JP2013066414A - Surface marker of stomach precursor cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface marker specific to a stomach precursor cell that can select a stomach precursor cell.SOLUTION: In this invention, three kinds of cell surface membrane proteins Adra2a, Fzd5, and Trpv6 that highly express at the precursor cell of stomach, can be identified, and any one of these proteins can be used as a surface marker of an excellent stomach specific precursor cell to evaluate the cell that differentiates into stomach appropriately at the differentiation process to the tissue cell of stomach using multipotent stem cells such as ES cells and iPS cells.

Description

  The present invention relates to cell surface markers that are specifically expressed in gastric progenitor cells.

In order to effectively and safely perform regenerative medicine using cells, it is important to selectively differentiate the cells of the tissue to be treated or to create precursor cells that differentiate only to the target tissue. is there. In particular, when stem cells such as ES cells and iPS cells are to be differentiated into specific tissue cells, cells of the target tissue and precursor cells that differentiate only into those cells can be purified, making it suitable for the transplant site. If a progenitor cell population can be separated, the stem cell therapeutic effect can be maximized and linked to a stable therapeutic result. As an effective means for selecting a stem cell population according to such a purpose, use of a cell surface marker specific to progenitor cells is considered.
So far, research has been widely attempted to create mesoderm tissue such as myocardium and endoderm tissue such as pancreatic β cells and liver parenchymal cells from stem cells such as ES cells and iPS cells, and some differentiation of β cells and hepatocytes A method of creating with efficiency has been developed (Non-Patent Documents 1 and 2). In the differentiation process into mesodermal and endoderm tissues, surface markers such as PDGFRβ and VEGFR2 (mesoderm tissue), CXCR4 and EpCAM (endoderm tissue) are known, respectively, and Plet-1 ( Non-patent document 3), pancreatic β cells such as NCAM1 (CD56) and DDR1 (patent documents 1 and 2), and hepatocytes such as c-Met, a6-integrin (CD49f), Dlk1, and Nope (neighbor of Punc E11) Cell surface markers specific to stem cells (Non-Patent Documents 4 and 5) have also been obtained. Furthermore, in the adult small intestine and large intestine, Lgr5 (Non-patent Document 6) and EphB2 (Non-patent Document 7) have been reported as intestinal progenitor cell markers. On the other hand, little research has been conducted on techniques for inducing tissue differentiation of the stomach, which is a tissue derived from the same digestive tract. Gastric cancer is the number one patient in Japan for cancer and the second highest mortality rate. Various symptoms such as digestion, malabsorption, dumping syndrome, reflux esophagitis and anemia occur as post-gastrectomy syndrome. Since the QOL is lowered, various methods for reconstructing the stomach after surgery have been studied. However, regenerative medicine of the stomach, including induction of differentiation of stomach tissue cells, has hardly been studied. In the first place, the analysis of the development of the stomach is greatly delayed for other organs (Non-patent Documents 8 and 9), and specific cell surface markers that can evaluate and select gastric progenitor cells in a living state have been found. Absent. The transcription factor Barx1 is known as a progenitor cell specific marker in the nascent stage (Non-patent Document 10), but it cannot be used for evaluation and selection in living cells because it is localized in the nucleus. Furthermore, the stomach is roughly divided into endoderm tissue covering the inside of the organ and mesenchymal tissue surrounding the outside, but no surface marker that can distinguish them has been identified. There are reports that the stomach of a dog was actually excised, covered with a biocompatible material, and regeneration of stomach tissue was reported, but regeneration of endoderm tissue covering the inside of the stomach was observed, but outside It has been reported that no regeneration of mesenchymal tissue surrounding the tissue is observed (Non-patent Document 11). Developmentally, the stomach is formed while the epithelium and mesenchymal tissue interact with each other, and there has been a method for appropriately evaluating both epithelial and mesenchymal cells in a living state. There wasn't. Expectations for regenerative medicine of gastric tissues are also increasing in order to improve the quality of life of patients after gastrectomy, and it is hoped to acquire a surface marker specific to gastric progenitor cells that can accurately evaluate and select gastric progenitor cells. It was rare.

U.S. Pat. No. 7,371,576 US Public 2011-001460 Japanese Patent Laid-Open No. 2003-9854

D'Amour, etal, Nature Biotech. (2006) 24,1392-1401. Soto-GutierrezA, et al, Nat Biotechnol. (2006) 24,1412-1419. DepreterMG, et al, Proc Natl Acad Sci U S A. (2008) 105,961-966. TanimizuN, et al, J. Cell. Sci. (2003) 116,1775-1786. Nierhoff D, et al, Hepatology (2007) 46,535-547. Baker N, etal, Nature (2007) 449,1003-1007. Merlos-Suarez, A. et al, Cell Stem Cell (2011) 8,511-524. Zorn, A.M., et al, Annu. Rev. Cell Dev. Biol. (2009) 25, 221-251. Fukuda, K., et al, Dev. Growth Differ. (2005) 47,375-382. Kim, B.M., et al, Dev. Cell (2005) 8,611-622. Kimiyasu Aikawa et al., Nissho Gaienkai (2009) 42,139. Keller, G M. Curr. Opin. Cell Biol. (1995) 7,862-869. Kubo, A. Development (2004) 131,1651-1662. D'Amour K.A. et al, Nature Biotechnol. (2005) 23,1534-1541. Spence J.R., et al, Nature (2011) 470,105-109.

Knowledge of the mechanism of early development important for differentiating stomach tissue cells from stem cells is greatly delayed in the stomach compared to other organs. Further, a stomach-specific marker that can evaluate the state of differentiation into the stomach, which is indispensable for accurately differentiating stomach tissue cells from stem cells, has not been provided. In particular, in regenerative medicine that needs to differentiate into gastric tissues accurately through multiple stages of differentiation from ES cells and iPS cells, by providing a marker gene that is specifically expressed during the development of the stomach, Differentiated cells can be evaluated, leading to the development of cell sorting technology.
The present invention is intended to provide a gastric progenitor cell-specific surface marker capable of selecting gastric progenitor cells based on such a technical background.

The present inventors focused on cell surface membrane proteins that are specifically expressed during the development process of the stomach, took out tissues at a time when the stomach tissues start to differentiate, and comprehensively expressed the genes expressed at that time using a microarray. As a result of analysis, three types of cell surface membrane proteins Adra2a, Fzd5, and Trpv6 that are highly expressed in the original cells of the stomach that differentiate into various cells that will constitute the stomach tissue in the future can be identified. It was.
The fetal gastrointestinal tract of the nascent fetus is only a single elongated tube, but as the developmental stage progresses, the esophagus, stomach, and intestines are divided into regions and differentiated into tissues with specific functions, Liver, pancreas, etc. also branch off from the digestive tract. The three identified markers are highly suitable as cell surface markers for cells that can differentiate into gastric tissue because they are expressed exclusively in the stomach in the gastrointestinal tract during the nascent stage.
In addition, we found that Adra2a is a surface marker that is specifically expressed in the entire mesenchymal tissue of the nascent stomach, whereas Fzd5 and Trv6 are surface markers that are specifically expressed in the latter half of the epithelial tissue of the nascent stomach. By combining these gastric progenitor cell-specific cell surface markers, it becomes possible to evaluate the differentiated state of the test cells and the detailed positioning at the developmental stage of the stomach in the state of living cells. Specifically, using antibodies that specifically recognize the above Adra2a, Fzd5 or Trv6 protein, etc., each in the process of differentiation into stomach tissue cells using pluripotent stem cells such as ES cells and iPS cells By observing the protein expression state, it became possible to appropriately evaluate cells that differentiate into epithelial and mesenchymal tissues of the stomach. Based on such findings, the present invention relating to an unprecedented method for evaluating progenitor cells of the stomach has been completed.

That is, the present invention is as follows.
[1] A polynucleotide reagent for detecting a gastric progenitor cell marker comprising a sequence selected from the following base sequences (1) to (5);
(1) a base sequence encoding any one of the amino acid sequences shown in SEQ ID NOs: 1 to 6 or a complementary base sequence thereof,
(2) a nucleotide sequence encoding an amino acid sequence in which one or several amino acids of any one of SEQ ID NOs: 1 to 6 are deleted, substituted or added, or a complementary nucleotide sequence thereof,
(3) any one of the nucleotide sequences represented by SEQ ID NOs: 7 to 12 or a complementary nucleotide sequence thereof,
(4) a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences represented by SEQ ID NOs: 7 to 12 or a complementary nucleotide sequence thereof;
(5) A base sequence comprising at least 15 consecutive bases in the base sequence of SEQ ID NOs: 7 to 12 or a complementary base sequence thereof.
[2] A primer set for detecting an Adra2a gene containing SEQ ID NOS: 13 and 14, a primer set for detecting a Trpv6 gene containing SEQ ID NOS: 15 and 16, or a primer for detecting an Fzd5 gene containing SEQ ID NOS: 17 and 18 The polynucleotide reagent according to the above [1], which is at least one primer set selected from the set.
[3] An antibody reagent for detecting a gastric progenitor cell marker comprising a polypeptide selected from the following (1) to (6);
(1) A polypeptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 1 to 6 (2) One or several amino acids are deleted, substituted or added in any amino acid sequence shown in SEQ ID NOs: 1 to 6 A polypeptide comprising the amino acid sequence (3) polypeptide encoded by any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 12 (4) complementary to any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 12 A polypeptide encoded by a base sequence that hybridizes under stringent conditions to the sequence (5) an antigenic fragment of the polypeptide of (1) to (4) above, which has at least 8 amino acid residues peptide.
[4] A method for detecting or determining gastric progenitor cells, comprising observing the presence or absence of expression of any one or more gastric progenitor cell markers selected from Adra2a, Trpv6 and Fzd5 genes in stem cells after differentiation induction treatment .
[5] The presence or absence of expression of the gastric progenitor cell marker is determined using the polynucleotide reagent according to [1] or [2] or the antibody reagent according to [3], The method for detecting or determining gastric progenitor cells according to [4] above.
[6] After differentiation induction treatment of stem cells, the presence or absence of expression of any one or more gastric progenitor cell markers selected from Adra2a, Trpv6 and Fzd5 genes is observed, and cells expressing the gastric progenitor cell markers are expressed A method for isolating gastric progenitor cells, comprising isolating.
[7] The presence or absence of expression of the gastric progenitor cell marker is determined using the polynucleotide reagent according to [1] or [2] or the antibody reagent according to [3]. A method for isolating gastric progenitor cells as described.
[8] A method for selecting gastric progenitor cells, the method comprising contacting the polynucleotide reagent according to [1] or [2] above and a cell sample considered to contain gastric progenitor cells.
[9] A method for selecting gastric progenitor cells, comprising the step of contacting the antibody according to [3] above with a cell sample considered to contain gastric progenitor cells.

  According to the present invention, cell surface markers that are specifically expressed in nascent gastric tissue cells could be identified. These cell surface markers can be used alone or in combination to determine exactly how much gastric progenitor cells are included in the various cells that emerge when differentiating gastric cells from stem cells such as ES cells and iPS cells. It can be applied as a cell evaluation kit for evaluation. Further, in order to differentiate gastric tissue cells from stem cells efficiently and with high accuracy, it can also be used as a surface marker for selecting gastric progenitor cells.

Identification of gastric surface markers that are specifically expressed in the pre-determined region of the stomach. Expression patterns of EpCAM, which is non-specifically expressed throughout the gastrointestinal endoderm in the nascent stage, and Barx1, a transcription factor that is specifically expressed in cells in the planned region of the stomach of E11.5 mouse embryos. B. Expected regions of the stomach in mouse 9.5 day embryos and 11.5 day embryos. C. Expression of each region-specific transcription factor in a sample of the esophagus, stomach, and intestinal regions excised from the mouse fetus. In the figure, Fr is the foregut, Md is the midgut, Hd is the hindgut, Es is the planned esophageal region, St is the planned stomach region, and In is the planned intestinal region. D. Search for cell surface markers that are specifically expressed in the planned region of the stomach in mouse 11.5 day embryos using microarrays. A-C. Expression of three cell surface markers Adra2a, Fzd5, and Trpv6 and other families in E9.5 day embryos and E11.5 day embryos. D. Expression of nuclear transcription factors specific to the planned region in E9.5 day embryos and E11.5 day embryos. EG. Data showing that the primers used in this experiment are positive control samples and can be used in PCR experiments. Expression analysis of Adra2a gene in mouse early embryo by in situ hybridization. A. Whole mount in situ hybridization of Adra2a gene in E9.5 day embryo. B. Section in situ hybridization of Adra2a gene in E11.5 day embryo. SS and GS refer to the area of the glandular stomach that has an intact abdominal stomach extending from the esophagus and the original gastric mucosa. C. Gastric expression of Adra2a gene in late development. Expression analysis of Fzd5 gene in mouse early embryo by in situ hybridization. A. Whole mount in situ hybridization of Fzd5 gene in E9.5 day embryo. B. Section in situ hybridization of Fzd5 gene in E11.5 day embryo. C. Gastric expression of Fzd5 gene in late development. Expression analysis of Trpv6 gene in mouse early embryo by in situ hybridization. A. Whole mount in situ hybridization of Trpv6 gene in E9.5 day embryo. B. Section in situ hybridization of Trpv6 gene in E11.5 day embryo. C. Gastric expression of Trpv6 gene in late development.

1. Gastric progenitor cell-specific surface marker of the present invention The gastric progenitor cell-specific surface marker of the present invention is Adra2a, Trpv6 and Fzd5 proteins or their genes. Both proteins are expressed specifically in a predetermined region of the stomach at the time when the morphogenesis of the stomach during the early developmental stage of gastrointestinal differentiation begins (E11.5 day embryo in mice) (FIG. 2A). The amino acid sequences of these proteins and the base sequences of the genes can be obtained from public databases. (For example, human-Adra2a (Protein: NP_000672: SEQ ID NO: 1) mouse-Adra2a (Protein: NP_031443: SEQ ID NO: 2), human-Trpv6 (Protein: NP_061116: SEQ ID NO: 3), mouse-Trpv6 (GenBank (NCBI) Protein: NP_071858: SEQ ID NO: 4), human-Fzd5 (Protein: NP_003459: SEQ ID NO: 5), mouse-Fzd5 (Protein: NP_073558: SEQ ID NO: 6), human-Adra2a gene (mRNA: NM_000681: SEQ ID NO: 7), mouse -Adra2a gene (mRNA: NM_007417: SEQ ID NO: 8), human-Trpv6 gene (mRNA: NM_018646: SEQ ID NO: 9), mouse-Trpv6 gene (mRNA: NM_022413: SEQ ID NO: 10), human-Fzd5 gene (mRNA: NM_003468: SEQ ID NO: 11), mouse-Fzd5 gene (mRNA: NM_022721: SEQ ID NO: 12))
When differentiating stem cells from the endoderm into the gastrointestinal tract, the expression of these Adra2a, Trpv6 and Fzd5 proteins in the test cell or test cell population can be detected at the gene level or by detecting the expressed protein. It can be evaluated whether or not it is a cell that will become a gastric cell in the future, or how many cells that will become a gastric cell in the future are contained in the test cell.

Adra2a is a kind of G protein-coupled adrenergic receptor. Among the adrenergic receptors α1, α2, and β (also referred to as Adra1, Adra2, and Adrb), it belongs to the adrenergic receptor α2 (Adra2), which is considered to be involved in platelet aggregation, lipolysis and various other nervous system effects. α2 (Adra2) is classified into three subtypes (Adra2a, 2b, and 2c), but it was expressed specifically in the planned region of the stomach during gastric morphogenesis (E11.5 day embryo) Only Adra2a (FIG. 2A).
The Adra2a protein can be represented as a protein comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence. 1 represents 20, preferably 1-10, and more preferably 1-5.) Here, homology at the amino acid sequence level of human Adra2a (SEQ ID NO: 1) and mouse Adra2a (SEQ ID NO: 2) Is about 92%, human Adra2a and human Adra2b are about 52%, human Adra2a and human Adra2c are about 53%, so that SEQ ID NO: 1 or 2 and 80% or more, preferably 90% or more, More preferably, the expression of Adra2a can be confirmed by detecting an amino acid sequence having 95% or more homology (identity).
In addition, the expression of Adra2a protein can be observed by increasing the amount of Adra2a mRNA transcribed, and the Adra2a gene to be detected at that time is the nucleotide sequence shown in SEQ ID NO: 7 or 8, or 90% or more thereof. Preferably, it can be expressed as a base sequence having 95% or more homology (identity). In order to observe the transcription amount of the Adra2a gene, it hybridizes under stringent conditions with a complementary nucleotide sequence of 90% or more, preferably 95% or more, with the nucleotide sequence shown in SEQ ID NO: 7 or 8. It is possible to detect by the method. Here, the stringent conditions, T.Maniatis et al., Eds., Molecular Cloning:. A Laboratory Manual 2 nd ed (1989) Cold Spring Harbor Laboratory, or Wilkinson, DG and Nieto, the method of MA (Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.Methods Enzymol (1993) 225, 361-373), etc., so-called specific hybrids are formed and non-specific hybrids are not formed. . For example, in the case of a method such as Northern hybridization to be detected on a membrane, 6 × SSC (1 × SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhardt's, 0.1 % Bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 μg / ml salmon sperm DNA, conditions of incubation at 50 ° C. for 4 hours to overnight, in tissue or in cells 5 x SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 μg / mL heparin, yeast RNA and 50% formamide specific for 100 ng / ml for in situ hybridization for simultaneous observation of gene expression location and expression level An example is a condition in which incubation is performed at 50 ° C. for 4 hours to overnight under the condition of adding a target probe.
The specific probe for Adra2a gene for in situ hybridization is a base corresponding to 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more at any position in the base sequence shown in SEQ ID NO: 7 or 8. It can be easily created by amplifying by a PCR reaction using a primer set designed to amplify the sequence. The primer used in the primer set at that time can be expressed as a base sequence of at least 15 bases or more, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 7 or 8, or a complementary sequence thereof.
Further, a primer set itself consisting of at least 15 bases, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 7 or 8 can also be used as a primer set for detecting Adra2a gene. Usually, these primer sets amplify the Adra2a gene by amplifying at least 50 bases or more, preferably 80 bases or more, more preferably 100 bases or more of the base sequence shown in SEQ ID NO: 7 or 8 by RT-PCR. It can be specifically detected. For example, it can detect using the primer of sequence number 13 and 14.
Expression of Adra2a protein can be detected by Adra2a antibody. In order to immunize mice, rabbits and the like when preparing Adra2a antibody, not only the full length of Adra2a protein but also an antigenic peptide consisting of a partial fragment that does not lose its antigenicity as Adra2a protein can be used. In this case, the antigenic peptide includes an amino acid sequence in which at least 8 amino acids, preferably 10 or more, more preferably 20 or more amino acids are continuously in common with the Adra2a protein.

Trpv6 is a calcium-selective cation channel belonging to the transient receptor potential (TRP) superfamily. Trpv5, another calcium-selective cation channel in the TRP superfamily, is expressed specifically in the stomach region during stomach morphogenesis (E11.5 day embryo). Only (FIG. 2B).
The Trpv6 protein can be represented as a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence.
Here, the homology at the amino acid sequence level of human Trpv6 (SEQ ID NO: 3) and mouse Trpv6 (SEQ ID NO: 4) is about 90%, whereas it is about 75% between human Trpv6 and human Trpv5. By detecting an amino acid sequence having a homology (identity) of 85% or more, preferably 90% or more, more preferably 95% or more with No. 3 or 4, the expression of Trpv6 can be confirmed.
In addition, although the expression of Trv6 protein can be observed by increasing the amount of mRNA transcription of Trpv6, the Trpv6 gene to be detected at that time is the nucleotide sequence shown in SEQ ID NO: 9 or 10, or 90% or more thereof, Preferably, it can be expressed as a base sequence having 95% or more homology (identity). In order to observe the amount of Trpv6 gene transcription, stringent conditions with a complementary base sequence paired with 90% or more, preferably 95% or more of the total length of the base sequence shown in SEQ ID NO: 9 or 10 It can detect by the method of hybridizing with.
Moreover, in order to observe the amount of Trpv6 gene transcription | transfer, it can also detect by the method of hybridizing by methods, such as in situ hybridization.
The specific probe for Trpv6 gene for in situ hybridization is a base corresponding to 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more at any position in the base sequence shown in SEQ ID NO: 9 or 10. It can be easily created by amplifying by a PCR reaction using a primer set designed to amplify the sequence. The primer used in the primer set at that time can be expressed as a base sequence of at least 15 bases or more, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 9 or 10, or a complementary sequence thereof.
In addition, a primer set itself consisting of at least 15 bases, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 9 or 10 can also be used as a primer set for detecting the Trpv6 gene. Usually, these primer sets amplify at least 50 bases, preferably 80 bases or more, more preferably 100 bases or more of the base sequence shown in SEQ ID NO: 9 or 10 by RT-PCR, thereby It can be specifically detected. For example, it can detect using the primer of sequence number 15 and 16.
Expression of Trpv6 protein can be detected by Trpv6 antibody. In order to immunize mice, rabbits, etc. when preparing Trpv6 antibodies, not only the full length of Trpv6 protein but also antigenic peptides consisting of partial fragments that do not lose their antigenicity as Trpv6 protein can be used . In this case, the antigenic peptide has a sequence of amino acids in which at least 8 or more, preferably 10 or more, more preferably 20 or more amino acids are continuously common to the Trpv6 protein.

Fzd5 is a seven-transmembrane Wnt signaling protein receptor with Wnt5A as a ligand. Examining the expression characteristics of the same family member at the time of gastrointestinal tract formation, Fzd5 is expressed specifically in the stomach in E11.5 day embryos, whereas Fzd8 is specifically expressed in the esophagus of E11.5 day embryos, Fzd7 was non-specifically expressed in the entire digestive tract of E9.5 day embryos and in E11.5 day embryos both in the esophagus and stomach, and Fzd10 expression was not detected (FIG. 2C).
The Fzd5 protein can be represented as a protein comprising the amino acid sequence shown in SEQ ID NO: 5 or 6, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence. Here, the homology at the amino acid sequence level of human Fzd5 (SEQ ID NO: 5) and mouse Fzd5 (SEQ ID NO: 6) is about 96%, whereas human Fzd5 and human Fzd8 having the highest homology in the family are close to each other. Since it is about 71%, Fzd5 expression is detected by detecting an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more of homology (identity) with SEQ ID NO: 5 or 6. Can be confirmed.
Further, the expression of the Fzd5 protein can be observed by increasing the amount of Fzd5 mRNA transcription. The Fzd5 gene to be detected at that time is the nucleotide sequence shown in SEQ ID NO: 11 or 12, or 90% or more thereof, Preferably, it can be expressed as a base sequence having 95% or more homology (identity). In order to observe the amount of Fzd5 gene transcription, stringent conditions with a complementary base sequence paired with 90% or more, preferably 95% or more, of the total length of the base sequence shown in SEQ ID NO: 11 or 12 are used. It can detect by the method of hybridizing with.
Moreover, in order to observe the transcription amount of Fzd5 gene, it can also detect by the method of hybridizing by methods, such as in situ hybridization.
The Fzd5 gene specific probe for in situ hybridization is a base corresponding to 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more at any position in the base sequence shown in SEQ ID NO: 11 or 12. It can be easily created by amplifying by a PCR reaction using a primer set designed to amplify the sequence. In addition, the primer used for the primer set in that case can be expressed as a base sequence of at least 15 bases, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 11 or 12, or a complementary sequence thereof.
In addition, a primer set itself consisting of at least 15 bases, preferably 20 bases or more of the base sequence shown in SEQ ID NO: 11 or 12 can be used as a primer set for detecting Fzd5 gene. Usually, these primer sets amplify Fzd5 gene by RT-PCR method by amplifying at least 50 bases or more, preferably 80 bases or more, more preferably 100 bases or more of the base sequence shown in SEQ ID NO: 11 or 12. It can be specifically detected. For example, it can detect using the primer of sequence number 17 and 18.
The expression of Fzd5 protein can be detected by Fzd5 antibody. In order to immunize mice, rabbits, etc. when preparing Fzd5 antibodies, not only the full length of Fzd5 protein, but also antigenic peptides consisting of partial fragments that do not lose their antigenicity as Fzd5 protein can be used. . In this case, the antigenic peptide is an Fzd5 protein that usually contains an amino acid sequence in which at least 8 or more, preferably 10 or more, more preferably 20 or more amino acids are continuously in common.

2. Method for Inducing Differentiation of Stem Cells into Gastric Cells In the present invention, the term “stem cells” refers to adults capable of differentiating not only into pluripotent embryonic stem cells (ES cells) or iPS cells but also into the digestive tract including the stomach. Of tissue stem cells.
Techniques for differentiating stem cells into endoderm cells and further into gastrointestinal cells are well known. For example, techniques for inducing differentiation by culturing ES cells in vitro and aggregating them to form cell clusters called embryoid bodies (EBs) to form endoderm cells have been widely used (non-patented). Literatures 12 to 14, Patent Literature 3). In particular, a method for efficiently producing endoderm cells by adding a high concentration of activin or the like is used (Non-patent Document 14).
Furthermore, in order to induce differentiation of endoderm cells into intestinal gastrointestinal cells, it was cultured with the addition of FGF4 and Wnt3a reported recently, then in a medium containing R-spondin, Noggin and EGF. A method of performing three-dimensional culture such as (Non-patent Document 15) can be applied. It is considered that differentiation can be induced in a similar manner by using the same gastrointestinal cells.

3. Gastric progenitor cell-specific surface marker detection method and measurement method of the present invention The test cells are subjected to gastric cancer in the state of living cells by immunostaining or flow cytometry using an antibody against the gastric progenitor cell-specific surface marker. Progenitor cells can be specifically measured. Similarly, the antibody can also be used for detection by a method such as Western blot or ELISA using an extract of a test cell.
Antibodies of Adra2a, Trpv6 and Fzd5 proteins can be obtained as appropriate according to a method for producing a normal monoclonal antibody using mice or the like, or a polyclonal antibody using rabbits or the like, using these proteins or antigenic peptides thereof as an immunogen. However, commercially available antibodies (Adra2: manufactured by Bioworld Technology, manufactured by Trpv6: abcam, manufactured by Fzd5: abcam) can be used.
In addition, the comprehensive analysis method of the protein using a mass spectrometer can also be used without using an antibody.
Furthermore, it can also be measured by a method for detecting gene expression such as RT-PCR or in situ hybridization using a specific primer or probe for the surface marker gene. Furthermore, it can also be measured by a comprehensive analysis method such as a microarray or a next-generation sequencer.

3. Use of gastric progenitor cell-specific surface marker of the present invention
A surface marker specific to gastric progenitor cells when differentiating gastric progenitor cells by combining various cell differentiation factors and drugs from stem cells such as ES cells and iPS cells via embryoid bodies and monolayer culture methods The amount of differentiated cells produced can be evaluated by immunofluorescent staining or Western blot. That is, it can be used as a cell evaluation kit for accurately evaluating the extent to which gastric progenitor cells are contained in various cells that appear when differentiating gastric cells from stem cells.
In addition, only the gastric progenitor cells can be survived by using flow cytometry or a magnetic column from the differentiated cell population using a fluorescent reagent or magnetic beads bound with the gastric progenitor cell-specific surface marker antibody of the present invention. In this state, it can be separated and purified.
The obtained gastric progenitor cells are adjusted to a shape that can be easily transplanted using a three-dimensional matrix with high biocompatibility. Can be used for transplantation treatment.

  Hereinafter, the present invention will be described in more detail with reference to examples. The technical scope of the present invention is not limited by these descriptions. The contents of technical documents cited in the present specification are considered to be part of the disclosure of the present specification.

Example 1 Identification of Cell Surface Markers Specifically Expressed in Early Stage Gastric Tissue Cells To identify cell surface markers specifically expressed in early stage gastric tissue cells, stomach morphogenesis from pregnant mice From the mouse fetus 9.5 day embryo before the start of gastric cancer and the 11.5 day embryo after the start of gastric morphogenesis, the planned region of the stomach and the esophagus and the intestinal region of the intestine before and after that were excised under a microscope. The excision was performed using as an index the expression of the nuclear transcription factor Barx1, which is expressed specifically in a predetermined region of the stomach (FIG. 1AB).
After excision, the specificity of the excised sample was confirmed by RT-PCR using the transcription factor expressed specifically in the planned region of the esophagus, stomach and intestine as an index. As a result, it was confirmed that the planned region of the stomach was correctly cut out along the expression of the specific nuclear transcription factor by this method (FIG. 1C).
Using these samples, microarray analysis using a mouse genome array was performed to search for cell surface markers expressed in a predetermined region of the stomach. As a result, when a gene that is highly expressed in the planned region of the stomach of the 11.5 day embryo than the planned region of the esophagus or intestine and that is localized in the cell surface membrane fraction was selected, 63 genes were identified. (FIG. 1D).
When a high-level gene showing a sufficient expression level was selected, three cell surface markers Adra2a, Fzd5, and Trpv6 could be identified.

(Example 2) Verification of specificity of cell surface marker The specificity of the cell surface marker identified in the above (Example 1) was verified by RT-PCR. Specifically, RNA was purified using RNeasy micro kit (QIAGEN) according to the instructions of the kit. Subsequently, cDNA was synthesized using Prime Script First Strand cDNA Synthesis Kit (Takara), and PCR reaction was performed using Ex Taq (Takara). PCR reaction was performed under the conditions of 95 ° C. for 1 min, 55-58 ° C. for 1 min, and 72 ° C. for 1 min.
In this example, the primer sequences used for detecting the Adra2a, Trpv6 and Fzd5 markers specific to gastric progenitor cells of the present invention by PCR are as follows.
<Adra2a gene amplification primer set>
Adra2a-F: 5'-cgaggttatgggttactggtact-3 '(SEQ ID NO: 13)
Adra2a-R: 5'-gtcaaggctgatggcgcacag-3 '(SEQ ID NO: 14)
<Trpv6 gene amplification primer set>
Trpv6-F: 5'-gcacaggccttccagcaaca-3 '(SEQ ID NO: 15)
Trpv6-R: 5'-gtactcccagccctccccatct-3 '(SEQ ID NO: 16)
<Fzd5 gene amplification primer set>
Fzd5-F: 5'-gtctgtgctgtgcttcatc-3 '(SEQ ID NO: 17)
Fzd5-R: 5'-agtgacacacacaggtagca-3 '(SEQ ID NO: 18)
In this example, the probes used for detecting the Adra2a, Trpv6 and Fzd5 markers specific to gastric progenitor cells of the present invention by in situ hybridization were prepared by PCR amplification using the following primers.
<Primer set for creating probe for Adra2a gene in situ hybridization>
Adra2a-F: 5'-gtgacactgacgctggtttg-3 '(SEQ ID NO: 19)
Adra2a-R: 5'-aggatcatgatgaggcaagg-3 '(SEQ ID NO: 20)
<Trpv6 gene in situ hybridization probe creation primer set>
Trpv6-F: 5'-ataacctggaggctgcaatg-3 '(SEQ ID NO: 21)
Trpv6-R: 5'-actggatgtgtttccgcttc-3 '(SEQ ID NO: 22)
<Primer set for preparing probes for Fzd5 gene in situ hybridization>
Fzd5-F: 5'-gcgcaccggccaagtgccc-3 '(SEQ ID NO: 23)
Fzd5-R: 5'-cggctgcaagcgacgctggc-3 '(SEQ ID NO: 24)

Among Adra2a, 2b, and 2c subtypes in the same adrenergic receptor α2 (Adra2) family as Adra2a, only Adra2a is expressed specifically in the planned region of the stomach in the E11.5 day embryo where gastric morphogenesis has begun (FIG. 2A).
Trpv6 is a calcium-selective cation channel belonging to the transient receptor potential (TRP) superfamily, and was expressed specifically in the planned region of the stomach in E11.5 day embryos. Trpv5, another calcium-selective cation channel of the TRP superfamily, is not expressed at this time (FIG. 2B).
Fzd5 is a 7-transmembrane Wnt receptor, and Fzd5 is expressed specifically in the stomach in E11.5 day embryos, whereas Fzd8 is E11. It is specifically expressed in the esophagus of day 5 embryos, and Fzd7 is non-specifically expressed in the entire digestive tract of day E9.5 embryos and in both the esophagus and stomach in day E11.5 embryos. Expression of Fzd10 was not detected (FIG. 2C).
Thus, it was confirmed that the three cell surface markers Adra2a, Fzd5, and Trpv6 identified this time were expressed specifically in the stomach during the gastrointestinal differentiation process in the early developmental stage.

(Example 3) Specificity of Adra2a
Adra2a is not expressed in the gastrointestinal tract in 9.5 day embryos before gastric morphogenesis (Fig. 3A), but in 11.5 day embryos, there is an aglandous stomach that continues from the esophagus and a glandular stomach with the original gastric mucosa. Expression is observed in both mesenchymal tissue parts (FIG. 3B). In addition, no expression was observed in the epithelium, and it was considered to be a surface marker expressed specifically in the mesenchymal tissue of the stomach. In addition, Adra2a expression disappears rapidly thereafter (FIG. 3C).

(Example 4) Specificity of Fzd5
Fzd5 is not expressed in the 9.5 day embryo before gastric morphogenesis other than in the vicinity of the chin (Fig. 4A), but in the 11.5 day embryo, it is specifically expressed in the epithelial tissue region near the glandular stomach. Is observed (FIG. 4B). This expression is not observed at all in the mesenchymal tissue part, and is considered to be a surface marker expressed specifically in the epithelial tissue below the glandular stomach. In addition, Fzd5 expression continues to be expressed specifically in epithelial tissue (FIG. 4C).

Example 5 Specificity of Trpv6
Trpv6 is not expressed in the digestive tract in the 9.5 day embryo before gastric morphogenesis (FIG. 5A), but in the 11.5 day embryo, specific expression is observed in the epithelial tissue portion of the glandular stomach (FIG. 5B). . This expression is not observed in the mesenchymal tissue part, and is considered to be a surface marker expressed specifically in the epithelial tissue of the stomach glandular stomach. In addition, Trpv6 expression continues to be expressed specifically in epithelial tissue (FIG. 5C).

[Sequence Listing Free Text]
Sequence number 1: h-Adra2a (Protein: NP_000672)
Sequence number 2: m-Adra2a (Protein: NP_031443)
Sequence number 3: h-Trpv6 (Protein: NP_061116)
Sequence number 4: m-Trpv6 (Protein: NP_071858)
Sequence number 5: h-Fzd5 (Protein: NP_003459)
Sequence number 6: m-Fzd5 (Protein: NP_073558)
Sequence number 7: h-Adra2a gene (mRNA: NM_000681)
Sequence number 8: m-Adra2a gene (mRNA: NM_007417)
Sequence number 9: h-Trpv6 gene (mRNA: NM_018646)
Sequence number 10: m-Trpv6 gene (mRNA: NM_022413)
Sequence number 11: h-Fzd5 gene (mRNA: NM_003468)
Sequence number 12: m-Fzd5 gene (mRNA: NM_022721)
Sequence number 13: m-Adra2a primer (f)
Sequence number 14: m-Adra2a primer (r)
SEQ ID NO: 15: m-Trpv6 primer (f)
SEQ ID NO: 16: m-Trpv6 primer (r)
SEQ ID NO: 17: m-Fzd5 primer (f)
SEQ ID NO: 18: m-Fzd5 primer (r)
Sequence number 19: m-Adra2a-probe
primer (f)
Sequence number 20: m-Adra2a-probe
primer (r)
SEQ ID NO: 21: m-Trpv6-probe primer (f)
SEQ ID NO: 22: m-Trpv6-probe primer (r)
Sequence number 23: m-Fzd5-probe primer (f)
Sequence number 24: m-Fzd5-probe primer (r)

Claims (9)

A polynucleotide reagent for detecting a gastric progenitor cell marker comprising a sequence selected from the following base sequences (1) to (5);
(1) a base sequence encoding any one of the amino acid sequences shown in SEQ ID NOs: 1 to 6 or a complementary base sequence thereof,
(2) a nucleotide sequence encoding an amino acid sequence in which one or several amino acids of any one of SEQ ID NOs: 1 to 6 are deleted, substituted or added, or a complementary nucleotide sequence thereof,
(3) any one of the nucleotide sequences represented by SEQ ID NOs: 7 to 12 or a complementary nucleotide sequence thereof,
(4) a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences represented by SEQ ID NOs: 7 to 12 or a complementary nucleotide sequence thereof;
(5) A base sequence comprising at least 15 consecutive bases in the base sequence of SEQ ID NOs: 7 to 12 or a complementary base sequence thereof.   The polynucleotide reagent is selected from an Adra2a gene detection primer set comprising SEQ ID NOs: 13 and 14, a Trpv6 gene detection primer set comprising SEQ ID NOs: 15 and 16, or an Fzd5 gene detection primer set comprising SEQ ID NOs: 17 and 18 The polynucleotide reagent according to claim 1, which is at least one primer set. An antibody reagent for detecting a gastric progenitor cell marker comprising a polypeptide selected from the following (1) to (6);
(1) A polypeptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 1 to 6 (2) One or several amino acids are deleted, substituted or added in any amino acid sequence shown in SEQ ID NOs: 1 to 6 A polypeptide comprising the amino acid sequence (3) polypeptide encoded by any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 12 (4) complementary to any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 12 A polypeptide encoded by a base sequence that hybridizes under stringent conditions to the sequence (5) an antigenic fragment of the polypeptide of (1) to (4) above, which has at least 8 amino acid residues peptide.   A method for detecting or determining gastric progenitor cells, comprising observing the presence or absence of expression of any one or more gastric progenitor cell markers selected from Adra2a, Trpv6 and Fzd5 genes in stem cells after differentiation induction treatment.   The stomach according to claim 4, wherein the presence or absence of expression of the gastric progenitor cell marker is determined using the polynucleotide reagent according to claim 1 or 2, or the antibody reagent according to claim 3. A method for detecting or determining progenitor cells.   After differentiation induction treatment of stem cells, the presence or absence of expression of any one or more gastric progenitor cell markers selected from Adra2a, Trpv6 and Fzd5 genes is observed, and cells expressing the gastric progenitor cell markers are isolated A method for isolating gastric progenitor cells.   The isolation of gastric progenitor cells according to claim 6, wherein the presence or absence of expression of the gastric progenitor cell marker is determined using the polynucleotide reagent according to claim 1 or 2 or the antibody reagent according to claim 3. Method.   A method for selecting gastric progenitor cells, comprising the step of contacting the polynucleotide reagent according to claim 1 or 2 with a cell sample considered to contain gastric progenitor cells.   A method for selecting gastric progenitor cells, comprising the step of contacting the antibody according to claim 3 with a cell sample considered to contain gastric progenitor cells.

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