JP2008206510A - Method for obtaining intestinal stem/precursor cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of acquiring stem/progenitor cells of intestinal epithelial cells and culturing the same, and to provide a method of inducing differentiation from the stem/progenitor cells of the intestinal epithelial cells to cells for configuring intestinal epithelial tissue. <P>SOLUTION: The method of acquiring the stem/progenitor cells of the intestinal epithelial cells and culturing the same includes culturing an aggregate of intestinal cells, preferably, culturing the aggregate in a culture broth containing EGF. The method of inducing the differentiation from the stem/progenitor cells of the intestinal epithelial cells to the cells for configuring the intestinal epithelial tissue includes removing the EGF from a culture broth of the cells. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for acquiring and efficiently culturing stem / progenitor cells of intestinal epithelial cells, and a stem / progenitor cell obtained by the method. The present invention further relates to a method for inducing differentiation of intestinal epithelial cells from stem / progenitor cells into cells that constitute intestinal epithelial tissue.

  Intestinal epithelial cells are often used in drug absorption studies and other pharmaceutical studies. In the conventional culture system, intestinal epithelial cells have been slaughtered for several days, and it has been necessary to always prepare and use new cells. Therefore, although a cell line derived from a tumor tissue is used, it is necessary to use normal cells in order to obtain highly reliable data, and it is desired that normal intestinal epithelial cells can be used easily. In this case, in order to maintain cells for a long period of time in culture, it can be said that in addition to mature intestinal epithelial cells, stem / progenitor cells that always produce them need to be maintained in culture.

  The intestinal tract, the organ responsible for the digestion and absorption of food, contains stem cells (intestinal stem cells) located at the root of intestinal epithelial cells with different functional roles, and the intestinal epithelial cells are continuously supplied by dividing and maintaining the stem cells. Is done. Thus, although intestinal stem cells have a vigorous proliferative ability, it is extremely difficult to keep the ability artificially in culture, and there has been no successful example of intestinal stem cell culture so far. If growth and differentiation of intestinal stem cells can be controlled in culture, they can be used not only as basic research tools for intestinal stem cell systems but also as screening tools in drug discovery processes such as drug absorption and metabolism tests. Further, intestinal epithelial tissue that can be transplanted by tissue engineering can be supplied.

On the other hand, embryonic stem cells (ES cells), which are pluripotent stem cell lines derived from early embryos, have pluripotency, and thus cells are gradually differentiated by a normal culture method. Therefore, in order to efficiently increase ES cells, it is necessary to proliferate ES cells in an undifferentiated state. In the case of mouse ES cells, it is known that an undifferentiated state can be maintained by adding leukemia inhibitory factor (LIF) to the medium (Non-patent Document 1), but intestinal stem cells which are tissue stem cells are an alternative to LIF. There are no known additive factors that would In addition, ES cells are generally known to produce cell aggregates called embryoid bodies upon differentiation induction, but a method for controlling the differentiation of intestinal stem cells has not yet been established.
Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified primers.Nature. 1988 Dec 15; 336 (6200): 688-90.

  The object of the present invention is to obtain stem / progenitor cells of intestinal epithelial cells, and further to efficiently culture the stem / progenitor cells. Still another object of the present invention is to provide a method for inducing differentiation of intestinal epithelial cells from stem / progenitor cells into cells that constitute intestinal epithelial tissue.

  As a result of intensive studies in view of the above problems, the present inventors obtained cell aggregates by suspension culture of cells derived from the intestinal tract (intestinal cells), and the aggregates were converted to epidermal growth factor (EGF). It was found that stem / progenitor cells of the intestinal tract can be selectively obtained and proliferated by culturing in a culture solution containing Furthermore, the present invention was completed by successfully differentiating the obtained stem / progenitor cells into cells that construct intestinal epithelial tissue under appropriate differentiation-inducing conditions. That is, the present invention is as follows.

[1] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates.
[2] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates in a culture solution containing EGF.
[3] The method according to [1] or [2] above, wherein the aggregate is obtained by suspension culture of intestinal cells.
[4] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising the following steps.
(1) Step of collecting intestinal cells from intestinal tract (2) Step of preparing cell mass (aggregate) by suspension culture of intestinal cells (3) Aggregation of intestinal cells in a culture solution containing EGF Step of culturing (4) Step of recovering cells expressing stem / progenitor cell markers of intestinal epithelial cells as stem / progenitor cells of intestinal epithelial cells [5] Stem / progenitor cell marker of intestinal epithelial cells is Musashi-1 The method according to [4] above, wherein
[6] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells in a culture solution containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol and EGF.
[7] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells at a high density in a culture solution containing EGF.
[8] A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising the following steps.
(1) Step of collecting intestinal cells from intestinal tract (2) Step of culturing intestinal cells at high density in a culture solution containing EGF (3) Cells expressing stem / progenitor cell markers of intestinal epithelial cells [9] The method according to [8] above, wherein the stem / progenitor cell marker of intestinal epithelial cells is Musashi-1.
[10] Stem / progenitor cells of intestinal epithelial cells obtained by the method according to any one of [1] to [9] above.
[11] A method for culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates.
[12] A method for culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates in a culture solution containing EGF.
[13] The method according to [11] or [12] above, wherein the aggregate is obtained by suspension culture of intestinal cells.
[14] A method for culturing intestinal epithelial cell stem / progenitor cells, comprising culturing intestinal cells in a culture solution containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol, and EGF. .
[15] A method for culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells at a high density in a culture solution containing EGF.
[16] A method for inducing differentiation of intestinal epithelial cells from stem / progenitor cells into cells that constitute intestinal epithelial tissue, wherein EGF is removed from a culture solution of the stem / progenitor cells.
[17] The method described in [16] above, wherein the cells constituting the intestinal epithelial tissue are enteroendocrine cells or mucus-secreting cells.
[18] A medium for intestinal epithelial cell stem / progenitor cell culture, containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol, and EGF.

  The method for inducing differentiation of intestinal epithelial cells obtained by the present invention into stem / progenitor cells and cells that form intestinal epithelial tissues is useful as a tool for basic research for understanding the development and differentiation of intestinal tissues. In addition, it enables the supply of intestinal epithelial tissue that can be transplanted by tissue engineering. Furthermore, it can be expected that an intestinal epithelial cell derived from the stem / progenitor cell can be used to efficiently conduct a drug absorption experiment in the intestinal tract.

  Unless defined otherwise in the text, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned in this specification are herein incorporated by reference for the purpose of describing and disclosing constructs and methodologies described in, for example, publications that may be used in connection with the described invention. Incorporated into the book.

  The stem / progenitor cells of intestinal epithelial cells to be subjected to the acquisition method and culture method of the present invention are derived from any mammalian intestinal cells. Here, the “mammal” specifically includes humans, cows, horses, dogs, guinea pigs, mice, rats and the like. Humans are preferred, but animals often used as laboratory animals such as mice and rats are also preferred when considering use as research tools such as drug absorption experiments.

  The intestine can be roughly divided into two types, the small intestine and the large intestine. The small intestine is further divided into the duodenum, jejunum, and ileum from the oral side, and the large intestine is divided into the cecum, colon (ascending colon, transverse colon, descending colon, sigmoid colon), and rectum. The adult intestine is composed of a lumen surrounded by epithelial layers and supporting mesenchymal tissue that are extensively folded into crypts and villi. Intestinal epithelial cells are constantly born, and old ones are detached. This is because the intestinal epithelial cell is born with a stem cell regenerative system in the intestinal epithelial tissue. Stem cells are located towards the bottom of the crypt where the roots of the villi of the small intestine are depressed. In the fetus, the endoderm cell layer is destined to become the intestinal epithelium, and the stratified cell layer is converted into a differentiated polarized epithelial monolayer. Intestinal epithelial morphology and homeostasis are the result of a highly controlled balance between cell proliferation, migration, differentiation and apoptosis. The “intestinal epithelial cell stem / progenitor cell” that is the subject of the acquisition method and culture method of the present invention is a cell that can differentiate into intestinal epithelial cells and / or is destined to differentiate. “Stem cells” are cells that have both self-renewal and pluripotency, and are not completely consistent with “progenitor cells” that have a limited differentiation fate, but they differentiate into intestinal epithelial cells. In the present invention, therefore, they are referred to as “stem / progenitor cells” without particular distinction. In addition, “stem / progenitor cell of intestinal epithelial cell” may be referred to as “intestinal stem / progenitor cell” for convenience.

  In the present specification, “intestinal cell” is a general term for cells derived from the intestine that are collected from adults or fetuses and that can become intestinal epithelial cells or intestinal epithelial cells having division ability. Specifically, the intestinal tract, preferably the small intestine portion, is taken out and minced, and by physical means such as vibration, by chemical treatment using EGTA or EDTA, or proteases such as collagenase, trypsin, chymotrypsin, pepsin It can be obtained by dispersing cells by enzymatic treatment with (preferably collagenase). The above process may be performed alone or in combination with a plurality of processes. When a fetal intestinal tract is used, it is preferable to use one having a cylindrical intestine structure at a time when irregularities begin to occur in the intestinal epithelial tissue. For example, in the case of mice, the fetus is collected from the fetus on the 12th to 17th day, preferably 14.5 days. The “intestinal cell” used in the present invention may be collected from the intestinal tract as described above, but is derived from a commercially available intestinal tract containing intestinal epithelial cells having division ability or cells that can become intestinal epithelial cells. It may be a cell. Such commercially available cells derived from the intestinal tract are separated by ACBRI (Applied Cell Biology Research Institute) (ACBRI 519) and available in Japan from Sumitomo Dainippon Pharma Co., Ltd. (human intestinal epithelial cells, cat No. CS-ABI-519).

  The first feature of the present invention resides in causing intestinal cells to form aggregates and culturing them. By forming an aggregate, cells can be placed in an environment closer to the living body. Here, the “aggregate” is a mass of cells having a three-dimensional structure (spherical shape or bunch of grapes) formed by collecting a large number of cells. For example, as will be described later, when seeded in a low-adhesion 96-well round bottom dish at a concentration of 20 to 400,000 cells / well, about 10 to 20 aggregates having a diameter of about 200 to 500 μm are formed. A sheet-like structure of cells is seen on the outermost part of the aggregate. The number and size of the cells constituting the aggregate can vary depending on the state of the cells and the culture conditions as long as an environment closer to the living body can be provided.

  Specifically, intestinal cell aggregates can be prepared as follows. First, intestinal cells are suspended in a suitable medium on a low-adhesion culture vessel. For example, when a 96-well round bottom dish is used as a low-adhesion culture vessel, 200 to 400,000 cells are seeded per well, and the formed aggregates are transferred to a low-adhesion 6 cm dish or the like. Incubate in an appropriate medium at 37 ° C. for 2 to 10 days, preferably 7 days. Examples of the low-adhesion culture vessel include those obtained by subjecting a culture vessel used in this field to a treatment that makes the adhesiveness low. Examples of the culture container include a culture dish, a culture flask, and a rotary culture instrument (such as a spinner flask). As the treatment for achieving low adhesion, a treatment for suppressing the adhesion of proteins and cells by covalently bonding hydrogel (coating) is used. Moreover, what is marketed can also be used.

Intestinal cells derived from adults are unlikely to form aggregates, but a similar effect (an effect of creating an environment close to the living body) can be obtained by culturing the intestinal cells at a high density. For example, a Petri dish having a diameter of 6 cm (amount of medium: 3.5 mL) (a normal Petri dish without treatment for adherent cells (but not having a low adhesion treatment); hereinafter also simply referred to as a normal Petri dish) ), 1-5 × 10 ⁶ cells are seeded and cultured at 37 ° C. for 1-5 days, preferably 3 days, in an appropriate medium to achieve high-density culture. it can.

  The medium that can be used in the above-described aggregate culture and high-density culture is not particularly limited as long as the intestinal cells can survive and can maintain the state in which the induction of differentiation and apoptosis is suppressed. Based on a cell culture medium (Dulbecco's modified Eagle medium, MEM, RPMI 1640 medium, Ham F-12 medium, etc.) usually used in the art, supplemented with various components as necessary is used. In particular, it is preferable to supplement components such as serum, antibiotics and growth factors.

Suitable components to be included in the medium include 5-20%, preferably 10% serum, 0.5-2 μg / mL, preferably 1 μg / mL insulin, 1 × 10 ⁻⁸ to 1 × 10 ^−6. mol / L, preferably 1 × 10 ⁻⁷ mol / L dexamethasone, 5-15 mmol / L, preferably 10 mmol / L nicotinamide, 1-5 mmol / L, preferably 2 mmol / L L-glutamine, Examples include 100 μmol / L, preferably 50 μmol / L β-mercaptoethanol, penicillin / streptomycin, and the like. Both are commercially available. As the serum, horse-derived or bovine-derived serum is used, and fetal serum is preferable. As the insulin, any of insulin produced from bovine and porcine pancreas and insulin produced by gene recombination technology can be used, but recombinant insulin is preferably used from the viewpoint of stable quality and supply.

  The second feature of the present invention is that intestinal cell culture is performed in the presence of EGF (epidermal growth factor) in order to selectively proliferate, obtain or culture intestinal stem / progenitor cells.

  Ehen was isolated and identified from the mouse submandibular gland as a substance having an early eye-cleaving action of the mouse fetus by Cohen et al., And then human EGF was discovered from urine (S. Cohen and G. Carpenteret al., Proc. Natl). Acad. Sci. USA., 72, 1317, 1975; H. Gregory, Nature, 257, 325, 1975). The gene structure has also been elucidated in detail (G. I. Bell et al., Nucleic Acids Res., 14, 8427, 1986). EGF is a protein of 6045 Da consisting of 53 amino acid residues and three intramolecular disulfide bonds, and binds to EGFR (epidermal growth factor receptor) existing on the cell surface as a ligand, which is important for the regulation of cell growth and proliferation Play an important role. The origin of EGF used in the present invention is not particularly limited as long as it makes it possible to selectively proliferate, obtain or culture intestinal stem / progenitor cells. It may be produced by synthesis or semi-synthesis based on information such as sequence. It may be commercially available. It is preferable to use mouse EGF for mouse stem / progenitor cells, and human EGF for human stem / progenitor cells, but it is not necessary to match animal species. EGF may be modified as long as intestinal stem / progenitor cells can be selectively proliferated, obtained or cultured. Such modifications include mutations, insertions, deletions and substitutions of one or more nucleotides or amino acids. Modified EGF can be prepared by means commonly practiced in the art. For example, EGF DNA is artificially modified by site-directed mutations such as a deletion mutant production method using exonuclease, a cassette mutation method, etc., and the modified EGF DNA is used. A desired protein can be prepared.

  EGF is preferably included in the medium continuously throughout the process of culturing intestinal cells in order to obtain intestinal stem / progenitor cells, but especially during the formation of aggregates and subsequent long-term culture and proliferation. Essentially included in the media. The concentration of EGF in the medium is 20 to 40 ng / mL, preferably about 20 ng / mL.

By culturing in a culture medium containing EGF, intestinal stem / progenitor cells can also be obtained from commercially available intestinal cells, for example, human small intestinal epithelial cells (Cat No. CS-ABI-519) described above. When commercially available human small intestinal epithelial cells are used, a culture solution containing EGF, particularly 20 to 40 ng / mL, preferably 20 ng / mL, is not required without forming aggregates or culturing at high density. In addition to mL of EGF, 5-20%, preferably 10% serum, 0.5-2 μg / mL, preferably 1 μg / mL insulin, 1 × 10 ⁻⁸ to 1 × 10 ⁻⁶ mol / L, Preferably 1 × 10 ⁻⁷ mol / L dexamethasone, 5-15 mmol / L, preferably 10 mmol / L nicotinamide, 1-5 mmol / L, preferably 2 mmol / L L-glutamine, 10-100 μmol / L, Preferably, intestinal stem / progenitor cells can be obtained by subculture using a culture solution containing 50 μmol / L β-mercaptoethanol.

10% fetal bovine serum, insulin (1 μg / mL), dexamethasone (1 × 10 ⁻⁷ mol / L), nicotinamide (10 mmol / L), L-glutamine (2 mmol / L), β-mercaptoethanol (50 μmol / L) ), Dulbecco's modified medium-Ham F12 mixed medium (1: 1) supplemented with penicillin (100 U / mL) + streptomycin (100 μg / mL) and EGF (20 ng / mL) is a medium for culturing intestinal cells (1: 1). A preferred example of the culture medium).

  The cell aggregate obtained above is replated in a normal Petri dish, and cultured at 37 ° C. in a medium (culture solution) containing EGF for 2 to 6 months, preferably about 3 months. Since cells derived from the adult intestine are difficult to form aggregates, cells are aggregated by high-density culture, which is equivalent to the aggregated state. Cells derived from the adult intestine are cultured at a high density in a culture solution containing EGF at 37 ° C. for 6 to 12 months, preferably about 8 months.

  When the aggregate culture or high-density culture is continued for a long period of time, many epithelial cell-like cells can be seen compared to fibroblast-like cells. If fibroblast-like cells remain, only fibroblast-like cells are killed by treatment with 1 to 10 μg / mL, preferably about 4 μg / mL of monoiodoacetic acid for about 3 to 5 hours. Can be made. If growth of epithelial cell-like cells is observed, the cells are then passaged in a cell culture vessel and used for analysis or stored frozen until use.

  Cells obtained by long-term culture of aggregates or high-density culture, or obtained by subculturing commercially available human small intestinal epithelial cells such as Cat No. CS-ABI-519 in a culture solution containing EGF Whether the cells to be obtained are intestinal epithelial cells or intestinal stem / progenitor cells can be determined by examining the presence or absence of marker protein expression in those cells. In the present specification, unless otherwise specified, “marker” means each of a group consisting of a series of proteins or genes encoding the same that exhibit an expression pattern specific to intestinal epithelial cells or intestinal stem / progenitor cells. To do. Such a marker protein may have different amino acid sequences depending on the type of mammal, but in the present invention, such a protein may be used as a marker protein as long as the expression pattern in the target cell is the same. it can. Examples of epithelial cell marker proteins include E-cadherin and Zo-1 (Laprise P, et al., J Biol Chem. 2004 Mar 12; 279 (11): 10157-66. Epub 2003 Dec 29. Escaffit F, et al., Exp Cell Res. 2005 Jan 15; 302 (2): 206-20.), A marker protein for intestinal epithelial cells is Villin (Maunoury, R. et al., EMBO). J. 7, 3321-3329 (1988).). Marker proteins for intestinal stem / progenitor cells include Musashi-1 (Potten CS, et al., Differentiation. 2003 Jan; 71 (1): 28-41.), Math-1 (Yang Q, et al., Science 294: 2155-2158), Neurogenin 3 (Ngn3, Jenny M, et al., EMBO J. 2002 Dec 2; 21 (23): 6338-47.).

  E-cadherin is a calcium-dependent intercellular adhesion molecule expressed on epithelial cells, and plays an important role in the formation and maintenance of epithelial tissues by homophilic adhesion that binds to each other. Zo-1 is known as a protein behind the tight junction (TJ), which is essential for epithelial cells and endothelial cells, and is involved in the formation of the adhesion device and the regulation of cell proliferation through it. It is considered a protein. Villin is a component of microvilli of small intestinal epithelial cells and has a function of bundling actin together with fibrin. Villin is found only in microvilli. Musashi-1 is an RNA-binding protein expressed by neural progenitor cells, and is reported to be expressed in intestinal stem / progenitor cells. Math-1 is a bHLH type transcription factor and is known to be necessary for differentiation into a secretory cell line in the mouse intestine, and is expressed in intestinal stem / progenitor cells. Similarly, Ngn3, which is a bHLH transcription factor, is important for the development of pancreatic endocrine cells and gastrointestinal tissues, and is expressed in intestinal stem / progenitor cells.

  In the present invention, confirmation of being an intestinal epithelial cell or an intestinal stem / progenitor cell is performed by specifically expressing the expression of these marker proteins and / or genes encoding them with respect to each marker protein or gene encoding the same. It can carry out by including the process of analyzing using the substance which has this. In the present specification, the method for the term “use” is not particularly limited, and specifically, for example, when a substance having specific affinity for the marker protein is used, the method may be used in combination with the marker protein antibody. Examples include a method using an antigen-antibody reaction, and a method using a hybridization reaction in the case of using a substance having specific affinity for a gene encoding a marker protein.

Examples of the substance having specific affinity for the marker protein include an antibody having a specific affinity for the protein or a fragment thereof, and the specific affinity specifically recognizes the protein by antigen-antibody reaction. The ability to combine. The antibody or a fragment thereof is not particularly limited as long as it can specifically bind to the protein, and may be a polyclonal antibody, a monoclonal antibody, or a functional fragment thereof. These antibodies or functional fragments thereof can be produced by methods generally performed in the art. For example, in the case of using a polyclonal antibody, there is a method in which the protein is injected subcutaneously into the back of the animal or into the abdominal cavity or vein of an animal such as a mouse or rabbit, and after waiting for the antibody titer to rise, antiserum is collected. In the case of using a monoclonal antibody, there is a method of preparing a hybridoma according to a conventional method and collecting the secreted solution. As a method for producing an antibody fragment, a method in which a cloned antibody gene fragment is expressed in a microorganism or the like is often used. The purity of the antibody, antibody fragment, etc. is not particularly limited as long as it retains specific affinity for the protein. These antibodies or fragments thereof may be labeled with a fluorescent substance, an enzyme, a radioisotope, or the like.
Further, these may be commercially available.

  Examples of the substance having specific affinity for the gene encoding the marker protein include an oligo or polynucleotide probe having specific affinity for the gene (hereinafter also simply referred to as a probe for convenience), and an oligo or polynucleotide primer pair ( The specific affinity means the property of hybridizing only to the gene of interest, and therefore is completely complementary to all or part of the gene, Alternatively, one to several mismatches may be included within the range satisfying the above properties. The probe and primer pair are not particularly limited as long as they have specific affinity for the gene. For example, all or a part of the base sequence of the gene, as well as oligo or polynucleotide containing their complementary sequences, etc. The selection is appropriately made according to the form of the gene to be detected. The origin of the oligo or polynucleotide is not particularly limited as long as it has a specific affinity for the gene, and even if it is synthesized, a necessary part is cut out from the gene and usually performed. It may be purified by. These oligos or polynucleotides may be labeled with a fluorescent substance, an enzyme, a radioisotope, or the like.

  Intestinal stem / progenitor cells have β-catenin transcription activity (He XC, et al., Nat Genet. 2004 Oct; 36 (10): 1117-21.). It can also be confirmed that it is a progenitor cell. For example, a method in which a construct (TOPGAL) in which a β-galactosidase gene is driven by TCF / b-catenin binding site is introduced into cells and positive cells are detected by X-gal staining can be used.

In the present invention, intestinal stem / progenitor cells can be selectively proliferated by culturing intestinal cell aggregates (or cells cultured at high density) in a culture solution containing EGF. Progenitor cells can be obtained efficiently. Furthermore, the self-renewal ability and pluripotency of the stem / progenitor cells are stably maintained for a long time. The fact that the intestinal stem / progenitor cells obtained by the method of the present invention maintain the self-replicating ability is based on the use of various known techniques capable of measuring cell proliferation, such as measurement of the number of cells and observation under an optical microscope. Thus, it can be easily determined by observing the proliferation of intestinal stem / progenitor cells in the culture vessel. Whether or not the intestinal stem / progenitor cells maintain pluripotency can be determined by whether or not a plurality of types of cells that construct intestinal epithelial tissue can be obtained when the stem / progenitor cells are induced to differentiate. Examples of cells that constitute intestinal epithelial tissues include enteroendocrine cells (gastrin-secreting cells, serotonin-secreting cells, chromogranin A-secreting cells, secretin-secreting cells, somatostatin-secreting cells, GIP (gastric inhibitory polypeptide) -secreting cells, and CCK (cholesterokinin) -secreting cells. Etc.) and mucus-secreting cells (goblet cells, panate cells, etc.), intestinal cells responsible for nutrient absorption. Determine whether the cell is the target cell by detecting the component specifically expressed in each cell using a substance with specific affinity for the component, or by directly analyzing the secretory component can do. Examples of the “substance with specific affinity” include an antibody having a specific affinity for each secretory component or a fragment thereof, and an oligo or polynucleotide probe having a specific affinity for a gene encoding each secretory component. It is done. Details of the “antibody or fragment thereof” and “oligo or polynucleotide probe” are as described above.
Goblet cells can also be determined by staining methods such as PAS staining and Alcian blue staining.

  Induction of differentiation of intestinal stem / progenitor cells into cells that constitute intestinal epithelial tissue can be easily carried out by removing EGF from the culture medium. Specifically, the culture can be performed by exchanging the culture medium containing EGF with a culture liquid not containing EGF. The induction of cell differentiation can be confirmed in about one week after medium replacement, but the period required for differentiation induction can vary depending on the state of the cell, the type of cell to be differentiated, and the like.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail according to an Example, these Examples do not limit the scope of the present invention at all. In addition, the reagents, devices, and materials used in the present invention are commercially available unless otherwise specified.

Example 1: Acquisition of intestinal stem / progenitor cells derived from fetal intestinal tract The intestinal tract was taken out from an embryonic day 14.5 mouse fetus and finely cut with a scalpel to obtain an intestinal tissue piece. The obtained piece of intestinal tissue was placed in a Hank's solution containing 1 mM EGTA, shaken in a water bath at 37 ° C. for 10 minutes, and then washed once with PBS by centrifugation. After centrifugation, intestinal tissue pieces were added with 1 mg / ml collagenase solution (dissolved in Hank's solution containing 5 mM CaCl ₂ ) and shaken in a 37 ° C. water bath for 15-20 minutes. After light pipetting, undigested tissue was removed through a mesh to obtain a cell dispersion (intestinal cells derived from mouse fetus).
The prepared mouse fetal intestinal cells were seeded in a low-adhesion 96-well round bottom dish (Low Cell Binding Plates, cat no: 145399; Nunc) at a concentration of 2 to 4 × 10 ⁵ cells / well, and Dulbecco's modified eagle Medium containing medium and F-12 medium at a ratio of 1: 1 [10% fetal bovine serum, 1 μg / mL insulin, 1 × 10 ⁻⁷ mol / L dexamethasone, 10 mmol / L nicotinamide, 2 mmol / L L -Containing glutamine, 50 μmmol / L β-mercaptoethanol, penicillin / streptomycin (100 U / mL (penicillin) +100 μg / mL (streptomycin)), 20 ng / mL EGF (Sigma, E9644)] at 37 ° C., 5% CO ₂ presence, an incubator In the culture. Hereinafter, this medium containing EGF is also referred to as EGF (+) medium or intestinal stem / progenitor cell culture medium.
Two days later, mouse fetal intestinal cells seeded in a 96-well round bottom dish gathered to form a cell mass (FIG. 1a). Thereafter, the formed cell mass is transferred to a low-adhesive 6 cm dish or the like, and further cultured for about 5 days. Then, the cell mass is transferred to a Petri dish (BD, 35-1007), and the above-mentioned EGF (+) medium is used. The culture was continued in an incubator at 37 ° C. in the presence of 5% CO ₂ (FIG. 1b). The Petri dish used here is an ordinary Petri dish that has not been subjected to treatment for adherent cells (but not low adhesion treatment). On the other hand, without using a low-adhesion dish, the cell dispersion was immediately seeded on an adhesive petri dish, and when cultured in the EGF (+) medium, only fibroblast-like cells remained in the dish (Fig. 1c).
When the aggregates were transferred to ordinary petri dishes and cultured for about 3 months, epithelial cell-like cells became more common than fibroblast-like cells. If fibroblast-like cells remain, the fibroblast-like cells can be killed by treatment with monoiodoacetic acid (4 μg / mL) for about 3 to 5 hours.
The obtained epithelial cell-like cells were passaged in a cell culture dish (FIG. 2) and used for analysis (described later) and cryopreservation.
As will be apparent from the examples described later, this epithelial cell-like cell contains stem / progenitor cells having self-replicating ability and pluripotency. Therefore, for convenience, FIP (Fetal Intestine progenitor) cells are used. Both will be referred to hereinafter.

Example 2: Acquisition of intestinal stem / progenitor cells derived from adult intestinal tract The inside of the intestinal tract taken out of an adult mouse was washed with PBS using a 23G needle, and then the scissors were put into the intestinal tract and the tube was opened. And further cut into 5 mm squares with scissors (intestinal tissue pieces). Using the obtained intestinal tissue piece, a cell dispersion was obtained in the same manner as in Example 1 by EGTA, collagenase and pipetting (adult mouse intestinal cells).
Since intestinal cells derived from adults are difficult to form cell clusters, they were aggregated in a single layer and cultured. That is, 1-5 × 10 ⁶ cells are seeded in a Petri dish having a diameter of 6 cm (ordinary petri dish not subjected to treatment for adherent cells (but not subjected to low adhesion treatment)), and high-density culture is performed. went. Using a culture solution having the same composition as in Example 1, the cells were cultured in an incubator at 37 ° C. in the presence of 5% CO ₂ .
When high-density culture was continued for about 8 months, many epithelial cell-like cells came to be seen with respect to fibroblast-like cells. If fibroblast-like cells remain, the fibroblast-like cells can be killed by treatment with monoiodoacetic acid (4 μg / mL) for about 3 to 5 hours.
The obtained epithelial cell-like cells were subcultured in a cell culture dish and used for analysis and cryopreservation.

Example 3: Proliferation ability The proliferation ability of FIP cells was examined. FIP cells were cultured in culture medium containing EGF at various concentrations (0 ng / mL to 80 ng / mL). The culture solution has the same composition as the culture solution used in Example 1 except that the concentration of EGF is different. A cell cultured for 2 days in a culture solution not containing EGF (FIG. 3a) and a cell cultured for 2 days in a culture solution containing EGF at a concentration of 20 ng / mL are shown (FIG. 3b). In addition, cells seeded in a 12-well culture plate and cultured for 1 day in a culture solution containing EGF at various concentrations were peeled off with trypsin-EDTA, and the number was counted with a hemocytometer (FIG. 3c).
Proliferation of intestinal cell-derived epithelial cell-like cells was shown to be EGF-dependent.

Example 4 Growth and Apoptosis FIP cells were cultured in EGF (+) medium (EGF concentration 20 ng / mL) on a cell culture dish for 2 days (FIG. 4a). Two sets of the same state were prepared. After culturing in the presence of EGF for 2 days, one medium was replaced with the same EGF (+) medium and further cultured for 1 day (FIG. 4b), and the other medium was replaced with a medium containing no EGF and further 1 Cultured for days (Fig. 4c). In the presence of EGF, the cells continued to grow, but in the absence of EGF, cell growth was suppressed and cells that died were confirmed. Changes in the proliferation ability of the cells in a series of processes were confirmed by BrdU immunostaining. Double staining was performed using DAPI, a nuclear stain (FIGS. 5a-f).
After culturing in the presence of EGF for 2 days, one medium was replaced with a medium containing no EGF and further cultured for 1 day, and the other medium was replaced with the same EGF (+) medium and further cultured for 1 day. Thereafter, BrdU (10 μM) (Sigma, B9285) was added to the medium, and the proportion of proliferating cells that incorporated BrdU after 1.5 hours was analyzed by flow cytometry and immunostaining. Flow cytometry analysis was performed using FACS Aria (BD) after labeling cells according to the manual of BrdU Flow Kit (BD, 552598). At this time, the gate was set using negative control as an index. BrdU immunostaining was performed using BrdU Flow Kit reagent and anti-BrdU antibody (x50) (BD, 347580), donkey anti-mouse IgG antibody conjugated with Cy3 (x200) (Jackson ImmunoResearch Laboratories, cat no. 715-165). -150), and DAPI (1 μg / mL) (Sigma, D9542), and observed with a fluorescence microscope. For cells cultured in the absence of EGF, BrdU incorporation was markedly reduced, indicating that the proliferative ability was reduced (FIGS. 5a-f).
In addition, when EGF was removed from the culture medium, dead cells were confirmed, and apoptosis induction in the absence of EGF was examined (FIGS. 5g to h).
Cells were cultured as in BrdU analysis. After the cells were collected from the dish, the cells were washed once with cold PBS, and a cell suspension was prepared with annexin V binding buffer (1 ×) (BD, 556454). 7-AAD (5 μL; BD, 559925) and APC-conjugated annexin V (5 μL; BD, 550474) were added thereto and reacted at room temperature for 15 minutes (shielded). Stained cells were analyzed using FACS Aria (BD). The gate was set using negative control as an index. Annexin V is a phospholipid binding protein. Apoptotic cells become exposed to phosphatidylserine on the surface of the cell membrane, and annexin V binds thereto. Annexin V therefore has a strong affinity for apoptotic cells. In the absence of EGF, cells to which annexin V binds markedly increased, indicating that apoptosis was induced (FIGS. 5g to h).
FIP cells actively divided and proliferated in the presence of EGF, but under the culture conditions not containing EGF, the proliferative activity was remarkably decreased and the number of cells causing apoptosis increased.

Example 5: Expression of marker proteins Expression of various marker proteins in FIP cells was examined. The expression of each marker protein was examined by immunostaining using the antibody of each protein. In order to show the presence of cells, double staining was performed with DAPI, a nuclear stain.
Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) (in PBS) for 5 minutes at room temperature. Subsequently, it was treated with 25% acetone (in methanol) for 1 minute at room temperature. After fixation, the cells were washed with PBS containing 0.05% polyoxyethylene (20) sorbitan monolaurate (Tween 20) (Wako) (PBS-Tween), followed by 0.2% Triton X-100. Treated with (Sigma) for 1 hour at room temperature. Nonspecific binding was blocked by treatment with 10% donkey serum (Sigma, D9663) for 1 hour at room temperature. Fixed cells were incubated with primary antibody in a humid chamber for 16 hours at 4 ° C. The cells were washed with PBS-Tween 20, blocked, and further incubated with the secondary antibody for 4 hours at 4 ° C. After incubation with DAPI for 5 minutes at room temperature and washing, the cells were observed with a fluorescence microscope.
The antibodies used are as follows.
(Primary antibody)
Anti-E-cadherin antibody: BD, 610181 (secondary antibody, for mouse); x100
Anti-Zo-1 antibody: Zymed, 61-7300 (secondary antibody, for rabbit); x100
Anti-villin antibody: Santa Cruz, sc-7672 (secondary antibody, for goat); × 10
Anti-Musashi-1 antibody: Chemicon, AB5977 (secondary antibody, for rabbits); × 100
(Secondary antibody)
For mice: Donkey anti-mouse IgG conjugated with Alexa 488 (Molecular Probes, A21202); × 200
For rabbits: Donkey anti-rabbit IgG conjugated with Alexa 488 (Molecular Probes, A21206); × 200
For goats: donkey anti-goat IgG conjugated with Alexa 488 (Molecular Probes, A11055); × 200
FIP cells expressed E-cadherin and Zo-1, which are epithelial markers, and villin, an intestinal epithelial cell marker, in all cells. Some cells expressed Musashi-1, which is considered an intestinal stem / progenitor cell marker (FIG. 6).

Example 6: Activation of β-catenin transcription A construct (TOPGAL) in which a β-galactosidase gene is driven by TCF / b-catenin binding site was introduced into cells using Lipofectamine 2000 (Invitrogen, 11668), and X-gal staining Detected positive cells. Specifically, the procedure was as follows.
(1) Wash cells with PBS (× 2)
(2) Fixing solution (2% PFA / 0.2% glutaraldehyde (GA) /0.05% NP-40 / PBS) is added (1.5 to 2 mL per well of a 6-well plate)
(3) Leave at room temperature for 1 minute (4) Wash with PBS (× 2)
(5) Add X-gal solution (1.5-2 mL per well of 6-well plate)
(6) Leave in an incubator at 37 ° C (at least 3 hours to overnight)
(7) Stop the reaction by washing with PBS (x2) (8) Observation (photographing), storage at 4 ° C in PBS (preparation of fixative)
Each component was mixed at the following ratio to prepare a fixative.
1% of 20% PFA / PBS (stored at -20 ° C)
25% GA 80μL (stored at -20 ℃)
5% NP-40 80μL (room temperature storage)
PBS 9mL (room temperature storage)
(Preparation of X-gal solution)
Each component was mixed in the following ratio to prepare an X-gal solution.
500 mM K ₃ Fe (CN) ₆ 50 μl (room temperature storage, shading storage)
500 mM K ₄ Fe (CN) ₆ 50 μl (room temperature storage, shading storage)
10 μl of 1M MgCl ₂ (stored at room temperature)
20 mg / ml X-gal 250 μl (stored at −20 ° C., protected from light)
4.7 ml of PBS (stored at room temperature)
X-gal positive cells were detected, confirming the transcriptional activation of β-catenin (FIG. 7).

Example 7: Induction of differentiation into cells that construct intestinal epithelial tissue FIP cells were cultured until they became confluent. When EGF was removed from the confluent state, there were cells that survived among the cells undergoing apoptosis. When these surviving cells were continuously cultured in the absence of EGF, cells showing the characteristics of differentiated intestinal epithelial cells appeared (FIG. 8).
When cells in this state were stained with PAS, a large number of cells that stained reddish purple appeared in cells that had been cultured in the absence of EGF (FIG. 9). PAS staining involves two reactions in which 1,2-glycol groups contained in carbohydrates are oxidized with periodic acid, and the resulting two aldehyde groups combine with one Schiff reagent molecule to form a red-purple compound. It is used to detect glycogen, neutral mucus polysaccharides, glycoproteins, mucus proteins, glycolipids, and the like. In intestinal epithelial tissue, it is known to dye goblet cells, which are one type of mucus-secreting cells. Specifically, the procedure was as follows. In addition, about each reagent, the reagent normally used when performing PAS dyeing | staining in this field | area can be used.
(1) Wash cells with PBS (× 1)
(2) Treated with 0.5% periodic acid for 7 minutes at room temperature (3) Washed with Milli-Q water (× 5)
(4) Treatment with Schiff reagent at room temperature for 7 to 10 minutes (5) Treatment with aqueous sulfite (treatment at room temperature for 1 minute is repeated 3 times)
(6) Wash with Milli-Q water (× 5)
(7) Treatment with hematoxylene (at room temperature for 1 minute)
(8) Wash with Milli-Q water (× 5)
In addition, immunostaining was performed using an antibody against a protein secreted and expressed in each intestinal epithelial tissue, and the expression state of the protein was analyzed. The antibodies of each protein used are as follows. In order to show the presence of cells, double staining was performed with DAPI, a nuclear stain.
(Primary antibody)
Anti-serotonin antibody: Immunostar, 20080 (secondary antibody, for rabbit); × 10
Anti-chromogranin A antibody: Santa Cruz, sc-1488 (secondary antibody, for goat); × 10
Anti-gastrin antibody: Santa Cruz, sc-7783 (secondary antibody, for goats); × 10
Anti-secretin antibody: Chemicon, AB981 (secondary antibody, for rabbits); x100
Anti-somatostatin antibody: AFFINITI, SA1268 (secondary antibody, for rabbit); × 10
Anti-GIP antibody: Chemicon, AB953 (secondary antibody, for rabbit); × 10
Anti-lysozyme antibody: Dako, A0099 (secondary antibody, for rabbit); x100
Anti-synaptophysin antibody: Dako, A0010 (secondary antibody, for rabbit); × 10
(Secondary antibody)
For rabbits: Donkey anti-rabbit IgG conjugated with Alexa 488 (Molecular Probes, A21206); × 200
For goats: donkey anti-goat IgG conjugated with Alexa 488 (Molecular Probes, A11055); × 200
As a result, when cultured in the absence of EGF, cells expressing serotonin, chromogranin A, gastrin, secretin, somatostatin, and GIP, which are characteristic of enteroendocrine cells, were detected, and Paneth cells, which are one type of mucus-secreting cells, were detected. Cells expressing characteristic lysozyme and synaptophysin were detected. From these results, it is understood that differentiation into intestinal endocrine cells was induced by removing EGF from the culture solution of FIP cells.

Example 8: Analysis by RT-PCR From the above examples, it was shown that FIP cells contain intestinal stem / progenitor cells, and the stem / progenitor cells continued to proliferate in the presence of EGF. It was found that, by removing EGF from the culture solution, the cells caused apoptosis and differentiated into cells constituting various intestinal epithelial tissues (schematic diagram is shown in FIG. 10).
Using FIP cells and the intestinal tissues of mice at each developmental stage (embryonic day 14.5, newborn, adult), the expression profile of genes considered to be involved in cell differentiation of the intestinal tract was analyzed by RT-PCR.
FIP cells were cultured in EGF (+) medium, and when they became confluent, the medium was replaced with a medium containing no EGF, and then cultured for 1 week. RNA was extracted from the cells (RNeasy Mini Kit; QIAGEN), and cDNA was obtained by reverse transcriptase reaction (SuperScript III). In RNA extraction from intestinal tissue, the tissue was disrupted using a homogenizer (polytron), and extraction and reverse transcriptase reaction were performed in the same manner. Subsequently, PCR was performed using these cDNAs, and the expression of various differentiation markers was analyzed.
The target markers and their primers are shown in Table 1.

  PCR cycle is 95 ° C. (4 minutes) → [94 ° C. (1 minute) → 56 ° C. (1 minute) → 72 ° C. (1 minute)] × 45 cycles → 72 ° C. (10 minutes). Nucleic acids produced by PCR were developed on a 2% agarose gel and analyzed. The results are shown in FIG.

Example 9: Analysis at the level of a single cell Since the experiments in Examples 3 to 7 described above were analyzes on a heterogeneous cell population, in order to strictly analyze the intestinal stem / progenitor cells, We considered that analysis at the single cell level was necessary. Therefore, FIP cells were clone-sorted using flow cytometry, and single cell culture was attempted in each well of a 96-well dish. Using one of the clones that had proliferated, the proliferation ability, the expression of the marker protein, and the induction of differentiation were examined in the same manner.
As a result, E-cadherin, Zo-1, and villin were also expressed in all cells for cells grown from a single colony, and Musashi-1 was expressed for some of the colonies. Furthermore, it showed EGF-dependent proliferation activity and cell death suppression, and also expressed multiple intestinal epithelial cell markers in the absence of EGF. Thus, it was confirmed that FIP cells include intestinal stem / progenitor cells by proliferating ability and pluripotency even in single cell level analysis.
From the above results, it was shown that the FIP cell population includes stem / progenitor cells having self-renewal ability and pluripotency and can be stably maintained for a long time. By using the method of the present invention, it has become possible to selectively proliferate intestinal epithelial stem / progenitor cells. By such selective growth, intestinal stem / progenitor cells can be efficiently obtained and cultured.

Example 10: Acquisition of intestinal stem / progenitor cells from human small intestinal epithelial cells In this example, human small intestinal epithelial cells (Cat No. CS-) separated by ACBRI and supplied in Japan from Dainippon Sumitomo Pharma Co., Ltd. ABI-519) was used. The cells were seeded and cultured in a culture dish according to the method described in the attached data sheet (FIG. 12a). As the culture dish, a type IV collagen-coated dish was used. As a culture solution, a medium described in the data sheet and attached thereto, that is, a CS-2.0 basal medium added with an additive (hereinafter, CS-2.0 + supplement) was used. The treatment was performed in the presence of 20 ng / mL EGF.
When cultured with CS-2.0 + supplement, the cells did not adhere to the culture dish when they were subcultured twice, and all cells were killed (FIGS. 12c and e).
Then, when subcultured in the same manner using the medium (intestinal stem / progenitor cell culture medium) containing EGF used for culturing intestinal cells derived from mouse fetuses prepared in Example 1, Cells that could adhere to the culture dish and maintain growth even after repeated generations were obtained (FIGS. 12b and 12d).
Since these cells were considered to be intestinal stem / progenitor cells contained in the purchased human epithelial cell population, they are hereinafter also referred to as hAIP (human adult intestine progenitor) cells for convenience.

Example 11: Marker protein expression and β-catenin transcriptional activation Marker protein expression was examined in the same manner as in Example 5 for hAIP cells.

hAIP cells expressed E-cadherin and Zo-1, which are epithelial markers, and villin, an intestinal epithelial cell marker, in all cells. Some cells expressed Musashi-1, which is considered to be an intestinal stem / progenitor cell marker (FIG. 13, upper panel).
Further, for hAIP cells, the presence or absence of β-catenin transcriptional activation was confirmed in the same manner as in Example 6. X-gal positive cells were detected, and β-catenin transcriptional activation was confirmed (FIG. 13, lower panel).

Example 12: Proliferative ability The proliferation ability of hAIP cells was examined in the same manner as in Example 3. The cells were cultured for 4 days in the EGF (+) medium used in Example 1 and the EGF (-) medium having the same composition except that EGF was not included. In the presence of EGF, hAIP cells actively divide and proliferate, but under the culture conditions not containing EGF, the proliferation activity decreased and the number of cells causing apoptosis increased (FIG. 14).
From this result, it can be seen that EGF promotes self-renewal of hAIP cells and suppresses cell death, thereby enabling maintenance in culture.

Example 13: Proliferation and apoptosis hAIP cells were cultured in EGF (+) medium (EGF concentration 20 ng / mL) for 4 days. Two sets of the same state were prepared. After culturing for 4 days in the presence of EGF, one medium was replaced with the same EGF (+) medium and further cultured for 1 day (FIG. 15A, left), and the other medium was replaced with a medium containing no EGF. The cells were further cultured for 1 day (FIG. 15A, right). In the presence of EGF, the cells continued to grow, but in the absence of EGF, cell growth was suppressed and cells that died were confirmed. Changes in the proliferation ability of the cells in a series of processes were confirmed by BrdU immunostaining. Double staining was performed using DAPI, which is a nuclear stain (FIG. 15A).
After culturing for 4 days in the presence of EGF, one medium was replaced with a medium containing no EGF and further cultured for 1 day, and the other medium was replaced with the same EGF (+) medium and further cultured for 1 day. Thereafter, BrdU (10 μM) (Sigma, B9285) was added to the medium, and the proportion of proliferating cells that incorporated BrdU after 1.5 hours was analyzed by flow cytometry and immunostaining. Flow cytometry analysis was performed according to the method described in Example 4 (FIG. 15B, left). Cells were cultured in the same manner as in the analysis of BrdU, and binding to annexin V was analyzed according to the method described in Example 4. Cells to which Annexin V binds were regarded as cells undergoing apoptosis, and the ratio was examined (FIG. 15B, right). It can be seen that by removing EGF from the culture solution, BrdU incorporation is reduced, that is, the proliferation ability is reduced. Furthermore, by removing EGF from the culture solution, the number of cells that undergo apoptosis increased.
In order to confirm the effect of EGF on the proliferation or apoptosis of hAIP cells, BrdU was similarly used in a culture solution containing AG1478, PD98059, or LY294002, which inhibits the EGF signaling system, in a culture solution containing EGF. Uptake and apoptosis induction were investigated.
PD98059 (2′-amino-3′-methoxyflavone) is a selective and cell-permeable inhibitor for MEK1 (MAP kinase kinase), resulting in inhibition of MEK1 activation and subsequent MAPK (ERK1 / 2) phosphorylation-activation is inhibited. Working concentration: 100 μM.
LY294002 ([2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one] is a potent and specific cell permeability inhibitor for phosphatidylinositol 3-kinase (PI3-kinase). Working concentration: 100 μM

Example 14: Induction of differentiation into cells constituting intestinal epithelial tissue hAIP cells are naturally induced to differentiate under the above-described culture conditions, and cells exhibiting the characteristics of intestinal epithelial cells appear. When cells in this state were subjected to PAS staining according to the method described in Example 7, many cells stained reddish purple appeared, so that the presence of goblet cells that were intestinal epithelial tissues could be confirmed (FIG. 16B). ).
Furthermore, differentiation induction into cells constituting intestinal epithelial tissue was examined by RT-PCR analysis in the same manner as in Example 8. The types of intestinal epithelial tissue induced are shown in FIG.
Using hAIP cells and tissues from the adult human small intestine, the expression profile of genes thought to be involved in intestinal cell differentiation was analyzed by RT-PCR. The target markers and the primers used therefor were those for humans shown in Table 2 below.

The results are shown in FIG. 16A.
During hAIP cell culture, a plurality of intestinal endocrine cells and mucus-secreting cells constituting the intestinal epithelial tissue were observed. Therefore, it was found that hAIP cells have pluripotency to differentiate into various cells constituting the intestine at the same time as they continue to grow in culture.

SEQ ID NO: 1: PCR primer (forward) for detecting villin (mouse).
SEQ ID NO: 2: PCR primer (reverse) for detecting villin (mouse).
SEQ ID NO: 3: PCR primer (forward) for detecting Lfabp (mouse).
SEQ ID NO: 4: PCR primer (reverse) for detecting Lfabp (mouse).
SEQ ID NO: 5: PCR primer (forward) for detecting chromogranin A (mouse).
SEQ ID NO: 6: PCR primer for detecting chromogranin A (mouse) (reverse)
SEQ ID NO: 7: PCR primer (forward) for detecting Trefoil factor-3 (mouse).
SEQ ID NO: 8: PCR primer (reverse) for detecting Trefoil factor-3 (mouse)
SEQ ID NO: 9: PCR primer (forward) for detecting lysozyme (mouse)
SEQ ID NO: 10: PCR primer (reverse) for detecting lysozyme (mouse)
SEQ ID NO: 11: PCR primer (forward) for detecting synaptophysin (mouse).
SEQ ID NO: 12: PCR primer (reverse) for detecting synaptophysin (mouse).
SEQ ID NO: 13: PCR primer for detecting Musashi-1 (mouse) (forward)
SEQ ID NO: 14: PCR primer (reverse) for detecting Musashi-1 (mouse)
SEQ ID NO: 15: PCR primer (forward) for detecting Math-1 (mouse)
SEQ ID NO: 16: PCR primer (reverse) for detecting Math-1 (mouse)
SEQ ID NO: 17: PCR primer (forward) for detecting villin (human)
SEQ ID NO: 18: PCR primer (reverse) for detecting villin (human)
SEQ ID NO: 19: PCR primer for detecting Lfabp (human) (forward)
SEQ ID NO: 20: PCR primer for detecting Lfabp (human) (reverse)
SEQ ID NO: 21: Primer for PCR (forward) for detecting schuclase-isomaltase (human)
SEQ ID NO: 22: Primer for PCR (reverse) for detecting schuclase-isomaltase (human)
SEQ ID NO: 23: PCR primer (forward) for detecting secretin (human)
SEQ ID NO: 24: PCR primer (reverse) for detecting secretin (human)
SEQ ID NO: 25: PCR primer for detection of GIP (human) (forward)
SEQ ID NO: 26: PCR primer for detecting GIP (human) (reverse)
SEQ ID NO: 27: Primer for PCR (forward) for detecting gastrin (human)
SEQ ID NO: 28: PCR primer (reverse) for detecting gastrin (human)
SEQ ID NO: 29: PCR primer for detecting chromogranin A (human) (forward)
SEQ ID NO: 30: PCR primer for detecting chromogranin A (human) (reverse)
SEQ ID NO: 31: PCR primer (forward) for detecting CCK (human)
SEQ ID NO: 32: PCR primer for detecting CCK (human) (reverse)
SEQ ID NO: 33: Primer for PCR (forward) for detecting somatostatin (human)
SEQ ID NO: 34: PCR primer for detecting somatostatin (human) (reverse)
SEQ ID NO: 35: PCR primer for detection of Trefoil factor-3 (human) (forward)
SEQ ID NO: 36: PCR primer (reverse) for detecting Trefoil factor-3 (human)
SEQ ID NO: 37: PCR primer for detection of lysozyme (human) (forward)
SEQ ID NO: 38: PCR primer for detecting lysozyme (human) (reverse)
SEQ ID NO: 39: PCR primer (forward) for detecting synaptophysin (human)
SEQ ID NO: 40: PCR primer (reverse) for detecting synaptophysin (human)
SEQ ID NO: 41: PCR primer (forward) for detecting Musashi-1 (human)
SEQ ID NO: 42: PCR primer (reverse) for detecting Musashi-1 (human)
SEQ ID NO: 43: PCR primer for detection of Math-1 (human) (forward)
SEQ ID NO: 44: Primer for PCR (reverse) for detecting Math-1 (human)
Sequence number 45: It is a primer for PCR (forward) for detecting Ngn3 (Neurogenin-3) (human).
SEQ ID NO: 46: PCR primer (reverse) for detecting Ngn3 (Neurogenin-3) (human)
SEQ ID NO: 47: PCR primer (forward) for detecting HPRT (human)
SEQ ID NO: 48: PCR primer for detecting HPRT (human) (reverse)

It is a figure which shows the simple procedure of intestinal cell culture. a is a figure (phase contrast image) which shows the aggregate formed on the non-adhesive culture container. b is a diagram (phase contrast image) showing a state in which the formed aggregates are cultured on an ordinary Petri dish not subjected to treatment for adherent cells (but not subjected to low adhesion treatment). c is a diagram (phase contrast image) showing a state in which intestinal cells are cultured on an adhesive culture vessel without forming aggregates. FIG. It is a figure (phase contrast image) which shows the state which subcultured and proliferated the epithelial cell-like cell derived from the intestinal cell obtained by a series of operation. It is the figure (phase contrast image) which showed the EGF-dependent proliferation ability of FIP cells. a shows the state of culturing for 2 days in the absence of EGF and b in the presence of EGF. c shows the result of removing cells with trypsin-EDTA and measuring with a hemocytometer. The horizontal axis indicates the concentration of added EGF, and the vertical axis indicates the number of cells. It is a figure (phase contrast image) which shows the state of a cell when the FIP cell cultured in the presence of EGF is cultured in the absence of EGF or subsequently in the presence of EGF. a shows a state in which FIP cells were cultured for 2 days in the presence of EGF. After culturing for 2 days in the presence of EGF, the cells were further cultured in a culture solution containing EGF for 1 day (b) or in a culture solution not containing EGF for 1 day (c). It is a figure which shows the state of the cell when a FIP cell cultured in the presence of EGF is subsequently cultured in the presence of EGF or in the absence of EGF. a and b are diagrams (fluorescence microscope images) showing the results of examining BrdU uptake of each cell by BrdU immunostaining. c and d are diagrams showing the results of staining each cell by BrdU / DAPI double staining (fluorescence microscope image). e and f are graphs showing the results of FACS analysis of BrdU incorporation in the presence or absence of EGF. g and h are graphs showing the results of FACS analysis of Annexin V binding in the presence or absence of EGF. It is a figure which shows the result of having investigated the expression of various marker proteins (E-cadherin, Zo-1, villin, and Musashi-1) in the FIP cell which continued culture | cultivation in EGF presence (fluorescence microscope image). It is a figure which shows the result of having detected the positive cell by introduce | transducing the construct (TOPGAL) in which a (beta) galactosidase gene is driven by TCF / b-catenin binding site into a cell, and X-gal staining. It is a figure (phase contrast image) which shows the state of the cell (b, d) which removed EGF from the FIP cell (a, c) cultured in the presence of EGF and further cultured for 7 days in the absence of EGF. It is the image which carried out PAS dyeing | staining of the cell (intestinal epithelial cell-like cell) which continued culturing FIP cell in EGF absence. a shows FIP cells in a confluent state, and b shows cells cultured for 7 days in the absence of EGF. It is the figure which showed typically the relationship between a FIP cell and EGF. It is a figure which shows the result of having investigated the various gene expression profiles in a FIP cell, embryonic day 14.5 mouse | mouth intestinal tissue, a newborn intestinal tissue, and an adult intestinal tissue by RT-PCR method. It is a figure which shows the mode of a cell at the time of subculture | cultivating a human small intestinal epithelial cell (Cat No. CS-ABI-519) by the culture medium for CS-2.0 + supplement culture medium or an intestinal stem / progenitor cell culture | cultivation. a shows the state of the first passage cell seeded in CS-2.0 + supplement medium, and b and d show the state of the cell subcultured twice in the medium for intestinal stem / progenitor cell culture. Show. It is a mode of the 1st day (b) and 5th day (d) after sowing. c and e show the state of cells subcultured twice in CS-2.0 + supplement medium. It is a mode of the 1st day (c) and 5th day (e) after sowing. It is a phase contrast image. The upper row is a diagram (fluorescence microscope image) showing the results of examining the expression of various marker proteins (E-cadherin, Zo-1, villin and Musashi-1) in hAIP cells that have been cultured in the presence of EGF. The lower panel shows the results of detecting positive cells by X-gal staining after introducing a construct (TOPGAL) in which a β-galactosidase gene is driven by TCF / b-catenin binding site. It is the figure (phase contrast image) which showed the EGF-dependent proliferation ability of the hAIP cell. a and c are in the presence of EGF, and b and d are in the absence of EGF for 4 days. c and d are high-magnification images of a and b, respectively. It is a figure which shows the result of having analyzed the influence of EGF on the proliferation and apoptosis of hAIP cells. FIG. 15A is a diagram showing the state of cells when hAIP cells that have been cultured in the presence of EGF are subsequently cultured in the presence of EGF or in the absence of EGF. The results of staining each cell by BrdU / DAPI double staining (fluorescence microscopic image) are shown. FIG. 15B is a graph showing the result of FACS analysis of BrdU incorporation in the presence or absence of EGF, or the use of various inhibitors, or the result of FACS analysis of apoptotic cells. FIG. 16A is a diagram showing the results of examining various gene expression profiles in hAIP cells and human adult intestinal tissue by RT-PCR. FIG. 16B is a PAS-stained image of hAIP cells.

Claims (18)

A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates. A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates in a culture solution containing EGF. The method according to claim 1 or 2, wherein the aggregate is obtained by suspension culture of intestinal cells. A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising the following steps.
(1) Step of collecting intestinal cells from intestinal tract (2) Step of preparing cell mass (aggregate) by suspension culture of intestinal cells (3) Aggregation of intestinal cells in a culture solution containing EGF Step of culturing (4) Step of recovering cells expressing intestinal epithelial cell stem / progenitor cell markers as stem / progenitor cells of intestinal epithelial cells The method according to claim 4, wherein the stem / progenitor cell marker of intestinal epithelial cells is Musashi-1. A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells in a culture solution containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol and EGF. A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells at a high density in a culture solution containing EGF. A method for obtaining stem / progenitor cells of intestinal epithelial cells, comprising the following steps.
(1) Step of collecting intestinal cells from intestinal tract (2) Step of culturing intestinal cells at high density in a culture solution containing EGF (3) Cells expressing stem / progenitor cell markers of intestinal epithelial cells Of recovering as stem / progenitor cells of intestinal epithelial cells The method according to claim 8, wherein the stem / progenitor cell marker of intestinal epithelial cells is Musashi-1. Stem / progenitor cells of intestinal epithelial cells obtained by the method according to any one of claims 1 to 9. A method of culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing aggregates of intestinal cells. A method for culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cell aggregates in a culture solution containing EGF. The method according to claim 11 or 12, wherein the aggregate is obtained by suspension culture of intestinal cells. A method for culturing stem / progenitor cells of intestinal epithelial cells, comprising culturing intestinal cells in a culture medium containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol and EGF. A method for culturing intestinal epithelial cell stem / progenitor cells, comprising culturing intestinal cells at a high density in a culture solution containing EGF. A method for inducing differentiation of intestinal epithelial cells from stem / progenitor cells into cells for constructing intestinal epithelial tissue, comprising removing EGF from a culture solution of the stem / progenitor cells. The method according to claim 16, wherein the cells constituting the intestinal epithelial tissue are enteroendocrine cells or mucus-secreting cells. A medium for culturing stem / progenitor cells of intestinal epithelial cells, containing serum, insulin, dexamethasone, nicotinamide, L-glutamine, β-mercaptoethanol and EGF.