WO2019093340A1 - Method for inducing primitive endoderm from naive pluripotent stem cells - Google Patents

Abstract

This method for preparing primitive endoderm from pluripotent stem cells in vitro includes a step in which naive pluripotent stem cells are cultured in a culture medium including at least one substance selected from bone morphogenetic protein (BMP) and fibroblast growth factor 4 (FGF4), and preferably also platelet-derived growth factor (PDGF), interleukine-6 (IL-6), TGFβ inhibitor, Wnt signal inhibitor, and retinoic acid, and primitive endoderm differentiation is induced.

Description

Primitive endoderm induction method from naive type pluripotent stem cells

The present invention relates to a method of primordial endoderm induction from naive pluripotent stem cells. The present invention also relates to a medium suitably used to derive primitive endoderm from naive type pluripotent stem cells. The present invention also relates to molecular markers for efficiently detecting and separating primitive endoderm. The invention further relates to methods of mimicking gastrulation by co-culture of primitive endoderm and epiblast and inducing mesendoderm differentiation.

At the developmental stage of mammals, an inner cell mass is formed inside the blastocyst, from which epiblasts and primitive endoderm appear, and the epiblasts undergo primitive intestinal formation and become embryonic mesendoderm While differentiating, primitive endoderm differentiates into extraembryonic tissues, in particular the yolk sac. Reproduction of these developmental processes by pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells is an analysis of the developmental mechanism, elucidation of the mechanisms of diseases at the developmental stage, etc. Useful for
However, human ES cells and iPS cells were classified into the more advanced primed type as compared to mouse pluripotent stem cells, and it was difficult to differentiate into epiblasts and primitive endoderm.
Therefore, Takashima, who is one of the present inventors, is a naive type pluripotent that resets human pluripotent stem cells to the same state as the basal state by expressing two genes of NANOG and KLF2 in human pluripotent stem cells. We have succeeded in obtaining sexual stem cells (Non-patent Document 1), and use this to analyze the induction and early development of primitive endoderm (Non-patent document 2).

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

An object of the present invention is to provide a method for efficiently and primitively inducing primitive endoderm from pluripotent stem cells. Another object of the present invention is to provide a method for efficiently inducing mesodermal differentiation using primitive endoderm and epiblast (primed pluripotent stem cells). Another object of the present invention is to provide a molecular marker for efficiently detecting and separating primitive endoderm.

The present inventors diligently studied to solve the above problems. As a result, it was 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, it was found that co-culture of the obtained primitive endoderm with primed pluripotent stem cells mimics gastrulation to induce mesendoderm differentiation. Furthermore, the present invention was completed based on the finding that CEACAM1 (carcinoembrytic antigen related cell adhesion molecule 1) and ANPEP (alanyl aminopeptidase, membrane) can be efficiently used for selection and detection of primitive endoderm as a specific marker of primitive endoderm. .

The present invention provides the following.
[1] A method for preparing primitive endoderm in vitro from pluripotent stem cells,
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 of preparing primitive endoderm according to [1], which comprises.
[3] The method for preparing primitive endoderm described in [1], wherein the medium comprises BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor.
[4] Any one of [1] to [3], wherein BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939 A method of preparing primitive endoderm as described in.
[5] The method for preparing primitive endoderm of 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 of [1] to [5], wherein the pluripotent stem cells are induced pluripotent stem cells.
[7] The method of preparing primitive endoderm according to any of [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 of [8] [1] to [7], and copriming the obtained primitive endoderm with primed pluripotent stem cells for primed pluripotency A method for preparing mesendoderm, comprising the step of differentiating an adult stem cell to mesendoderm.
[10] A myocardium including the steps of preparing mesendoderm by the method described in [9] [8], and further culturing the obtained mesendoderm to differentiate it into myocardial progenitor cells and / or pancreatic progenitor cells. Method of preparing precursor cells and / or pancreatic precursor cells.
[10] Providing primitive endoderm, and co-culturing the primitive endoderm with primed pluripotent stem cells to differentiate primed pluripotent stem cells to mesoderm. Method of preparation.
[11] Primitive endoderm cells prepared by the method according to any one of [1] to [7].
[12] A method for separating 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 containing primitive endoderm cells, comprising the step of detecting primitive endoderm cells using CEACAM1 or ANPEP.
[14] A reagent for detecting primitive endoderm cells, which comprises a molecule that specifically binds to CEACAM1 or ANPEP.
[15] A medium for naive type pluripotent stem cells comprising BMP and FGF4.
[16] The medium for naive type pluripotent stem cells according to [15], further comprising one or more selected from PDGF, IL-6, a TGFβ inhibitor, a Wnt signal inhibitor and retinoic acid.
[17] The medium for naive type pluripotent stem cells according to [15], which comprises BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor.
[18] Any of [15] to [17], wherein BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939 The culture medium for naive type pluripotent stem cells described in 4.

According to the present invention, primitive endoderm can be easily derived from pluripotent stem cells simply by culture procedures without forced expression of a gene. Furthermore, co-culture of primordial endoderm with primed pluripotent stem cells can mimic gastrulation in physiological developmental processes to achieve mesendoderm differentiation, and furthermore, from mesendoderm Differentiation can be induced to myocardial progenitor cells and pancreatic progenitor cells. The method of the present invention realizes differentiation induction to primitive endoderm, which is an extraembryonic cell which is difficult in conventional prime type pluripotent stem cells, by a simple procedure, and is functionally functional using pluripotent stem cells. It is an innovative method that can be a basic technology to derive mature cells and tissues. The method of the present invention is useful for elucidation of developmental mechanism, regenerative medicine, elucidation of the mechanism of disease at the developmental stage, treatment and the like. In addition, the method for sorting / detecting primitive endoderm using the molecular marker of the present invention is useful because it can selectively sort / detect primitive endoderm cells.

FIG. 2 shows the procedure for inducing expression of GATA gene or SOX gene in pluripotent stem cells (PSC). Photomicrograph showing colony morphology of naive PSC or primed PSC overexpressing GATA gene or SOX gene. The graph which shows the time-dependent change of the expression level of the endogenous GATA4, GATA6, SOX17 gene in naive type PSC or prime type PSC which overexpressed GATA gene or SOX gene. The graph which shows the time-dependent change of the expression level of the endogenous PDGFRA gene in naive type PSC or primed PSC which overexpressed GATA gene or SOX gene. The figure which shows the result of having analyzed the time-dependent change of the expression level of the endogenous PDGFRA gene in naive type PSC or prime type PSC which overexpressed GATA gene or SOX gene by flow cytometry. The naive type PSC which overexpressed GATA6 gene or GATA4 gene, it divided into a PDGFRA positive cell and a PDGFRA negative cell, and the graph which showed the time-dependent change of the expression level of the endogenous gene in each. The graph which compared the expression level of the endogenous gene in Day0 and Day 3 (PDGFRA positive cell) in naive type PSC or prime type PSC which overexpressed GATA6 gene. The graph which compared the expression level of the endogenous gene in Day0 and Day 3 (PDGFRA positive cell) in naive type PSC or prime type PSC which overexpressed GATA4 gene. The graph which shows the result of having analyzed the time-dependent change of the expression level of the endogenous PDGFRA gene in the naive type PSC (H9) which overexpressed GATA6 gene and culture | cultivated by adding FGF4 by flow cytometry. The graph which showed the expression level of the endogenous gene in Day0 and Day 3 (PDGFRA positive cell) in naive PSC (H9) which overexpressed GATA6 gene and was culture | cultivated by non-serum medium (SFO3) or serum medium (GMEM). The results of immunostaining analysis of expression of GATA6, GATA4, SOX17 or NANOG in Day 0 and Day 3 (PDGFRA positive cells) in naive PSC (H9) cultured in non-serum medium (SFO3) with GATA6 gene overexpressed and cultured in non-serum medium (SFO3) Show photo. Blue indicates DAPI (nuclear) staining. The figure which shows the result of having conducted the PCA analysis using the whole gene in PDGFRA positive cells (Day1, Day 3) induced by overexpressing GATA6 in naive type PSC or primed PSC (Day 1, Day 3). The results of Day 0 are also shown. The figure which shows the result of having analyzed the expression level of a primitive streak related gene or an endoderm related gene in PDGFRA positive cells (Day1, Day 3) induced by overexpressing GATA6 in naive type PSC or primed PSC (N = 2). The results of Day 0 are also shown. The results of comparison of the expression of Top100 gene for Epiblast and primitive endoderm (PrE) in human embryos in cells (H0-naive-GATA6) (Day 0, Day 1 and Day 3) induced by expressing GATA6 in naive PSC are shown. 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 naive PSC or primed PSC Figure showing. Figure: Results of ChIP-seq (BMP2, BMP6, IL6ST or FRZB) in cells (H9-naive-GATA6 and H9-primed-GATA6) induced by expressing GATA6 in naive PSC or primed PSC . The figure which shows the result of having analyzed the expression level of BMP2, BMP6, PDGFRA, LEFTY1, IL6ST or FRZB gene in PDGFRA positive cells (Day 1, Day 3) induced by overexpressing GATA6 in naive type PSC (N = 2). The results of Day 0 are also shown. Photographs showing the results of analysis of phosphorylation of SMAD1 / 5/8, MAPK, STAT3 and SMAD2 by Western blot. The figure which shows the result of having analyzed the time-dependent change of the expression level of the endogenous PDGFRA gene in the naive type | mold PSC in which differentiation induction was carried out in the culture medium containing 7 factors (BMP is BMP2) by flow cytometry. The figure which shows the result of having analyzed the time-dependent change of the expression level of the endogenous PDGFRA gene in naive type | mold PSC differentiation-induced by the culture medium containing 7 factors (BMP is BMP6) by flow cytometry. The figure which shows the result of having analyzed the time-dependent change of the expression level of the endogenous PDGFRA gene in the naive type | mold PSC in which differentiation induction was carried out in the culture medium containing 7 factors (BMP is BMP4) by flow cytometry. The graph which compared the expression level of the endogenous gene in Day 3 (PDGFRA positive cell) in naive type PSC differentiation-induced by GATA6 forced expression or 7 factors (BMP is BMP4) culture. The results of Day 0 are also shown. The figure which shows the result of having analyzed the expression of GATA6 and GATA4 or SOX17 in Day 3 (PDGFRA positive cell) in naive type PSC differentiation-induced by GATA6 forced expression or 7 factor (BMP is BMP4) culture by an immunostain (photograph). The figure which shows the result of having analyzed the expression level of CD57 and CD75 in the prime type | mold PSC which forcedly expressed differentiation of naive type PSC or GATA6 in which the differentiation induction was carried out by the culture medium containing seven factors by flow cytometry. The figure which shows the result of having compared the expression level of CEACAM1 gene in a naive type PSC origin PDGFRA positive cell and a prime type PSC origin PDGFRA positive cell. The expression level of CEACAM1 in naive PSC (before differentiation induction), GATA6 forced expression naive type PSC, 7 factor culture naive type PSC, primed PSC (before differentiation induction) or GATA6 forced expression primed PSC was analyzed by flow cytometry FIG. The figure which shows the result of having analyzed the expression level of ANPEP and CEACAM1 in naive type PSC (before differentiation induction), GATA6 forced expression naive type PSC, and 7 factor culture naive type PSC by flow cytometry. The expression level (Day 0, Day 3, reculture 13 days) of visceral endoderm gene, yolk sac gene and extraembryonic mesoderm gene in naive PSC-derived PDGFRA positive cells and primed PSC-derived PDGFRA positive cells is compared. Figure. The figure which shows the result of having analyzed the expression of T and OCT3 / 4 by an immunostaining by coculturing a naive type PSC origin PDGFRA positive cell (primitive endoderm cell) and prime type PSC, and analyzing it. When naive PSC is subtracted from factor 7 or factor 7 and factor 1 is subtracted, and differentiation is induced with factor 6, PD03 (MEC inhibitor), LDN193189 (BMP inhibitor), JaKi (JaK inhibitor), or 6 factor The figure which shows the result of having analyzed the expression level of PDGFRA at the time of inducing differentiation by adding each of Activin and CH (Wnt activating agent) by flow cytometry. The results of flow cytometric analysis of the expression level of PDGFRA when differentiation was induced by G-CSF activation of naive PSC into which GP130Y118F chimeric receptor had been introduced, and by addition of 6 factors excluding IL-6 from 7 factors Figure. The figure which shows the result of having analyzed the expression level of CEACAM1 when knocking down GATA6 using shGATA6 in naive type PSC, and inducing differentiation using 7 factors by flow cytometry.

<Method of Preparing Primitive Endoderm>
The method of preparing primitive endoderm of the present invention from pluripotent stem cells in vitro comprises
Naive pluripotent stem cells,
BMP (Bone morphogenetic protein) and FGF4 (Fibroblast growth factor 4)
Culturing in a medium containing S. and inducing primitive endoderm differentiation.
The medium is at least one selected from PDGF, IL-6, a TGFβ inhibitor, a Wnt signal inhibitor and retinoic acid in addition to BMP and FGF4, preferably two or more, more preferably three or more, further preferably Preferably, four or more, particularly preferably all five) are contained.
In a more preferred embodiment, the medium comprises, in addition to BMP and FGF4, a TGFβ inhibitor and a Wnt signal inhibitor.

<Pluripotent stem cells>
In the present invention, a pluripotent stem cell is a stem cell having pluripotency capable of differentiating into many cells existing in a living body, and also having proliferation ability, any of which can be induced to primitive endoderm. Cells are included. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, cloned embryonic derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatozoa Stem cells ("GS cells"), embryonic germ cells ("EG cells"), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells), and the like are included. Preferred pluripotent stem cells are iPS cells and ES cells. The pluripotent stem cell is preferably derived from a mammal, more preferably from a primate, and even more preferably from a human.

Methods for producing iPS cells are known in the art and can be produced by introducing a reprogramming factor into any somatic cell. Here, the reprogramming factor includes, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, 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 and other genes or gene products are exemplified, and these reprogramming factors may be used alone or in combination. Also good. As a combination of initialization factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 01084 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 05 626, 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.S. L. Judson et al. , (2009), Nat. Biotechnol. , 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem 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 as described in is illustrated.

Somatic cells include, but are not limited to, fetal (child) somatic cells, neonatal (child) somatic cells, and any mature healthy or diseased somatic cells, and also primary culture cells. , Passage cells, and cell lines are also 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 precursor cells, (3) blood cells (peripheral Blood cells, cord blood cells etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells etc.), hair cells, hepatocytes, gastric mucous 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>
Although naive type pluripotent stem cells are pluripotent stem cells having properties similar to those of pre-implantation embryo, specifically, they have the following features (Cytometry Research 27 (1): 19 ~ 24, 2017).
It shows a domed colony morphology, and the colony size is smaller than that of the primed one.
One or more of CD75, KLF4 and TFCP2L1 are expressed as markers.
The genome is demethylated.

Naive pluripotent stem cells can be prepared, for example, by the following method.
Method using overexpression of NANOG and KLF2 (Takashima et al., Cell 158: 1254-1269, 2014)
Method using 5i LFA condition (Theunissen et al., Cell Stem Cell. 2016 Oct 6; 19 (4): 502-515.)
Method using HDAC (histone deacetylase) inhibitor (Guo, G. et al. (2017). Development 144 (15): 2748-2763.)
Alternatively, it can be obtained by culturing primed pluripotent stem cells using a commercially available culture medium for preparing naive pluripotent stem cells such as t2iLGo (Ndiff 227 [Takara Bio, Cat. Y40002]).

<Prime-type pluripotent stem cells>
Although primed pluripotent stem cells are pluripotent stem cells having properties similar to postimplantation embryo epiblasts, they are generally induced pluripotency obtained by introducing a reprogramming factor into somatic cells Stem cells and human ES cells fall under this category and have not been subjected to the above-described naive treatment.
Primed pluripotent stem cells have the following features.
It shows flat colony morphology and colony size is larger than naive type.
As markers, CD75, KLF4 and TFCP2L1 are negative.
The genome is methylated.

<TGFβ inhibitor>
A TGFβ inhibitor is a substance that inhibits signal transduction from binding of TGFβ to the receptor to SMAD, and a substance that inhibits binding to the receptor ALK (activin receptor-like kinase) family, or ALK Substances that inhibit the phosphorylation of SMAD by a family can be mentioned, for example, Lefty-1 (NCBI Accession No. is exemplified by mouse: NM — 010094, human: NM — 020997), SB431542, SB202190 (above, RKLindemann et al., Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), SB-525334, GW6604, NPC30345, SD093, SD908, SD208 (Scios), LY2109762, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408) 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 solution, the concentration that exerts the TGFβ inhibitory effect can be appropriately selected according to the type of the TGFβ inhibitor. For example, when using A83-01, it is usually 0.025 to It is in the range of 100 μM, preferably 0.075 to 50 μM, more preferably 0.25 to 10 μM.

<Wnt signal inhibitor>
The Wnt signal inhibitor is not particularly limited as long as it can suppress the Wnt signaling pathway involved in gene expression and cytoskeleton regulation, and specifically, XAV939 (tankyrase inhibitor), IWP-1, IWP- 2, low molecular weight compounds such as IWP-3, IWP-4, IWR-1, 53AH (more porcupine inhibitors), KY02111 and derivatives thereof, proteins such as IGFBP4, DKK1, Wnt-C59, Wnt and Wnt signals Antisense nucleic acid, RNA interference inducing nucleic acid (eg, siRNA), competitive peptide, antagonist peptide, inhibitory antibody, antibody-ScFV fragment, dominant negative mutant, and expression vectors for suppressing protein expression or function constituting pathway Low molecular weight Wnt signaling inhibitor preferably compounds, it can be preferably exemplified a XAV939 and IWP-4.
As the concentration of the Wnt signal inhibitor contained in the culture solution, the concentration that exerts the Wnt signal inhibitory effect can be appropriately selected according to the type of Wnt signal inhibitor, and, for example, when using XAV 939, it is usually 0.025 to It is in the range of 100 μM, preferably 0.075 to 50 μM, more preferably 0.25 to 10 μM.

<PDGF>
The PDGF used in step (i) is preferably a protein having a dimer structure, such as dimers with type A monomers (PDGF-AA), dimers with type B monomers (PDGF-BB), A Examples are dimers of type monomers and type B monomers (PDGF-AB) and the like, with preference given to PDGF-AA.
PDGF is preferably of mammalian origin, preferably of human origin. As human PDGF-A, for example, a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM_002607 can be mentioned. PDGF includes its fragments and functional variants as long as it has the desired differentiation inducing activity. PDGF may be commercially available, or proteins purified from cells or recombinantly produced proteins may be used. The concentration of PDGF dimer contained in the culture solution 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>
The BMP includes at least one BMP selected from the group consisting of BMP2, BMP4 and BMP6, but is preferably BMP4. The BMP is preferably derived from a mammal, and is preferably derived from a human. The human BMP4 includes, for example, a protein having the amino acid sequence of the accession number: AAH20546.1 of the National Center for Biotechnology Information (NCBI). As long as BMP has a desired differentiation inducing activity, fragments and functional variants thereof are included. BMPs may be commercially available, or proteins purified from cells or recombinantly produced proteins 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 may be 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 ( AM 580) (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), and retinol palmitate, retinol, retinal, 3-dehydroretinol, 3-dehydroretinal and the like.
The concentration of retinoic acid or a derivative thereof contained in the culture solution is, for example, 1 nM to 1000 nM, preferably 5 nM to 500 nM, more preferably 10 nM to 250 nM.

<FGF4>
The FGF4 is preferably derived from a mammal, and is preferably derived from a human. As human FGF4, for example, a protein having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: NM_002007 can be mentioned. As far as FGF4 has a desired differentiation inducing activity, fragments and functional variants thereof are included. As FGF4, a commercially available one may be used, or a protein purified from cells or a recombinantly produced protein 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. Examples of human IL-6 include a protein having the amino acid sequence of accession number: M18403 of National Center for Biotechnology Information (NCBI). As long as IL-6 has a desired differentiation inducing activity, fragments and functional variants thereof are included. As IL-6, a commercially available one may be used, or a protein purified from cells or a recombinantly produced protein may be used. The concentration of IL-6 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.

The culture solution used in the primitive endoderm induction step is not particularly limited, but it is possible to use BMP and FGF4, preferably further PDGF, IL-6, TGFβ inhibitor, Wnt signal inhibitor and retinoin in naive pluripotent stem cell maintenance medium. It can be prepared by adding one or more types (preferably two or more types, more preferably three types or more, further preferably four types or more, particularly preferably all five types) selected from acids. These factors may be added at different times. For example, IL-6 can also be added 48 hours after initiation of induction. As a naive type 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 (1μM) + CHIR99021 (1μM) + SB590085 (0.5μM) + WH-4-023 (1μM) + Y-27632 (10μM) + LIF + Activin A
Theunissen, T. W., 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 serum free may be used. For example, albumin, insulin, transferrin, selenium, fatty acid, trace element, 2-mercaptoethanol, thiolglycerol, lipid, amino acid, L-glutamine, non-essential amino acid, vitamin, growth factor, low molecular weight compound, antibiotic It may also contain one or more substances such as substances, antioxidants, pyruvate, buffers, mineral salts, cytokines and the like.

In the primitive endoderm induction step, naive type pluripotent stem cells may be adherent culture or suspension culture, and in the case of adherent culture, the culture vessel may be coated and used, or coculture with a feeder cell etc. Good. Examples of feeder cells to be cocultured include primary fibroblasts (MEF) derived from mouse embryos treated with mitomycin C, STO cells, SNL cells, OP9 cells, C3H10T1 / 2 cells and the like.

When culture is carried out by suspension culture in the primitive endoderm induction step, it is desirable to form the aggregates (also referred to as spheres) in a non-adherent state to the culture vessel and culture the cells, and such culture is particularly preferable. Although not limited, a culture vessel which has not been artificially treated (eg, coated with an extracellular matrix etc.) for the purpose of improving adhesion to cells, or a treatment which artificially suppresses adhesion (eg, poly Hydroxyethyl methacrylate (poly-HEMA), nonionic surface-active polyol (Pluronic F-127 etc.) or phospholipid analogue structure (eg, water-soluble polymer having 2-methacryloyloxyethyl phosphorylcholine as a structural unit (Lipidure) It can be carried out by using a culture vessel coated with.

In the primitive endoderm induction step, when adherent culture is performed, it can be performed by culturing extracellular matrix using a coated culture vessel. The coating treatment may be carried out by appropriately removing the solution after the solution containing the extracellular matrix is put in the culture vessel. Here, the extracellular matrix is a supramolecular structure existing outside the cell, and may be naturally derived or artificial (recombinant). For example, substances such as polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin and fragments thereof can be mentioned. These extracellular matrices may be used in combination, for example, preparations from cells such as BD MatrigelTM.

The culture temperature conditions for culturing naive type pluripotent stem cells in the primitive endoderm induction step are not particularly limited, and for example, about 37 ° C. to about 42 ° C., about 37 to about 39 ° C. is preferable. The culture period can be appropriately determined by those 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 is obtained, and is, for example, at least 1 day or more, preferably 2 to 5 days.

<Primitive endoderm>
By performing the above-described culture step, primitive endoderm can be obtained.
Primitive endoderm can be 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 Receptor Receptor) It is characterized by the expression of one or more of primitive endoderm markers such as COL4A1 (alpha-1 subunit of collagen type IV), SPARC (Secreted protein acidic and rich in cysteine) and the like. In addition to these one or more markers, cells expressing CEACAM1 or ANPEP described later are more preferable.

In order to enrich primitive endoderm cells, a step of selecting primitive endoderm can be performed after performing a primitive endoderm induction step. The sorted primitive endoderm can be further differentiated into visceral endoderm and yolk sac by continuing the culture.
Sorting can be performed using the expression of one or more types of primitive endoderm-specific markers as described above as an indicator.
When primitive endoderm cells are extracted using primitive endoderm markers, expression of each marker protein may be used as an index, or the gene encoding each of the above marker proteins is expressed (mRNA is expressed) What you do may be an indicator.

As a reagent used for sorting primitive endoderm cells (as in extraction or detection) from a cell population containing primitive endoderm cells, a reagent having a specific affinity to the above-mentioned primitive endoderm marker Anything can be used, and antibodies, aptamers, peptides or compounds that specifically recognize can be used, preferably antibodies or fragments thereof. In addition, when examining gene expression of these markers, primers and probes that hybridize to these marker genes can be used.

The antibodies may be polyclonal or monoclonal antibodies. These antibodies can be made 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, non-human animals such as rabbits are synthesized by synthesizing a marker protein expressed and purified in E. coli according to a conventional method, or an oligopeptide having a partial amino acid sequence. And can be obtained according to a conventional method from the serum of the immunized animal. On the other hand, in the case of a monoclonal antibody, it can be obtained from hybridoma cells prepared by fusing the spleen cells obtained from the above-mentioned immunized non-human animal with myeloma cells (Current protocols in Molecular Biology) Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11). Examples of antibody fragments include a portion of an antibody (eg, a Fab fragment) or a synthetic antibody fragment (eg, a single chain Fv fragment “ScFv”). Fragments of antibodies such as Fab and F (ab) ₂ fragments can also be produced by methods well known in the art of genetic engineering. Where the marker is a membrane protein, it is preferably an antibody to the extracellular domain. The antibody may be a commercially available antibody.

In order to distinguish and separate bound cells, a reagent such as an antibody having the affinity is, for example, a fluorescent label, a radioactive label, a chemiluminescent label, an enzyme, a detectable substance such as biotin or streptavidin, or protein A, It may be bound or conjugated to a substance that enables isolated extraction such as protein G, beads, magnetic beads and the like.

Examples of methods for sorting (as well as extraction or detection) primitive endoderm cells include methods using a flow cytometer. In addition, a method of sedimentation using an antibody bound to a carrier, a method of sorting cells by magnetism using magnetic beads (for example, MACS), a method of using a cell sorter using a fluorescent label, or immobilization of antibodies etc. The method etc. which use the carrier (for example, cell concentration column), etc. are illustrated.

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

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

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

In the present invention, “cell population containing primitive endoderm cells” is a collection of cells containing primitive endoderm cells, regardless of the origin, although naive pluripotent stem cells ( It is preferable that it is a cell population containing primitive endoderm cells obtained by inducing differentiation of human naive type pluripotent stem cells) to primitive endoderm. In addition, the method of differentiation induction from naive type pluripotent stem cells to primitive endoderm is not limited to the above, and may be obtained by a differentiation induction method by gene transfer such as GATA6.

In the present invention, “sorting (separation) of primitive endoderm cells” means to increase the proportion of primitive endoderm cells as compared to that before sorting (separation), preferably 50 primitive endoderm cells. %, 60%, 70%, 80% or 90% or more. More preferably, it is to obtain cells consisting of 100% primitive endoderm cells.

When selecting (separating) primitive endoderm cells from a cell population containing primitive endoderm cells using CEACAM1 or ANPEP as an index, it may be used in combination with primitive endoderm markers such as GATA4, GATA6, SOX17, FOXA2 described above, etc. Good. This enhances the enrichment rate of primitive endoderm as compared to using CEACAM1 or ANPEP alone.

In addition, selectively selecting (separating) cells that are positive for the marker may be selecting (separating) all cells that are positive for the marker, but selecting cells having a certain amount or more of the marker expression amount It may be (separated). For example, in the primitive endoderm-containing cell population, cells expressing the marker within the top 50%, cells within the top 40%, cells within the top 33%, cells within the top 30%, within the top 20% Cells, or cells within the top 10%, can be selectively harvested.

<Kit for Sorting or Detection of Primitive Endoderm Cells Using CEACAM1 or ANPEP as an Index>
The present invention also provides a kit for sorting or detecting primitive endoderm cells, which comprises a reagent that specifically binds to CEACAM1 or ANPEP. The detection reagents contained in the present extraction kit are as described above, and include antibodies against CEACAM1 or ANPEP, and polynucleotides that hybridize to the CEACAM1 gene or ANPEP gene. The extraction kit in the present invention may also contain, together with a reagent that specifically binds to CEACAM1 or ANPEP, instructions describing how to use the detection reagent.

<Method for preparing mesendoderm and myocardial progenitor cells and / or pancreatic progenitor cells>
The present invention also provides a method for preparing mesendoderm, which comprises co-culturing primitive endoderm with primed pluripotent stem cells to differentiate primed pluripotent stem cells into mesoderm. Furthermore, there is provided a method for preparing cardiac muscle progenitor cells and / or pancreatic progenitor cells, comprising the step of culturing the obtained mesendoderm to differentiate into cultured cardiac progenitor cells and / or pancreatic progenitor cells. The primitive endoderm may be obtained by the method as described above, or may be obtained by other methods such as forced expression of GATA6 gene or the like in naive pluripotent stem cells.

In ontogeny, epiblasts cause gastrulation and form meso- and endoderm, which differentiate and develop. Primitive endoderm is present in contact with epiblasts 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 the state of gastrulation in vitro, which is close to the physiological early implantation stage development. .

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

For example, coculture of primitive endoderm and primed pluripotent stem cells can be performed by seeding cultured primitive endoderm cells with primed pluripotent stem cells. Preferably, the medium is a medium for iPS / ES cells such as AK03, mTeSR1 or F12 / KSR, and may be cultured by adding an extracellular matrix component such as Matrigel. The ratio of the numbers of primitive endoderm and primed pluripotent stem cells to be subjected to co-culture is preferably 1: 3 to 3: 1, more preferably 1: 2 to 2: 1, and particularly preferably 1: 1. The culture temperature conditions for co-cultivation are not particularly limited, but for example, about 37 ° C. to about 42 ° C., about 37 to about 39 ° C. is preferable. Also, the culture period is not particularly limited, and for example, it is at least 12 hours or more, preferably 24 to 48 hours.

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

<Media for Induction of Differentiation to Primitive Endoderm>
The present invention is also directed to a culture medium for naive pluripotent stem cells for inducing differentiation into primitive endoderm including BMP and FGF4, preferably BMP and FGF4 and PDGF, IL-6, TGFβ inhibitor, Wnt signal inhibitor and For induction of differentiation to primitive endoderm comprising one or more types (preferably 2 or more types, more preferably 3 types or more, still more preferably 4 types or more, particularly preferably all 5 types) selected from retinoic acid Provided is a medium for naive type pluripotent stem cells. These may be media prepared in advance such that each component is contained at a concentration effective for inducing differentiation into primitive endoderm, or may be prepared and used by adding each component immediately before use. Good. These factors may be added at different times. For example, IL-6 can also be added 48 hours after initiation of induction. Thus, some factors may be provided separately. The medium (kit) may be accompanied by an instruction manual describing the method of use and preparation. The medium for inducing differentiation into primitive endoderm can further contain other components necessary for the culture of naive type pluripotent stem cells.

Hereinafter, the present invention will be more specifically described by way of examples, but the present invention is not limited to the following embodiments.

Material and method
Cell culture
Human primed pluripotent stem cell (PSC) line (H9 ES (embryonic stem) cells, H1 ES cells, AdiPS cells) is a conventional condition (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. M3148] was used and maintained on γ-irradiated MEF. Cells were collected every 5 to 7 days with 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 2. _{Using 2} ), it was peeled off and passaged like a small clamp.

Human naive pluripotent stem cell line (H9 ES cells, H1 ES cells, Adi PS cells derived) is t2iLGo (Ndiff 227 [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]) were used to maintain on MEF. Cells were detached and passaged every 3 to 5 days using Accutase (Sigma-Aldrich, Cat. A6964).

The naive H9 PSC was established by a method using an HDAC inhibitor (Guo, G. et al. (2017). Development 144 (15): 2748-2763.). At the time of establishment, EOS plasmid was introduced into primed H9 PSC by electroporation (primed H9 EOS). A gene that is a drug resistance marker (puromycin resistance) is incorporated into the plasmid, and after introduction into cells, the cells into which the plasmid has been introduced by drug selection using Puromycin Dihydrochloride (Thermo Fisher Scientific, Cat. A1113802) Was selected. The primed H9 EOS is detached with trypsin / EDTA (Nacalai Tesque, Cat. 32777-15) into single cells, and 1 × 10 ⁵ cells / cm ² of cells are treated with 10 μM Y-27632 (Wako, Cat. 034) In the F12 / KSR medium to which 24024) was added, it was seeded on MEF. From the next day, culture with cRM1 (Ndiff, 1 μM PD03, 10 ng / ml hLIF, 1 mM Valproic acid sodium salt [VPA; Sigma-Aldrich, Cat. P4543]) for 48 hours, and then cRM2 (Ndiff, 1 μM PD03, 10 ng / ml hLIF, Cells were maintained at 2 μM Go, 2 μM XAV 939 [Sigma-Aldrich, Cat. X 3004]. Most of the cells form colonies with a domed morphology from about 3-5 passages. At this point, it was established by switching culture to t2iLGo medium.

In some experiments, naive H9 was established using 5i LFA conditions (Theunissen et al., Cell Stem Cell. 2016 Oct 6; 19 (4): 502-515.). Primed H9 was detached with trypsin / EDTA into single cells, and 1 × 10 ⁵ cells / cm ² of cells were seeded onto MEFs in F12 / KSR medium supplemented with 10 μM Y-27632. From the next day, 5i LFA medium (Ndiff, 1 μM PD03, 1 μM CH, 1 μM WH-4-023 [A Chemtek H620061], 0.5 μM SB 590885 [R and D 2650], 10 μM Y-27632, 10 ng / ml hLIF, 20 ng / ml Activin A The culture was changed to [R & D, Cat. 388-AC], 8 ng / ml bFGF). When most of the cells formed colonies showing domed morphology (3-5 passages), they were established by switching to t2iLGo and maintaining.

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

After naive PSC cultured on primitive endoderm-derived MEF are detached by Accutase and recovered, it is seeded on a gelatin-coated dish, and t2iLGo supplemented with 10 μM Y-27632 (ROCK inhibitor) at 37 ° C., 1 to 2 Incubate for time to remove MEF. Then resuspend in each induction medium and start induction simultaneously with seeding.

When induced by gene overexpression, 1 × 10 ⁵ cells / cm ² were seeded on Fibronectin (Millipore, Cat. FC010). For induction media, serum media (Glasgow Minimum Essential Medium [GMEM; Sigma-Aldrich, Cat. G5154], 15% FBS [Thermo Fisher Scientific, Cat. 10437028], 2 mM L-Glutamine [Thermo Fisher Scientific, Cat. 25030081], 0.1 mM bovine serum albumin (BSA; Wako, 1 mM Sodium Pyruvate [Thermo Fisher Scientific, Cat. 11360-070], NEAA, 0.1 mM 2-mercaptoethanol) or S. Clon SFO-3 (Adea, Cat. SS 1303) Cat.012-2381), 50 μM 2-mercaptoethanol, 25 ng / ml recombinant human FGF4 (FGF4; Peprotech, Cat. 100-31), 1 μg / ml heparin sodium (Wako, Cat. 081-00131) is used. did. 0.1 μg / ml Dox was added up to 48 hours after initiation of induction.

When induced with compounds, cells were seeded at 5 × 10 ⁴ cells / cm ² on iMatrix-511 silk (MAX, Cat. 89 2021). For induction medium, Ndiff 227 medium, 25 ng / ml FGF4, 1 μg / ml heparin sodium, 10-200 ng / ml recombinant human BMP-4 (BMP-4; R & D, Cat. 314-BP), 10 ng / ml recombinant human PDGF-AA (PDGF-AA, Peprotech, Cat. 100-13A), 10 ng / ml recombinant human IL-6 (IL-6; Oriental Yeast, Cat. 47066000), 1 μM XAV939, 3 μM A83-01 (Tocris, Cat. 2939), What added 0.1 micromol retinoic acid (RA; Sigma-Aldrich, Cat.R2625) was used. IL-6 was added 48 hours after initiation of induction. Also, in some experiments, 10-500 ng / ml recombinant human BMP-2 (BMP-2; Oriental Yeast, Cat. 47304000) instead of BMP-4, or 50 ng / ml recombinant human BMP-6 (BMP- 6; Peprotech, Cat. 120-06) was used.

FACS analysis / sorting
Primitive endoderm-like cells and primed PSCs were detached by trypsin / EDTA, and naive PSCs were exfoliated into single cells by Accutase and recovered. Thereafter, blocking was performed for 30 minutes on ice using HBSS (Thermo Fisher Scientific, Cat. 14185052) added with 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), Dylight 650-anti-CD75 antibody (novusbio, Cat. NBP2-47890) , BV421-anti-CD57 antibody (BD, Cat. 563896) was added in each combination and incubated on ice for 30 minutes. After washing, Streptavidin-APC (Biolegend, Cat. 405207), Streptavidin-PE (eBioscience, Cat. 12-4317-87), 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 Aria II (BD) was used for sorting. Also, Flow Jo V10.2 software was used for data analysis.

Derivation from primordial endoderm to visceral endoderm and induction into yolk sac cells. On day 3, cells were purified by flow cytometry using PDGFRA or CEACAM1 antibody. Thereafter, culture was continued on MEF using DMEM + 10% FBS medium. At day 13, RNA was extracted, and it was confirmed that visceral endoderm and yolk sac cells were differentiated.

Reverse Transcription Quantitive Real-time PCR
Total RNA was extracted with the RNeasy kit (Qiagen, Cat. 74106), and cDNA was synthesized from 1000 ng of RNA using SuperScript IV (Thermo Fisher Scientific, Cat. 18090050) and an oligo-dT primer. For real-time PCR, use 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 for PCR amplification QuantStudio 3 ( Thermo Fisher Scientific) was used. Analysis after Real-time RT-PCR reaction was performed using QuantStudio Design & Analysis Software v 1.4.1.

Immunostaining
The 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. The cells were blocked for 2 hours with PBS + 1% BSA + 0.05% Tween-20 (PBS-BT). Primary antibodies were added after dilution in PBS-BT and incubated for 2 hours at room temperature. After washing, the secondary antibody was diluted 1: 2000 in PBS-BT and incubated for 2 hours at room temperature. Nuclei were stained using DAPI (Sigma- Aldrich, Cat. D 9542).

PDGFRA-positive cells were recovered by FACS sorting from cells co-cultured with primitive endoderm cells and primed PSC GATA6 over-expressing or compound-induced cells. The recovered cells were replated at a density of 3 × 10 ⁵ cells / cm ^{2 on} an iMatrix 511-coated slide glass. The following day, primed PSC was exfoliated with DB (cell dissociation solution mixed with collagenase and trypsin) in a small clamp manner, and seeded on the cells replated on the slide. The cells were cultured in primed ES cell culture medium (AK03, mTeSR1, F12 / KSR, etc.) alone or in a culture medium obtained by adding matrigel (5%) to primed ES cell culture medium.

result
Human naive PSC and primed PSC-derived PDGFRA-positive cells are different populations to generate human naive PSC, human primed ES cells (H9-primed cells) and human iPS cells (AdiPS-primed cells). Human primed pluripotent stem cells (H9-EOS cells, AiPS-EOS cells) into which EOS vector had been introduced were treated with HDAC inhibitor, human naive PSC was induced and maintained in t2iLGo medium, and reset cells were established ( H9-naive cells, AiPS-naive cells).
When GATA6, GATA4 and SOX17 are overexpressed in mouse ES cells, they can be induced to primitive endoderm (PrE), and XEN cells can be established. Overexpression of GATA6, GATA4 and SOX17 in mouse ES cells is known to induce mouse PrE. Therefore, plasmids expressing GATA6, GATA4 or SOX17 were introduced into H9-naive and H9-primed cell lines under DOX induction (FIG. 1A). When DOX was added to H9-naive and differentiated in serum, naive dome-like colonies became flat and differentiated in any overexpression (FIG. 1B). When gene expression was confirmed, it was found that GATA4, GATA6 and SOX17, which are markers of PrE from human naive type, are expressed from day 2 and rise during day 4 in the cell line overexpressing GATA 6 (FIG. 1C). . Overexpression of GATA6 also induced expression of GATA4, 6, SOX17 (FIG. 1C, lower). Overexpression of GATA4 was also found to induce gene expression, although not as much as GATA6. On the other hand, cells overexpressing SOX17 could not express GATA4, GATA6, SOX17 (Endo) in the naive type. Even in the Primed type, expression was not able to be induced as much as GATA6 was overexpressed with only slight induction of the gene.

In mouse blast cysts, PrE is PDGFRA positive. It is also known that cells differentiated from mouse ES cells to PrE express PDGFRA. It is reported that PDGFRA is expressed in PrE of human embryo in analysis using single cell RNA sequence (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 cells induced there, it was found that expression of PDGFRA is increased when GATA4, 6 is expressed similarly to the naive type and prime type (FIG. 1D). Therefore, expression of PDGFRA was confirmed by flow cytometry (FIG. 1E). When GATA6 and GATA4 were overexpressed, expression of PDGFRA was observed from Day 1 and 41% of GATA6 and 20% of GATA4 cells were PDGFRA positive on Day 3. On the other hand, when SOX17 was overexpressed, expression of PDGFRA was not observed until day 3 (FIG. 1D, E). Subsequently, gene expression of PDGFRA positive cells was examined. When naive-derived PDGFRA-positive cells were purified and their expression was examined, H9-naive-GATA6 PDGFRA-positive cells expressed PrA markers GATA3, 4, SOX17, HNF4A, FOXA2, COL4A1, while being pluripotent The expression of sex markers OCT3 / 4 and NANOG decreased. On the other hand, GATA6 and HNF4A were also induced from H9-naive-GATA4, but the PrE marker could not be induced as much as GATA6 (FIG. 1F).

On the other hand, gene expression of PDGFRA-positive cells derived from H9-primed-GATA6 was examined and compared to the naive type, PrA genes such as HNF4A, FOXA2, CER1 and OTX2 including GATA4 and SOX17 were not expressed, and PDGFRB, Genes related to mesoderm and mesenchymal system such as KDR, SNAI2, CDH11 and VIMENTIN were expressed (FIG. 1G). When H9-naive-GATA4 and H9-primed-GATA4 were compared, Pr9 related genes were not expressed from H9-primed, but genes of mesoderm were expressed (FIG. 1H). From the above, it was found that cells expressing the PrE-related gene are derived from the naive type, while cells expressing the mesodermal gene are derived from the prime type.

Human naive PSC-derived PDGFRA-positive cells were induced using a non-serum medium, SFO3 medium or N2B27 medium, in order to clearly see differentiation signals equivalent to PrE. When GATA6 was overexpressed and induced by adding FGF4 in H9-naive-GATA6, about 30% of cells became PDGFRA positive in Day 1, and 80% of cells were PDGFRA positive in Day 3 (FIG. 2A) . PDGFRA-positive cells were sorted, and their expression was confirmed. As a result, PrE-related genes such as GATA4, SOX17, FOXA2, HNF4A, COL4A1, and SPARC were almost comparable under serum condition (GMEM) and non-serum condition (SFO3) (Figure 2B). The immunostaining confirmed that overexpression of GATA6 induces GATA4 and SOX17 (FIG. 2C). In order to further analyze the properties of PDGFRA cells, comprehensive analysis using RNA sequences was performed. When PCA analysis was performed using all genes, H9-naive pluripotent stem cells and primed pluripotent stem cells were different in PC1, and PDGFRA-positive cells (Day 1, Day 3) induced by overexpressing GATA 6 were Similar to the naive and primed undifferentiated state, PC1 was different and PC2 was changed in the same direction (FIG. 2D). That is, it is suggested that they are different cell populations. Since it was suggested that the cells derived from H9-primed are mesodermal cells from FIG. 1G, when the expression of mesodermal genes was observed, in D1, the gene related to the early primitive streak was It is speculated to be expressed and induced into mesodermal cells (FIG. 2E). On the other hand, in naive, such mesodermal genes were not induced. When the expression of Top100 gene for Epiblast and PrE in cells derived from H9-naive and human embryos was compared, it was found that Day 0 is close to epiblast and Day 3 is close to PrE (FIG. 2F). From the above, it is considered that PDGFRA positive cells derived from human naive type pluripotent stem cells are cells close to PrE.

GATA6 Modifies the Signal As a result of overexpression of GATA6, the naive form differentiates into PrE, while the primed form induces different cells expressing mesodermal genes, so to investigate the direct role of GATA6 ChIP-seq was performed using H9-naive-GATA6 and H9-primed-GATA6. GATA6 was actually bound to genes considered important for PrE such as GATA6, GATA4, SOX17, HNF4A (Fig. 3A). At the same time, it was found to bind to PDGFRA. Furthermore, it was bound to the secretion factor or receptor called BMP2, BMP6, IL6ST, 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 PSC and continued to be expressed after induction of differentiation. In addition, after overexpression of GATA6, protein activation was examined, and phosphorylation of SMAD1 / 5/8, phosphorylation of MAPK, phosphorylation of STAT3, and phosphorylation of SMAD2 were suppressed (FIG. 3D). From this, GATA6 induces the secretion of BMP and PDGFA simultaneously with the induction of PrE gene group, while the induction of FRZB may suppress the Wnt signal.

From the results of Chock sequencing and gene expression after overexpression of signal cocktail-induced GATA6, in H9-naive cells, BMP secretion and STAT3 signal activation, PDGFRA induction, activin signal and Wnt signal suppression were observed. Then, PDGF-AA, BMP2, IL-6, A83-01, XAV939 were added without overexpression of GATA6, and it was examined whether PrE was induced. In addition, since it is reported that FGF4 and RA work positively for induction of PrE in mice, it was induced by adding 7 factors obtained by adding FGF4 and RA (retinoic acid) to the above-mentioned factors (FIG. 4A). As a result, about 28.6% of PDGFRA positive cells were induced from the naive type (Fig. 4 B, C). Induction was also seen when BMP4, 6 in the same family as BMP2 was changed (Fig. 4B, C). Because BMP4 induces the most PDGFRA-positive cells as compared to BMP2 and BMP6, experiments were conducted with BMP4 added. When the induced PDGFRA-positive cells were purified and gene expression was confirmed, it was confirmed that PrE-related genes such as GATA4, 6, SOX17, PDGFRA, HNF4A, and FOXA2 were expressed and PrE could be induced (FIG. 4D) ). In addition, it was found that immunostaining can also induce GATA6 + GATA4 + SOX17 + cells as GATA6 overexpression (FIG. 4E).
In the H1-naive type PSC and AdiPS, PDGFRA-positive cells were also induced by the 7-factor-containing medium, and it was similarly found that they express PrE-related genes and can be induced by the other two strains. Furthermore, when RNA sequencing was performed and exhaustive analysis was performed, it was found that a gene related to PrE was induced as in GATA6 overexpressing cells.

When the novel human PrE-specific surface marker naive PSC is cultured with bFGF + ACTIVIN (TGFB), it converts to the primed form in about 10 days. It was reported that the naive PSC expresses the surface antigen CD75 and the primed one expresses the surface CD57. When naive type to PrE was induced with chemical (the above 7 factors), PDGFRA positive cells gradually decreased the expression of the naive marker CD75 in PDGFRA positive cells and did not express the prime marker CD57 (Fig. 5A). Conversely, when PDGFA-positive cells were induced by overexpressing GATA6 in primed form, PDGFRA-positive cells were CD57 + CD75- (FIG. 5A). From the above, naive derived PrE was PDGFRA + CD75 +/− CD57− and prime derived cells were successfully separated by surface antigen as PDGFRA + CD75−CD57 +.

On the other hand, CD75 can not completely separate the naive pluripotent stem cells that are epiblasts and the PDGFRA-positive cells that are PrE. Since PDGFRA is not specific for PrE, we searched for surface antigens that were specifically expressed by PrE and not in epiblast and mesoderm. Specific surface antigens were searched on the condition that GATA6 did not bind directly in ChIP sequence, was expressed in PDGFRA-positive PrE in RNA sequence, not in primed form, and was expressed in a published paper. By the way, CECAM1 was identified. When RNA of naive and primed PDGFRA-positive cells was compared by qPCR, CEACAM1 was observed to be expressed only by naive-derived PrE (FIG. 5B). When co-stained with PDGFRA on Day 3 and compared by flow cytometry, PDGFRA positive cells were CECAM 1 positive (FIG. 5C). On the other hand, primed PDGFA-positive cells do not express CEACAM1. From this, it became clear that CEACAM1 can be used as a PrE marker.

In addition, when differentiation was induced from naive PSC to PrE with chemical (the above-mentioned 7 factors), expression of ANPEP was also increased in PDGFRA positive cells, as in CEACAM1 (FIG. 5D). Overexpression of GATA6 in naive PSC gave similar results. From this, it became clear that ANPEP can also be used as a PrE marker.

PrE's re-culture and function
When H9-naive and H9-primed induced PDGFRA-positive cells were sorted and recultured, visceral endoderm (FOXA1, CER1), yolk sac marker gene (AFP, VIL1, PDPN) were observed from H9-naive. , GPC3) but not from H9-primed except for PDPN (FIG. 6A). Similarly, the expression of yolk sac marker genes AFP, VIL1, GPC3, FOXA1, PDPN, and DAB2 was also observed from PDGFRA-positive PrE cells induced with 7 factors (note that PDGFRA-positive PrE cells induced with 7 factors are the same as those in 7). The cells can be maintained and cultured for more than 10 passages in a medium containing the factor, and the maintained cells expressed genes related to visceral endoderm / yolk sac cells and genes for extraembryonic mesenchymal cells). When gene expression was confirmed by RNA sequencing, naive type derived PDGFRA positive PrE cells actually express the yolk sac marker, and from H9-naive type, VE / YE can be induced via PrE but induced from primed Can not.

In human development where PrE induces primed PSC into mesoderm, there is PrE in the lower layer of bilaminar epiblast, and bilaminar epiblast forms a primitive streak and differentiates into mesodermal cells. At this time, PrE is considered to play an important role. It was co-cultured with PSC and observed whether the induced PrE cells exhibited such ability. After 24 hours PSC became T positive cells and after 48 hours T positive cells migrated towards PrE (FIG. 6B). This indicates that PrE induced primed PSCs into mesoderm and was able to replicate the process of gastrulation in the early developmental stages in vitro.
From the above, naive-type-derived PDGRA-positive cells not only express similar genes as PrE, but also may have functionally the same ability as PrE.

BMP, FGF and GATA6 cooperate to induce PrE.
In order to examine the importance of each signal in PrE induction with 7 factors, 1 factor was subtracted from each of the 7 factors and differentiation was induced with 6 factors (FIG. 7A). Alternatively, each of PD03 (MEC inhibitor), LDN193189 (BMP inhibitor), JaK inhibitor (JaKi), Activin, CH (Wnt activator) was added to 6 factors (FIG. 7A). As a result, when FGF4 or BMP was removed, almost no PDGFRA ⁺ cells were expressed, and it was found that FGF and BMP are essential for PrE induction. On the other hand, PDGFRA-positive cells decreased only slightly by 31.5% when ACTIVIN inhibitor was removed and 33.2% when Wnt signal inhibitor was removed, but conversely, when ACTIVIN and Wnt stimulation were performed, primitive endoderm The cells were found to disappear. From the above, it was found that activin and Wnt inhibit primitive endoderm differentiation (FIG. 7A, lower). Although no data is shown, PDGFRA, CEACAM1 and ANPEP positive cells can not be induced with one factor of FGF alone or BMP4 alone, whereas PDGFRA, CEACAM1 and ANPEP positive cells when induced with two factors (FGF, BMP4) Was obtained 9.1%. When differentiation was induced by four factors (FGF4, BMP4, XAV939, A83-01), 22.3% of PDGFRA, CEACAM1 and ANPEP positive cells were obtained. From these results, it was suggested that FGF and BMP are essential for PrE induction, and in addition to these, it is preferable to use a TGFβ inhibitor and a Wnt signal inhibitor.
On the other hand, when IL-6 was removed, a decrease in PDGFRA-positive cells was observed 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 lower).
GP130 was activated and GP130Y118F chimeric receptor activating STAT3 was introduced into naive PSC to induce PrE. It was found that expression of chimeric receptor from D0 reduces expression of PDGFRA positive cells, whereas expression from D2 effectively induces PDGFRA positive cells (FIG. 7B). This indicates that in humans, JAK-STAT signal is important for maintenance of naive pluripotent stem cells and at the same time plays an important role in PrE (FIG. 7B).

On the other hand, GATA6 was knocked down using shGATA6, and PDGFRA positive cells were induced using 7 factors. However, neither PDGFRA-positive cells nor CEACAM1-positive cells were induced, and GATA6 was found to be a key gene essential for PrE induction (FIG. 7C).

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

Claims (18)

A method of 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 (Bone morphogenetic protein) and FGF4 (Fibroblast growth factor 4) to induce primitive endoderm differentiation. The medium further comprises one or more selected from PDGF (Platelet-Derived Growth Factor), IL-6 (Interleukin 6), TGF (Transforming Growth Factor) β inhibitor, a Wnt signal inhibitor and retinoic acid. Item 2. A method for preparing primitive endoderm according to Item 1. The method for preparing primitive endoderm according to claim 1, wherein the medium comprises BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor. The BMP according to any one of claims 1 to 3, wherein BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939. Method of preparation of primitive endoderm. The primitive endoderm according to any one of claims 1 to 4, further comprising the step of purifying the primitive endoderm cells using CEACAM1 (carcinoembryonic antigen related cell adhesion molecule 1) or ANPEP (alanyl aminopeptidase, membrane). How to prepare. 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 obtain primed pluripotent stem cells. A method for preparing mesendoderm, comprising the step of differentiating to mesendoderm. A myocardial progenitor cell comprising the steps of preparing mesoderm by the method according to claim 8, and further culturing the resulting mesoderm to differentiate it into myocardial progenitor cells and / or pancreatic progenitor cells. And / or a method of preparing pancreatic precursor cells. A method for preparing mesendoderm, comprising the steps of providing primitive endoderm and co-culturing said primitive endoderm with primed pluripotent stem cells to differentiate primed pluripotent stem cells into mesoderm. Primitive endoderm cells prepared by the method according to any one of claims 1 to 7. A method of separating primitive endoderm cells from a cell population comprising primitive endoderm cells, comprising the step of selecting primitive endoderm cells using CEACAM1 or ANPEP. A method of detecting primitive endoderm cells in a cell population comprising primitive endoderm cells, comprising the step of detecting primitive endoderm cells using CEACAM1 or ANPEP. A reagent for detecting primitive endoderm cells, which comprises a molecule that specifically binds to CEACAM1 or ANPEP. Medium for naive type pluripotent stem cells containing BMP and FGF4. Furthermore, the medium for naive type pluripotent stem cells according to claim 15, further comprising one or more selected from PDGF, IL-6, a TGFβ inhibitor, a Wnt signal inhibitor and retinoic acid. The medium for naive type pluripotent stem cells according to claim 15, comprising BMP, FGF4, a TGFβ inhibitor and a Wnt signal inhibitor. 18. The method according to any one of claims 15 to 17, wherein the BMP is BMP4, BMP2 or BMP6, PDGF is PDGF-AA, the TGFβ inhibitor is A83-01, and the Wnt signal inhibitor is XAV939. Medium for naive type pluripotent stem cells.

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