS sorting from cells induced by GATA6 overexpression or compounds. Harvested cells were replated on iMatrix511-coated glass slides at a density of 3×10 ⁵ cells/cm ² . The next day, primed PSCs were detached with DB (a mixture of collagenase and trypsin) in small clumps and plated on top of the reseeded cells on the slide. The cells were cultured in a prime ES cell medium (AK03, mTeSR1, F12/KSR, etc.) alone or in a prime ES cell medium supplemented with Matrigel (5%).

result
Human naive and primed PSC-derived PDGFRA-positive cells are distinct populations
Human primed pluripotent stem cells (H9-EOS cells) transfected with EOS vector into human primed ES cells (H9-primed cells) and human iPS cells (AdiPS-primed cells), AiPS-EOS cells) were treated with HDAC inhibitors, human naive PSCs were induced and maintained in t2iLGo medium, and reset cells were established (H9-naive cells, AiPS-naive cells).
Overexpression of GATA6, GATA4, and SOX17 in mouse ES cells can induce primitive endoderm (PrE) and establish XEN cells. Overexpression of GATA6, GATA4 and SOX17 in mouse ES cells is known to induce mouse PrE. Therefore, plasmids expressing GATA6, GATA4 or SOX17 under DOX induction were introduced into H9-naive and H9-primed cell lines (Fig. 1A). When DOX was added to H9-naive and differentiated in serum, the naive dome-shaped colonies became flat and differentiated in both cases of overexpression (Fig. 1B). As a result of confirming gene expression, it was found that in the cell line overexpressing GATA6, PrE markers GATA4, GATA6, and SOX17 were expressed from the human naive type from day 2 and increased until day 4 (Fig. 1C, top). . When GATA6 was overexpressed in the primed type, the expression of GATA4, 6, and SOX17 was induced (Fig. 1C, bottom). It was found that overexpression of GATA4 also induced gene expression, though not as much as GATA6. On the other hand, cells overexpressing SOX17 could not express GATA4, GATA6 and SOX17(Endo) in the naive type. Even in the primed type, only slight gene induction was observed, but the expression could not be induced to the extent that GATA6 was overexpressed.

In mouse blastocysts, PrE is PDGFRA positive. Cells differentiated from mouse ES cells to PrE are also known to express PDGFRA. It has been reported that PDGFRA is expressed in human embryo PrE by single cell RNA sequencing analysis (Petropoulos, S. et al. (2016). Cell 165(4): 1012-1026. and Blakeley, P. et al. (2015). Development 142(18): 3151-3165.). When the expression of PDGFRA was confirmed by qPCR in the induced cells, it was found that expression of GATA4 and 6 increased the expression of PDGFRA in the same manner as in the naive and primed cells (Fig. 1D). Therefore, the expression of PDGFRA was confirmed by flow cytometry (Fig. 1E). When GATA6 and GATA4 were overexpressed, PDGFRA expression was observed from day 1, and on day 3, 41% of GATA6 and 20% of GATA4 cells were PDGFRA positive. On the other hand, when SOX17 was overexpressed, PDGFRA expression was not observed until day 3 (Fig. 1D, E). Subsequently, gene expression in PDGFRA-positive cells was examined. When naive-derived PDGFRA-positive cells were purified and examined for their expression, PrE markers GATA3, 4, SOX17, HNF4A, FOXA2, and COL4A1 were expressed in PDGFRA-positive cells of H9-naive-GATA6, while pluripotent cells were expressed. Expression of sex markers OCT3/4 and NANOG decreased. On the other hand, H9-naive-GATA4 also induced GATA6 and HNF4A, but failed to induce the PrE marker as much as GATA6 (Fig. 1F).

On the other hand, when we examined the gene expression of PDGFRA-positive cells induced by H9-primed-GATA6, we found that PrE genes such as GATA4, SOX17, HNF4A, FOXA2, CER1, and OTX2 were not expressed, and PDGFRB, Mesoderm- and mesenchymal-related genes such as KDR, SNAI2, CDH11, and VIMENTIN were expressed (Fig. 1G). When H9-naive-GATA4 and H9-primed-GATA4 were compared, PrE-related genes were not expressed in H9-primed, but mesodermal genes were expressed (Fig. 1H). From the above, it was found that the naive type induces cells expressing PrE-related genes, while the prime type induces cells expressing mesodermal genes.

Human naive PSC-derived PDGFRA-positive cells were induced using serum-free SFO3 medium or N2B27 medium in order to more clearly see differentiation signals equivalent to PrE. In H9-naive-GATA6, when GATA6 was overexpressed and induced by adding FGF4, about 30% of the cells became PDGFRA-positive on Day 1, and 80% of the cells were PDGFRA-positive on Day 3 (Fig. 2A). . When PDGFRA-positive cells were sorted and expression was confirmed, PrE-related genes such as GATA4, SOX17, FOXA2, HNF4A, COL4A1, and SPARC were expressed at almost the same level in serum (GMEM) and non-serum (SFO3) conditions. (Figure 2B). Immunostaining confirmed that overexpression of GATA6 induces GATA4 and SOX17 (Fig. 2C). In order to analyze the properties of PDGFRA cells more deeply, we performed a comprehensive analysis using RNA sequencing. When PCA analysis was performed using all genes, H9-naive and primed pluripotent stem cells differed in PC1, and PDGFRA-positive cells induced by overexpressing GATA6 (Day1, Day3) Similar to the undifferentiated state, PC1 was different between the naive type and the primed type, and PC2 changed in the same direction (Fig. 2D). That is, it is suggested that they are different cell populations. From Figure 1G, it was suggested that the cells induced from H9-primed are mesodermal cells. It is assumed to be expressed and induced to mesoderm cells (Fig. 2E). On the other hand, naïve mice did not induce such mesodermal genes. Comparing the expression of Top100 genes for Epiblasts and PrE in H9-naive induced cells and human embryos, day 0 was closer to epiblasts and day 3 was closer to PrE (Fig. 2F). From the above, it is considered that PDGFRA-positive cells induced from human naive pluripotent stem cells are cells close to PrE.

GATA6 modifies the signal
Overexpression of GATA6 resulted in the differentiation of the naive type to PrE, whereas the primed type induced different cells expressing mesodermal genes. and H9-primed-GATA6 were used to perform ChIP-seq. GATA6 actually binds to genes important for PrE, such as GATA6, GATA4, SOX17 and HNF4A (Fig. 3A). It was also found to bind to PDGFRA at the same time. Furthermore, it bound to secreted factors or receptors of BMP2, BMP6, IL6ST, and FRZB (Fig. 3B). These were also confirmed to bind in ChIP-qPCR.
When the gene expression of these signal-related factors was examined after induction, BMP2, BMP6, and FRZB were indeed elevated in PDGFRA after induction (Fig. 3C). On the other hand, LEFTY and IL6ST were expressed from PSCs and continued to be expressed after induction of differentiation. When the protein activation was examined after GATA6 overexpression, suppression of SMAD1/5/8 phosphorylation, MAPK phosphorylation, STAT3 phosphorylation, and SMAD2 phosphorylation was observed (Fig. 3D). This suggests the possibility that GATA6 induces the PrE gene group and at the same time induces the secretion of BMP and PDGFA, and on the other hand suppresses the Wnt signal by inducing FRZB.

signal cocktail induction
Gene expression after GATA6 overexpression and ChIP sequencing revealed BMP secretion, activation of STAT3 signaling, induction of PDGFRA, and suppression of activin and Wnt signaling in H9-naive cells. PDGF-AA, BMP2, IL-6, A83-01, and XAV939 were added without overexpressing PrE to examine whether PrE was induced. In addition, since it has been reported that FGF4 and RA act positively on PrE induction in mice, 7 factors including FGF4 and RA (retinoic acid) were added to the above factors to induce PrE (Fig. 4A). As a result, approximately 28.6% of PDGFRA-positive cells were induced from the naive type (Fig. 4B, C). Induction was observed even when BMP4 and 6, which belong to the same family as BMP2, were substituted (Fig. 4B, C). Compared with BMP2 and BMP6, since BMP4 induces the most PDGFRA-positive cells, experiments were performed with BMP4 added. When the induced PDGFRA-positive cells were purified and the gene expression was confirmed, PrE-related genes such as GATA4, 6, SOX17, PDGFRA, HNF4A, and FOXA2 were expressed, confirming that they could be induced into PrE (Fig. 4D). ). Immunostaining also revealed that GATA6+GATA4+SOX17+ cells could be induced similarly to GATA6 overexpression (FIG. 4E).
PDGFRA-positive cells were also induced in H1 naive PSCs and AdiPS by the 7-factor-containing medium, and PrE-related genes were similarly expressed. Furthermore, comprehensive analysis by RNA sequencing revealed that PrE-related genes were induced in GATA6-overexpressing cells as well.

When novel human PrE-specific surface marker naive PSCs are cultured with bFGF+ACTIVIN (TGFB), they transition to the prime type in about 10 days. It was reported that naïve PSCs express the surface antigen CD75 and primed express surface CD57. When the naive type was induced with PrE chemical (above 7 factors), the expression of the naive marker CD75 in the PDGFRA-positive cells gradually decreased, and the prime marker CD57 was not expressed (Fig. 5A). Conversely, when GATA6 was overexpressed in the primed form to induce PDGFRA-positive cells, the PDGFRA-positive cells were CD57+CD75- (Fig. 5A). From the above, naive-derived PrE was PDGFRA+CD75+/-CD57-, and prime-derived cells were PDGFRA+CD75-CD57+, and were successfully separated according to surface antigens.

On the other hand, CD75 cannot completely separate naive pluripotent stem cells, which are epiblasts, from PDGFRA-positive cells, which are PrE. Since PDGFRA is not PrE-specific, we searched for surface antigens that are specifically expressed by PrE and not in the epiblast and mesoderm. We searched for specific surface antigens on the condition that GATA6 did not bind directly in ChIP sequencing, was expressed in PDGFRA-positive PrE in RNA sequencing, was not expressed in the primed type, and was expressed in a published paper. However, CECAM1 was identified. When the RNAs of naive and primed PDGFRA-positive cells were compared by qPCR, expression of CEACAM1 was observed only in naive-derived PrE (Fig. 5B). When co-stained with PDGFRA on Day 3 and compared by flow cytometry, PDGFRA-positive cells were CECAM1-positive (Fig. 5C). On the other hand, prime-derived PDGFA-positive cells do not express CEACAM1. From this, it became clear that CEACAM1 can be used as a PrE marker.

In addition, when naive PSCs were induced to differentiate into PrE by chemical (the seven factors mentioned above), the expression of ANPEP was also increased in PDGFRA-positive cells as well as CEACAM1 (Fig. 5D). Similar results were obtained when GATA6 was overexpressed in naive PSCs. From this, it became clear that ANPEP can also be used as a PrE marker.

PrE re-culture and function
PDGFRA-positive cells induced by H9-naive and H9-primed were sorted and recultured. , GPC3), but not from H9-primed except for PDPN (Fig. 6A). Similarly, PDGFRA-positive PrE cells induced with 7 factors expressed the yolk sac marker genes AFP, VIL1, GPC3, FOXA1, PDPN, and DAB2. They could be maintained and cultured for more than 10 passages in medium containing the factor, and the maintained cells expressed genes associated with visceral endoderm-yolk sac cells and genes associated with extra-embryonic mesenchymal cells). When gene expression was confirmed by RNA sequencing, PDGFRA-positive PrE cells derived from the naive type actually expressed yolk sac markers, and VE/YE could be induced from the H9-naive type via PrE, but induced from the primed type. Can not.

PrE induces prime-type PSCs to mesendoderm In human development, PrE exists in the lower layer of the bilaminar epiblast, and the bilaminar epiblast forms a primitive streak and differentiates into mesoderm cells. In this case, PrE is considered to play an important role. Whether the induced PrE cells exhibited such ability was observed by co-culture with PSCs. After 24 hours, PSCs became T-positive cells, and after 48 hours, T-positive cells migrated towards PrE (Fig. 6B). This indicates that PrE was able to induce primed PSCs into the mesendoderm and recapitulate the process of gastrulation during early development in vitro.
Based on the above, it is possible that the naive-derived PDGRA-positive cells not only express genes similar to PrE, but also have functional capabilities equivalent to PrE.

BMP, FGF, and GATA6 cooperate to induce PrE.
In order to investigate the importance of each signal in PrE induction by 7 factors, 1 factor was subtracted from each of the above 7 factors, and differentiation was induced by 6 factors (Fig. 7A). Alternatively, PD03 (MEC inhibitor), LDN193189 (BMP inhibitor), JaK inhibitor (JaKi), Activin, and CH (Wnt activator) were added to the 6 factors (Fig. 7A). As a result, when FGF4 or BMP was excluded, almost no PDGFRA ⁺ cells were expressed, indicating that FGF and BMP are essential for PrE induction. On the other hand, the number of PDGFRA-positive cells decreased slightly to 31.5% when the ACTIVIN inhibitor was removed and 33.2% when the Wnt signal inhibitor was removed. The cells were found to disappear. From the above, it was found that activin and Wnt inhibit primitive endoderm differentiation (FIG. 7A, bottom). Although the data are not shown, PDGFRA, CEACAM1 and ANPEP positive cells cannot be induced by FGF alone or BMP4 alone. was 9.1%. When differentiation was induced with four factors (FGF4, BMP4, XAV939, A83-01), 22.3% of PDGFRA, CEACAM1 and ANPEP positive cells were obtained. These results suggest that FGF and BMP are essential for PrE induction, and that TGFβ inhibitor and Wnt signaling inhibitor should be used in addition to these.
On the other hand, when IL-6 was removed, PDGFRA-positive cells decreased to 33.2%, but when JAKi was added, the cells did not proliferate, and almost no cells were present in D3. Therefore, when a low concentration of 100 nM JAKi was added, PDGFRA-positive cells almost disappeared (FIG. 7A, bottom).
GP130Y118F chimeric receptor, which activates GP130 and activates STAT3, was introduced into naive PSCs to induce PrE. It was found that the expression of the chimeric receptor from D0 decreased the expression of PDGFRA-positive cells, whereas the expression from D2 effectively induced PDGFRA-positive cells (Fig. 7B). From this, it was found that the JAK-STAT signal is important for the maintenance of naive pluripotent stem cells in humans and at the same time plays an important role in PrE (Fig. 7B).

On the other hand, shGATA6 was used to knock down GATA6, and 7 factors were used to induce PDGFRA-positive cells. However, neither PDGFRA-positive cells nor CEACAM1-positive cells were induced, indicating that GATA6 is a key gene essential for PrE induction (Fig. 7C).

Although not shown in the data, when the inner cell mass (ICM) was isolated from preimplantation marmoset embryos and induced with 7 factors, a large amount of SOX17-positive primitive endoderm was induced. From this, it can be seen that it can be used not only for humans but also for non-human primates.

Claims (18)

A method for preparing primitive endoderm in vitro from pluripotent stem cells, comprising:
A method comprising the step of inducing primitive endoderm differentiation by culturing naive pluripotent stem cells in a medium containing BMP (Bone morphogenetic protein) and FGF4 (Fibroblast growth factor 4). The medium further contains one or more selected from PDGF (Platelet-Derived Growth Factor), IL-6 (Interleukine-6), TGF (Transforming Growth Factor) β inhibitor, Wnt signal inhibitor and retinoic acid. Item 1. The method for preparing primitive endoderm according to item 1. The method for preparing primitive endoderm according to claim 1, wherein the medium contains BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor. BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, TGFβ inhibitor is A83-01, Wnt signal inhibitor is XAV939, according to any one of claims 1 to 3 A method for preparing primitive endoderm. 5. The primitive endoderm according to any one of claims 1 to 4, further comprising a step of purifying primitive endoderm cells using CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1) or ANPEP (alanyl aminopeptidase, membrane). preparation method. The method for preparing primitive endoderm according to any one of claims 1 to 5, wherein the pluripotent stem cells are induced pluripotent stem cells. The method for preparing primitive endoderm according to any one of claims 1 to 6, wherein the pluripotent stem cells are human pluripotent stem cells. A step of preparing primitive endoderm by the method according to any one of claims 1 to 7, and co-culturing the obtained primitive endoderm with primed pluripotent stem cells to produce primed pluripotent stem cells A method for preparing mesendoderm, comprising a step of differentiating to mesendoderm. Myocardial progenitor cells and / Or a method for preparing pancreatic progenitor cells. A method for preparing mesendoderm, comprising the steps of providing primitive endoderm, and co-culturing the primitive endoderm with primed pluripotent stem cells to differentiate the primed pluripotent stem cells to mesendoderm. Primitive endoderm cells prepared by the method according to any one of claims 1-7. A method of isolating primitive endoderm cells from a cell population comprising primitive endoderm cells, the method comprising sorting primitive endoderm cells using CEACAM1 or ANPEP. A method of detecting primitive endoderm cells in a cell population comprising primitive endoderm cells, the method comprising detecting primitive endoderm cells using CEACAM1 or ANPEP. A reagent for detecting primitive endoderm cells, comprising a molecule that specifically binds to CEACAM1 or ANPEP. Naive pluripotent stem cell medium containing BMP and FGF4. 16. The medium for naive pluripotent stem cells according to claim 15, further comprising one or more selected from PDGF, IL-6, TGFβ inhibitors, Wnt signal inhibitors and retinoic acid. 16. The medium for naive pluripotent stem cells according to claim 15, comprising BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor. BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, TGFβ inhibitor is A83-01, Wnt signal inhibitor is XAV939, according to any one of claims 15-17 Medium for naive pluripotent stem cells.

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