WO2017175866A1 - Method for selectively inducing endodermal cells from pluripotent stem cells - Google Patents

Abstract

This method is for selectively inducing a sub-population of definitive endoderm cells selected from the group consisting anterior-domain-of-anterior-definitive-endoderm (AADE) cells, posterior-domain-of-anterior-definitive-endoderm (PADE) cells, and posterior-definitive-endoderm (PDE) cells, from pluripotent stem cells, and the method comprises a step (i) for culturing the pluripotent stem cells in a culture medium containing activin A and a GSK3β inhibitor, to obtain early anterior primitive streak cells or late anterior primitive streak cells, and a step (ii) for culturing the obtained early anterior primitive streak cells or late anterior primitive streak cells in a culture medium that contains activin A, and contains or does not contain a BMP inhibitor, to obtain anterior-domain-of-anterior-definitive-endoderm cells, posterior-domain-of-anterior-definitive-endoderm cells, or posterior-definitive-endoderm cells. The sub-population of definitive endoderm cells can be differentiated and induced into desired endodermal cells.

Description

A method for selectively inducing endoderm cells from pluripotent stem cells

The present application selectively induces definitive endoderm subtypes that are deeply involved in the differentiation of pluripotent stem cells into specific endoderm lineage cells, thereby leading to 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 further than any one of anterior foregut, posterior foregut and middle hindgut.

Human pluripotent stem cells (hPSC), such as human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC), differentiate into cells that make up endoderm organs such as pancreas, liver, lung and small intestine It has been reported in recent years (Non-Patent Documents 5-7, 14, 17, 18, 21, 25, 27). In these reports, human pluripotent stem cells are induced into definitive endoderm cells via primitive streak (PS), and then induced into various endoderm cells. It is said that definitive endoderm cell induction from human pluripotent stem cells can be confirmed by the expression of two known definitive endoderm marker genes, SOX17 and FOXA2.

In the development of vertebrates, it is known that definitive endoderm cells are classified into two different populations, anterior definitive endoderm cells (Anterior Definitive Endoderm) and posterior definitive endoderm cells (Posterior Definitive Endoderm). These cells are further suggested to be patterned along the anteroposterior axis of the embryo and differentiated into separate cells (28).

Definitive endoderm cells are formed through primitive streak cells (Non-patent Documents 1, 20, and 23). The formation of primitive streak is an indispensable step for the differentiation of definitive endoderm, and if there is no formation of primitive streak, it is said that a defect occurs in the formation of definitive endoderm (Non-patent Documents 3 and 26). Prototype fate map experiments using mouse embryos indicate that definitive endoderm progenitor cells are found in anterior primitive streak cells that express the T-box transcription factor, Eomesodermin (Eomes) (non- Patent Documents 4, 10, and 23). There are reports suggesting that anterior definitive endoderm cells and posterior definitive endoderm cells are derived from primitive streak at different developmental stages (Non-patent Document 13).

Until now, it has been reported to induce definitive endoderm cells from human pluripotent stem cells using various methods as shown in Table 1.

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.

The present application aims to provide a method of selectively inducing a definitive endoderm subtype that differentiates from a mammalian pluripotent stem cell into a specific endoderm cell. It is another object of the present application to provide a method for selectively inducing the anterior foregut and posterior foregut and the middle foregut through each definitive endoderm subtype of pluripotent stem cells. It is another object of the present application to provide a method for selectively inducing differentiation from pluripotent stem cells to desired endoderm cells.

The present application differentiates anterior and primitive developmental anterior streak cells (Anterior Primitive Streak, APS) from pluripotent stem cells, and three different types of definitive endoderm cell subpopulations: anterior to the definitive endoderm Selective differentiation into domain (Anterior domain of Anterior Definitive Endoderm, AADE) cells, anterior definitive endoderm posterior domain (Posterior domain of Anterior Definitive Endoderm, PADE) cells, and posterior definitive endoderm (Posterior Definitive Endoderm, PDE) cells Provide a way to make it happen. The present application further includes a source selected from the group consisting of Posterior 前 Foregut (PFG) cells, Anterior 腸 Foregut (AFG) cells and Midhindgut (MHG) cells from these definitive endoderm cell populations, respectively. A method for inducing intestinal (Gut Tube, GT) cells is provided.

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

The present application further includes a subpopulation of definitive endoderm cells selected from the group consisting of anterior definitive endoderm anterior domain (AADE) cells, anterior definitive endoderm posterior domain (PADE) cells, and posterior definitive endoderm (PDE) cells. Gastrointestinal tract selected from the group consisting of anterior foregut (AFG) cells, posterior foregut (PFG) cells, and midhindgut (MHG) cells, comprising a step of culturing in a medium containing no stimulating factor GT) A method of inducing into a cell is provided.

The present application provides a method for inducing anterior foregut cells, posterior foregut cells, and middle and foregut cells from pluripotent stem cells via three patterns of definitive endoderm cells. The anterior foregut is differentiated into the middle ear, thymus, thyroid, trachea, lungs, and esophagus, the posterior foregut is differentiated into the stomach, duodenum, pancreas, and liver, and the midhindgut cells are differentiated into the small and large intestines It is known. That is, differentiation induction from human pluripotent stem cells to desired cells can be selectively and reliably performed by the method of the present application.

Also included in the present application is a method of further differentiation-inducing cells from the anterior foregut cells, posterior foregut cells or mid-hind enterocytes obtained by the method of the present application to obtain cells constituting each endoderm organ.

Two hypothetical models of definitive endoderm (DE) differentiation and pattern formation leading to differences in endoderm cells derived from DE are shown. Schematic diagram of two differentiation methods (Methods 1 and 2) for inducing differentiation from human induced pluripotent stem cells (hiPSC) via anterior primitive streak (APS) and DE to late gastrulation (LGT). hiPSC cell line 585A1 was differentiated into definitive endoderm (DE) by two differentiation methods. Immunostaining was performed for DE markers SOX17 and FOXA2. Representative images of 3 independent experiments are shown. Scale bar, 100 μm. hiPSC cell line 585A1 was differentiated into late gastrulation (LGT) by two differentiation methods. Immunostaining was performed for AFP, a marker for hepatoblasts. Representative images of 3 independent experiments are shown. Scale bar, 100 μm. Two procedures for inducing differentiation from human induced pluripotent stem cells (hiPSC) to the early gastrulation (LGT) via the anterior primitive streak (APS), definitive endoderm (DE) and gastrulation (GT) (Method A and Schematic of B). Immunostaining was performed for HNF1β, HNF4α and SOX2 in gastrointestinal (GT) stage cells derived from hiPSC in two ways. In addition, SOX2 and AFP were immunostained for cells in the late gastrointestinal (LGT) stage induced thereafter. Scale bar 100 μm. QRT-PCR analysis results of SOX2 mRNA expression at each differentiation stage when differentiation was induced from human induced pluripotent stem cells by Method B. APS: anterior primitive streak, AFG: anterior foregut, LAFG: anterior foregut region of late gastrulation. 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. A heat map showing gene expression for each marker of primitive streak (PS) and two types of definitive endoderm (DE). Principal component analysis (PCA) for the differentiation of human induced pluripotent stem cells (hiPSC) into the anterior foregut region (LAFG) of the late gastrulation and the posterior foregut region (LPFG) of the late gastrulation. A heat map showing the difference in gene expression profiles between the anterior definitive endoderm anterior domain (AADE) and the anterior definitive endoderm posterior domain (PADE). APS: anterior primitive streak, AFG: anterior foregut, PFG: posterior foregut. BRACHYURY, EOMES and CDX2 were immunostained for early and late anterior primitive streak (APS) cells. 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 gastrointestinal (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 differentiation stages. LAPS: Late anterior primitive streak, PDE: Posterior definitive endoderm, MHG: Middle hindgut, LMHG: Middle hindgut region (A)-(C) of late gastrulation are data from 3 independent experiments Are expressed as mean ± SEM (n = 3). Differentiation procedure from hiPSC into three definitive endoderm (DE) lineages. At each stage, marker genes that characterize the cells are shown at the bottom of each stage. The anterior definitive endoderm anterior domain (AADE), the anterior definitive endoderm posterior domain (PADE) and the posterior definitive endoderm (PDE) are the posterior foregut (PFG), anterior foregut (AFG), and midhind gut (respectively) MHG). APS: anterior primitive streak, GT: gastrulation.

Several differentiation protocols using different sets of inducers essential for early embryogenesis have been proposed for inducing definitive endoderm cells from human pluripotent stem cells (Table 1). One common feature of induced definitive endoderm cells is that they express two marker genes, SOX17 and FOXA2, regardless of the differentiation method. From the following tests, three subpopulations of definitive endoderm that are further differentiated into anterior foregut, posterior foregut and mid-hind enterocytes by controlling the induction method from pluripotent stem cells to definitive endoderm Was able to be selectively induced (FIG. 5A).

During chicken development, Brachyury (+) Eomes (+) cells are known to be present in APS during Hamburger Hamilton (HH) stages 3-5 (A Chicken Embryo Gene Expression Database; http: / /geisha.arizona.edu/geisha ( confirmed 03.03.2016) Expression of two BMP antagonists, Noggin and Chordin, in the primitive node is observed from HH stage 4. Therefore, Eomes after HH stage 4 (+) APS assumed to be affected by Noggin and Chordin.

By adding a BMP inhibitor in the induction step from the anterior primitive streak to definitive endoderm, the definitive endoderm cell subpopulation (Posterior domain of Anterior Definitive) that differentiates into the anterior foregut but not into the posterior foregut Endoderm, PADE cells), but if a BMP inhibitor is not included, confirms that it induces a DE cell subpopulation (Anterior domain of Anterior Definitive Endoderm, AADE cells) that differentiates into the posterior foregut (FIG. 2). Therefore, AADE produced from human iPS cells is the cell that will be the future posterior foregut region formed before Noggin and Chordin are expressed in the primitive nodules, while PADE is the effect of Noggin and Chordin from the primitive nodules It is the cell that will be the future anterior foregut region formed below. Human iPS cell-derived posterior definitive endoderm (Posterior Definitive Endoderm, PDE) is induced from late APS and differentiates into mid-hind enterocytes (Fig. 5B).

In the present specification and claims, a pluripotent stem cell is a stem cell that has pluripotency that can be differentiated into all cells existing in a living body and also has proliferative ability. Embryonic stem (ES) cells (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55: 254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165), clones obtained by nuclear transfer Embryonic-derived embryonic stem (ntES) cells (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), sperm 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; JL Resnick et al. (1992), Nature, 359: 550-551 ), 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.NBiotechnol. 26: 101-1062008 (2008); WO2007 / 069666), many derived from cultured fibroblasts and bone marrow stem cells These include functional cells (Muse cells) (WO2011 / 007900). In the present application, human pluripotent stem cells are used.

The pluripotent stem cells used in the method of the present application may be cultured in a single cell state by being substantially separated (or dissociated) by any method, or cell aggregates in which cells adhere to each other You may culture in a state. More preferably, the cells are cultured in a single cell state. Examples of the separation method include mechanical separation and separation solution having protease activity and collagenase activity (for example, trypsin and collagenase-containing solution Accutase (TM) and Accumax (TM) (Innovative Cell Technologies, Inc) ) Or separation using a separation solution having only collagenase activity. Pluripotent stem cells can be adherently cultured using a coated culture dish.

In each step of the method of the present application, the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C. The culture is performed in an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is Preferably it is about 2 to 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 a basal medium used for culturing animal cells. As the basal medium, for example, 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, RPMI Examples include 1640 medium, Fischer's medium, and mixed media thereof. The basal medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum free. As needed, for example, albumin, transferrin, KnockOut Serum Replacement (KSR) (serum substitute for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acid , Insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol and other serum replacements, including lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), In one or more substances, such as non-essential amino acids (NEAA), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like, or other normal animal culture media It may contain one or more substances to be added. In one embodiment, the basal medium is RPMI 1640 medium containing B27 supplement and fetal calf serum.

The medium used in step (i) may further contain a ROCK inhibitor. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK). For example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000) Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg, Uenata et al., Nature 389: 990-994 (1997)), SR3677 (eg, Feng Y et al. , J Med Chem. 51: 6642-6645 (2008)), GSK269962 (eg, Stevenger RA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), H-1152 (example) , Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (eg, Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)) and These derivatives, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants, and their expression vectors. Other known low-molecular compounds can also be used as ROCK inhibitors (for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508). 2004/0002507, 2003/0125344, 2003/0087919, and International Publications 2003/062227, 2003/059913, 2003/062225, 2002/076976 No., 2004/039796). In the present invention, one or more ROCK inhibitors may be used. Preferred ROCK inhibitors used in this step include Y-27632, Fasudil / HA1077, SR3677, GSK269962 and H-1152.

When using Y-27632 as the 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, for example, about 10 μM. In the specification and claims of the present application, when “about” is accompanied by a numerical value, it includes up to ± 30%, ± 20%, or ± 10% of the numerical value.

As activin A, activin derived from mammals such as humans and mice can be used. As activin used in the present invention, it is preferable to use activin derived from the same animal species as the pluripotent stem cell used for differentiation. For example, when human-derived pluripotent stem cell is used as a starting material, human-derived activin is used. It is preferable to use it. These activins are commercially available.

The concentration of activin A in the medium is usually 0.1 to 200 ng / ml, for example 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, phosphorylation ability for β-catenin), and many are already known. For example, BIO (indirubin derivative BIO ( Also known as GSK-3β inhibitor IX; 6-bromoindirubin 3′-oxime), a maleimide derivative 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 (also known as GSK-3β peptide inhibitor; Myr-N-GKEAPPAPPQSpP-) NH2) and CHI with high selectivity R99021 (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 used easily. The GSK-3β inhibitor used in the present invention may preferably be CHIR99021.

In step (i), a relatively low concentration of GSK3β inhibitor is used in order to obtain Early Early APS cells. “Relatively low concentration” means that the concentration is lower than that in the case of inducing the late front primitive streak (LateLAPS) described below. When CHIR99021 is used, the concentration in the medium may be 1 μM to less than 4 μM, for example, 1.5 μM to 3.5 μM, preferably about 3 μM.

The generation of the early primordial streak can be confirmed by the fact that the cells express BRACHYURY and EOMES and do not express CDX2.

In step (i), a relatively high concentration of GSK3β inhibitor is used to obtain late anterior primitive streak (LateLAPS) cells. When CHIR99021 is used 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.

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

The culture days in step (i) may be 12 hours to 36 hours, for example, about 1 day.
Depending on the concentration of the GSK 3β inhibitor in step (i), the pluripotent stem cells are expressed in early anterior primitive streak cells (cultured in the presence of a relatively low concentration of GSK3β inhibitor) or late anterior primitive streak cells (comparison) 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 and containing or not containing a BMP inhibitor.
What is necessary is just to select suitably from the well-known basal medium for animal cultures similar to process (i) as a basal medium used for process (ii). The basal media may be the same or different in steps (i) and (ii). For example, activin A may be added to a medium obtained by adding fetal calf serum to RPMI1640 as a basal medium. 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; PB 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), and LDN-193189 is preferably used.

When LDN-193189 is used as a BMP inhibitor, the concentration may be, for example, 100-1000 nM, 300-700 nM, for example, about 500 nM.
The culture period in step (ii) may be 36 to 60 hours, for example, about 2 days.
In step (ii), three types of subpopulations of definitive endoderm cells can be selectively obtained. That is,
(1) When the early anterior primitive streak cells are cultured in the presence of activin A and a BMP inhibitor, posterior domain (PADE) cells of the anterior definitive endoderm are obtained.
(2) When the anterior primordial streak cells are cultured under conditions where activin A is present but no BMP inhibitor is present, anterior definitive endoderm anterior domain (AADE) cells are obtained.
(3) When the late anterior primitive streak cells are cultured under conditions where activin A is present but no BMP inhibitor is present, posterior definitive endoderm (PDE) cells are obtained.
All definitive endoderm cells express SOX17 and FOXA2.

The present application includes an anterior foregut (AFG) cell, anterior posterior, comprising the step (iii) of culturing the PADE cell, AADE cell, or PDE cell obtained in step (ii) in a medium not containing a differentiation-inducing factor. Further provided is a method of obtaining intestinal (PFG) cells, or middle hindgut (MHG) cells.

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

That is, the present application includes the following three aspects:
1 First Embodiment A method for producing an anterior foregut cell from a human pluripotent stem cell via a posterior domain cell of the anterior definitive endoderm, 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 the step (i-1) are activin A step of culturing in a medium containing A and a BMP inhibitor, and (iii-1) a step of culturing the cells obtained in the step (ii-1) in a medium containing no differentiation-inducing factor.

2 Second aspect A method for producing posterior foregut cells from human pluripotent stem cells via anterior definitive endoderm anterior domain cells, comprising the following steps (i-2) to (iii-2) :
(I-2) culturing pluripotent stem cells in a medium containing activin A and a relatively low concentration of GSK3β inhibitor;
(Ii-2) culturing the cells obtained in the step (i-2) in a medium containing activin A, and (iii-2) differentiating the cells obtained in the step (ii-2). A step of culturing in a medium containing no inducer.

3 Third Embodiment A method for producing a middle hindgut cell from a pluripotent stem cell via a posterior definitive endoderm cell, 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 GSK3β inhibitor, and (ii-3) the cells obtained in the step (i-3), A step of culturing in a medium containing activin A, and (iii-3) a step of culturing the cells obtained in the step (ii-3) in a medium containing no differentiation-inducing factor.

AFG cells differentiate into cells that make up the middle ear, thymus, thyroid, trachea, lungs, and esophagus. PFG is differentiated into cells that make up the stomach, duodenum, pancreas and liver. It is known that MHG is differentiated into cells constituting the small and large intestines. Methods for inducing differentiation into each cell are known, and those skilled in the art can appropriately select from known methods. A method for obtaining the cells by further inducing differentiation of each cell obtained by the method of the present application is also included in the present application.

Hereinafter, the present application will be described in more detail based on examples.
1. Materials and methods 1.1. Cell culture To obtain a feeder-free culture, hiPSC (585A1 cells) (Okita K et al., 2011, Kajiwara M et al., 2012) was obtained according to the manufacturer's instructions. 8 Maintained in medium (Thermo Fisher Scientific, Waltham, MA). For routine subculture, hiPSC colonies were isolated by enzymatic method using 0.5 mM EDTA (Wako, Osaka, Japan) and added at a ratio of 1: 100 by adding 10 μM Y-27632 (Wako). divided.

1.2. Differentiation into anterior primitive streak (APS) HiPSC colonies cultured to 80% confluence were separated into single cells by 0.5 mM EDTA by an enzymatic method. The cells were added 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) with / ml activin A and 8 μM CHIR99021 ) And 10 μM Y-27632 in RPMI 1640 medium (NACALAI TESQUE, Kyoto, Japan) and 9 × 10 ⁴ cells / cm ² on a plate coated with Matrigel (Becton Dickinson, Franklin Lakes, NJ) Seeding and culturing for 1 day.

1.3. Differentiation into definitive endoderm (DE) Early differentiation from APS to DE cells 2% (vol / vol) B27 supplement (GFR-B27), 50 U / ml P / S for induction into AADE cells And RPMI 1640 medium supplemented with 100 ng / ml activin A and cultured for 2 days in RPMI 1640 medium supplemented with 100 ng / ml activin A and 500 nM LDN193189 (Axon Medchem) for induction into PADE cells Thus, the progenitor APS was induced into the anterior definitive endoderm anterior domain (AADE) cells or the anterior definitive endoderm posterior domain (PADE).
Differentiation of late APS into DE cells Late APS cells were treated with 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 gastrointestinal (Gut Tube, GT) cells and late gastrointestinal (Late Gut Tube, LGT) cells For the induction of GT cells, three types of DE cells (AADE, PADE and PDE) were introduced into Improved MEM. The cells were cultured in a Zinc Option (iMEM) medium (Thermo Fisher Scientific) for 3 days. Thereafter, the GT cells were cultured for 3 days using the same medium and differentiated into LGT cells.

1.5. Differentiation into liver and lung lineage cells The obtained LGT cells were transformed into the previously reported differentiation protocol (Kajiwara M et al., 2012, Proc Natl Acad Sci USA 109, 12538-12543, Gotoh S et al., 2014, Stem Cell Reports 3, 394-403.) Were further differentiated into hepatocyte-like cells and alveolar epithelial progenitor cells.
Differentiation into hepatocyte-like cells Using hiPSC-derived LGT cells obtained in 1.4, 20 ng / mL recombinant human hepatocyte growth factor (HGF; PeproTech, Rocky Hill, NJ) and 20 ng / mL recombinant human oncostatin M ( The cells were cultured in a hepatocyte culture medium containing OsM; PeproTech) for 6 days to induce differentiation into hepatocyte-like cells.
Differentiation into alveolar epithelial progenitor cells HiPSC-derived LGT cells obtained in 1.4 were obtained from 1 × B27 and N2 supplements (Thermo Fisher Scientific), 50 U / ml P / S, 0.05 mg / ml L-ascorbic acid (Sigma- Aldrich, Tokyo, Japan), 0.4 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 The cells were cultured for 4 days in DMEM / F12 medium (Thermo Fisher Scientific) containing Glutamax containing 3.5 μM CHIR99021.

1.6. Immunostaining Cells were fixed with 4% paraformaldehyde (PFA) / PBS for 20 minutes at 4 ° C. After washing with PBS, the cells were blocked with 5% normal donkey serum (Funakoshi, Tokyo, Japan) / PBST (PBS / 0.1% Triton X-100) for 1 hour at room temperature. The primary antibody was diluted with blocking solution and incubated with the sample overnight at 4 ° C. After washing 3 times with PBS, the cells were incubated with the 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 and were used at a 1: 200 dilution. For nuclear staining, Hoechst 33342 trihydrochloride trihydrate (Thermo Fisher Scientific) was used at a 1: 200 dilution. The primary antibodies used are shown in Table 2.

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 sample is subjected to PCR amplification using a thermal cycler (Veriti 96-well Thermal Cycler, Applied Biosystems, Waltham, Mass.) And using an Ex-Taq PCR kit (Takara, Shiga, Japan) according to the manufacturer's instructions. PCR was performed. The PCR cycle was as follows: for the housekeeping gene β-ACTIN, initial denaturation for 2.5 minutes at 94 ° C, followed by 94 ° C for 30 seconds, 60 ° C for 1 minute, 72 ° C for 30 seconds. 25 cycles and a final extension reaction were performed at 72 ° C. for 10 minutes. For other genes, initial denaturation for 2.5 minutes at 94 ° C, 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 final The extension reaction was performed in a cycle of 10 minutes at 72 ° C. qPCR was performed using 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. As recommended by the manufacturer, the threshold cycle method was used to analyze the gene expression level data and calibrate to the gene expression level of β-ACTIN. PCR reactions were performed in triplicate for each sample. Table 3 shows the primer sequences 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, Mass.) According to the manufacturer's instructions. The library was sequenced in HiSeq2500 100-cycle single-read mode. All sequence read data was extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in CASAVA 1.8.2 pipeline. Map the sequence reading data to the hg19 reference gene downloaded on April 25, 2014 using Tophat v2.0.14. Calculation of gene expression values, and normalize it by RPKMforgenes (10th December 2012). The level was expressed as log ₂ (RPKM + 1). A heat map of gene expression was created by the heatmap.2 function of the R 3.2.1 gplots library. Two-way hierarchical clustering of gene expression of tissue and cell samples was performed using the hclust function of R3.2.1.

2. Results
2.1. There are two models showing the characteristics of definitive endoderm cell populations that have been confirmed to produce different types of definitive endoderm (DE) cells with different differentiation protocols: 1) DE cells are A single cell type pluripotent endoderm progenitor cell that can differentiate into any endoderm cell (FIG. 1A, left), 2) DE cells contain multiple types of lineage-determined endoderm progenitor cell populations (FIG. 1A, right). If the DE cells are a single type of pluripotent endoderm progenitor cells, even if the DE cells are induced by different differentiation protocols, they are uniformly endodermal cells, for example It was hypothesized that it could be differentiated into late fetal (LGT) stage alpha-fetoprotein (AFP) (+) cells, ie hepatoblasts.

In order to examine this hypothesis, we modified AFP (+) from hiPSC by modifying a previously reported induction protocol for liver lineage cells (Takebe, T. et al., 2014, Nat Protoc 9, 396-409.). ) A direct differentiation method into cells was developed. Induction of APS by treatment with activin A and CHIR99021, followed by treatment with activin A alone, is very efficient in SOX17 (+) FOXA2 (+) DE cells (FIG. 1B Method 1, FIG. 1C top). Induced. Furthermore, LGT-stage AFP (+) cells were generated from DE cells by treatment with BMP4 and fibroblast growth factor 2 (FGF2) (upper figure in FIG. 1D).

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

2.2. Differentiation directionality of the two DE cell subtypes To investigate whether the differentiation potential of the DE cell subpopulation is restricted to specific endoderm cells, it was produced by the two methods of FIG. 1B. The DE subpopulation was further differentiated by culturing for 3-6 days without the addition of inducer (FIG. 2A). After 3 days in culture, Method A cells differentiated into HNF1β and HNF4α positive cells, markers of the posterior foregut region of GT cells. Few cells expressed SOX2, which is a marker of the anterior foregut region of GT cells (FIG. 2A, 2B upper diagram). When HNF1β (+) HNF4α (+) cells were further cultured, they differentiated into AGT (+) cells at the LGT stage (upper figure in FIG. 2B).

This result indicates that the DE cells induced by the method A differentiate into cells in the posterior foregut region of the late gastrulation, that is, hepatoblasts, without adding an exogenous inducer. On the other hand, the cells of Method B were SOX2 (+) at the LGT stage and differentiated into cells in the anterior foregut region of the late gastrulation. HNF1β, HNF4α and AFP were negative (bottom of FIG. 2B). It confirmed by qRT-PCR analysis about SOX2 (RNA) expression in each step (FIG. 2C). These results suggest that the possibility of future differentiation into a specific endoderm lineage is already restricted to the DE stage.

In order to evaluate the developmental potential of hiPSC-derived LGT cells in vitro, hepatocyte-like cells and alveolar epithelium were used from the LGT cells produced by Method A or B using the 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 not differentiated into ventral anterior foregut cells of NKX2.1 (+) (FIG. 2D, 2E top). In contrast, LGT cells induced by Method B differentiated into NKX2.1 (+) cells without differentiating into AFP (+) or ALB (+) cells (FIG. 2D, 2E lower panel). These results show that DE cells induced by Method A are anterior definitive endoderm anterior domain (AADE) cells that produce the posterior foregut region of GT and LGT cells, whereas DE cells induced by Method B Suggests that the anterior definitive endoderm posterior domain (PADE) cells 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 systems. RNA sequencing analysis was performed to reveal the differences in gene expression patterns between the two types of DE cells, AADE and PADE cells, and GT and LGT cells derived from them. As a result, there was almost no 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 a relatively similar gene expression profile between AADE and PADE cells (FIG. 3B). However, 273 genes that were differentially expressed between AADE and PADE cell populations were identified (FIG. 3C and Table 4). These data suggest that although AADE and PADE have similar expression patterns for the well-known DE and PS markers, there are candidate markers that can distinguish these DE subpopulations.

2.4 Establishing methods for inducing late anterior primitive streak (late APS), posterior definitive endoderm (PDE) and midhindgut (MHG) cells Novel differentiation of PDE cells with limited differentiation potential to midhindgut cells To establish the protocol, the differentiation from hiPSC to APS was further investigated. From the report that pattern formation to mesoderm subtype correlates with the timing of mesoderm induction in PS (Non-patent Document 15), it is assumed that DE pattern formation will also correspond to the timing of DE cell differentiation in APS did. Under this assumption, the characteristics of hiPSC-derived APS cells induced by treatment with 100 ng / ml activin A and 3 μM CHIR99021 were examined (FIG. 2A). By immunostaining, these cells were positive for the APS markers BRACHYURY and EOMES, but there were few cells positive for CDX2, which is a marker for the late primitive streak (upper figure in FIG. 4A). These data show 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 hiPSC was developed by modifying the differentiation protocol for late primitive streak (Non-patent Document 15). The culture was carried out for 1 day in the presence of 100 ng / ml activin A and 8 μM CHIR99021. It was confirmed by immunostaining of the obtained cells that late APS cells, which are BRACHYURY (+) EOMES (+) CDX2 (+), were generated (lower figure in FIG. 4A).

In order to induce DE cells from late-stage APS cells, the cells were further cultured for 2 days in the presence of 100 ng / ml of activin A. The obtained cells were positive for SOX17 (+) FOXA2 (+), and definitive endoderm cells were It was confirmed that it was generated (lower figure in FIG. 4B). The cells were further cultured for 3 days without adding exogenous inducers. The ability of the resulting cells to develop into the endoderm system was examined, and differentiated cells that were positively stained for HNF1β, HNF4α, and CDX2, which are markers of the middle hindgut, were found (lower panel in FIG. 4C). 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 hiPSC via prophase APS and AADE did not differentiate into the CDX2 (+) midhind gut region of GT cells (on FIGS. 4A, 4B, 4C). Figure). These results indicate that an efficient differentiation method from hiPSCs to middle hindgut cells via PDE cells can be established by generating late APS cells, and patterning into DE subtypes This suggests 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 a plurality of subtypes, and each differentiates into a specific endoderm lineage (FIG. 5B). Moreover, the method of selectively deriving each subtype of definitive endoderm cells from pluripotent stem cells was established (FIG. 5A).

Claims (13)

1. A sub-layer of definitive endoderm cells selected from the group consisting of pluripotent stem cells to anterior definitive endoderm anterior domain (AADE) cells, anterior definitive endoderm definitive domain (PADE) cells, and posterior definitive endoderm (PDE) cells A method of selectively directing to a population comprising the following steps (i) and (ii):
   (I) culturing pluripotent stem cells in a medium containing activin A and a GSK3β inhibitor to obtain early anterior primitive streak cells or late anterior primitive streak cells, and (ii) the obtained early anterior primitive Striatal cells, or late anterior primitive streak cells, are cultured in a medium containing activin A and with or without a BMP inhibitor to produce anterior definitive endoderm anterior domain cells, anterior definitive endoderm Obtaining domain cells or posterior definitive endoderm cells.
2. The anterior foregut (AFG) cell or the posterior foregut further comprising the step of culturing the subpopulation of definitive endoderm cells obtained in step (ii) in a medium not containing a differentiation-inducing factor according to claim 1. A method of selectively inducing gastrointestinal cells selected from the group consisting of (PFG) cells and middle hindgut (MHG) cells.
3. The subpopulation of definitive endoderm is the anterior definitive endoderm posterior domain (PADE) cells, which comprises the following steps (i) and (ii):
   (I) a step of culturing pluripotent stem cells in a medium containing activin A and a relatively low concentration of a GSK3β inhibitor to obtain anterior primordial streak cells; and (ii) an early phase obtained in step (i). The method according to claim 1 or 2, comprising a step of culturing anterior primitive streak cells in a medium containing activin A and a BMP inhibitor.
4. 4. The method according to claim 3, further comprising the step of culturing the obtained posterior domain cells of the anterior definitive endoderm in a medium not containing a differentiation-inducing factor to obtain an anterior foregut (AFG) cell.
5. The method according to claim 3 or 4, wherein the BMP inhibitor is LDN-193189.
6. A subpopulation of definitive endoderm is anterior definitive endoderm anterior domain (AADE) cells,
   (I) a step of culturing pluripotent stem cells in a medium containing activin A and a relatively low concentration of a GSK3β inhibitor to obtain anterior primordial streak cells, and (ii) obtained in step (i) above The method of Claim 1 or 2 including the process of culture | cultivating an anterior primitive primitive streak cell in the culture medium containing an activin A.
7. The method according to claim 6, further comprising the step of culturing the obtained anterior definitive endoderm anterior domain cells in a medium not containing a differentiation-inducing factor to obtain posterior foregut (PFG) cells.
8. The method according to any of claims 3 to 7, wherein the GSK3β inhibitor is CHIR99021, and the concentration of CHIR99021 in the medium is about 3 µM.
9. The subpopulation of definitive endoderm is posterior definitive endoderm (PDE) cells, the following steps (i) and (ii):
   (I) culturing pluripotent stem cells in a medium containing activin A and a relatively high concentration of GSK3β inhibitor; and (ii) culturing the cells obtained in step (i) in a medium containing activin A. The method of Claim 1 or 2 including the process of culture | cultivating.
10. The method according to claim 9, further comprising culturing the obtained posterior definitive endoderm (PDE) cells in a medium not containing a differentiation-inducing factor to obtain middle hindgut (MHG) cells.
11. The method according to claim 9 or 10, wherein the GSK3β inhibitor is CHIR99021, and the concentration of CHIR99021 in the medium is 4 µM or more.
12. 12. The method of claim 11, wherein the concentration of the CHIR99021 in the medium is about 8 [mu] M.
13. The method according to any one of claims 1 to 12, wherein the pluripotent stem cells are human iPS cells or human ES cells.

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