JP7201223B2 - Method for selectively inducing endoderm cells from pluripotent stem cells - Google Patents

Description

By selectively inducing definitive endoderm subtypes that are intimately involved in the differentiation of pluripotent stem cells into cells of specific endodermal lineages, the present application provides an anterior and posterior foregut from pluripotent stem cells, and The present invention relates to a method for selectively inducing the middle hindgut. The present application also relates to a method for selectively inducing desired endoderm cells from any of the anterior foregut, posterior foregut and mid-hingegut.

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), differentiate into cells that make up endodermal organs such as the pancreas, liver, lung and small intestine. It has been reported in recent years to obtain In these reports, human pluripotent stem cells are induced into definitive endoderm cells via the primitive streak (PS) and then into various endoderm lineage cells. Definitive endoderm cell induction from human pluripotent stem cells is said to be confirmed by the expression of two known definitive endoderm marker genes, SOX17 and FOXA2.

During vertebrate development, definitive endoderm cells are known to fall into two distinct populations, the Anterior Definitive Endoderm and the Posterior Definitive Endoderm. These cells are further suggested to be patterned along the anterior-posterior axis of the embryo and to differentiate into distinct cells (28).

Definitive endoderm cells are formed via primitive streak cells (Non-Patent Documents 1, 20, 23). The formation of the primitive streak is an essential step in the differentiation of the definitive endoderm, and without the formation of the primitive streak, the formation of the definitive endoderm is said to be defective (Non-Patent Documents 3, 26). Presumptive fate mapping experiments using mouse embryos have shown that definitive endoderm progenitors are found within anterior primitive streak cells that express the T-box transcription factor, Eomesodermin (Eomes). Patent documents 4, 10, 23). There are reports suggesting that anterior and posterior definitive endoderm cells are derived from different developmental stages of the primitive streak (13).

Various methods as shown in Table 1 have been used to derive definitive endoderm cells from human pluripotent stem cells.

FBS:ウシ胎児血清、WNT3A：Wingless-type MMTV integration site family, member 3A、BMP:骨形成タンパク質、FGF:繊維芽細胞増殖因子、PI3K阻害剤: イノシトールリン脂質3キナーゼ阻害剤。

FBS: fetal bovine serum, WNT3A: Wingless-type MMTV integration site family, member 3A, BMP: bone morphogenetic protein, FGF: fibroblast growth factor, PI3K inhibitor: inositol phospholipid 3 kinase inhibitor.

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An object of the present application is to provide a method for selectively inducing mammalian pluripotent stem cells into definitive endoderm subtypes that differentiate into specific endoderm cells. The present application also aims to provide a method for selectively inducing pluripotent stem cells through each definitive endoderm subtype into the anterior and posterior foregut and the mid-hingegut. A further object of the present application is to provide a method for selectively inducing the differentiation of pluripotent stem cells into desired endoderm cells.

The present application differentiates pro- and late-stage Anterior Primitive Streak (APS) cells from pluripotent stem cells into three distinct types of definitive endoderm cell subpopulations: anterior to anterior definitive endoderm; Selectively differentiate into Anterior domain of Anterior Definitive Endoderm (AADE), Posterior domain of Anterior Definitive Endoderm (PADE), and Posterior Definitive Endoderm (PDE) cells provide a way to The present application further provides progenitors selected from the group consisting of Posterior Foregut (PFG) cells, Anterior Foregut (AFG) cells and Midhindgut (MHG) cells from these definitive endoderm cell populations, respectively. A method for inducing gut (Gut Tube, GT) cells is provided.

Thus, the present application provides pluripotent stem cells to definitive endoderm cells selected from the group consisting of anterior domain of anterior definitive endoderm (AADE) cells, posterior domain of anterior definitive endoderm (PADE) cells, and posterior definitive endoderm (PDE) cells. A method for selectively inducing a subpopulation of germ layer cells is provided comprising the following steps (i) and (ii):
(i) culturing the pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor to obtain prophase anterior primitive streak cells or late anterior primitive streak cells, and (ii) the obtained prophase anterior primitive streak cells Striatal cells, or late anterior primitive streak cells, are cultured in medium containing activin A and with or without BMP inhibitors to form anterior domain cells of anterior definitive endoderm, posterior to anterior definitive endoderm. Obtaining domain cells or posterior definitive endoderm cells.

The present application further provides a subpopulation of definitive endoderm cells selected from the group consisting of anterior domain of anterior definitive endoderm (AADE) cells, posterior domain of anterior definitive endoderm (PADE) cells, and posterior definitive endoderm (PDE) cells. Gastrula selected from the group consisting of anterior foregut (AFG) cells, posterior foregut (PFG) cells and midhindgut (MHG) cells, comprising the step of culturing the gastrulation ( GT) cells.

The present application provides methods for deriving anterior and posterior foregut cells and mid-heptal enterocytes from pluripotent stem cells via three patterns of definitive endoderm cells. The anterior foregut differentiates into the middle ear, thymus, thyroid, trachea, lung and esophagus, the posterior foregut into the stomach, duodenum, pancreas and liver, and the middle hindgut cells into the small and large intestine. It is known. That is, the method of the present application makes it possible to selectively and reliably induce the differentiation of human pluripotent stem cells into desired cells.

The present application also includes a method for obtaining cells constituting each endodermal organ by further inducing differentiation from anterior foregut cells, posterior foregut cells, or middle hindgut cells obtained by the method of the present application.

Two hypothetical models of definitive endoderm (DE) differentiation and patterning leading to differentiation of endodermal lineage cells derived from DE are shown. Schematic representation of two differentiation methods (Methods 1 and 2) to induce differentiation from human induced pluripotent stem cells (hiPSCs) to late gastrula (LGT) via the anterior primitive streak (APS) and DE. The hiPSC cell line 585A1 was differentiated into definitive endoderm (DE) by two differentiation methods. Immunostaining was performed for SOX17 and FOXA2, which are DE markers. Representative images of three independent experiments are shown. Scale bar, 100 μm. The hiPSC cell line 585A1 was differentiated into late gastrulation (LGT) by two differentiation methods. Immunostaining was performed for AFP, a hepatoblast marker. Representative images of three independent experiments are shown. Scale bar, 100 μm. Two procedures to induce differentiation from human induced pluripotent stem cells (hiPSCs) to late gastrula (LGT) via anterior primitive streak (APS), definitive endoderm (DE) and gastrulation (GT) (Method A and B) Schematic diagram. Gastrula (GT) stage cells derived from hiPSCs by two methods were immunostained for HNF1β, HNF4α and SOX2. In addition, cells at the late gastrulation (LGT) stage induced thereafter were immunostained for SOX2 and AFP. Scale bar 100 μm. qRT-PCR analysis results for SOX2 mRNA expression at each stage of differentiation when human induced pluripotent stem cells were induced to differentiate by Method B. APS: anterior primitive streak, AFG: anterior foregut, LAFG: anterior foregut region of late gastrula. hiPSC-derived late gastrulation (LGT) cells were differentiated into ALB(+) hepatocyte-like cells. Scale bar, 100 μm. hiPSC-derived late gastrulation (LGT) cells were differentiated into NKX2.1(+) alveolar progenitor cells. Scale bar, 100 μm. Heatmap showing gene expression for each marker of the primitive streak (PS) and two types of definitive endoderm (DE). Principal component analysis (PCA) for the differentiation of human induced pluripotent stem cells (hiPSCs) into late gastrulation anterior foregut region (LAFG) and late gastrula posterior foregut region (LPFG), respectively. Heat map showing differences in gene expression profiles between the anterior domain of the anterior definitive endoderm (AADE) and the posterior domain of the anterior definitive endoderm (PADE). APS: anterior primitive streak, AFG: anterior foregut, PFG: posterior foregut. Immunostaining of prophase and anaphase anterior primitive streak (APS) cells was performed for BRACHYURY, EOMES and CDX2. Scale bar, 100 μm. Two types of definitive endoderm cells were immunostained for SOX17 and FOXA2 (B). Scale bar, 100 μm. AADE: anterior domain of anterior definitive endoderm, PDE: posterior definitive endoderm Immunostaining images of gastrula (GT) stage cells for HNF1β, HNF4α and CDX2. Scale bar, 100 μm. PFG: posterior foregut, MHG: middle hindgut Results of qRT-PCR analysis of CDX2 mRNA expression at various stages of differentiation. LAPS: late anterior primitive streak, PDE: posterior definitive endoderm, MHG: mid-hingegut, LMHG: mid-hingegut region (A)-(C) are data from three independent experiments. are expressed as mean±S.E.M. (n=3). Differentiation procedure from hiPSCs to three definitive endoderm (DE) lineages. At each stage, marker genes that characterize the cells are indicated at the bottom of each stage. The anterior domain of the anterior definitive endoderm (AADE), the posterior domain of the anterior definitive endoderm (PADE) and the posterior definitive endoderm (PDE) are divided into the posterior foregut (PFG), anterior foregut (AFG) and middle hindgut (PDE), respectively. MHG). APS: anterior primitive streak, GT: gastrula.

Several differentiation protocols using different sets of inducers essential for early embryonic development have been proposed to derive definitive endoderm cells from human pluripotent stem cells (Table 1). One of the common features of induced definitive endoderm cells is that they express two marker genes, SOX17 and FOXA2, regardless of differentiation method. From the studies presented below, three subpopulations of definitive endoderm that can be further differentiated into anterior foregut, posterior foregut and mid-hingegut cells by controlling the method of induction from pluripotent stem cells to definitive endoderm. It was possible to selectively induce ration (Fig. 5A).

During chicken development, Brachyury (+) Eomes (+) cells are known to reside in APS during stages 3 to 5 of Hamburger Hamilton (HH) (A Chicken Embryo Gene Expression Database; http:/ /geisha.arizona.edu/geisha ( confirmed 2016.03.03) Expression of two BMP antagonists, Noggin and Chordin, in primitive nodes from HH stage 4. Therefore, Eomes after HH stage 4 (+) APS assumed to be influenced by Noggin and Chordin.

Addition of a BMP inhibitor in the anterior primitive streak to definitive endoderm induction step resulted in a subpopulation of definitive endoderm cells that differentiated into the anterior foregut but not the posterior foregut (Posterior domain of anterior definitive endoderm). On the other hand, when BMP inhibitor was not included, it was confirmed that DE cell subpopulation (Anterior domain of Anterior Definitive Endoderm, AADE cells) that differentiated into the posterior foregut was induced. (Fig. 2). Therefore, AADE produced from human iPS cells is a cell that will become the future posterior foregut region formed before the expression of Noggin and Chordin in the primitive nodule, whereas PADE is the cell that is formed from the primitive nodule to the influence of Noggin and Chordin. It is the cells that will become the future anterior foregut region formed below. Human iPS cell-derived posterior definitive endoderm (PDE) is induced from late APS and differentiates into middle hindgut cells (Fig. 5B).

In the specification and claims of the present application, pluripotent stem cells are stem cells that have pluripotency capable of differentiating into all cells existing in a living body and that also have proliferative potential. Embryonic stem (ES) cells (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165), clones obtained by nuclear transfer Embryonic stem (ntES) cells derived from embryos (T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502), spermatogonial stem cells (“GS cells”) (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012), embryonic germ cells ("EG cells") (Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551), induced pluripotent stem (iPS) cells (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. /069666), pluripotent cells derived from cultured fibroblasts and bone marrow stem cells (Muse cells) (WO2011/007900). Human pluripotent stem cells are used in the present application.

The pluripotent stem cells used in the method of the present application may be cultured as single cells by substantially separating (or dissociating) by any method, or may be cultured as cell aggregates in which cells are adhered to each other. It can be cultured under conditions. More preferably, it is separated into single cells and cultured. Methods of separation include, for example, mechanical separation and separation solutions having protease activity and collagenase activity (e.g., solutions containing trypsin and collagenase Accutase (TM) and Accumax (TM) (Innovative Cell Technologies, Inc)). ) or separation using a separation solution that has only collagenase activity. Pluripotent stem cells can be adherently cultured using coated culture dishes.

In each step of the method of the present application, the culture temperature is, but is not limited to, about 30 to 40°C, preferably about 37°C, culture is performed in an atmosphere of air containing _CO2 , and the _CO2 concentration is Preferably about 2-5%.

Step (i) of the present application is a step of culturing pluripotent stem cells in a medium containing activin A and a GSK3 inhibitor.
The medium used in step (i) can be prepared by appropriately adding activin A to the basal medium used for culturing animal cells. Examples of basal media include MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, and RPMI. 1640 medium, Fischer's medium, mixed medium of these and the like are included. The basal medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free. If necessary, e.g. albumin, transferrin, KnockOut Serum Replacement (KSR) (serum replacement for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids , insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), One or more substances such as non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and their equivalents, or other normal animal culture media It may contain one or more substances added. In one embodiment, the basal medium is RPMI 1640 medium with B27 supplement and fetal bovine serum.

The medium used in step (i) may further contain a ROCK inhibitor. ROCK inhibitors are not particularly limited as long as they can suppress the function of Rho-kinase (ROCK), for example, Y-27632 (e.g., Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000) Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil/HA1077 (e.g. Uenata et al., Nature 389: 990-994 (1997)), SR3677 (e.g. Feng Y et al. , J Med Chem. 51: 6642-6645 (2008)), GSK269962 (e.g. Stavenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005/037197), H-1152 (e.g. Ther. 93: 225-232 (2002)), Wf-536 (e.g. Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)) and Included are derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNA), dominant-negative mutants, and expression vectors thereof. In addition, other known low-molecular-weight compounds can also be used as ROCK inhibitors (e.g., US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508). , WO 2004/0002507, WO 2003/0125344, WO 2003/0087919, and WO 2003/062227, WO 2003/059913, WO 2003/062225, WO 2002/076976. 2004/039796). One or more ROCK inhibitors may be used in the present invention. Preferred ROCK inhibitors for use in this step include Y-27632, Fasudil/HA1077, SR3677, GSK269962 and H-1152.

When using Y-27632 as a ROCK inhibitor, the concentration in the medium is 0.1 μM to 100 μM, preferably 1 μM to 500 μM, more preferably 5 μM to 200 μM, eg about 10 μM. In the specification and claims of the present application, the term "about" attached to a numerical value includes values up to ±30%, ±20%, or ±10% of the numerical value.

As activin A, any mammal-derived activin such as human or mouse can be used. As the activin used in the present invention, it is preferable to use activin derived from the same animal species as the pluripotent stem cells used for differentiation. For example, when human-derived pluripotent stem cells are used as starting materials, human-derived activin It is preferable to use These activins are commercially available.

The concentration of activin A in the medium is usually 0.1 to 200 ng/ml, such as 5 to 150 ng/ml, preferably 80 to 120 ng/ml, particularly preferably about 100 ng/ml.

A GSK3 inhibitor is defined as a substance that inhibits the kinase activity of GSK-3β protein (for example, the ability to phosphorylate β-catenin), and many of them are already known. SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl )-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione) , GSK-3β inhibitor VII (4-dibromoacetophenone), a phenyl α-bromomethyl ketone compound, L803-mts (a.k.a. GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP- NH2) and the highly selective CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)
pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile). These compounds are commercially available from, for example, Calbiochem and Biomol, and can be easily used. A GSK-3β inhibitor for use in the present invention may preferably be CHIR99021.

In step (i), a relatively low concentration of GSK3β inhibitor is used to obtain early anterior primitive streak (Early APS) cells. By "relatively low concentration" is meant a concentration lower than that for inducing the late anterior primitive streak (Late APS) as described below. When using CHIR99021, the concentration in the medium may be from 1 μM to less than 4 μM, such as from 1.5 μM to 3.5 μM, preferably about 3 μM.

Generation of the prophase anterior primitive streak can be confirmed by the cells expressing BRACHYURY, EOMES and not CDX2.

In step (i), a relatively high concentration of GSK3β inhibitor is used to obtain late anterior primitive streak (Late APS) cells. When using CHIR99021 as a GSK3β inhibitor, the concentration in the medium for obtaining late anterior primitive streak (Late APS) cells may be 4 μM or more, for example 4 μM to 15 μM, preferably about 8 μM.

Generation of late anterior primitive streak cells can be confirmed by the cells expressing BRACHYURY, EOMES and CDX2.

The number of days of culture in step (i) may be 12 to 36 hours, eg, about 1 day.
Depending on the concentration of the GSK 3β inhibitor in step (i), pluripotent stem cells can either form early anterior primitive streak cells (cultured in the presence of relatively low concentrations of GSK3β inhibitor) or late anterior primitive streak cells (comparative cultured in the presence of a high concentration of GSK3β inhibitor).

In step (ii), the cells obtained in step (i) are cultured in a medium containing activin A with or without BMP inhibitors.
The basal medium used in step (ii) may be appropriately selected from known animal culture basal mediums similar to those used in step (i). The basal media in steps (i) and (ii) may be the same or different. For example, a medium obtained by adding fetal bovine serum to RPMI1640 as a basal medium may be used as a basal medium, and activin A may be added thereto. The type and amount of activin A may be the same as in step (i).

BMP inhibitors include proteinaceous inhibitors such as Chordin, Noggin, Follistatin, Dorsomorphin 6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1, 5-a]pyrimidine, its derivatives (P. B. Yu et al. (2007), Circulation, 116:II_60; P.B. Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3- yl) quinoline) is exemplified, and LDN-193189 is preferably used.

When using LDN-193189 as a BMP inhibitor, its concentration may be, for example, 100-1000 nM, 300-700 nM, eg about 500 nM.
The culture period of step (ii) may be 36 hours to 60 hours, for example about 2 days.
In step (ii), three subpopulations of definitive endoderm cells can be selectively obtained. Namely
(1) When prophase anterior primitive streak cells are cultured in the presence of activin A and a BMP inhibitor, anterior definitive endoderm posterior domain (PADE) cells are obtained.
(2) When prophase anterior primitive streak cells are cultured in the presence of activin A but in the absence of BMP inhibitors, anterior domain of the anterior definitive endoderm (AADE) cells are obtained.
(3) Posterior definitive endoderm (PDE) cells are obtained when late anterior primitive streak cells are cultured in the presence of activin A but in the absence of BMP inhibitors.
Both definitive endoderm cells express SOX17 and FOXA2.

The present application provides anterior foregut (AFG) cells, posterior anterior Further provided is a method of obtaining intestinal (PFG) cells or mid-heptal gut (MHG) cells.

The basal medium used in this step may be appropriately selected from those described in step (i). Examples of basal medium include IMEM Zinc Option medium. The basal medium is used without adding substances such as avitin, GSK3β inhibitor, BMP inhibitor and other differentiation-inducing factors. The basal medium used may contain one or more of the substances listed in step (i) above and other substances usually added to animal cell culture media. The culture period of step (iii) may be 3-8 days, for example about 6 days.
Step (iii) induces PADE cells into anterior foregut (AFG) cells, AADE cells into posterior foregut (PFG) cells, and PDE cells into mid-hestgut (MHG) cells.

Thus, the present application includes the following three aspects:
1 First embodiment A method for producing anterior foregut cells from human pluripotent stem cells via posterior domain cells of anterior definitive endoderm, the method comprising the following steps (i-1) to (iii-1) :
(i-1) a step of culturing pluripotent stem cells in a medium containing activin A and a relatively low concentration of a GSK3β inhibitor, and (ii-1) the cells obtained in step (i-1) with activin and (iii-1) culturing the cells obtained in step (ii-1) in a medium containing no differentiation-inducing factor.

2 Second embodiment A method for producing posterior foregut cells from human pluripotent stem cells via anterior domain cells of anterior definitive endoderm, the method comprising the following steps (i-2) to (iii-2) :
(i-2) culturing the pluripotent stem cells in a medium containing activin A and a relatively low concentration of a GSK3β inhibitor;
(ii-2) culturing the cells obtained in step (i-2) in a medium containing activin A, and (iii-2) the cells obtained in step (ii-2) are differentiated culturing in medium without inducer.

3 Third Aspect A method for producing mid-hinge enterocytes from pluripotent stem cells via retro-definitive endoderm cells, the method comprising the following steps (i-3) to (iii-3):
(i-3) a step of culturing pluripotent stem cells in a medium containing activin A and a relatively high concentration of a GSK3β inhibitor, and (ii-3) the cells obtained in step (i-3), a step of culturing in a medium containing activin A; and (iii-3) a step of culturing the cells obtained in step (ii-3) in a medium containing no differentiation inducer.

AFG cells differentiate into cells that make up the middle ear, thymus, thyroid, trachea, lung and esophagus. PFG is differentiated into cells that make up the stomach, duodenum, pancreas and liver. and MHG are known to differentiate into cells that constitute the small and large intestine. Methods for inducing differentiation into each cell are known, and those skilled in the art can appropriately select from known methods. The present application also includes a method for obtaining the above cells by further inducing differentiation of each cell obtained by the method of the present application.

The present application will be described in more detail based on the following examples.
1. Materials and methods
1.1. Cell Culture To obtain feeder-free cultures, hiPSCs (585A1 cells) (Okita K et al., 2011, Kajiwara M et al., 2012) were grown in Essential 8 medium (Thermo Fisher Scientific, Waltham, MA). For routine subculturing, hiPSC colonies were isolated enzymatically with 0.5 mM EDTA (Wako, Osaka, Japan) and expanded at a ratio of 1:100 by adding 10 μM Y-27632 (Wako). divided.

1.2. Differentiation into the anterior primitive streak (APS)
80% confluent hiPSC colonies were enzymatically dissociated into single cells with 0.5 mM EDTA. The cells were supplemented with 100 ng/ml recombinant human/mouse/rat activin A and 3 μM CHIR99021 (Axon Medchem, Groningen, Netherlands) for early APS induction and 100 ng for late APS induction. 2% (vol/vol) B27 supplement (GFR-B27, Growth Factor Reduced, Thermo Fisher Scientific), 50 U/ml Penicillin/Streptomycin (P/S, Thermo Fisher Scientific), supplemented with /ml Activin A and 8 μM CHIR99021 ) and 10 μM Y-27632 in RPMI 1640 medium (NACALAI TESQUE, Kyoto, Japan) and plated at 9×10 ⁴ cells/cm ² on Matrigel (Becton Dickinson, Franklin Lakes, NJ) coated plates. Seeded and cultured for 1 day.

1.3. Differentiation to definitive endoderm (DE) Differentiation from prophase APS to DE cells
RPMI 1640 medium supplemented with 2% (vol/vol) B27 supplement (GFR-B27), 50 U/ml P/S and 100 ng/ml activin A for induction into AADE cells. Prophase APS to anterior definitive endoderm (AADE) cells or anterior domain of the anterior definitive endoderm (AADE) cells were cultured for 2 days in RPMI 1640 medium supplemented with 100 ng/ml activin A and 500 nM LDN193189 (Axon Medchem) for induction. It was directed to the posterior domain of the definitive endoderm (PADE).
Differentiation of Late APS to DE Cells Late APS cells were incubated in RPMI 1640 medium supplemented with 2% (vol/vol) B27 supplement (GFR-B27), 50 U/ml P/S and 100 ng/ml Activin A for 2 days. Cultured and induced into posterior definitive endoderm (PDE) cells.

1.4. Differentiation into gastrula (Gut Tube, GT) and late gastrula (Late Gut Tube, LGT) cells
For GT cell induction, three types of DE cells (AADE, PADE and PDE) were cultured in Improved MEM Zinc Option (iMEM) medium (Thermo Fisher Scientific) for 3 days. The GT cells were then cultured in the same medium for 3 days to differentiate into LGT cells.

1.5. Differentiation into Liver and Lung Lineage Cells They were further differentiated into hepatocyte-like cells and alveolar epithelial progenitor cells according to Cell Reports 3, 394-403.).
Differentiation into hepatocyte-like cells
hiPSC-derived LGT cells obtained in 1.4 were treated with 20 ng/mL recombinant human hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ) and 20 ng/mL recombinant human oncostatin M (OsM; PeproTech). Differentiation into hepatocyte-like cells was induced by culturing in hepatocyte culture medium for 6 days.
Differentiation into alveolar epithelial progenitor cells
hiPSC-derived LGT cells obtained in 1.4 were treated with 1×B27 and N2 supplement (Thermo Fisher Scientific), 50 U/ml P/S, 0.05 mg/ml L-ascorbic acid (Sigma-Aldrich, Tokyo, Japan), 0.4 Glutamax containing mM monothioglycerol (Wako), 100 ng/ml recombinant human bone morphogenetic protein (BMP)4 (R&D Systems), 0.5 μM all-trans retinoic acid (ATRA; Sigma-Aldrich) and 3.5 μM CHIR99021 was added to DMEM/F12 medium (Thermo Fisher Scientific) for 4 days.

1.6. Immunostaining Cells were fixed with 4% paraformaldehyde (PFA)/PBS for 20 minutes at 4°C. After washing with PBS, cells were blocked with 5% normal donkey serum (Funakoshi, Tokyo, Japan)/PBST (PBS/0.1% Triton X-100) for 1 hour at room temperature. Primary antibodies were diluted in blocking solution and incubated with samples overnight at 4°C. After washing three times with PBS, cells were incubated with secondary antibody for 1 hour at room temperature. Secondary antibodies used in this study included Alexa Fluor 488-, 546- or 647-conjugated anti-mouse, rabbit, or goat IgG donkey antibodies, used at a 1:200 dilution. For nuclear staining, Hoechst 33342 trihydrochloride trihydrate (Thermo Fisher Scientific) was used at a 1:200 dilution. Table 2 shows the primary antibodies used.

1.7. RT-PCR and real-time quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using the RNeasy kit (Qiagen, Tokyo, Japan) according to the manufacturer's recommendations, followed by cDNA synthesis using standard protocols (Mae S et al., 2013). Briefly, first-strand cDNA was synthesized from 1 μg of total RNA using ReverTra Ace (TOYOBO, Osaka, Japan). The cDNA samples were subjected to PCR amplification using a thermal cycler (Veriti 96-well Thermal Cycler, Applied Biosystems, Waltham, Mass.) and Ex-Taq PCR kit (Takara, Shiga, Japan) according to the manufacturer's instructions. , PCR was performed. The PCR cycles were as follows: initial denaturation at 94°C for 2.5 minutes, followed by 94°C for 30 seconds, 60°C for 1 minute, and 72°C for 30 seconds for the housekeeping gene β-ACTIN. 25 cycles and a final extension reaction were performed at 72°C for 10 minutes. For other genes, initial denaturation at 94°C for 2.5 minutes, followed by 30-40 cycles of 94°C for 30 seconds, 58°C-50°C for 30-60 seconds, 72°C for 30 seconds, and a final The extension reaction was cycled at 72°C for 10 minutes. qPCR was performed using the Step One Plus Real-Time PCR System (Applied Biosystems) and SYBR Green PCR Master Mix (Takara). Denaturation was performed at 95°C for 30 seconds, followed by 24 cycles of 95°C for 5 seconds and 60°C for 30 seconds. A threshold cycle method was used to analyze the gene expression level data and calibrate to the gene expression levels of β-ACTIN as recommended by the manufacturer. PCR reactions were performed in triplicate for each sample. Table 3 shows the sequences of the primers used.

1.8. RNA Sequencing 100 ng of total RNA from cultured cells was subjected to library preparation using KAPA stranded mRNA-seq Kits (KAPA Biosystems, Woburn, MA) according to the manufacturer's instructions. The library was sequenced on the HiSeq2500 in Single-Read mode for 100 cycles. All sequence reads were extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in the CASAVA 1.8.2 pipeline. Sequence reads were mapped to the hg19 reference gene downloaded on April 25, 2014 using Tophat v2.0.14. Levels were expressed as log ₂ (RPKM+1). Gene expression heatmaps were generated by the heatmap.2 function of the gplots library in R 3.2.1. Two-way hierarchical clustering of gene expression in tissue and cell samples was performed using the hclust function in R3.2.1.

2. Results
2.1.Different Differentiation Protocols Confirmed Generation of Different Types of Definitive Endoderm (DE) Cells There are two models that characterize definitive endoderm cell populations: 1) DE cells are 2) DE cells contain multiple types of lineage-committed endoderm progenitor populations, which are a single-cell type of multipotent endoderm progenitor cells capable of differentiating into any endoderm lineage cell (Fig. 1A, left); (Fig. 1A, right). Given that DE cells are a single type of pluripotent endoderm progenitor cells, the present inventors have found that even DE cells induced by different differentiation protocols are uniformly endoderm lineage cells, e.g. It was hypothesized that they could be differentiated into α-fetoprotein (AFP) (+) cells at the late gastrulation (LGT) stage, ie hepatoblasts.

To test this hypothesis, we derived AFP (+ ) developed a direct differentiation method to cells. Induction of APS by treatment with activin A and CHIR99021 followed by treatment with activin A alone induced SOX17(+) FOXA2(+) DE cells (Fig. 1B method 1, Fig. 1C top panel) very efficiently. induced. Furthermore, treatment with BMP4 and fibroblast growth factor 2 (FGF2) generated AFP(+) cells at the LGT stage from DE cells (Fig. 1D upper panel).

By removing extrinsic mesodermal differentiation signals such as BMPs and by neutralizing endogenous BMPs with the BMP antagonist noggin or the BMP receptor inhibitor LDN193189, DE cells It has been reported that it can be induced from APS cells (Non-Patent Document 11). We confirmed that treatment with activin A and LDN193189 also reliably generated SOX17(+)FOXA2(+)DE cells from APS cells (Fig. 1B, Method 2, Fig. 1C, lower panel). However, the latter cells could not be differentiated into AFP(+) cells by treatment with BMP4 and FGF2 (FIG. 1D, lower panel). These data demonstrate that DE cells derived from hiPSCs using different protocols differ in their ability to differentiate into AFP-positive late gastrula foregut region cells. That is, it was found that the fate of differentiating into various endoderm cells was already restricted at the DE stage, and the model diagram shown on the right side of FIG. 1A was found to be correct.

2.2. Differentiation tropism of the two DE cell subtypes
To investigate whether the differentiation potential of the DE cell subpopulations is restricted to specific endoderm lineage cells, the DE subpopulations produced by the two methods of FIG. The cells were cultured for an additional 3-6 days for differentiation (Fig. 2A). After 3 days of culture, the cells of Method A differentiated into HNF1β and HNF4α positive cells, markers of the posterior foregut region of GT cells. Almost no cells expressing SOX2, which is a marker for the anterior foregut region of GT cells, were present (Figures 2A and 2B, upper figures). When HNF1β(+) HNF4α(+) cells were further cultured, they differentiated into AFP(+) cells at the LGT stage (Fig. 2B, upper panel).

This result indicates that DE cells induced by Method A differentiate into cells of the posterior foregut region of late gastrulation, ie, hepatoblasts, without the addition of exogenous inducers. On the other hand, the cells of Method B were SOX2(+) at the LGT stage and differentiated into cells of the anterior foregut region of late gastrula. HNF1β, HNF4α and AFP were negative (FIG. 2B lower panel). SOX2 mRNA expression at each stage was confirmed by qRT-PCR analysis (Fig. 2C). These results suggest that the potential for future differentiation toward specific endoderm lineages is already restricted to the DE stage.

To evaluate the developmental potential of hiPSC-derived LGT cells in vitro, hepatocyte-like cells and alveolar epithelial cells were isolated from LGT cells produced by Method A or B using previously reported methods (Non-Patent Documents 6 and 7). Differentiation into progenitor cells was induced. It was confirmed that hiPSC-derived LGT cells induced by method A differentiated into ALBUMIN (ALB) (+) cells, but did not differentiate into NKX2.1 (+) ventral anterior foregut cells (Fig. 2D, 2E upper diagram). In contrast, LGT cells induced by Method B differentiated into NKX2.1(+) cells without differentiating into AFP(+) or ALB(+) cells (FIGS. 2D, 2E lower panels). These results indicate that DE cells induced with method A are anterior domain of anterior definitive endoderm (AADE) cells that give rise to the posterior foregut region of GT and LGT cells, whereas DE cells induced with method B are the posterior domain of the anterior definitive endoderm (PADE) cells that differentiate into the anterior foregut region of GT and LGT cells.

2.3. Comparison of gene expression profiles of AADE and PADE The above results suggest that DE cells generated by different protocols have the potential to differentiate into specific endoderm lineages. RNA sequencing analysis was performed to reveal differences in gene expression patterns between the two types of DE cells, AADE and PADE cells and their derived GT and LGT cells. As a result, there was little difference in the expression levels of well-known PS and DE markers between AADE and PADE cells (Fig. 3A). Principal component analysis (PCA) also confirmed relatively similar gene expression profiles between AADE and PADE cells (Fig. 3B). However, we identified 273 differentially expressed genes between the AADE and PADE cell populations (Fig. 3C and Table 4). These data suggest that AADE and PADE have similar expression patterns to well-known DE and PS markers, but that candidate markers exist that can distinguish these DE subpopulations.

2.4. Establishment of a method for inducing late anterior primitive streak (late APS), posterior definitive endoderm (PDE) and middle hindgut (MHG) cells Novel differentiation of PDE cells with restricted differentiation potential to middle hindgut cells To establish the protocol, differentiation from hiPSCs to APS was further investigated. Based on reports that patterning into mesoderm subtypes correlates with the timing of mesoderm induction in PS (15), we postulate that DE patterning may also correspond to the timing of DE cell differentiation in APS. bottom. Under this assumption, hiPSC-derived APS cells induced by treatment with 100 ng/ml activin A and 3 μM CHIR99021 were characterized (Fig. 2A). By immunostaining, these cells were positive for the APS markers BRACHYURY and EOMES, but few cells were positive for CDX2, a marker for the late primitive streak (FIG. 4A, upper panel). These data demonstrate that treatment with 100 ng/ml activin A and 3 μM CHIR99021 differentiates hiPSCs into prophase APS cells. Therefore, a novel method for differentiating late APS cells from hiPSCs was developed by modifying the late primitive streak differentiation protocol (Non-Patent Document 15). Culture was performed for 1 day in the presence of 100 ng/ml activin A and 8 μM CHIR99021. By immunostaining the obtained cells, it was confirmed that late stage APS cells, which are BRACHYURY (+) EOMES (+) CDX2 (+), were generated (FIG. 4A, lower diagram).

For derivation of DE cells from late APS cells, they were further cultured in the presence of 100 ng/ml activin A for 2 more days. It was confirmed that it was generated (lower diagram in FIG. 4B). The cells were cultured for an additional 3 days without the addition of exogenous inducers. The developmental potential of the obtained cells toward the endodermal lineage was investigated, and differentiated cells stained positively for HNF1β, HNF4α and CDX2, which are mid-hintestinal markers, were found (FIG. 4C, lower diagram). These findings were also confirmed by qRT-PCR analysis for CDX2 mRNA expression. In contrast, the hiPSC-derived posterior foregut region of GT cells generated from hiPSCs through prophase APS and AADE did not differentiate into the CDX2(+) middle hindgut region of GT cells (Figures 4A, 4B, 4C top). figure). These results indicate that an efficient differentiation method from hiPSCs to mid-heptal enterocytes, via PDE cells, can be established by generating late APS cells, and that patterning into DE subtypes suggesting that it correlates with the timing of DE induction in APS (Fig. 5A).

From the above results, it was confirmed that definitive endoderm cells contain multiple subtypes, each of which differentiates into a specific endoderm lineage (Fig. 5B). We also established a method for selectively inducing each subtype of definitive endoderm cells from pluripotent stem cells (Fig. 5A).

Claims (8)

A method for selectively inducing anterior foregut (AFG) cells from pluripotent stem cells, the method comprising the following steps (i), (ii) and (iii):
(i) culturing the pluripotent stem cells in medium containing activin A and a relatively low concentration of a GSK3β inhibitor having activity equivalent to CHIR99021 from 1 μM to less than 4 μM to obtain prophase anterior primitive streak cells; ,
(ii) culturing the prophase anterior primitive streak cells obtained in step (i) in medium containing activin A and a BMP inhibitor to obtain anterior definitive endoderm posterior domain (PADE) cells; and
(iii) culturing the obtained anterior definitive endoderm posterior domain (PADE) cells in a differentiation-inducing agent-free medium to obtain anterior foregut (AFG) cells; 2. The method of claim 1 , wherein said BMP inhibitor is LDN-193189. A method of selectively inducing pluripotent stem cells into posterior foregut (PFG) cells, the method comprising the following steps (i), (ii) and (iii):
(i) culturing the pluripotent stem cells in medium containing activin A and a relatively low concentration of a GSK3β inhibitor having activity equivalent to CHIR99021 from 1 μM to less than 4 μM to obtain prophase anterior primitive streak cells; ,
(ii) culturing the prophase anterior primitive streak cells obtained in step (i) above in medium containing activin A to obtain anterior domain of the anterior definitive endoderm (AADE) cells; and (iii) above Culturing the anterior domain of anterior definitive endoderm (AADE) cells obtained in step (ii) in a medium free of differentiation inducers to obtain posterior foregut (PFG) cells . The method of any of claims 1-3 , wherein the GSK3β inhibitor is CHIR99021 and the concentration of CHIR99021 in the medium is about 3 μM. A method for selectively inducing pluripotent stem cells into mid-heptal gut (MHG) cells, the method comprising the following steps (i), (ii) and (iii):
(i) culturing the pluripotent stem cells in a medium containing activin A and a relatively high concentration of a GSK3β inhibitor having an activity equivalent to 4 μM or more of CHIR99021 to obtain late anterior primitive streak cells ;
(ii) culturing the late anterior primitive streak cells obtained in step (i) in a medium containing activin A to obtain posterior definitive endoderm (PDE) cells; and
(iii) culturing the obtained posterior definitive endoderm (PDE) cells in a differentiation-inducing factor-free medium to obtain mid-hinggut (MHG) cells; 6. The method of claim 5 , wherein the GSK3[beta] inhibitor is CHIR99021, and the concentration of CHIR99021 in the medium is 4 [mu]M or higher. 7. The method of claim 6 , wherein the concentration of said CHIR99021 in culture medium is about 8 [mu]M. The method according to any one of claims 1 to 7 , wherein the pluripotent stem cells are human iPS cells or human ES cells.

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