JP5092124B2 - ES cell differentiation induction method - Google Patents

Description

  The present invention relates to a method for inducing differentiation of ES cells. More specifically, the present invention relates to a method for inducing differentiation of ES cells into endoderm using a cell line derived from mesoderm as a support cell.

  Due to its pluripotency, ES cells are a useful model system for studying gene function in the nascent stage, and may be a transplantable cell source for medical use. In order to use ES cells for cell replacement therapy in diseases such as diabetes, it is necessary to control differentiation, which remains a major challenge. Although understanding of the differentiation of ES cells into neural tissue, hematopoietic tissue, and heart tissue is quite advanced (Yamashita, J., Itoh, H., Hirashima, M., et al. (2000). Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408, 92-6; and Ying, QL, Stavridis, M., Griffiths, D., Li, M., and Smith, A. (2003). Conversion of embryonic stem cells Nat Biotechnol 21, 183-6), little is known about the differentiation of ES cells into endoderm tissues.

  In 2001, it was reported that insulin-containing pancreatic cells were formed from ES cells in vitro by adding certain factors after culturing under conditions that enriched nestin-positive neuronal populations (Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., and McKay, R. (2001). Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.Science 292, 1389-94). However, this insulin positive cell does not express Pdx1, a marker of functional pancreatic beta cells in both pancreatic and duodenal endoderm. The cells did not express insulin I transcript, but expressed trace amounts of insulin II transcript. These cells also stained positively for insulin but were not stained with anti-C-peptide antibodies, which are a byproduct of insulin de novo synthesis (Rajagopal, J., Anderson, WJ, Kume, S., Martinez, OI, and Melton, DA (2003). Insulin staining of ES cell progeny from insulin uptake. Science 299, 363). These findings indicate that insulin staining is due to insulin uptake from the medium and the cells themselves do not synthesize insulin, and whether nestin-positive cells give rise to beta cells that produce insulin Met. However, other researchers have reported that insulin-secreting cells are produced from ES cells using similar conditions that are selective for nestin-positive cells, and this problem has not yet been elucidated (Blyszczuk, P., Czyz, J., Kania, G., et al. (2003). Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci USA 100, 998-1003; Hori, Y., Rulifson, IC, Tsai, BC, Heit, JJ, Cahoy, JD, and Kim, SK (2002). Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells.Proc Natl Acad Sci USA 99, 16105- 10; and Moritoh, Y., Yamato, E., Yasui, Y., Miyazaki, S., and Miyazaki, J. (2003). Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes 52, 1163-8). It has recently been reported that when glucose is added to the medium, nestin-positive progenitor cells give rise to a cell population that releases insulin, but notably no C-peptide release is detected (Hansson, M., Tonning, A., Frandsen, U., et al. (2004). Artifactual insulin release from differentiated embryonic stem cells. Diabetes 53, 2603-9). This suggests that the cells are not functional insulin secreting cells. Therefore, the problem of manipulating ES cells to produce endocrine pancreatic β cells remains unresolved.

  In mouse embryos, Pdx1 expression is the first differentiation signal and is detected in the dorsal endoderm of the intestine in day 8.5 embryos. In day 9.5 embryos, Pdx1 expression is found in dorsal and ventral pancreatic buds and duodenal endoderm. In adults, Pdx1 expression is maintained in the duodenal epithelium and pancreatic islet β cells secreting insulin and plays an important role in the transcriptional regulation of insulin genes (Offield, MF, Jetton, TL, Labosky, PA, et al (1996). PDX1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122, 983-95). Targeted mutagenesis of Pdx1 in mice has shown that Pdx1 is required for pancreatic and rostral duodenal development (Ahlgren, U., Jonsson, J., and Edlund, H. (1996). The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1 / PDX1-deficient mice. Development 122, 1409-16). Therefore, Pdx1 is an essential molecule for the development of the pancreas, and is also useful as an initial marker for pancreatic progenitor cells and other endoderm-derived tissues such as the stomach, duodenum, and bile ducts.

  We have previously reported a protocol for efficiently generating Pdx1-positive cells using an ES cell line with a lacZ reporter gene at the Pdx1 locus. Co-cultured ES cells with embryonic pancreatic primordia or pancreatic mesenchyme showed that a significant increase in the number of cells induced to differentiate into Pdx1-expressing cells was induced. We screened for growth factors that promote the differentiation of ES cells into pancreas and found that TGFβ2 partially mimics the differentiation-inducing activity of pancreatic primordia. Genetic manipulation such as overexpression of chicken mix family (cmix) genes (Peale, FV, Jr., Sugden, L., and Bothwell, M. (1998). Characterization of CMIX, a chicken homeobox gene related to the Xenopus gene mix.1. Mech Dev 75, 167-70; and Stein, S., Roeser, T., and Kessel, M. (1998). CMIX, a paired-type homeobox gene expressed before and during formation of the avian primitive streak Mech Dev 75, 163-5) specifically promoted the differentiation of ES cells into the endoderm system. However, when a large amount of induction source is required, application of embryonic pancreatic primordia or pancreatic mesenchyme to induce ES cell differentiation is difficult.

  As described above, it has been reported that cells stained with an insulin antibody increase by using a method of changing the culture conditions of ES cells. However, the cells detected by this method are not cells that have been produced through the process of normally producing insulin-producing β cells in vivo. Moreover, it has been pointed out that this is an apparent phenomenon by the cells adsorbing insulin in the medium (Rajagopal, J. et al., Science 299, 363, 2003). In order to solve this, a culture method that induces the expression of an early marker of pancreatic development is effective. An object of the present invention is to provide a novel method for inducing differentiation of an ES cell that enables differentiation of even a large amount of ES cells into the endoderm system.

  Any type of cell promotes the growth of endoderm cells, leading to differentiation into specific cells on the anteroposterior axis, and these cells replace the signal from pancreatic primordia or pancreatic mesenchyme. The inventors speculated. Therefore, the present inventors screened cultured cell lines for the purpose of discovering such an induction source, and the mesoderm-derived cell line shows a strong activity of inducing differentiation of ES cells into endoderm. In addition, it was found that the development of pancreas or liver is promoted. The present invention has been completed based on these findings.

That is, according to the present invention, there is provided a method for inducing differentiation from ES cells into endoderm cells, comprising culturing mammalian-derived ES cells in the presence of supporting cells.
Preferably, the feeder cells are cells derived from mesoderm.
Preferably, the feeder cells are M15 cells, MEF cells or ST2 cells.

Preferably, differentiation is induced from ES cells to any of undifferentiated endoderm progenitor cells, immature cells of endoderm-derived organs, or mature cells of endoderm-derived organs.
Preferably, the endoderm-derived organ is the pancreas, liver, lung, pharynx, or small intestine.
Preferably, when culturing ES cells in the presence of feeder cells, actin bin, basic fibroblast growth growth factor (bFGF), and / or noggin are added and cultured.
Preferably, when culturing ES cells in the presence of feeder cells, nicotinamide is further added and cultured.
Preferably, the mammal-derived ES cells are mouse, monkey or human-derived ES cells.

  According to another aspect of the present invention, there is provided an endoderm cell obtained by the above-described method of the present invention and differentiated from an ES cell.

  According to still another aspect of the present invention, a step of inducing differentiation from an ES cell into an endoderm cell by the above-described method of the present invention, and a flow cytometry (FACS) using a fluorescent label for the differentiation-induced endoderm cell. The method of obtaining the endoderm type | system | group cell which differentiation-induced from ES cell including the process isolate | separated by this is provided.

According to still another aspect of the present invention, culturing endoderm cells differentiated from ES cells obtained by the above-described method of the present invention on a plate coated with 2-methacryloxyethyl phosphorylcholine. A method for maintaining and culturing endoderm cells induced to differentiate from ES cells is provided.
Preferably, in the maintenance culture method described above, the culture is performed in the presence of knockout serum replacement (KSR).

  According to still another aspect of the present invention, when ES cells derived from mammals are cultured in the presence of supporting cells to induce differentiation from ES cells to endoderm cells, ES cells are present in the presence of a test substance. When cells are cultured and ES cells are cultured in the absence of the test substance, the degree of differentiation induction into endoderm cells and differentiation into endoderm cells when ES cells are cultured in the presence of the test substance A method is provided for screening for a substance that promotes or inhibits differentiation induction from ES cells to endoderm cells, comprising comparing the degree of induction.

Preferably, the test substance is a growth factor or a low molecular weight compound.
Preferably, the degree of differentiation induction into endoderm cells is measured using the expression level of the marker expressed in the endoderm as an index.
Preferably, the mammal-derived ES cells are mouse, monkey or human-derived ES cells.

Hereinafter, embodiments of the present invention will be described in more detail.
The present invention relates to a method for inducing differentiation from ES cells into endoderm cells, and in particular, is a method characterized by culturing mammalian-derived ES cells in the presence of supporting cells.

  In the differentiation-inducing method of the present invention, cells of digestive organs derived from endoderm such as pancreas can be induced from ES cells using feeder cells. Inducing differentiation of endoderm cells using feeder cells has not been reported so far and has been achieved for the first time by the present invention. In the examples of the present invention, expression of the gene marker gene (Pdx1) of pancreatic stem cells was visualized with a fluorescent protein, enabling rapid and sensitive detection. In the present invention, ES cells are easily induced to differentiate by co-culture with supporting cells. Therefore, when a specific growth growth factor is added, differentiation induction is further promoted. Therefore, the method of the present invention can also be used as a screening for unknown differentiation-inducing factors.

  The ES cell used in the present invention is not particularly limited as long as it is a mammal-derived ES cell. For example, mouse, monkey, or human-derived ES cells can be used. As the ES cell, for example, a cell having a reporter gene introduced in the vicinity of the Pdx1 gene can be used in order to facilitate confirmation of the degree of differentiation. For example, as used in the examples below, the 129 / Sv-derived ES cell line R1, J1 or ES cell SK7 line having a GFP reporter transgene under the control of the Pdx1 promoter, which has incorporated the lacZ gene at the P dx1 locus Etc. can be used. Alternatively, an ES cell PH3 strain having an mRFP1 reporter transgene under the control of a Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of a Pdx1 promoter can also be used.

  Mammal-derived ES cells can be cultured by a conventional method. For example, in the presence of mitomycin C-treated mouse embryo fibroblasts (MEF) as feeder cells, LIF (ESGRO 1000 units / ml, Maintained in differentiation medium supplemented with Chemicon) (Dulbecco's modified Eagle medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS), 0.1 mM 2-mercaptoethanol, 100 μM non-essential amino acids, 2 mM L-glutamine) can do.

  In the present invention, ES cells are cultured in the presence of feeder cells, that is, ES cells are co-cultured with feeder cells. The feeder cells used in the present invention are not particularly limited as long as ES cells can be induced to differentiate into endoderm cells, but preferably cells derived from mesoderm can be used as feeder cells. Specific examples of cells derived from mesoderm that can induce differentiation of ES cells into endoderm cells include M15 cells, MEF cells, and ST2 cells.

  M15 cells (mouse, mesonephros) are registered in the cell bank (CAMR Center for Applied Microbiology & Research (ECACC, Salisbury, Wiltshire)) as registration number ECACC 95102517. M15 cells are described in the literature (Larsson, SH, Charlieu, JP, Miyagawa, K., et al. (1995). Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 81, 391-401) Is available according to Bank information for M15 is listed below.

Version 4.200201
M15 (mouse, mesonephros)
ECACC 95102517
Morphology: Epithelial
Mouse mesonephric epithelium, polyoma virus large T transformed
Depositor: Prof V van Heyningen, MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK (Originator)
No restrictions. Patent: None Specified By Depositor
Properties: Products: WT1 (expressed gene) Applications: Gene expression and protein studies connected to kidney development and Wilms' tumourigenesis.
Available in the following LABORATORY:
CAMR Center for Applied Microbiology & Research (ECACC, Salisbury, Wiltshire)
DMEM + 2mM Glutamine + 10% Fetal Bovine Serum (FBS). Split confluent cultures 1: 5 to 1:10 ie seeding at 5x1,000 to 1x10,000 cells / cm2 using 0.25% trypsin or trypsin / EDTA; 5% CO2; 37C [cell growth impaired at lower places]. Karyotype: Hyperdiploid
Hazard: CZ-II
The WT1-expressing mesonephric cell line M15 (alias Meso15) was established from mouse mesonephros transgenically expressing the large T protein of polyoma virus under the control of the early viral enhancer.As a tumour suppresser gene with a key role in urogenital development, WT1 is implicated as predisposition gene in the pathogenesis of Wilms' tumour (WT).
Further information
Research council deposit: Yes
Price_code: C
Bibliographic references:
Cell 1995; 81: 391
By Beatrice ...
TITLE: M15
DATE: 2005/04/24 00:32
URL: http://www.biotech.ist.unige.it/cldb/cl3312.html
European Collection of Cell Cultures,
Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK
June Poulton
European Collection of Cell Cultures
Health Protection Agency,
Porton down
SP40JG Salisbury, Wiltshire UK
Phone: + 44-1980-612512
Fax: + 44-1980-611315
E-mail: ecacc@hpa.org.uk
URL: http://www.ecacc.org.uk/

  MEF (from ICR mouse) is registered with ATCC as catalog number ATCC # SCRC-1046. MEF cells can be obtained according to the description in the literature (Nagy A, et al. Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition Cold Spring Harbor Press; 2003).

  ST2 cells are registered as RCB0224 in the RIKEN Tsukuba BioResource Center. In addition, ST2 cells can be used in the literature (Ogawa, M., Nishikawa, S., Ikuta, K., Yamamura, F., Naito, M., Takahashi, K. and Nishikawa, S. EMBO J 1988; 7: 1337- 1343).

  These feeder cells can be cultured according to a conventional method using a normal medium for animal cells supplemented with serum or the like (for example, RPMI medium or DMEM medium).

  The method for culturing mammalian-derived ES cells in the presence of feeder cells is not particularly limited. For example, ES cells can be cultured using feeder cells as feeder cells. For example, undifferentiated ES cells are dissociated with trypsin and cultured in suspension in an untreated culture dish in the presence of LIF in a differentiation medium to form embryoid bodies. Embryoid bodies that have been differentiated for 2 days are treated with trypsin, and seeded in a differentiation medium on a plate previously encapsulated (precoated) with feeder cells (supporting cells). Thereafter, differentiation can be induced into endoderm cells by culturing for several days.

  In the differentiation-inducing method of the present invention, when culturing ES cells in the presence of supporting cells, other substances (eg, growth factors or low molecular compounds) can be added and cultured. For example, differentiation induction from ES cells to endoderm cells can be further promoted by adding and culturing with actin bin, bFGF and / or noggin. In addition, when adding actinbin, bFGF, and / or noggin, differentiation induction from ES cells to endoderm cells can be promoted by adding nicotinamide and culturing.

  According to the ES cell differentiation induction method using the feeder cells according to the present invention, any of ES cells, undifferentiated endoderm progenitor cells, immature cells of endoderm-derived organs, or mature cells of endoderm-derived organs Differentiation can be induced. Examples of the endoderm-derived organ include, but are not limited to, the pancreas, liver, lung, pharynx, or small intestine. The differentiation from ES cells to endoderm cells can be confirmed by measuring the expression level of a marker specific to endoderm.

Furthermore, according to the present invention, the ES cells are cultured in the presence of a test substance when differentiation induction from ES cells to endoderm cells is performed by culturing mammalian-derived ES cells in the presence of supporting cells. The degree of differentiation induction into endoderm cells when ES cells are cultured in the absence of the test substance and the degree of differentiation induction into endoderm cells when ES cells are cultured in the presence of the test substance A method for screening for a substance that promotes or inhibits differentiation induction from ES cells to endoderm cells is provided. As a test substance, a growth factor or a low molecular weight compound can be used. At this time, it is possible to measure the degree of differentiation induction into endoderm cells using the expression level of the marker expressed in the endoderm as an index.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(A) Experimental method (1) ES cell line
The 129 / Sv-derived ES cell line R1 incorporating the lacZ gene at the Pdx1 locus was provided by Dr. Christopher Wright (Vanderbilt University). The Pdx1 / LacZ ES cell line is a differentiation medium (10% fetal bovine serum (10% fetal bovine serum) supplemented with LIF (ESGRO 1000 units / ml, manufactured by Chemicon) in the presence of mitomycin C-treated mouse embryonic fibroblasts (MEF) as feeder cells. FBS), Dulbecco's modified Eagle medium (DMEM, Sigma) supplemented with 0.1 mM 2-mercaptoethanol, 100 μM non-essential amino acids, 2 mM L-glutamine.

  The ES cell line SK7 having an EGFP reporter transgene under the control of the Pdx1 promoter was established from a transgenic mouse homozygous with the Pdx1 / EGFP gene. ES cell SK7 strain is the smallest Glasgow supplemented with LIF (1000 units / ml), 15% Knock-out Serum Replacement (KSR, Invitrogen), 1% FBS, 100 μM non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate Maintain on MEF in essential medium (GMEM, Sigma).

  The ES cell PH3 strain, which has an mRFP1 reporter transgene under the control of the Hnf3β endoderm-specific enhancer fragment and an EGFP reporter transgene under the control of the Pdx1 promoter, is a transgenic mouse heterozygous with the Hnf3β / mRFP1 gene, and the Pdx1 / EGFP gene. And a blastocyst obtained by crossing a homozygous transgenic mouse. The ES cell PH3 line is maintained in the same manner as the ES cell SK7 line except that it is maintained without MEF.

  Cynomolgus monkey ES cell CMK6 strain was purchased from Asahi Techno Glass Co., Ltd. Cynomolgus monkey ES cell line is 20% KnockOut Serum Replacement (KSR, Gibco BRL), 0.1 mM 2-mercaptoethanol, 100 μM non-essential amino acids, 2 mM in the presence of mitomycin C-treated mouse embryonic fibroblasts (MEF) as feeder cells L-glutamine and Dulbecco's modified Eagle's medium supplemented with 1 mM sodium pyruvate and F-12 nutrient mixture (one-to-one mixed medium (DMEM / F12, Sigma)).

  Human ES cell strain KhES-1 was provided by the Institute of Regenerative Medicine, Kyoto University. Human ES cell line is 20% KnockOut Serum Replacement (KSR, Gibco BRL), 0.1 mM 2-mercaptoethanol, 100 μM non-essential amino acids, 2 mM in the presence of mitomycin C-treated mouse embryonic fibroblasts (MEF) as feeder cells Dulbecco's modified Eagle's medium supplemented with L-glutamine and F-12 nutrient mixture (1: 1 mixed medium (DMEM / F12, Sigma)).

(2) Differentiation of ES cells For differentiation studies, Pdx1 / LacZ ES cells once passaged on gelatin-coated plates were used. Dissociate undifferentiated Pdx1 / LacZ ES cells with trypsin, and culture in suspension in an untreated culture dish (bacterial grade, Iwaki Glass) at a concentration of 3.0 × 10 ⁵ cells / 90 mm plate in the absence of LIF in the differentiation medium. Embryoid bodies were formed. Embryoid bodies that had been differentiated for 2 days were treated with trypsin, and seeded at 5000 per well in a differentiation medium in a 24-well plate pre-coated with feeder cells. Control wells were precoated with gelatin alone. After ES cells seeded in control gelatin-coated wells or wells coated with feeder cells were differentiated in differentiation medium for 12 days, the cells were fixed and analyzed for X-Gal staining.

  The ES cell strain SK7 passaged on MEF was used once for differentiation studies without forming embryoid bodies after passage once on gelatin-coated plates. In a 24-well plate pre-coated with feeder cells, 500 ES cells SK7 strain per well were seeded in a differentiation medium and cultured for 3 to 4 days. On day 3 or 4, FBS was replaced with KSR at the indicated times and growth factors were added. The medium was replaced only once on the 6th day with KSR-substituted differentiation medium supplemented with growth factors. The exact date on which growth factors were added, or the exact date on which the differentiation medium was replaced with KSR-substituted differentiation medium, is as noted in each figure. Fluorescence photographs of ES cells were taken every 24 hours under a stereomicroscope (Leica MZFLIII).

Method of differentiation culture of monkey ES cells Day 0 of culture: Seed 20,000 monkey ES cells per well in a differentiation medium in a 24-well plate. The medium is changed on days 1, 3, 5, 7, and 9 of the culture. The differentiation medium is the same as the differentiation medium for mouse ES cells.

Differentiation culture method of human ES cells The differentiation culture conditions of human ES cells are as follows. On day 0 of culture, 24-well plates were seeded with human ES cells at a concentration of 20,000 cells / well, and the medium was changed on days 1, 3, 5, 7, 9, and 11 of culture. The medium used was a KSR replacement differentiation medium (10% KSR / DMEM (high glucose)).

(3) Feeder cell line M15 cells derived from embryonic kidney were provided by Dr. T. Noce of Mitsubishi Chemical Life Science Institute. ST2 cells are stromal cells known to have the ability to promote differentiation from ES cells to blood cells. MEFs were isolated from embryonic day 12.5-14.5 day embryos. ST2 cells were cultured in RPMI medium supplemented with 5% FBS and 0.1 mM 2-mercaptoethanol, and other cells were cultured in DMEM medium supplemented with 10% FBS. Feeder cells other than ST2 cells were treated with 200 μg / ml mitomycin C for 2.5 hours before use. M15 cells were seeded in gelatin-coated 24-well plates at a concentration of 2 × 10 ⁵ per well. MEF cells were used at 1 × 10 ⁵ cells per well. ST2 cells were used without mitomycin C treatment, and were seeded at 5 × 10 ⁴ cells / well in a 24-well plate 3 to 4 days before seeding of ES cells.

(4) Growth factor Recombinant human follistatin 300 (GT, # 2669) was purchased from GT. Recombinant human activin A (GT, # 2338) was used at 10 ng / ml. Recombinant mouse noggin / Fc chimera (R & D, # 719-NG) was used at 100 ng / ml. Recombinant human bone morphogenetic protein (BMP) 2 (Osteogenetics GmbH, Wurzburg, Germany) was used at 5 ng / ml or 25 ng / ml. Nicotinamide was purchased from Sigma.

(5) X-gal staining for detecting β-galactosidase activity
ES cell cultures were fixed in 4% paraformaldehyde in PBS for 20 minutes at room temperature, rinsed several times with PBS, and stained for β-galactosidase activity. For X-gal staining, cells were washed with 20 mM K ₃ Fe (CN) ₆ , 20 mM K ₄ Fe (CN) ₆ -3H ₂ O, 2 mM MgCl _{2 in} PBS in the presence of 1 mg / ml X-gal. Incubate overnight at 37 ° C. in a buffer containing 0.02% Nonidet-P40, 0.01% deoxycholic acid. The number of LacZ positive cells in each well was counted under a microscope. DNA was extracted from the cells after X-gal staining and quantified. The total number of cells in each well was measured from the DNA standard and the percentage of X-gal positive cells in each well was calculated. The total number of cells differentiated for 12 days was 3 × 10 ⁵ to 1 × 10 ⁶ .

(6) Quantification of GFP positive cells
Fluorescence photographs of ES cells were taken every 24 hours under a stereomicroscope (Leica MZFLIII), and fluorescence intensity was quantified with Lumina Vision software (Mitani Corporation, Japan). Immunofluorescence photographs were taken with an Olympus IX70 using a CCD camera (DP50).

(7) Analysis by reverse transcription polymerase chain reaction (RT-PCR)
RNA was extracted from ES cells using TRIZOL reagent (Gibco BRL) followed by DNase (Sigma). For reverse transcription (RT) reaction, 3 μg of RNA was reverse transcribed using M-MLV reverse transcriptase (Toyobo) and oligo dT primer (Gibco BRL). 1 μl of 5-fold diluted cDNA (corresponding to 1/100 of reverse transcript) was used for PCR analysis.

  For each primer combination, the number of cycles and the amount of cDNA required for RT-PCR in the linear range were determined empirically. Table 1 shows the primer sequences and the number of cycles used for each primer pair. 1/5 of the RT-PCR product was loaded on 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed with FluoroImager (Molecular Dynamics) using the DNA sensitive dye SYBRGreenI (Molecular Probes).

PCR conditions:
Iapp, Ins2 and PP:
96 ° C (denaturation) for 30 seconds, 60 ° C (annealing) for 2 seconds, and 72 ° C (extension) for 45 seconds (Hot start)
Amylase, Ins1 and Kir6.2
30 seconds at 96 ° C (denaturation) and 30 seconds at 70 ° C (annealing and elongation) (Hot start)
Other:
30 seconds at 96 ° C (denaturation), 2 seconds at 60 ° C (annealing), and 45 seconds at 72 ° C (extension)

(8) Immunohistochemical examination (experimental method shown in FIGS.
When performing a full mount immunohistochemistry, mesoderm cells or ES cells were placed on a Nunc Thermanox Colerslips 24-well type (Nunc, # 174950). Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and washed thoroughly with 0.1% tween-containing phosphate buffered saline (PBS-T). Infiltrate with PBS containing 1% TritonX-100 at room temperature for 10 minutes, incubate with blocking solution (x5 Blocking One, Nacalai Tesque) for 1 hour, then add the primary antibody in blocking solution to the sample and overnight at 4 ° C Incubated. The sample was thoroughly washed with PBS-T and mounted with PermaFluor Aqueous Mounting Medium (IMMUNON). Confocal images were acquired using a Leica Spectral Confocal Scanning System, TCS-SP2 (Leica).

  The antibodies used for detection were rabbit anti-AFP (Biomeda, # A02), goat anti-albumin (Sigma, # A1151), mouse anti-Cdx2 (BioGenex, # MU392-UC), mouse anti-CK19 (DAKO, # M0888), goat Anti-HNF3β (Santa Cruz, # sc-9187), rabbit anti-Pdx1 (Chemicon, # AB5754), and rabbit anti-T (Santa Cruz, # sc-20109), rabbit anti-Nkx2.1 (Santa Cruz, # sc-13040) Rabbit anti-GFP (MBL, # 598). Secondary antibodies used were Allexa 568-conjugated goat anti-rabbit and mouse antibody (Molecular Probes, # A11036 and # A11019), Allexa 488-conjugated goat anti-rabbit and mouse antibody (Molecular Probes, # A11070), Cy3-conjugated donkey anti-goat. Antibody (Jackson Lab, # 705-166-147).

(9) Flow cytometry with fluorescent labeling (FACS) (experimental method of FIG. 12)
M15 cells were seeded at 2 × 10 ⁵ cells per well and cultured before use. 5,000 SK7 ES cells were seeded on the mesodermal cell layer in differentiation medium and allowed to differentiate for 8 days. On the 8th day of differentiation, SK7 ES cells were dissociated using Cell dissociation buffer (GIBCO BRL) at 37 ° C. for 20 minutes. Cells were stained with a combination of monoclonal antibodies (mAb). The antibodies used in this experiment were biotin-conjugated anti-E-cadherin monoclonal antibody ECCD2 (provided by Prof. Akiyoshi Nagahama and Prof. Taro Ogawa, Kumamoto University; unlabeled antibody can be purchased from Takara Bio Inc.), PE-conjugated anti-Cxcr4 monoclonal Antibody 2B11 (Becton, Dickinson and Company). Cells were incubated with a mixture of labeled monoclonal antibodies for surface staining. Stained cells were filtered through a 40 μm mesh, resuspended in HBSS-1% BSA containing propidium iodide and analyzed by FAC S Canto (Becton, Dickinson and Company). Data was recorded using CellQuest Prosoftware (Becton, Dickinson and Company) and analyzed using Flowjo program (Tree Star).

(10) Flow cytometry by fluorescence labeling (FACS) (experimental method of FIG. 13)
M15 cells treated with mitomycin were cultured overnight at 6 × 10 ⁶ cells per 90 mm plate before use. 3 × 10 ⁵ ES cells, strain SK7, were seeded on the M15 cell layer in differentiation medium and allowed to differentiate for 9 days. On day 9 of differentiation, ES cell SK7 strain was trypsinized with 0.25% trypsin-EDTA for 10 minutes, filtered through a 40 μm mesh, and propidium iodide at a concentration of 1 × 10 ⁶ to 2 × 10 ⁶ cells / ml It was resuspended in HBSS-1% BSA containing and analyzed by FACS Vantage SE. Undifferentiated ES cell line SK7 was used as a negative control. Differentiated ES cells were divided into 5 fractions according to the intensity of green fluorescence, and RNA extraction and RT-PCR analysis were performed.

(11) Method for maintaining culture of ES cell-derived differentiated cells Differentiated ES (dES) cells were cultured for 8 days in a medium supplemented with 10% FBS, with both activin and bFGF added under M15. After culturing ES cells for 8 days, dES was trypsinized with 0.05% trypsin / EDTA and coated with 2-Methacryloyloxyethyl PhosphorylCholine (MPC) (low cell binding dish and plate) Pdx1-positive cells can be maintained and cultured by seeding again in Nalgenunc, # 145389).

(12) Transplantation under the mouse kidney capsule
(The Pdx1-positive differentiated cells on the first day of maintenance culture were repaired in the above section and used for transplantation experiments (FIG. 14). Under normal anesthesia, dES (about 3 × 10 ⁶ cells under the left kidney capsule of mice) One week after transplantation, mice transplanted with ES cells were killed, kidneys were removed and fixed with 4% paraformaldehyde, and frozen sections of the grafts (10 μm) were analyzed by immunohistochemistry.

(13) Differentiation of ES cells (experimental method of FIG. 17)
For differentiation under serum-free conditions, SK7 ES cells were seeded at 5,000 cells per well in 24-well plates pre-coated with mesoderm cells in differentiation medium and cultured for 8 days. The medium was changed every 2 days. ES cells or Pdx1 / EGFP cells differentiated into embryonic endoderm (E-cadherin + / CXCR4 + cells) were compared. ES cells were 10 μg / ml insulin (Sigma), 5.5 μg / ml transferrin (Sigma), 6.7 pg / ml selenium in 10% FBS (FBS) or with and without 20 ng / ml activin. Differentiation was performed in serum-free basal medium (DMEM, Gibco BRL) (ITS) supplemented with (Sigma) and 0.25% albumin (Albmax, GIBCO).

(B) Results (1) Production of factors that promote the differentiation of ES cells into Pdx1-expressing intestinal endoderm cells by mesoderm cells About the activity of promoting the endoderm differentiation of ES cells using a co-culture system Cultured cells and cell lines were screened. Since an ES cell line in which the lacZ reporter gene was inserted at the Pdx1 locus was used, the expression of the Pdx1 gene could be visualized by in situ X-Gal staining. As a result, the middle kidney cell line M15 (Larsson, SH, Charlieu, JP, Miyagawa, K., et al. (1995). Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing. Cell 81, 391- 401), mouse embryo fibroblasts (MEF) (Nagy A, et al. (2003) Manipulating The Mouse Embryo: A Laboratory Manual. Third Edition Cold Spring Harbor Press), and ST2 cells (Ogawa, M., Nishikawa, S) ., Ikuta, K., Yamamura, F., Naito, M., Takahashi, K. and Nishikawa, S. (1988) EMBO J, 7: 1337-1343) It was found that differentiation of ES cells into Pdx1 / β-gal expressing cells was promoted when co-cultured with ES cells. The M15 cell line significantly promoted the differentiation of ES cells into Pdx1 / β-gal expressing endoderm cells. When cultured on the M15 cell layer, 0.1% of all differentiated ES cells became Pdx1 / β-gal expressing cells (FIG. 1). This is similar to the proportion of Pdx1 / β-gal expressing cells induced by co-culture with embryonic pancreatic primordia. MEF moderately promoted differentiation of ES cells into Pdx1 / β-gal expressing endoderm cells. Since ES cells cultured on M15 cells produced the highest number of Pdx1 / β-gal expressing cells with good reproducibility, the M15 cell line was used in subsequent experiments.

  The time-dependent changes of Pdx1 / β-gal expressing cells on the M15 feeder cell layer were monitored. FIG. 2 shows that the number of Pdx1 / β-gal expressing cells peaked on day 12 and then decreased. When differentiated ES cells were trypsinized on day 7 and passaged on a freshly prepared M15 cell layer, Pdx1 / β-gal positive cells reappeared on day 15 (equivalent to day 8 after passage). Appeared. The short time required for the reappearance of Pdx1 / β-gal positive cells means that when ES cells are cultured on the M15 feeder cell layer, undifferentiated cells or certain endoderm progenitor cells are present in the culture. Indicates that it exists. When subculture was performed up to 10 times, reappearance of Pdx1 / β-gal positive cells was also observed.

(2) Monitoring of endoderm differentiation in living ES cells using Pdx1 / GFP expression In order to track pdx1 expression in living ES cells using fluorescent dyes, a plasmid containing Pdx1 / GFP was introduced. # 48.9 transgenic mice (Gu, G., Wells, JM, Dombkowski, D., Preffer, F., Aronow, B., and Melton, DA (2004) .Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development 131, 165-79), an ES cell line was established. The P # 48.9 strain of transgenic mice has been shown to reproduce Pdx1 expression through embryogenesis. Initially, eight ES cell lines were established, all of which showed similar Pdx1 / GFP expression patterns during in vitro differentiation. Among them, the ES cell line named SK7 proliferated well and was used for the subsequent experiments. Pdx1 / GFP-expressing ES cells (SK7) were differentiated on M15 cells treated with mitomycin, and the time-dependent changes in Pdx1 / GFP expression were monitored.

  A schematic diagram of the differentiation procedure is shown in FIG. 3A. The ES cell SK7 strain was seeded at a low density of 500 cells / well in a 24-well plate containing a differentiation medium containing 10% FBS. The culture conditions were compared between a control differentiation medium and a medium in which FBS in the differentiation medium after 4th day was replaced with Knocked-out Serum Replacement (KSR-substituted differentiation medium). In the low-density culture, it was revealed that the number of Pdx1 / EGFP positive cells after day 4 increased when ES cells were cultured in KSR-substituted differentiation medium (FIG. 3A). The morphological changes of ES cell colonies during differentiation are shown in FIG. 3C. Many of the colonies remain swelled until the fifth day of differentiation, and cell migration from the center of the colonies is observed around the sixth day (FIG. 3C). As the cells migrate, Pdx1 / EGFP expression can be detected on the 6th day, and the number of expressed cells reaches the maximum value on the 8th day and then decreases (FIG. 3B). In the Pdx1 / EGFP positive colony, the cells migrated from the center of the colony, and then the Pdx1 / EGFP positive cells were in the periphery of the colony to form a round large flat colony. Pdx1 / EGFP negative colonies showed features of raised and dense colonies. These results indicate that it is necessary to acquire migration performance for pancreatic differentiation.

(3) Detection of expression of pancreatic endocrine markers and other endoderm-derived tissue markers in ES cells differentiated on the M15 feeder cell layer by RT-PCR analysis
In order to characterize the differentiation state of ES cell line SK7 on M15 feeder cells, the expression of pancreatic endocrine markers was analyzed. Pdx1 expression was detected on day 4 and peaked on day 8 (FIG. 4). Other endocrine progenitor markers such as Nkx2.2, Nkx6.1, Pax 4, Pax6, Isl1, NeuroD, Ngn3 were also detected. Expression of Pax4 and Isl1 is also detected in control ES cells differentiated in gelatin-coated wells, but expression of other markers is specifically induced in ES cells differentiated on the M1 5 feeder cell layer. Insulin 2 expression in ES cells differentiated on the M15 feeder cell layer was detected on day 8. Other endocrine molecular markers and exocrine markers such as glucagon, pancreatic polypeptide (Pp) and somatostatin (Sst) are also specifically detected in ES cells differentiated on M15 feeder cells. Maturation / cell markers such as Iapp, glucokinase, Kir6.2 are detected.

  In addition, immature liver markers (albumin, α-fetoprotein, Met), lung markers (Sftpc (Nkx2.1 expression is shown in the immunostaining in Fig. 5)), and oral marker (Pax9) are detected. . That is, by the induction method using M15, the endoderm organ is induced in the liver, lungs and oral cavity in addition to the pancreas. Furthermore, since the small intestine is also induced (FIG. 5), it is shown that it has a wide organ-inducing ability. Therefore, the differentiation induction method of the present invention is useful for inducing differentiation of endoderm organs including pancreas, liver, lung, small intestine and oral cavity (Pax9, RT-PCR).

The above results support that the M15 feeder cells specifically promote the differentiation of ES cells into endoderm-derived organs such as pancreas and liver. The active M esodermal D erived I nducing A ctivity ( mesoderm-derived differentiation-inducing activity; mdia) was named.

  Furthermore, the effect of MDIA (mesoderm-derived differentiation inducing activity) on other ES cells (R1 and J1 ES cells) was examined. Expression of Pdx1 or endocrine markers (insulin, pancreatic polypeptide, and somatostatin) was elevated in RIA and J1 ES cells treated with MDIA (right diagram in FIG. 4). Therefore, it was shown that MDIA can be applied to various types of ES cells.

(4) Detection of expression of endoderm-related marker in differentiated cells derived from SK7 cells FIG. 5 shows the results of detecting the expression of the endoderm-related marker in differentiated cells derived from SK7 cells by immunohistochemical examination. As shown in Figure 5, HNF3β, a common marker of endoderm cells, various endoderm organ-related genes: albumin of liver marker gene, Nkx2.1 of lung marker gene, Cdx2 gene of small intestine marker (red) Was recognized. Hnf3β-expressing cells overlap with Pdx1 / EGFP positive cells, but cells expressing endoderm organ-related genes other than pancreas such as lung and liver are different from Pdx1 / EGFP positive cells (green). It was.

(5) Endoderm precursor cells induced by MDIA treatment
The results of RT-PCR analysis indicated that other endoderm-derived tissues (such as liver) were also induced by MDIA treatment of ES cells. This shows that MDIA treatment specifically induces differentiation from ES cells to endoderm. To monitor the differentiation of endoderm progenitors, we used an endoderm-specific enhancer fragment of the Hnf3β gene (Nishizaki, Y., Shimazu, K., Kondoh, H., Sasaki, H. (2001) Identification of essential sequence motifs in the node / notochord enhancer of Foxa2 (Hnf3beta) gene that are conserved across vertebrate species. Mech Dev 102, 57-66), to control the expression of monomeric red fluorescent protein I reporter gene (mRFP1) A transgenic mouse into which the designed Hnf3β / mRFP1 gene was introduced was prepared. Hnf3β / mRFP1 transgenic mice reproduced the expression pattern of Hnf3β as previously reported (Nishizaki et al., 2001). The Hnf3β / mRFP1 transgenic mouse is a Pdx1 / EGFP transgenic mouse strain P # 48.9 (Gu, G., Wells, JM, Dombkowski, D., Preffer, F., Aronow, B., and Melton, DA (2004) Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development 131, 165-79). An ES cell line carrying both Hnf3β / mRFP1 and Pdx1 / EGFP transgenes was established, and the ES cell line designated PH3 was used in subsequent experiments. The ES cell PH3 line was differentiated on M15 cells treated with mitomycin and the time-dependent kinetics of expression of Pdx1 / EGFP and Hnf3β / mRFP1 were monitored. FIG. 6 shows that Hnf3β / mRFP1 expression is observed on day 7 of differentiation, which is one day ahead of Pdx1 / EGFP expression. The population of ES cells that express Hnf3β is larger than the population that expresses Pdx1. A population of ES cells that express time-dependent expression and Hnf3β and Pdx1 reproduces normal embryonic development. From these results, it was shown that endoderm precursor is induced by MDIA treatment of ES cells.

(6) Part of mesoderm-derived differentiation-inducing activity (MDIA) is the action of activin. To clarify the molecular mechanism of mesoderm-derived differentiation-inducing activity (MDIA), activin βA in the feeder cell line used in the experiment Expression of several growth factors such as activin βB and follistatin was screened. Activin βA and activin βB are expressed at high levels in M15 cells and at moderate levels in MEF cells. Follistatin is not expressed in M15 cells, but is expressed at high levels in MEF cells and ST2 cells (FIG. 7A). The effect of activin A addition in ES cell differentiation cultures was analyzed. Addition of 10 ng / ml activin A significantly promoted Pdx1 / EGFP expression on M15 cells (FIG. 7B, Student t-test, * p <0.01). When follistatin was added at 25 ng / ml, Pdx1 / EGFP positive cells decreased, and when added at 250 ng / ml, it decreased further significantly. MDIA promotion by external addition of activin A (10 ng / ml) is inhibited by 250 ng / ml follistatin. These results indicate that studies on MDIA-mediated molecules are useful in studying the inductive signals required for ES cell differentiation into pancreas. The above results indicate that activin is one of the molecules that mediate MDIA. Since MDIA is not completely inhibited by follistatin, M15 cells involved in MDIA, which differentiate ES cells into endoderm cells, may contain other molecules. By studying secreted or membrane-bound molecules expressed in M15 cells or other feeder cells exhibiting MDIA, information on the molecular mechanism of ES cell differentiation into endoderm can be obtained. .

(7) MDIA promotes the differentiation of ES cells into the endoderm system and can be used for screening of inducers or culture conditions that promote this differentiation.
We further examined whether MDIA could be used to screen for induced molecules that further promote the differentiation of ES cells into endoderm lineage when added to ES cells cultured on M15 cell layers. Since it was shown from FIG. 3A that ES cells cultured in KSR-substituted differentiation medium produced more Pdx1 / EGFP-expressing cells, the possibility of the presence of an inhibitor in serum was estimated. Noggin, a BMP inhibitor, was added at various times to test its effect on MDIA. As a result, it was revealed that 100 ng / ml noggin added on days 5-8 enhanced MDIA. As shown in FIG. 8, the enhancement effect of Noggin can be observed using a KSR-substituted differentiation medium instead of the differentiation medium. However, the effect of noggin addition was not limited to the late stages of differentiation. Addition of 100 ng / ml noggin on days 0-4 enhanced MDIA. Additional experiments in which noggin was added in a shorter period of time show that noggin addition on days 3-4 is similar to that on days 0-4 (Figure 8). Next, it was examined whether BMP in the serum has an inhibitory effect on the differentiation of ES cells into the endoderm system. FIG. 9 shows that the addition of noggin to the differentiation medium on days 3-4 produced Pdx1 / EGFP-expressing cells of the same level as when cultured in KSR-substituted differentiation medium without addition of noggin. The number of Pdx1 / EGFP-expressing cells in ES cells differentiated in KSR-substituted differentiation medium decreased with the addition of BMP2, which was offset by the addition of noggin (FIG. 9). However, the differentiation of ES cells in the serum-free state on days 0-4 revealed that Pdx1 / EGFP expression was low due to poor cell growth. Therefore, in subsequent experiments, noggin was added on the 3rd to 4th days in order to promote the differentiation of ES cells into the endoderm system, and after the 4th day of differentiation, KSR-substituted differentiation medium was used instead of the differentiation medium. ing.

  Furthermore, the effect of adding nicotinamide (NA) with noggin or activin A was examined. FIG. 10 shows that the addition of noggin and activin or noggin and nicotinamide showed no additive effect. However, the addition of noggin, activin, and nicotinamide promoted Pdx1 / EGFP expression. The inset shows time-series data when any of noggin, activin, or nicotinamide is added.

  Since BMP2 and nicotinamide are not expressed in M15 cells or other cell lines, these results indicate that MDIA can be used to screen for growth factors or compounds that promote endoderm differentiation.

(8) Promotion of differentiation induction into pancreas by addition of growth growth factor
It was shown that the number of Pdx1 / EGFP-expressing cells increased by adding activin, bFGF, or activin + bFGF into a differentiation medium containing 10% FBS. FIG. 11A shows a Pdx1 / EGFP image on day 8. Activin was added to 20 ng / ml and bFGF 5 ng / ml. In FIG. 11A, the morphology of Pdx1 / EGFP positive colonies was changed by adding activin and FGF. The cell clumps at the center of the positive colony disappeared, and as a result, the number of cells expressing Pdx1 / EGFP increased. Moreover, the synergistic effect was shown by making both act on ES cell simultaneously. In addition, the uppermost figure of d8 / M15 shows that the Pdx1 / EGFP positive colony expanded when M15 was used for differentiation, the cell mass appears to be small in the center, and the Pdx1 / EGFP positive colony is around the colony. Located at the edge (arrow). In the Pdx1-negative colony, a large round colony is observed in the center (arrow head). + In activin (20), 20 ng / ml of activin was added from day 0 to day 8 of differentiation. A large number of Pdx1 / EGFP positive pancreatic stem cells appear, showing a form of expanded colonies, and a decrease in Pdx1 / EGFP negative colonies. The small mass of cells that was visible in the center of the Pdx1 / EGFP positive colony disappeared. In + bFGF (5), 5 ng / ml of bFGF is added from the 0th day to the 8th day of differentiation. The migration of cells in the center is promoted, showing an expanded colony morphology. As a result, Pdx1 / EGFP positive cells are accumulated by the marginal part of the colony. In addition, in activin (20) + bFGF (5), activin and bFGF are added from the 0th day to the 8th day of differentiation. The two further synergized and more Pdx1 / EGFP positive pancreatic stem cells appeared.

  FIG. 11B shows the result of staining the differentiated cells obtained with the addition of both activin and bFGF in FIG. 11A with an antibody against C-peptide. Since C-peptide occurs as a by-product during insulin biosynthesis, positive cells are shown to actually biosynthesize insulin. Pdx1 / EGFP positive cells are green. Among the differentiated cells, cells positive for C-peptide alone (red) were also observed, but many cells were positive for both C-peptide / Pdx1 (yellow). Enlarged photos are shown in A and B.

  Furthermore, differentiation was evaluated using FACS. In FIG. 12A, it is assumed that the endoderm (definitive endoderm) is both E-cadherin and Cxcr4 positive cells. Pancreatic progenitor cells are E-cadherin and Pdx1 / EGFP positive cells. On M15, the endoderm occupies 8% of all cells on day 8. Under the conditions where activin and bFGF were added, the endoderm ratio increased, accounting for 47% of all cells. In addition, pancreatic progenitor cells accounted for 22% of the endoderm on M15, but the ratio of pancreatic progenitor cells in the endoderm increased to 67% under the conditions of activin and bFGF.

  Figure 12B also shows ES cells, M15 cells, and differentiated ES using indicators of side scattered light (SSC) that reflects the complexity of the cell's internal structure and forward scattered light (FSC) that reflects the size of the cell. The FACS development image of a cell is shown. Using the SSC and FSC indices, M15 cells can be removed by sorting out the part enclosed by the polygon in the figure. FIG. 12B shows an example under the conditions where activin and bFGF were added, and it can be seen that this M15-free fraction contains Pdx1 / EGFP positive cells.

(9) Isolation and expression analysis of ES cell-derived Pdx1 / EGFP positive cells by FACS
In order to investigate the molecular characteristics of ES cell-derived Pdx1 / EGFP-expressing cells, the differentiated ES cell population was analyzed by FACS. FIGS. 13A and B show that the population of Pdx1 / EGPF positive cells is shifted to the GFP positive side in ES cells differentiated on M15 cells compared to undifferentiated ES cells. Differentiated ES cells were divided into 5 fractions according to the expression level of GFP, and the molecular marker expression of pancreatic differentiation was analyzed by RT-PCR for each fraction. RT-PCR analysis shows that Pdx1 expression is high in the no.4 fraction with strong GFP positive signal (FIG. 13C). This fraction represents approximately 1% of the total differentiated ES cells (FIG. 13B). In this fraction with a strong GFP-positive signal, expression of NeuroD, insulin, PP, and Sst was also observed, indicating that the cells of this fraction are of pancreatic lineage.

(10) Maintenance culture of differentiated ES cells Differentiated ES cells can be maintained by the method shown in FIG. That is, it was possible to maintain and culture Pdx1-positive cells by redifferentiating the differentiated ES cells in a low cell binding dish coated with MPC under the addition of both activin and bFGF under M15 (FIG. 14). ).

(11) Maintenance of Pdx1 / EGFP and insulin expression in ES cells transplanted under the kidney capsule of mice Differentiated ES cells were transplanted under the kidney capsule. The transplanted mice were killed one week later and the frozen sections of the grafts were analyzed by immunohistochemistry. FIG. 15 shows that Pdx1 / EGFP expression is maintained and insulin expression is also seen. These results indicate that Pdx1 / EGFP positive cells can proliferate and differentiate into insulin producing cells when transplanted into mice.

(12) Differentiation induction using monkey ES cells

FIG. 16 shows a stained image of cells induced to differentiate on M15 using monkey ES cells in the same manner as mouse ES cells. The culture conditions of monkey ES cells (purchased from Asahi Techno Glass Co., Ltd.) are as follows. On day 0 of culture, monkey ES cells were seeded in a 24-well plate at a concentration of 20,000 cells / well, and the medium was changed on days 1, 3, 5, 7, and 9 of culture. The culture medium used was a differentiation medium (10% FBS / DMEM (high glucose). A and B in FIG. 16 are stained images on the 4th and 8th days after induction of differentiation, and are markers for mesoderm progenitor cells. The results of staining with HNF3β and mesoderm marker T (Brachyury) showed strong expression of HNF3β on both day 4 and day 8. C, D, and E in FIG. It expresses the pancreatic progenitor cell marker Pdx1, the small intestine marker Cdx2, the liver marker albumin, and the bile duct marker CK19, so that the endoderm cells (pancreas, liver, bile duct, small intestine) Differentiation was shown.

(13) Differentiation induction using human ES cells FIG. 17 shows a stained image of cells induced to differentiate on M15 using human ES cells in the same manner as mouse ES cells. Differentiation culture conditions for human ES cells are as follows. On day 0 of culture, 24-well plates were seeded with human ES cells at a concentration of 20,000 cells / well, and the medium was changed on days 1, 3, 5, 7, 9, and 11 of culture. As a medium, differentiation medium (10% KSR / DMEM (high glucose) was used. In FIGS. 17A and 17B, on day 12, endoderm cell marker HNF3β, small intestine marker Cdx2, liver marker The expression of albumin, α-fetoprotein, and bile duct marker CK19 indicated differentiation into endoderm cells (liver, bile duct, small intestine).

(14) Induction of pancreatic progenitor cells using serum-free medium on M15 feeder cells
FIG. 18 shows the results of evaluating differentiation potential using SK7 cells on M15 feeder cells. The definitive endoderm is a positive cell for both E-cadherin and Cxcr4. Serum factors are considered necessary for endoderm maintenance and differentiation into Pdx1 / EGFP positive cells. In serum-free medium alone, differentiation into endoderm and pancreatic progenitor cells was low on M15 cells, but when activin was added, the ability to differentiate into endoderm and pancreatic progenitor cells increased (FIG. 18). In particular, 2% of the cells differentiated into pancreatic progenitor cells, and a fairly high efficiency was achieved.

  According to the method of the present invention, endoderm cells such as pancreatic stem cells can be efficiently induced to differentiate from ES cells. In addition, by using the culture method of the present invention, the differentiation induction effect of an unknown substance on the pancreas can be measured with high sensitivity. According to the present invention, differentiated ES cell-derived pancreatic stem cells or hepatic stem cells can be purified and maintained and cultured in vitro. In addition, the method of the present invention can be applied to a plurality of types of ES cell lines. The method of the present invention can also be applied to cells of endoderm origin that have the same development.

FIG. 1 shows screening for pancreatic differentiation-inducing ability in various cell lines. A: Schematic diagram of differentiation induction method. After seeding ES cells in which LacZ gene was knocked into the Pdx1 promoter at a density of 5000 cells / well (24 wells) on cultured cells, they were cultured in the presence of serum and X-gal stained on the 12th day to obtain pancreatic cells from various cell lines. The differentiation induction effect was evaluated. Since the cells are seeded on the cultured cells on the second day after the formation of the floating embryoid bodies (EB), the evaluation by X-gal staining was performed on the 14th day of the culture. B: X-gal stained image. Each photograph shows the cell name used as a feeder cell. C: The ratio of Pdx1 / β-gal positive cells when each cell line is used as a feeder cell is shown on the vertical axis. Inductive effects were observed for M15, MEF, and ST2. Student's t-test was significantly different from ES cells differentiated on the control gel: **, p <0.01. FIG. 2 shows evaluation of Pdx1 / β-gal positive cells over time from ES cells using M15. A: Using M15, which showed the highest pancreatic differentiation-inducing effect by screening, differentiation induction experiments from ES cells were performed and evaluated with X-gal staining over time. The photo shows the number of days after the start of differentiation induction. The number of days in parentheses is the number of days after passage. B: Time course of the appearance of Pdx1 / β-gal positive cells by passage on M15 feeder cells. The number of days after the start of differentiation induction is shown on the horizontal axis, and the number of Pdx1 / β-gal positive cells is shown on the vertical axis. In differentiation induction using M15, Pdx1 / β-gal positive cells first appear at the peak on day 12, but then decrease. When subcultured onto M15 feeder cells, Pdx1 / β-gal positive cells peaked again at 7-8 days after passage. The first passage is indicated by a black circle, and the second passage is indicated by a black square. FIG. 3 shows evaluation of differentiation into Pdx1 / EGFP positive cells in real time using ES cells (SK7 cells) in which GFP is introduced under the Pdx1 promoter. A: A schematic diagram of the differentiation induction method is shown. In the differentiation induction method using SK7, seeding is carried out on M15 without forming EB. In both figures, after seeding at 500 cells / well (24 wells), the cells were cultured in a 10% serum medium until the fourth day of culture, and were cultured in a 10% KSR medium from the fourth day of culture. Appearance of Pdx1 / EGFP positive cells over 6 to 8 days after differentiation induction was evaluated. At high density (5000 cells / well), differentiation occurs only in 10% serum medium, but at low density (500 cells / well), differentiation efficiency is poor. When replaced with 10% KSR medium, Pdx1 / EGFP positive cells appear from day 6 Come on. It reaches its peak on the 8th day. Evaluation of differentiation into Pdx1 / EGFP positive cells was performed on the fluorescence photographs by the sum of fluorescence intensities using Luminavision software. B: The Pdx1 / EGFP positive colony when differentiated using M15 shows an expanded form, and the Pdx1 / EGFP positive cell is located at the marginal part of the colony. Pdx1 / EGFP negative colonies show round colony morphology. The differentiation image of the 7th day after differentiation induction is shown. C: Time-lapse culture image using SK7 cells and appearance of Pdx1 / EGFP positive pancreatic stem cells. A round colony is shown until the 5th day after differentiation induction, but on the 6th day after the differentiation induction, cell migration occurs rapidly and shows a spread colony morphology. Pdx1 / EGFP positive cells are located at the margin of the colony. On day 9 after differentiation induction, Pdx1 / EGFP positive cells decrease. FIG. 4 shows the results of RT-PCR analysis. Expressions of various pancreatic progenitor cell-related genes, pancreatic endocrine cell marker genes, exocrine cell marker genes, differentiated β-cell marker genes, and liver marker genes were observed. Although the SK7ES cell line was established from ICR mice, the same promotion of differentiation induction by M15 support cells was detected for other R1, J1 ES cell lines. In cells induced to differentiate on M15 feeder cells, expression of the pdx1 gene or endocrine marker genes such as insulin, pancreatic peptide, and somatostatin was detected on day 8. FIG. 5 shows the results of detection of endoderm-related marker expression in differentiated cells derived from SK7 cells by immunohistochemical examination. Expression of HNF3β, albumin, Nkx2.1, and Cdx2 was observed. FIG. 6 shows that endoderm progenitor cells are derived from ES cells. In order to further investigate the dynamics of endoderm progenitor cells differentiated from ES cells, the endoderm-specific expression enhancer region of the mouse Hnf3β gene was used. An ES cell line was established from Pdx1 / GFP and Hnf3β / mRFP1 double transgenic mice so that the expression of the Hnf3β gene could be visualized with a mRFP1 (monomeric Red Fluorescent Protein 1) reporter protein. In the established Pdx1 / EGFP-Hnf3β / mRFP1 ES cell line, Pdx1 / EGFP expression is detected from the 8th day, whereas Hnf3β / mRFP1 expression is detected from the 7th day (1 day earlier). Moreover, expression of Hnf3β / mRFP1 is induced in a wider range of cells than Pdx1 / EGFP expressing cells. Analysis of transgenic mice shows that the Hnf3β gene is expressed along the anteroposterior axis in the intestinal epithelium at the time when the region of the early endoderm is determined (embryonic day 8.5), compared to the pdx1 gene Since it was confirmed that it was expressed in a wider range as early as 1 day, it was suggested that this HNF3β-positive cell corresponds to an endoderm progenitor cell in the stage before it was localized. Therefore, differentiation induction on M15 supporting cells is considered to reflect the normal development process. FIG. 7 shows that part of the mechanism of differentiation induction by M15 is mediated by activin. A: Exhaustive gene expression analysis was performed on cultured cells used for screening to elucidate the mechanism of pancreatic differentiation induction by M15 using Affymetrix Gene Chip, and the expression of genes showing bioactive effects was examined. As a result, it was clarified that the follistatin inhibitor of activin involved in pancreatic differentiation induction in normal development is extremely low at M15. In addition, activin is expressed to some extent in M15 cells, MEF, and ST2 cells. B: Activation of pancreatic differentiation was promoted by activin, and differentiation induction was inhibited by follistatin. Activin and follistatin were added on the third day after differentiation induction. The culture was carried out in a medium containing FBS until the third day, and a medium substituted with KSR was used until the third to the eighth day. The appearance of Pdx1 / EGFP positive pancreatic stem cells on day 8 was evaluated. When follistatin was added during differentiation induction to evaluate the effect of follistatin, pancreatic differentiation induction was inhibited in a concentration-dependent manner. (Significant Student's t-test compared to Cont, * p <0.05, n = 3). However, pancreatic differentiation induction effect by M15 could not be inhibited 100%. In addition, activation of pancreatic differentiation was promoted by activin (10 ng / ml), but the effect could be inhibited by adding an excessive amount of follistatin (student's t Significant for -test, ** p <0.01, n = 3). FIG. 8 shows the results of examining the effect on differentiation induction by adding noggin to the medium. In the early differentiation induction, a medium containing FBS was used, and the inhibitors contained therein were considered. Noggin was added for various periods, and Pdx1 / EGFP positive cells appearing in real time were evaluated. Induction of pancreatic differentiation was promoted by adding Noggin at the early stage of differentiation induction. The number of days that promoted by adding noggin could be identified on days 2-4, or on days 3-4 as a minimum. FIG. 9 shows that the promotion of pancreatic differentiation induction by noggin is due to inhibition of BMP2 contained in the serum. On day 3-4 of differentiation induction, 100 ng / ml of noggin or a predetermined concentration of BMP2 was added. When there is a mark, FBS in the medium on the 3rd to 4th days is replaced with KSR. On day 6, Pdx1 / EGFP positive cells were evaluated. In the FBS-containing differentiation medium, there was a significant difference with the addition of noggin compared to the non-addition group (Student's t-test, ** p <0.01). When replaced with KSR, pancreatic differentiation induction was promoted (Comparison with Noggin (-). Student's t-test, * p <0.05,). This promotion was inhibited by the addition of BMP2 (comparison between the KSR-substituted differentiation medium noggin (-) and the BMP addition group (* p <0.05, ** p <0.01). The inhibition by BMP2 was eliminated again by the addition of noggin ( **, p <0.01). FIG. 10 shows that noggin and nicotinamide, or activin and nicotinamide synergistically promote pancreatic differentiation induction. Nicotinamide alone did not promote pancreatic differentiation but synergistically induced pancreatic differentiation when combined with noggin or activin. The combination of noggin, activin, and nicotinamide shifted the appearance of Pdx1 / EGFP positive cells to an early stage, and peaked on the 6th day after induction of differentiation. (Student's t-test, ** p <0.01 compared with the control group of KSR-substituted differentiation medium in each day) FIG. 11 shows the promotion of differentiation induction into the pancreas by the addition of activin and bFGF. FIG. 11A shows that the number of Pdx1 / EGFP-expressing cells greatly increased by the addition of activin, bFGF, or both activin and bFGF in a differentiation medium containing 10% FBS. Show. FIG. 11B shows the result of staining a differentiated cell obtained with the addition of both activin and bFGF in FIG. 11A with an antibody against C-peptide. FIG. 12 shows that the promotion of differentiation induction into the pancreas by the addition of activin and bFGF can be quantitatively evaluated using FACS, and the degree thereof is very large. Moreover, it shows that M15 supporting cells can be selectively removed from differentiated ES cells using FACS. FIG. 12A shows that the endoderm (definitive endoderm) is a positive cell for both E-cadherin and Cxcr4. Figure 12B selects M15 cells from a population of differentiated ES cells using side-scattered light (SSC) that reflects the complexity of the cell's internal structure and forward-scattered light (FSC) that reflects the size of the cell. 2 shows FACS development images of differentiated ES cells before and after removal. FIG. 13 shows the purification of ES cell-derived pancreatic stem cells using a cell sorter and the analysis of molecular markers that are expressed. SK7 ES cells were induced to differentiate in FBS-containing medium in 300,000 cells / 90 mm dish on M15 cells, and FACS was performed on the 9th day. Due to differentiation induction, the cell population of the fraction of GFP positive cells increased. Fractions 1 to 4 were collected. Fraction 4 is strongly GFP positive, and cells expressing Pdx1 gene are concentrated in this fraction. In addition, NeuroD, Somatostatin, and insulin are expressed. FIG. 14 shows a method for maintaining and culturing differentiation-induced Pdx1-positive pancreatic progenitor cells. Pdx1-expressing cells can be maintained by culturing the differentiated ES cells by adding both activin and bFGF under M15 and seeding again on a low cell binding dish coated with MPC. FIG. 15 shows transplantation of differentiated ES cells under the adult mouse kidney capsule. The Pdx1-positive pancreatic progenitor cells derived from ES cells are transplanted under the adult mouse capsule by the method of FIG. FIGS. 15A and 15B show fluorescence micrograph images, in which Pdx1 / EGFP positive cells are retained. FIG. 15C shows the presence of insulin, Pdx1 / EGFP double positive cells by immunohistochemical analysis of the graft. FIG. 16 shows the results of induction of differentiation into endoderm progenitor cells and hepatopancreas cells using cynomolgus monkey ES cells. In FIG. A, on the fourth day of differentiation induction, many endoderm progenitor cells and mesoderm progenitor cells are induced to differentiate. Images of cells on day 4 (A) and day 8 (B) after differentiation of HNF3β (red: endoderm marker), T (green: mesoderm marker) and DAPI nuclear staining. In FIG. B, many endoderm cells remain on the 8th day of differentiation induction. Mesodermal progenitor cells can no longer be detected. In FIG. C, Pdx1-positive cells (red) of pancreatic progenitor cells and Cdx2-positive cells (green) of small intestinal progenitor cells were detected on day 10 of differentiation induction. Both are different cell populations. In FIG. D, albumin positive cells (red) and α-fetoprotein positive cells (green) of liver progenitor cells were detected on day 10 of differentiation induction. There were many cells that were expressed independently, but co-expressed cells were also observed. In FIG. E, CK19 positive cells (red) and α-fetoprotein positive cells (green) of bile duct cells were detected on day 10 of differentiation induction. Cells expressing each independently were observed. FIG. 17 shows the results of induction of differentiation into endoderm organs using human ES cells. In FIG. A, on the 12th day of differentiation induction, Hnf3β positive cells (red) of endoderm cells and Cdx2 positive cells (green) of small intestinal progenitor cells were detected. Although many cells co-expressed, Cdx2-only positive cells were also observed. In FIG. B, on the 12th day of differentiation induction, albumin positive cells (red) and α-fetoprotein positive cells (green) of liver progenitor cells were detected. In FIG. C, CK19 positive cells (green) and α-fetoprotein positive cells (red) of bile duct cells were detected on the 12th day of differentiation induction. FIG. 18 shows the results of induction of pancreatic progenitor cells by differentiation induction using a serum-free medium on M15 feeder cells.

Claims (8)

Induction of differentiation from ES cells to endoderm cells, including culturing mammalian-derived ES cells with the addition of actin bin, basic fibroblast growth growth factor (bFGF) and noggin in the presence of M15 cells how to. The method according to claim 1, wherein nicotinamide is further added when culturing ES cells in the presence of M15 cells. The method according to claim 1 or 2 , wherein the mammal-derived ES cells are mouse, monkey or human-derived ES cells. A step of inducing differentiation from an ES cell into an endoderm cell by the method according to any one of claims 1 to 3 , and a step of separating the differentiation-induced endoderm cell by flow cytometry (FACS) using a fluorescent label. A method of obtaining endoderm cells induced to differentiate from ES cells. Inducing differentiation from ES cells to endoderm cells by culturing mammalian-derived ES cells with the addition of actin bin, basic fibroblast growth growth factor (bFGF) and Noggin in the presence of M15 cells In addition, the ES cells were cultured in the presence of the test substance, and the ES cells were cultured in the presence of the test substance and the degree of differentiation induction into endoderm cells when the ES cells were cultured in the absence of the test substance. A method of screening for a substance that promotes or inhibits differentiation induction from ES cells to endoderm cells, comprising comparing the degree of differentiation induction to endoderm cells in the case. The screening method according to claim 5 , wherein the test substance is a growth factor or a low molecular compound. The screening method according to claim 5 or 6 , wherein the degree of differentiation induction into endoderm cells is measured using the expression level of a marker expressed in the endoderm as an index. The screening method according to claim 5 , wherein the mammal-derived ES cells are mouse, monkey, or human-derived ES cells.