JP2011072231A - Method for detecting endoderm cell, intestinal tract cell or pancreatic cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting endoderm cells, intestinal tract cells or pancreatic cells by identifying the marker molecules of the endoderm cells, intestinal tract cells or pancreatic cells and utilizing them, and also a method for separating the endoderm cells, intestinal tract cells and pancreatic cells. <P>SOLUTION: This method for detecting and separating the endoderm cells, intestinal tract cells or pancreatic cells is provided by including the detection of expression of at least one of a gene selected from among a decay-accelerating factor [DAF, CD55] gene, Tmem184a gene, Akr1c19 gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4 gene, Pbxip1 gene, Pcbd1 gene and Pcdh1 gene. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for detecting and separating endoderm cells, intestinal cells or pancreatic cells. More specifically, the present invention relates to a method for detecting and separating endoderm cells, intestinal cells or pancreatic cells using the expression of a novel marker molecule of endoderm cells, intestinal cells or pancreatic cells as an index.

The digestive organs and respiratory organs, such as the pancreas, liver, stomach, intestine, and lung, which are essential for life support, originate from the embryonic endoderm. In the planned fate tracking experiment, the definitive endoderm
The fate of erm: DE) has been found to begin to be determined at embryonic day 6-6.5 in mouse embryos.
The earliest pancreatic marker gene currently known in mice, Pdx1 (Pancreatic and duoden
al homeobox gene 1) begins to be expressed in the ventral endoderm of the intestine on the 8.5th day of embryogenesis (Non-patent Document 1). However, only undeveloped pancreatic buds that do not grow or differentiate are formed in Pdx1 knockout mice. Despite the importance of inducing pancreatic bud signaling in specific regions of the foregut endoderm in understanding pancreatic development, many molecular mechanisms have not been elucidated.

In order to induce differentiation of specific cells from ES cells, it is very important to identify a marker gene that is not expressed in other regions but expressed only in the target region. However, in reality, differentiated cells are often evaluated by combining a plurality of marker genes. For endoderm, E-
A method for identifying embryonic endoderm by using cadherin and chemokine receptor Cxcr4 has been reported (Non-patent Document 2). However, the present inventors have confirmed that the expression of Cxcr4 decreases as the development proceeds in the pancreas, and in order to study the localization of endoderm, a marker gene that is expressed until late differentiation is required. It has become. In addition, there is no report on genes that work earlier than Pdx1 in the planned pancreatic region of the foregut endoderm. further,
Cell surface markers that are specifically expressed in the pancreas region have not been found at the time when endoderm regionization occurs, and this is an obstacle to analysis using a flow cytometer in pancreatic differentiation studies. On the other hand, many genes expressed in early pancreatic buds are not known, and it is very useful to identify novel genes.

We have already in vitro on ES cells from Pdx1
It has succeeded in inducing differentiation of positive cells (Non-patent Document 3). Furthermore, it has succeeded in efficiently inducing differentiation of not only endoderm but also ectoderm and mesoderm by changing humoral factors and differentiation conditions (Non-patent Document 4). In addition, research using microarrays for comprehensive gene expression analysis is also conducted (Non-patent Documents 5 and 6).

Offield MF, Jetton TL, Labosky PA, et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.Development. 1996; 122: 983-995. Yasunaga M, Tada S, Torikai-Nishikawa S, et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotechnol. 2005; 23: 1542-1550. Shiraki N, Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm. Stem Cells. 2008; 26: 874-885. Shiraki N, Higuchi Y, Harada S, et al. Differentiation and characterization of embryonic stem cells into three germ layers. Biochem BiophysRes Commun. 2009; 381: 694-699. Yoshida T, Murata K, Shiraki N, et al. Analysis of gene expressions of embryonic stem-derived Pdx1-expressing cells: Implications of genes involved in pancreas differentiation. Dev Growth Differ. 2009; 51: 463-472. Yoshida T, Shiraki N, Baba H, et al. Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas. Genes Cells. 2008; 13: 667-678.

Cxcr4 is used as a marker gene for the conventional endoderm and intestinal tract.
It was observed that Cdcr4 gene expression in the intestinal tract was decreased, and Pdx1-positive cells on embryonic day 9.5 were present in the fraction of Cxcr4-negative cells. Therefore, identification of molecular markers of the endoderm and intestinal tract at a later stage of development is desired. The present invention relates to a method for identifying an endoderm cell, intestinal cell or pancreatic cell marker molecule, a method for detecting endoderm cell, intestinal cell or pancreatic cell using the same, and a method for isolating endoderm cell, intestinal cell or pancreatic cell. It was set as the problem which should be solved to provide.

The present inventors have succeeded in inducing differentiation of Pdx1-positive cells from ES cells in vitro. In the present invention, microarray analysis was performed on embryonic endoderm and pancreas differentiated cells derived from ES cells, and candidate genes specifically expressed in embryonic endoderm and pancreas were searched. As a result of microarray analysis, we succeeded in identifying a decay accelerating factor (also referred to as DAF or CD55) as a new surface marker of embryonic endoderm, intestine and pancreas. Furthermore, by the same method, we found genes that are expressed in several early pancreatic buds. These genes can be used as very useful markers in the identification of pancreatic progenitor cells. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) Decay accelerating factor (DAF, CD55) gene, Tmem184a gene, Akr1c19 gene, 3300001A
09 detecting an expression of at least one gene selected from Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene, A method for detecting cells or pancreatic cells.
(2) Decay accelerating factor (DAF, CD55) gene, Tmem184a gene, Akr1c19 in target cells
Detecting the expression of at least one gene selected from a gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene, and said at least one gene The method according to (1), wherein a cell expressing the cerebellum is identified as an endoderm cell, intestinal cell or pancreatic cell.
(3) at least detecting the expression of a decay accelerating factor (DAF, CD55) gene (1
Or a method for detecting an endoderm cell, intestinal cell or pancreatic cell according to (2).
(4) The method according to (3), wherein the expression of a decay accelerating factor (DAF, CD55) gene is detected using an antibody against the decay accelerating factor (DAF, CD55).
(5) The method according to (3) or (4), further comprising detecting the expression of the E-cadherin gene.

(6) A method for isolating endoderm cells, intestinal cells or pancreatic cells, comprising selecting cells that express a decay accelerating factor (DAF, CD55) gene.
(7) (a) preparing a cell sample containing endoderm cells, intestinal cells or pancreatic cells, (b)
An endoderm cell, intestinal cell or pancreas according to (6), comprising the step of adding an antibody against a decay accelerating factor (DAF, CD55) to the cell sample, and (c) isolating the cell bound to the antibody. A method of separating cells.
(8) The method for isolating endoderm cells, intestinal cells or pancreatic cells according to (6) or (7), wherein cells expressing E-cadherin gene in addition to decay accelerating factor (DAF, CD55) gene are selected. .
(9) A reagent for detecting or separating endoderm cells, intestinal cells or pancreatic cells, which contains an antibody against a decay accelerating factor (DAF, CD55).

Decay accelerating factor (DAF, CD55) is expressed in embryonic endoderm and mesoderm regions in normal mouse embryos. From analysis by flow cytometer, CD55 is Cxcr
It was found that it was expressed in a narrower range compared to 4, and in the differentiation induction experiment using ES cells, the expression level did not decrease until the late stage of culture. Furthermore, from the analysis with a flow cytometer using Pdx1 / GFP mouse embryos, Pdx1-positive cells are present in the E-cadherin (+) / Cxcr4 (-) fraction, but CD55 is a Pdx1-positive pancreatic progenitor cell. It was found to be useful as a pancreatic endoderm marker. From these results, CD55 is very useful when used in combination with E-cadherin as a marker for identifying embryonic endoderm, intestine and pancreas. Since expression of CD55 is detected until later than Cxcr4, it can be used as a late intestinal marker. CD55 is also expressed in pancreatic progenitor cells, and is therefore superior in that cells differentiated into pancreas can be purified by expression of surface layer markers without genetic modification of ES cells. This antibody that recognizes CD55 is useful as a reagent for studying cells of the intestinal tract and pancreas.

FIG. 1 shows a differentiation induction method. A shows differentiation induction conditions. The conditions for inducing differentiation from ES cells to three germ layers are shown. All differentiation induction was performed using M15 cells derived from mouse middle kidney. Activin and bFGF were added to induce differentiation of mesendoderm (Mesendoderm) and endoderm (Endoderm). To induce differentiation of ectoderm, SB203580 was added and cultured for 5 days. BMP7 was added to induce differentiation of mesoderm and cultured for 5 days. B shows a schematic diagram of a sample used for microarray analysis. D in the schematic diagram indicates the number of days from the start of differentiation induction. A sample induced by adding the humoral factor shown in FIG. 1A to each germ layer was collected using a flow cytometer. The marker used for sorting is shown in the lower left. FIG. 2 shows the expression pattern of CD55. A shows the expression level in CD55 microarray analysis. The expression pattern in each germ layer of CD55 obtained by microarray analysis is shown. The red line is for CD55 and the number of lines matches the number of probes set in the array. From this graph, it can be seen that CD55 is highly expressed in the endoderm region and maintains a constant expression level until 8 days after the start of differentiation induction. B shows real time PCR of CD55. The expression pattern in each germ layer of CD55 obtained by real-time PCR analysis is shown. The same sample as used in the microarray analysis was used. From this result, it was reconfirmed that the gene was highly expressed in the endoderm region. FIG. 3 shows the analysis by the in situ hybridization method. A shows the expression pattern (Whole-mount) in embryonic day 8.5 mouse embryos. An RNA probe for CD55 was prepared, and the expression pattern in normal embryos was examined using whole-mount in situ hybridization. A-1,3,4) Pictures showing a signal specific to the foregut (AIP) are shown. Our previous studies on cell fate using DiI strongly suggest that ventral pancreatic progenitor cells originate from the region of the foregut, and CD55 is very early in the pancreas. The possibility of becoming a marker of A-2) A signal was also confirmed in the hindgut. B shows the expression pattern (Whole-mount) in embryonic day 9.5 mouse embryo. Analysis was performed in the same manner as in FIG. 3A. B-1) As shown by the arrowhead, a signal was confirmed in the intestinal tract. B-2,3,4) B-1 embryos were sectioned and observed to confirm the expression of signals in the intestinal tract. C shows the expression pattern (section) in embryonic day 8.5 mouse embryos. For further detailed analysis of the expression position, in situ hybridization was performed on the section. The photos of C-a, b, c, d are from the same embryo. From the photographs of C-a and b, expression was confirmed in the amniotic membrane in addition to the hindgut region. Expression in the foregut region was confirmed from the photographs of C-c and d. D shows the expression pattern (section) in embryonic day 9.5 mouse embryo. Photos of D-a, b, c, d, e are from the same embryo. From D-b, expression was confirmed in the lung primordium. Moreover, the expression in the mesenchyme near the hindgut was confirmed from the photograph of De. FIG. 4 shows the analysis by a flow cytometer. A shows the expression analysis of CD55 in ES cells. A-1) An analysis diagram using cells on the fifth day after initiation of differentiation induction is shown. The upper figure is a diagram developed with E-cadherin and Cxcr4 and then developed again with CD55, and the lower figure is developed with E-cadherin and CD55 and then developed with Cxcr4. A-2) The transition of double-positive cells that change with the differentiation induction period when E-cadherin is taken on the vertical axis and Cxcr4 and CD55 are taken on the horizontal axis, respectively. A-3) E-cadherin and CD55 double positive fractions were collected using a flow cytometer and re-cultured on M15 cells. GFP fluorescence was confirmed on the 4th day after the start of re-culture, and on the 5th day, more intense GFP fluorescence was observed. B shows the expression analysis of CD55 in normal embryos. Analysis was performed using whole embryos of Pdx1 / GFP mice on embryonic day 9.5. After developing with E-cadherin and GFP, double positive fractions were re-developed with Cxcr4 and CD55, respectively. The fraction re-developed with the upper Cxcr4 contains Cxcr4-negative cells, whereas no such negative fraction was observed with the lower CD55. FIG. 5 shows microarray analysis using E-cadherin and CD55 double positive cells. A shows a scattered plot. Microarray analysis was performed to examine the elevated genes in E-cadherin / CD55 and E-cadherin / Cxcr4 double positive fractions, and the results are shown in the figure. From this figure, it was found that E-cadherin / CD55 and E-cadherin / Cxcr4 double positive cells showed very similar expression profiles. B indicates Clustering. In addition to the two fractions shown in A, the data of undifferentiated ES cells (ES), mesendoderm (Mes), ectoderm (Ect), and paraxial mesoderm (PAM) are added, The results of clustering analysis are shown. From this result, it was found that the expression profiles of E-cadherin / CD55 and E-cadherin / Cxcr4 double positive cells were very similar to those of other germ layers. FIG. 6 shows a summary of the microarray analysis. The result of the microarray analysis shown in FIG. 4 is schematically represented by a Venn diagram. The gene extraction conditions were such that the signal intensity in embryonic endoderm cells was 50 or more and the normalization value was 5 or more. There were 207 genes whose expression was commonly increased in the two fractions. As a percentage, it was about 70% for the entire E-cadherin / CD55 double positive fraction and about 80% for the entire E-cadherin / Cxcr4 double positive fraction. Among the 207 genes, genes that have been reported to be upregulated in endoderm are shown in light blue squares. FIG. 7 shows the expression pattern of candidate genes in mouse embryos with embryonic days (E) 8.5 and E9.5. Tmem184a expression was examined by whole mount in situ hybridization in E8.5 (A, B) and E9.5 (C) embryos. Tmem184a was detected in the anterior intestinal portal (AIP) (A, B, arrowhead) and intestinal epithelium (C, arrowhead). FIG. 8 shows expression patterns of candidate genes in mouse embryos with embryonic days (E) 9.5 and E12.5. Tmem184a expression was examined by section in situ hybridization at E 9.5 (A, B and C) and E12.5 (D, E). Tmem184a was detected in intestinal epithelium (A, B, C) and E12.5 pancreatic bud (D, E). FIG. 9 shows the expression pattern of candidate genes in mouse embryos with embryonic days (E) 8.5 and E9.5. The expression of Akr1c19 was examined by whole mount in situ hybridization in E8.5 (A, B and C) and E9.5 (D) embryos. Akr1c19 was detected in the anterior intestinal portal (AIP) (A arrowhead). FIG. 10 shows the expression pattern of candidate genes in mouse embryos with embryonic days (E) 9.5, 12.5 and E14.5. Akr1c19 expression was examined by section in situ hybridization at E9.5 (A, B, C and D), E12.5 (E) and E14.5 (F). Akr1c19 was detected in the intestinal epithelium (A, B, C, D) and E12.5 and E14.5 pancreas (E, F). FIG. 11 shows the expression pattern of candidate genes in E14.5 mouse pancreas. The expression of these genes was examined by section in situ hybridization.

Hereinafter, the present invention will be described in more detail.
(List of abbreviations)
Pdx1: Pancreas duodenum homeobox 1
Cxcr4: Chemokine (CXC motif) receptor 4
ES cell: Embryonic stem cell
Ect: Ectoderm
LPM: Lateral plate mesoderm
PAM: Paraxial mesoderm
MES: Mesendoderm
DE: Definitive endoderm
MEF: Mouse enbryonic fibroblasts
FGF: Fibroblast growth factor
LIF: Leukemia inhibitory factor
AIP: Anterior intestinal portal

In the present invention, the expression pattern in early embryos and differentiated ES cells was analyzed in detail for the CD55 gene specifically expressed in the endoderm identified by the ES cell differentiation induction system and microarray. The CD55 gene was not known to be expressed in the endoderm, but has been reported as a cell surface marker, and antibodies are also commercially available. Therefore, ES cell differentiation induction system and embryonic day 9.5
Pdx1 / GFP mouse embryos were used for analysis with a flow cytometer. In addition, in order to see what gene expression is elevated in CD55 positive cells, the conventional E-cadher
The endoderm fraction defined by in (+) / Cxcr4 (+) and the endoderm fraction defined by this gene, E-cadherin (+) / CD55 (+) fraction, were collected respectively for microarray analysis. Performed comparative analysis. As a result, the CD55 gene could be isolated as a novel marker gene for identifying embryonic endoderm. However, as a result of the expression analysis, in addition to the expression in the endoderm, the expression in the mesoderm was confirmed, it may be necessary to identify the embryonic endoderm by combining multiple markers as in the existing method. It was revealed. However, the existing marker Cx
It was possible to show that the embryonic endoderm fraction can be concentrated as in cr4, and that it is expressed until the late stage of endoderm development. Furthermore, regarding the genes expressed in ES cell-derived Pdx1-positive cells, whether they are expressed in early pancreatic buds on sections and whol
When we searched by e mount in situ hybridization, we identified several new genes.

In the method of detecting endoderm cells, intestinal cells or pancreatic cells according to the present invention, decay accelerating factor (DAF, CD55) gene, Tmem184a gene, Akr1c19 gene, 3300001A09Rik gene, Aebp2
Gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4 gene, Pbxip1 gene, Pc
The expression of at least one gene selected from bd1 gene and Pcdh1 gene is used as an index. The decay-accelerating factor (DAF, CD55) acts only on the C3 / C5 convertase on the cells in which it exists and inhibits C3 / C5 convertase, thereby suppressing complement activation. It is a regulatory protein.

The decay-accelerating factor (DAF, CD55) in the present invention is derived from a vertebrate, preferably from a mammal, unless otherwise specified. For example, the nucleotide sequence and amino acid sequence of the human decay accelerating factor (DAF, CD55) gene are shown in NCBI GenBank (NM_000574.3), and the nucleotide sequence and amino acid sequence of the rat decay accelerating factor (DAF, CD55) gene are It is shown in NCBI GenBank (NM_0022269.2), and the nucleotide sequence and amino acid sequence of mouse decay accelerating factor (DAF, CD55) gene are shown in NCBI GenBank (NM_010016.2).

Examples of the human decay promoting factor (DAF, CD55) gene in the present invention include (a) a nucleic acid comprising a base sequence encoding the human decay promoting factor (DAF, CD55) protein shown above, and (b) a human decay promoting factor. (DAF, CD55) a nucleic acid encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the protein, (c) human decay accelerating factor (DAF, CD55
) 60% or more, preferably 70% or more, more preferably 8% of the nucleotide sequence encoding the protein
A nucleic acid comprising a base sequence having a homology of 0% or more, more preferably 90% or more, (d) 60% or more, preferably 70% or more of the amino acid sequence of the human decay accelerating factor (DAF, CD55) protein
More preferably, it hybridizes under stringent conditions with a nucleic acid encoding an amino acid sequence having an identity of 80% or more, more preferably 90% or more, or (e) a nucleic acid encoding a human decay accelerating factor (DAF, CD55) protein. Nucleic acids that soy are included. In the above (b), the number of amino acids to be modified is usually 1 to 15, preferably 1 to 10, more preferably 1 to 7, and more preferably 1 to 5. In addition, the above-mentioned nucleic acid (e) is prepared by preparing a probe from either a nucleic acid containing a protein coding sequence of a human decay accelerating factor (DAF, CD55) gene such as human or a target nucleic acid, It can be identified by detecting whether it hybridizes to a nucleic acid. For example, human decay promoting factor (DAF) such as human
, CD55) may include a nucleic acid that hybridizes under stringent conditions with a nucleic acid (DNA or RNA) consisting of any continuous partial sequence or full length in the protein coding sequence of the gene. Alternatively, it may contain a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a protein coding sequence of a human decay accelerating factor (DAF, CD55) gene such as human. Examples of stringent hybridization conditions include, for example, 5 × S.
Hybridization was performed at a temperature of about 48 ° C. to 52 ° C. in a solution containing SC, 7% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, 5 × Denhardt's solution,
48 ° C. to 68 ° C. 2 × SSC (or in 1 × SSC, or in 0.5 × SSC, or 0.1
In xSSC), conditions for washing for 1 hour can be mentioned. Tmem184a gene, Akr1c19 gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene,
About Hipk2 gene, Lass4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene,
The same as in the case of the human decay accelerating factor (DAF, CD55) gene.

“Detection of endoderm cells, intestinal cells or pancreatic cells” includes, for example, detecting whether the cell fraction contains endoderm cells, intestinal cells or pancreatic cells, or quantifying the ratio. It is. The detection of endoderm cells, intestinal cells or pancreatic cells also includes “identification” of the cells. In addition, “separating” endoderm cells, intestinal cells or pancreatic cells means separating the cell population into the cells or a cell population containing the cells and other cell populations. In the present invention, the separation of endoderm cells, intestinal cells or pancreatic cells may be to increase the proportion of the cells in the cell population.

Endoderm cells are cells that can be differentiated into endoderm-derived tissues such as pancreas, liver, lung, stomach, intestine and thyroid rather than differentiated into tissues produced from ectoderm or mesoderm, or endoderm-derived tissues It is a differentiated cell.
Intestinal cells are cells that can differentiate into intestinal epithelial cells such as the small intestine and large intestine, or cells that have differentiated into intestinal epithelial cells.
Pancreatic cells are cells that can differentiate into mature pancreatic cells, such as endocrine cells, exocrine cells, and pancreatic duct cells, or cells that have differentiated into mature pancreatic cells.

The present invention relates to human decay accelerating factor (DAF, CD55) gene, Tmem184a gene, Akr1c19 gene,
3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Las
Provided are a method for detecting and separating endoderm cells, intestinal cells or pancreatic cells using as an index the expression of at least one gene selected from the s4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene. The present inventors have included a decay accelerating factor (DAF, CD55) gene, a Tmem184a gene, an Akr
1c19 gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk
It was found that at least one gene selected from 2 genes, Lass4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene is a marker for endoderm cells, intestinal cells or pancreatic cells. By detecting or selecting cells that express the decay accelerating factor (DAF, CD55) gene (decay accelerating factor (DAF, CD55) positive cells), the endoderm cells, intestinal cells or pancreatic cells can be detected and identified. Germ cells, intestinal cells or pancreatic cells can be specifically isolated. In the present invention, the expression of decay accelerating factor (DAF, CD55) gene may be production of decay accelerating factor (DAF, CD55) mRNA and / or production of decay accelerating factor (DAF, CD55) protein. That is, by detecting decay accelerating factor (DAF, CD55) mRNA or decay accelerating factor (DAF, CD55) protein, the expression of decay accelerating factor (DAF, CD55) gene can be detected. For example, decay accelerating factor (DAF, CD55) mRNA is detected by known methods such as Northern hybridization using decay accelerating factor (DAF, CD55) cDNA fragment or oligonucleotide, RNA protection assay, or RT-PCR. Is possible. Detection of decay accelerating factor (DAF, CD55) protein is performed by Western blotting using an anti-decay accelerating factor (DAF, CD55) antibody, immunoprecipitation, ELISA, immunohistochemistry, FACS (fluoresc)
It can be detected by a known method such as a method using ence activated cell sorting (fluorescence activated cell separation). Tmem184a gene, Akr1c19 gene,
3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Las
Human decay-promoting factors for s4 gene, Pbxip1 gene, Pcbd1 gene, and Pcdh1 gene
The same as for the (DAF, CD55) gene.

In the detection method and the separation method of the present invention, it is preferable to detect the expression of a decay accelerating factor (DAF, CD55) gene by detecting a decay accelerating factor (DAF, CD55) protein. In addition, cells can be efficiently detected or fractionated by using a cell sorter such as FACS.

There is no restriction | limiting in particular as a cell used for the detection or isolation | separation of this invention, The tissue or cell etc. which are estimated to contain an endoderm cell, an intestinal cell, or a pancreatic cell can be used. The cell is a vertebrate-derived cell, preferably a mammalian cell (for example, a rodent such as a mouse or a rat, a cell such as a primate such as a monkey or a human). When considering application to humans, human cells are preferred.

Cell samples can be prepared according to known methods. For example, from tissue to endoderm cells,
In order to prepare a sample containing intestinal cells or pancreatic cells, tissues are collected and rinsed, and then treated with an enzyme solution containing collagenase and dispase to disperse the cells. Endoderm cells, intestinal cells or pancreatic cells can be detected and separated from the cells thus prepared.

The method of the present invention can also be applied to cell lines. Cells prepared from the intestinal tract or pancreas of a mammalian fetus or newborn can be immortalized, and for example, endoderm cells, intestinal cells or pancreatic cells can be selected by the method of the present invention. Disintegration promoting factor using cells derived from ES cells
It is also possible to isolate (DAF, CD55) positive cells.

Specifically, the method of the present invention for detecting endoderm cells, intestinal cells or pancreatic cells comprises (a) a step of detecting the expression of a decay accelerating factor (DAF, CD55) gene in a cell, and (b) an acceleration of decay. It can be carried out by identifying cells expressing the factor (DAF, CD55) gene as endoderm cells, intestinal cells or pancreatic cells. For example, the presence or ratio of endoderm cells, intestinal cells or pancreatic cells can be determined by measuring the presence or ratio of decay accelerating factor (DAF, CD55) positive cells, respectively. A cell population containing a high percentage of decay accelerating factor (DAF, CD55) positive cells is judged to contain a high percentage of endoderm cells, intestinal cells or pancreatic cells.
Therefore, endoderm cells, intestinal cells or pancreatic cells can be selected by the step of selecting cells that express the decay accelerating factor (DAF, CD55) gene. For example, (a) detecting the expression of a decay accelerating factor (DAF, CD55) gene in a cell, and (b) a decay accelerating factor (DAF, CD
55) An endoderm cell, an intestinal cell or a pancreatic cell can be isolated by the step of isolating a cell expressing the gene. Alternatively, the cells are fractionated in advance, the expression of the decay accelerating factor (DAF, CD55) gene is detected in the fractionated cells, and the cells expressing the decay accelerating factor (DAF, CD55) gene are selected. Germ cells, intestinal cells or pancreatic cells can be isolated and selected. Tmem184a gene, Akr1c19 gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4 gene, Pbxip1 gene, Pcbd1 gene, and Pcd
The h1 gene is the same as that of the human decay accelerating factor (DAF, CD55) gene.

Separation of endoderm cells, intestinal cells or pancreatic cells is preferably carried out by using an antibody that binds to a decay accelerating factor (DAF, CD55) protein expressed on the cell surface. A step of preparing a cell sample, (b) a step of adding an antibody against a decay accelerating factor (DAF, CD55) to the cell sample, and (c) a step of separating cells bound with the antibody.

By recovering the separated cells, endoderm cells, intestinal cells or pancreatic cells can be recovered. For example, anti-collapse promoting factors (DAF, CD
55) Immobilizing ligands that bind to antibodies or decay accelerating factor (DAF, CD55) proteins, and binding cells directly or indirectly to them, separation using immunosorbent columns, fluorescent antibody-labeled cell separation methods, immunomagnetics There are separation methods using beads. In addition, the above step (c)
It can be performed using a cell sorter such as CS. Separation of cells using a cell sorter can be performed by a known method.

The cells prepared in the present invention can be cultured or stored using an appropriate medium. The medium can be supplemented with serum, growth / differentiation factors, and the like. Examples of the medium include, but are not limited to, DMEM containing about 10% fetal calf serum (FCS) or an equivalent supplement. For example, activin 20ng / ml and bFGF 50ng / ml (in 10% FB
It can be cultured in the presence of S / DMEM or 15% KSR / DMEM) or in the following medium.

Cell culture may be cell maintenance, incubation, growth, storage, or the like. For example, in vitro, incubation in a suitable medium at 37 ° C., 5% CO _{2 in} a humid environment. Can be mentioned.

In addition, since decay-accelerating factors (DAF, CD55) are markers for endoderm cells, intestinal cells or pancreatic cells, the expression of decay-accelerating factor (DAF, CD55) gene is used as an index to indicate endoderm cells, intestinal cells or pancreatic cells. Cells can be monitored. Based on this, for example, it is possible to evaluate the effects of various drugs, gene expression, and other stimuli on cell differentiation induction. For example, various agents that act on endoderm cells, intestinal cells, or pancreatic cells can be assayed by detecting the effect of a test sample on the expression of decay accelerating factors (DAF, CD55).

The present invention further relates to a reagent for detecting or separating endoderm cells, intestinal cells or pancreatic cells, comprising an antibody against a decay accelerating factor (DAF, CD55). Antibodies against decay accelerating factors (DAF, CD55) can be obtained by using decay accelerating factor (DAF, CD55) protein or its partial peptides as immunogens, or cells expressing decay accelerating factor (DAF, CD55) proteins as immunogens. It can be manufactured by using.

Disintegration accelerating factor as an immunogen (DAF, CD55) There are no particular restrictions on the type of animal from which the protein is derived. Disintegration promoting factors derived from humans, monkeys, mice, rats, cows, rabbits, and other vertebrates
(DAF, CD55) protein can be used. The antibody can be prepared according to a known method. For example, a monoclonal antibody can be produced by a known cell fusion method.

The above antibody can be used as a reagent for detecting or separating endoderm cells, intestinal cells, or pancreatic cells by appropriately combining with physiological saline, buffer solution, salt, stabilizer and the like. For example, this antibody can be used to examine the distribution and amount of endoderm cells, intestinal cells or pancreatic cells in tissues, and to measure the concentration of endoderm cells, intestinal cells or pancreatic cells in a cell sample. . The antibody may be fluorescently labeled.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

(1) Method (cell culture)
(1) ES cell culture
Pdx1 / GFP ES cells (SK7 ES cells) were established from blastocysts of transgenic mice having homozygous recombinant genes introduced with GFP under the Pdx1 promoter. ES cells were mouse embryonic fibroblast (MEF) dependent and cultured using ES cell maintenance medium.

(ES cell maintenance medium for SK7 ES cells)
Glasgow minimum essential medium (GMEM) (Invitrogen)
15% Knock-out Serum Replacement (KSR) (Invitrogen)
1% FBS (Hyclone)
100 μM nonessential amino acids (Invitrogen)
2 mM L-glutamine (Nacalai tesque)
1 mM sodium pyruvate (Invitrogen)
50 units / ml penicillin and 50 μg / ml streptomycin (Nacalai tesque)
100 μM β-mercaptoethanol (Sigma)
1000 units / ml LIF (Chemicon)

(2) Culture of feeder cells
M15 cells are produced by Prof. Toshiaki Nose (Mitsubishi Chemical Life Science Institute) and Dr. M. Rassoulzadegan (
Granted by the University of Nice-Sophia Antipolis) (Larsson SH, Charlieu JP, M
iyagawa K, et al. Subnuclear localization of WT1 in splicing or transcription
factor domains is regulated by alternative splicing. Cell. 1995; 81: 391-401). The cells were cultured in the following medium. M15 cells were cultured until confluent, and treated with mitomycin C (Sigma) (200 μg / ml at 37 ° C. for 2.5 hours) were used for differentiation induction experiments. The day before differentiation induction, 0.5 ml, 6 well pl on a gelatin-coated 24-well plate at a concentration of 4 x 10 ⁵ cells / ml
Seed ate in 2 ml volume.

(M15 medium)
DMEM (Invitrogen)
10% FBS (Hyclone)
2 mM L-glutamine (Nacalai tesque)
50 units / ml penicillin and 50 μg / ml streptomycin (Nacalai tesque)
100 μM β-mercaptoethanol (Sigma)

(3) Differentiation induction method Co-culture with supporting cells When differentiation induction was performed, ES cells cultured on a gelatin-coated dish were used in order to reduce the carry-in of MEF. In this case, ES cell maintenance medium was used. The ES cells were detached with 0.25% trypsin / EDTA solution and centrifuged at 4 ° C. and 1000 rpm to collect the cells. The cells were redissolved in a differentiation induction medium to a concentration of 10000 cells / ml. The prepared ES cell lysate was seeded in a volume of 0.5 ml in a 24-well plate and 2 ml in a 6-well plate in 24 well and 6 well plates that had been cultured until the feeder cells were confluent. The differentiation induction medium was changed every 2 days. The composition of the differentiation induction medium is shown below.

(Differentiation induction medium)
DMEM (Invitrogen)
10% FBS (Hyclone)
100 μM nonessential amino acids (Invitrogen)
2 mM L-glutamine (Nacalai tesque)
50 units / ml penicillin and 50 μg / ml streptomycin (Nacalai tesque)
100 μM β-mercaptoethanol (Sigma)

(4) Humoral factor and inhibitor The various humoral factors and inhibitors were used at the following concentrations.
Recombinant human Activin A (R & D) 20 ng / ml
human bFGF (PEPROTECH) 50 ng / ml
Recombinant human BMP 7 (R & D) 25 ng / ml
SB203580 (CALBIOCHEM) 1 μM;

(Analysis using a flow cytometer)
The cells were incubated with Cell Dissociation Buffer (Invitrogen) at 37 ° C. for 20 minutes, detached from the plate, adjusted to a concentration of 1 × 10 ⁶ cells / sample (50 μl), and stained with the following antibody. Stained cells are Hank's buffered salt containing 1% FBS and propidium iodide.
After redissolving in solution (Sigma), the solution was filtered using a 40 μm mesh to obtain a sample for analysis. Samples were analyzed using FACS Canto (Becton Dickinson) and data were analyzed using BD FACS
Acquired using Diva Software (Becton Dickinson). Analysis was also performed on normal embryos on day 9.5 of the embryo in the same manner as described above. Flowjo program for analysis of the obtained data
(Tree Star) was used. For microarray analysis, FACS Aria or Aria II (
Cells were sorted using Becton Dickinson).

The antibodies used are listed below.
biotin-conjugated anti-E-cadherin monoclonal antibody (mAb) ECCD2, phycoeryt
hrin (PE) -conjugated anti-Cxcr4 mAb 2B11 (BD Biosciences Pharmingen), PE-conju
gated anti-FLK1 mAb AVAS12 (BD Biosciences Pharmingen), biotin-conjugated anti
-PDGFRα mAb APA5.Streptavidin-Allophycocyanin (BD Biosciences Pharmingen).

(Reculture)
E-cadherin and CD55 double positive fractions from cells induced to differentiate for 5 days on M15 cells
Sorted using ia. The sorted cells were 24 well plasted with M15 cells.
Te was seeded at a density of 2.0 × 10 ⁵ cells / well. Re-culture is 4500 mg / l glucose, 10% FBS, A
ctivin, bFGF (refer to the section of cell culture 4) Nicotineamide was added. The medium was changed every day from the start of re-culture.

(Microarray analysis)
Total RNA was extracted from the collected cells using a flow cytometer, and a sample for analysis was prepared according to the protocol of Affymetrix. The quality of the final sample
After confirmation with Bioanalyzer 2100 (Agilent), the sample was injected into Affymetrix MOE430 2.0 and hybridization was performed at 45 ° C. for 16 hours. Flud after hybridization
Using ics Station 450, washing and antibody reaction are performed, then GeneChip scanner 3000
Was used to scan the array. The obtained data was analyzed using GeneSpring GX (Agilent).

(RNA extraction from cells and cDNA synthesis)
Samples from which support cells were removed using FACS from the cell population after differentiation induction,
Total RNA was extracted using RNeasy mini kit (Qiagen). RNA 3 μg to oligo dT pr
cDNA was synthesized in a 20 μl system using 10 pmol of imers (Toyobo). The obtained cDNA was diluted 5-fold, and 1 μl thereof was used as a template for real-time PCR.

(Real-time PCR)
For primer design, the Perfect Real Time Primer support system (Takara Bio Inc., http://www.takara-bio.co.jp/prt/intro.htm) and Primer3 (http: // f
rodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). In addition to the primer set of the target gene, β-actin was used as an internal standard marker for correcting the RNA amount between samples. As a procedure, first, a cDNA dilution solution for a calibration curve was prepared for each primer. The sample for the calibration curve includes Pdx1-positive embryonic endoderm cells 8 days after the start of differentiation induction
Add 5 times diluted cDNA synthesized from RNA extracted from (D8 DE GFP (+)) to 3, 10, 30, 100
It was used after diluting twice. As a negative control, sterilized water was used. Samples include ES cells, ectoderm, mesoderm (mixed side plate and axial mesoderm), mesendoderm, embryonic endoderm on day 5 after initiation of differentiation, and embryonicity on day 7 after initiation of differentiation. A 5-fold diluted solution of cDNA synthesized from RNA extracted from endoderm and Pdx1-positive and negative embryonic endoderm cells 8 days after initiation of differentiation induction was used. Reagent is Fast SYBR Green Master Mix, instrument is 7500 Fast Real-time PCR system
(Both were ABI). All samples were run at N = 2. The data after the analysis was analyzed with Excel, and the relative value when D8 DE GFP (+) was set to 100 was calculated.

Relative Value Correction Method Since the relative amount (Qty) was calculated from the calibration curve, the value of Mean Qty was used when N = 2 or more.
Mean Qty (relative amount) / β-actin value in each germ layer × 100 (1)
Value calculated in (1) / D8 DE GFP (+) fraction value Correction value

(Whole-mount in situ hybridization)
The samples used were normal embryos from embryonic day 9.5 and fetal day 8.5. Samples were fixed with 4% paraformaldehyde overnight at 4 ° C. and dehydrated with methanol. 10 μg / ml Proteinase K
After treatment with (nacalai tesque, # 29442-85) for 10 minutes, it was fixed with 0.2% glutaraldehyde / 4% paraformaldehyde for about 30 minutes. 70 ° C after washing with PBST (1xPBS (-), 0.1% Tween20)
Prehybridization is performed for 6 hours at 60 to 60 ° C., and 1 μg / ml of Digoxigenin (DIG) -labeled antisense RNA probe (Roche, # 11-277-073-910) is allowed to hybridize overnight at 70 to 60 ° C. It was. Hybridization buffer is 50% formamide (nacalai
tesque, # 16345-65), 0.5M NaCl, 1xPE, 50μg / ml Yeast torula RNA (Roche, # 109-495)
, 500μg / ml heparin (Sigma, # H-3393-250KU), 2.5% 3-((3-Cholamidopropyl) dimethy
lammonio] -1-propanesulfonate (CHAPS), 0.1% Tween20. Then Wash1 (0.
3M NaCl, 1xPE (10 mM Piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES) (pH 6.8
), 1 mM EDTA), 0.1% Tween20), Wash2 (0.05M NaCl, 1xPE, 0.1% Tween20), Wash3 (50% formamide, 0.3M NaCl, 1xPE, 0.1% Tween20), Wash4 (50% formamide, 0.15M NaCl, 1xP
E, 0.1% Tween20), Wash5 (0.5M NaCl, 1xPE, 0.1% Tween20) at 70 ° C for 30 minutes, then MABT at room temperature (1xMAB (100 mM maleic acid, 150 mM NaCl (pH 7.5))), 0.1% Washed with Tween20). Block for about 6 hours with 2% Blocking solution (Roche, # 1-096-176, sheep serum (Sigma, # S-2263), dissolved in MABT) and anti-DIG antibody (Anti Digoxigenin- AP Fab fragments
, Roche, # 11-093-274-910). Wash again with MABT, NTMT (0.1M NaCl, 0.1
M Tris-HCl (pH 9.5), 0.05M MgCl ₂ , 0.1% Tween 20) after shaking for 1 hour, 4-nitroblue tetrazolium chloride (NBT) (Roche, # 1-383-213), 5-bromo-4 -Chloro-3-indolyl
-Color developed with phosphoric acid (BCIP) (Roche, # 1-383-221). Plasmids used for RNA probe preparation were purchased from Open Biosystems and the RIKEN Tsukuba Research Institute Bioresource Center.

(Paraffin section preparation)
Paraffin sections were prepared from mouse embryos for which specific signals were obtained by whole-mount in situ hybridization. During the replacement and embedding operations, the mouse embryos were processed in Uni-cassette biopsy six (Sakura Finetech Japan). Whole-
After mount in situ hybridization, 100% methanol-substituted sample is once in 50% ethanol / PBS, 70% ethanol / PBS, 90% ethanol / PBS, once every 30 minutes, twice in 100% ethanol, 100% xylene bath Substitute for 2 hours to overnight, then xylene / paraffin (2: 1
) Once, overnight, twice in a paraffin bath and overnight, and embedded. For the embedded sample, sections were prepared with a thickness of 10-20 μm depending on the intensity of the signal. The prepared paraffin sections were dried overnight at 37 ° C, then twice in a 100% xylene bath, twice in a 100% ethanol bath for 5 minutes, 90% ethanol, 70% ethanol, 50% ethanol bath, milli-Q water 1 time for 5 minutes, then 50% ethanol, 70% ethanol, 90% ethanol bath once for 5 minutes, 100% ethanol bath twice for 5 minutes, then 100% xylene bath twice After
Encapsulation was performed.

(Create frozen section)
Samples were fixed with 4% paraformaldehyde overnight at 4 ° C. and shaken overnight in 15% sucrose and 30% sucrose at 4 ° C. overnight. Then, it was embedded with an OCT compound (Tissue-Tec), a section was prepared with a thickness of 10 μm, and dried overnight at 37 ° C.

(In situ hybridization on the section (frozen section))
Immerse the slide glass in PBST, hydrate the OCT compound, and add 1 μg / ml Proteinase K.
After 5 minutes of treatment, the cells were fixed with 0.2% glutaraldehyde / 4% paraformaldehyde for about 30 minutes. After washing with PBST, prehybridization is performed at 70 ° C to 60 ° C for 5-6 hours.
DIG-labeled antisense RNA probe 1 μg / ml was hybridized overnight at 70 ° C. to 60 ° C. Hybridization buffer is 50% formamide, 5xSSC (citrate buffer
pH7.0), 1% sodium lauryl sulfate (SDS), 50μg / ml Yeast torula RNA, 50μg / ml h
eparin was used. 50% formamide 5xSSC (pH7.0), 1% SDS, 50% formamide, 2xSSC at 70 ° C
It was then washed at 60 ° C. for 30 minutes each and washed with MABT at room temperature. Blocking was performed for about 6 hours with the Blocking solution, and the anti-DIG antibody was reacted overnight at 4 ° C. After washing again with MABT, shaking with NTMT for 30 minutes, the color was developed with NBT and BCIP.

(2) Results (2-1) Microarray analysis and extraction of candidate genes The present inventors have previously induced differentiation of endoderm cells efficiently from ES cells by combining the use of feeder cells and addition of humoral factors. (Shiraki N, Yoshida)
T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-e
xpressing regional-specific definitive endoderm. Stem Cells. 2008; 26: 874-885;
And Shiraki N, Umeda K, Sakashita N, et al. Differentiation of mouse and human
embryonic stem cells into hepatic lineages. Genes Cells. 2008; 13: 731-746). In the process of constructing the differentiation induction method, it has been found that various signals are involved in the differentiation of ES cells. With reference to these findings, by changing the combination of humoral factors added as shown in FIG. 1A, it has succeeded in efficiently inducing differentiation of not only endoderm but also mesoderm and ectoderm (Shiraki N, Higuchi Y, Harada S, et al. Diffe
rentiation and characterization of embryonic stem cells into three germ layers
Biochem Biophys Res Commun. 2009; 381: 694-699).

In the present invention, using the differentiation inducing method of FIG. 1A, expression profiles of various differentiated cells as shown in FIG. 1B were created, and the endoderm markers were searched by comparing them. First, specific cell populations were collected from cells differentiated into their lineages using a flow cytometer. The antibody combinations used for sorting are described in FIG. 1B. About 1.0 × 10 ⁶ cells were collected from each cell population, and samples for microarray analysis were prepared. For the analysis, Afflymetrix GeneChip MOE430 2.0 in which all mouse gene sets were integrated was used.

Signal intensity> 5 in early embryonic endoderm cells (D5 DE) on day 5 from the start of differentiation induction
0, Flag = Presence, ES5, known undifferentiated ectoderm, lateral plate mesoderm, paraxial mesoderm, mesoderm with 5 times more expression in D5 DE The analysis was conducted mainly on

Of these genes, 27 genes with transcription factors and domain structures were selected as candidate genes for expression analysis in normal mouse embryos in order to analyze expression in the endoderm.

(2-2) Analysis results by whole-mount in situ hybridization To examine the expression of the candidate gene selected in (2-1) in normal embryos,
Whole-mount in situ hybridization was performed using embryos on day 9.5.
A novel candidate CD55 gene (see the following literature) was found that confirmed specific expression in the endoderm at both stages of embryonic day 9.5 and fetal day 8.5.

Lublin DM, Atkinson JP. Decay-accelerating factor: biochemistry, molecular biol
ogy, andfunction. Annu Rev Immunol. 1989; 7: 35-58.
Miwa T, Song WC.Membrane complement regulatory proteins: insight from animal s
tudies and relevance to human diseases.Int Immunopharmacol. 2001; 1: 445-459.
Miwa T, Sun X, Ohta R, et al. Characterization of glycosylphosphatidylinositol-
anchored decay accelerating factor (GPI-DAF) and transmembrane DAF gene express
ion in wild-type and GPI-DAF gene knockout mice using polyclonal and monoclonal
antibodies with dual or single specificity. Immunology. 2001; 104: 207-214.
Song WC, Deng C, Raszmann K, et al. Mouse decay-accelerating factor: selective
and tissue-specific induction by estrogen of the gene encoding the glycosylphos
phatidylinositol-anchored form. J Immunol. 1996; 157: 4166-4172.
Spicer AP, Seldin MF, Gendler SJ. Molecular cloning and chromosomal localizati
on of the mouse decay-accelerating factor genes. Duplicated genes encode glyco
sylphosphatidylinositol-anchored and transmembrane forms.J Immunol. 1995; 155:
3079-3091

In the following experiment, detailed analysis of this CD55 was advanced. From the results of microarray analysis, it was confirmed that CD55 shows a high expression level in the embryonic endoderm fraction after the 5th day of differentiation induction (FIG. 2A). Moreover, the same result was shown also in the real-time PCR analysis performed in order to confirm the expression level (FIG. 2B). From these results, it was confirmed that the expression level of CD55 was increased from the embryonic endoderm fraction on the fifth day after the start of differentiation induction, and a constant expression level was maintained until the eighth day after the start of differentiation induction. did. Furthermore, analysis by whole-mount in situ hybridization confirmed specific expression in the foregutal region in normal embryos of day 8.5 and in the intestinal region in normal embryos of day 9.5. (FIGS. 3A and B). Subsequently, a paraffin section was prepared for the above sample, and a region where CD55 was expressed was confirmed. Signal is embryonic
It was confirmed in the foregut endoderm region in the 8.5 day mouse normal embryo and in the hind intestinal endoderm region in the embryonic day 9.5 mouse normal embryo (FIGS. 3A and B). In order to perform further detailed analysis, frozen sections were prepared, and the sections were analyzed by in situ hybridization. As a result, embryo 8
In the normal embryo of day 5, the expression was confirmed in the amniotic membrane region in addition to the foregut and hindgut endoderm regions,
On day 9.5 of the embryo, signals were confirmed in the lung primordia and mesenchyme (FIGS. 3C and D). The above results revealed that CD55 is expressed in the early endoderm region but also in the mesoderm.

(2-3) Flow cytometry analysis (cultured cells)
CD55 is a known gene and has been reported to be expressed on the cell surface. Furthermore, since the antibody is commercially available, the analysis was performed using a flow cytometer. As a method for identification as embryonic endoderm, it has been reported that the double positive part is defined as an embryonic endoderm fraction using two marker genes of E-cadherin and chemokine receptor Cxcr4. The method is currently used. Although the original goal was to search for a single marker to identify embryonic endoderm, whole-mount in situ hybridization and in situ in sections
As a result of the analysis by hybridization, it was found that CD55 is expressed not only in the endoderm but also in the mesoderm. Therefore, in order to remove the mesoderm fraction, flow cytometry analysis was performed using E-cadherin together with CD55. In the analysis, cells (D5 DE) induced to differentiate into endoderm by the differentiation induction method shown in FIG. 1A were used. First, FSC and SSC were used to remove supporting cells M15 cells, then PI negative fractions were identified, and the fractions were further developed with E-cadherin, Cxcr4 and CD55 positive fractions. First, after developing with E-cadherin and Cxcr4 which are the current marker genes, the double positive fraction was re-developed with CD55. The other was developed with E-cadherin and CD55, and the double positive fraction was re-developed with Cxcr4 (FIG. 4A). From this result, it was found that the fraction developed with E-cadherin and Cxcr4 and then re-developed with CD55 contains CD55 negative cells. On the other hand, after deploying with E-cadherin and CD55, Cx
It was found that the fraction re-developed with cr4 did not contain Cxcr4-negative cells. From the above results, it was suggested that both E-cadherin and CD55 positive cells are cell populations defining a narrower range than both E-cadherin and Cxcr4 positive embryonic endoderm (FIG. 4A).

Further, a double positive fraction of E-cadherin and CD55 was collected from cells induced to differentiate on M15 cells for 5 days using a flow cytometer, and re-cultured on M15 cells. Re-culture start 4
On the day (day 9 of differentiation induction), fluorescence of GFP begins to be observed, and day 5 (day 10 of differentiation induction started)
On day 2), stronger GFP fluorescence was confirmed. This result strongly suggested that the double positive fraction of E-cadherin and CD55 regulates embryonic endoderm.

Furthermore, since the present inventors have confirmed in the previous experiment that the expression level of Cxcr4 decreases as endoderm differentiation proceeds, next, on the 8th day, 10th day, and 12th day after the start of differentiation induction. The time course of E-cadherin and Cxcr4 double positive fraction and E-cadherin and CD55 double positive fraction was examined (FIG. 4A). In the embryonic endoderm fraction on the 8th day after the initiation of differentiation induction, high expression was observed in any of the marker genes. However, on the 10th day, E-cadherin
It was confirmed that the cell numbers of E-cadherin and Cxcr4 double positive fractions were decreased, while the number of cells in CD55 double positive fractions did not change. Furthermore, on the 12th day, E-cadherin and CD
While the number of cells in the 55 double positive fraction remains unchanged, E-cadherin and Cxcr
It was confirmed that the number of cells of 4 double positive fractions was greatly reduced. This suggests that CD55 can also be used as a marker for endoderm with advanced differentiation.

(2-4) Flow cytometry analysis (normal embryo)
Next, using a Pdx1 / GFP mouse embryo of embryonic day 9.5, the expression of CD55 in a normal embryo was analyzed by a flow cytometer (FIG. 4B). As analysis methods, development was performed with E-cadherin and GFP, and double positive fractions were re-developed with Cxcr4 or CD55. From this analysis, Pdx1-positive cells
When expanded with xcr4, both Cxcr4 positive and negative cells were included, but when expanded with CD55, cells completely negative for the marker CD55 were not included. From this,
The Pdx1-positive pancreatic progenitor cells on day 9.5 of embryonic day were found to be CD55 positive.

(2-5) Microarray analysis Based on the results of the above experiment, E-cadherin and Cxcr4 double positive fraction or E-cadherin and CD5
In order to examine what kind of gene expression was increased in 5 double positive fractions, each fraction was fractionated and subjected to microarray analysis (FIG. 5). As shown in FIG. 5A, it was revealed that E-cadherin and Cxcr4 double positive cells and E-cadherin and CD55 double positive cells showed very similar expression profiles. In addition, similar results were obtained by clustering analysis compared with other germ layers (FIG. 5B). Furthermore, the gene whose expression was elevated in each fraction was also analyzed in detail (FIG. 6). E-cadherin and Cxcr4 double positive fraction and E-cadheri
In both n and CD55 double positive fractions, 2 genes have increased expression compared to other germ layers
There were many genes that had already been reported to be elevated in the endoderm, among them, and among them as candidate genes that express endoderm-specific or pancreas-specific, Of those analyzed for expression in normal mouse embryos, 14 genes were included. An example of 207 genes is shown in FIG. These genes include all endoderm markers such as Foxa2 and Sox17, indicating that CD55 can be used as an endoderm marker.
The non-overlapping part also contained previously reported endoderm markers such as Lefty1 and Sfrp1.

(2-6) Identification of a novel gene expressed in pancreatic buds at the early stage of development Further, among the genes expressed in Pdx1-positive cells derived from ES cells, whether or not it is expressed in pancreatic buds at the early stage of development is examined. On the section and whole mount in situ h
When searching by the ybridization method, several novel genes were identified (FIGS. 7, 8, 9 and 10).

FIG. 7 shows whole mount in situ hybridization analysis of the Tmem184a gene. In E8.5 mouse embryos, expression was observed in the anterior intestinal portal (AIP) (A, B, arrowhead), and in E9.5, expression was observed in the intestinal epithelium including pancreatic buds ( C, arrowhead).
FIG. 8 shows the expression pattern of the Tmem184a gene in the intestinal epithelium. In E9.5, expression was observed in the intestinal epithelium. In E12.5, expression was observed in pancreatic buds.

FIG. 9 shows whole mount in situ hybridization analysis of the Akr1c19 gene. In E8.5 mouse embryos, expression was observed in the anterior intestinal portal (AIP) (A arrowhead), and in E9.5, expression was observed in the intestinal epithelium including pancreatic bud (D, arrowhead). .
FIG. 10 shows the expression pattern of Akr1c19 gene in E9.5 intestinal epithelium and E12.5, E14.5 fetal pancreatic epithelium. In E9.5, the Akr1c19 gene was expressed in the intestinal epithelium. E12.5,
In E14.5, the Akr1c19 gene was expressed in the pancreatic epithelium. Its expression pattern is very similar to the Pdx1 gene.

Table 1 shows a list of genes whose expression was observed in the pancreas of E14.5 in addition to the above. For these genes, expression in the pancreas was not known at all: 3300001A09Rik, Ae
There are seven genes, bp2, AI464131, Akr1c19, Foxp4, Lass4, and Pbxip1, and two genes, Pcbd1 and Pcdh1, are present only in Gene Paint for expression in the pancreas.

FIG. 11 shows the expression pattern in the pancreatic epithelium of the gene whose expression was observed in the pancreas of E14.5. The image of in situ hybridization in the section is shown.

(3) Discussion To date, reports on the search for new marker genes specific to embryonic endoderm have been made (Hou J, Charters AM, Lee SC, et al. A systematic screen for genes expressed i).
n definitive endoderm by Serial Analysis of Gene Expression (SAGE) .BMC Dev Bi
ol. 2007; 7: 92 .; and Sherwood RI, Jitianu C, Cleaver O, et al. Prospective isol
ation and global gene expression analysis of definitive and visceral endoderm.
Dev Biol. 2007; 304: 541-555). However, none of the papers showed a novel gene expressed specifically in the embryonic endoderm. Based on the above report, the present invention was carried out for the purpose of isolating a novel marker gene specific for embryonic endoderm or pancreas. In the present invention, attention has been focused on CD55 as a novel candidate gene specific for embryonic endoderm or pancreas. From the above results, it was found that CD55 is expressed in embryonic endoderm and mesoderm regions in normal mouse embryos (FIG. 3). Thus, it is possible to identify embryonic endoderm by using CD55 in combination with E-cadherin that recognizes cells other than mesoderm, rather than using it as a single marker to identify embryonic endoderm. It became clear. With E-cadherin
Cxcr4 or CD55 combined with the results of analysis by flow cytometer, C
It was suggested that D55 is expressed in a narrower range than Cxcr4 and maintains high expression until late in culture (FIG. 4A). In addition, E-cadherin and CD55 double positive fractions were collected,
GFP fluorescence was observed by re-culturing.

Furthermore, from the results of analysis using a flow cytometer using Pdx1 / GFP mouse normal embryos on embryonic day 9.5, Cdcr4-negative cells are present in Pdx1-positive cells on embryonic day 9.5.
It was found that when extracted with in and Cxcr4, Pdx1-positive cells could be identified without being missed when extracted with E-cadherin and CD55 (FIG. 4B). Finally, E-cadherin and Cx
The results of microarray analysis of each of cr4, E-cadherin and CD55 double positive cells showed that CD55 is useful as a marker for identifying embryonic endoderm.

Claims (9)

Decay accelerating factor (DAF, CD55) gene, Tmem184a gene, Akr1c19 gene, 33
00001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hipk2 gene, Lass4
A method for detecting endoderm cells, intestinal cells or pancreatic cells, comprising detecting the expression of at least one gene selected from a gene, a Pbxip1 gene, a Pcbd1 gene, and a Pcdh1 gene. Disintegration accelerating factor (DAF, CD55) gene, Tmem184a gene, A in target cells
kr1c19 gene, 3300001A09Rik gene, Aebp2 gene, AI464131 gene, Foxp4 gene, Hi
Detecting the expression of at least one gene selected from the pk2 gene, the Lass4 gene, the Pbxip1 gene, the Pcbd1 gene, and the Pcdh1 gene, and detecting cells expressing the at least one gene as endoderm cells, intestinal cells or The method according to claim 1, wherein the method is identified as a pancreatic cell. The method for detecting endoderm cells, intestinal cells or pancreatic cells according to claim 1 or 2, comprising detecting expression of at least a decay accelerating factor (DAF, CD55) gene. Decay-accelerating factors (DAF, CD55) using antibodies against decay-accelerating factors (DAF, CD55)
55) The method according to claim 3, wherein the expression of the gene is detected. The method according to claim 3 or 4, further comprising detecting expression of the E-cadherin gene. Selecting cells that express the decay accelerating factor (DAF, CD55) gene,
A method for separating endoderm cells, intestinal cells or pancreatic cells. (A) preparing a cell sample containing endoderm cells, intestinal cells or pancreatic cells,
(B) adding an antibody against a decay accelerating factor (DAF, CD55) to the cell sample; and (c)
The method for separating endoderm cells, intestinal cells or pancreatic cells according to claim 6, comprising a step of separating cells bound with the antibody. The method for isolating endoderm cells, intestinal cells or pancreatic cells according to claim 6 or 7, wherein cells expressing the E-cadherin gene in addition to the decay accelerating factor (DAF, CD55) gene are selected. A reagent for detecting or separating endoderm cells, intestinal cells or pancreatic cells, comprising an antibody against a decay accelerating factor (DAF, CD55).