JP2009229455A - Method for detecting, preparing, and culturing pancreas precursor cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide identification of cell membrane antigen which can fractionate pancreatic cells, a method for providing pancreatic cells by using the identified cell membrane antigen, and a method for culturing Langerhans islets and a method for forming and culturing pancreatic ducts using cells fractionated by the cell membrane antigen. <P>SOLUTION: Dlk and PCLPI have been found as an pancreatic surface antigen in developmental process. It has been proved that pancreas precursor cell can easily be fractionated by using these antigens as a marker. In addition, a new cultivation system for utilizing those cells fractionated by this invented method to build three-dimensional structure of Langerhans islets and pancreatic ducts has been developed. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting and preparing pancreatic progenitor cells such as pancreatic parenchymal progenitor cells and pancreatic duct progenitor cells including islets using cell surface antigens. Furthermore, the present invention relates to a culture method for inducing islets and / or pancreatic ducts from a cell sample containing pancreatic progenitor cells.

  The pancreas is composed of an acinus that exoculates pancreatic juice containing digestive enzymes into the intestine, an islet that secretes hormones that regulate blood sugar in the blood, and a pancreatic duct that releases pancreatic juice into the intestine. The acinar forms a glandular structure, and the islets form a spherical structure, which exists independently of each other. In the acinar, one type of cell secretes digestive enzymes such as amylase, lipase, and trypsin. On the other hand, in the islets, α cells secrete glucagon, β cells secrete insulin, δ cells secrete somatostatin, and PP cells secrete pancreatic polypeptide (PP). The pancreatic islet has a characteristic structure in which β cells are present in the center and α, δ, and PP cells surround the periphery, and this structure is particularly remarkable in rodents. This three-dimensional structure is considered to be important for exerting the function of islets (Non-patent Documents 1 and 2). As described above, islets are independent of the acini and endocrine hormones to regulate blood sugar. Furthermore, islets are the only tissues in the body that secrete insulin that lowers blood sugar, and blood sugar level control can be performed with a small number.

  The pancreas is an organ derived from the endoderm. In the mouse, the pancreatic primordium is formed on the dorsal and ventral sides from a specific part of the inner lung lobe of the foregut around E9.5, and later connected with the rotation of the duodenum. To do. Later, the dorsal pancreatic buds are thought to mature while rapidly growing and enlarged. Regarding the development of the pancreas, analysis at the cellular level has not progressed due to the absence of an appropriate culture system, and there is only limited information. It has been known that glucagon is expressed from around E13.5 and then insulin, amylase, etc. are expressed by immunostaining of fetal tissue. At this stage, expression of secreted protein is observed, but it is scattered in the pancreas, and the three-dimensional structure of the acinar and islet is not formed. It is known that an acini and pancreatic duct are formed just before birth, but the details are unknown. In addition, there is a long-standing theory that islets arise from the pancreatic duct in terms of immunohistochemistry (Non-patent Documents 3 and 4). In order to prove this, culture of the pancreatic duct part was tried (Non-Patent Documents 5 to 7), but it is accompanied by dedifferentiation from the adult pancreatic duct and long-term culture, so it is considered that it always reflects the physiological state. Hateful. On the other hand, transcription factors that are essential for the development of the pancreas have been identified based on the results of knockout mouse knockout mice and lineage tracing. Ngn3 is expressed in islet progenitor cells (Non-Patent Documents 8 to 10), and p48 is expressed in acinar progenitor cells (Non-Patent Documents 11 to 13). In addition, the expression of many transcription factors necessary for each differentiation stage is known, and various theories about cell lineage have been proposed based on these (Non-Patent Documents 14 to 19). Analysis at the cellular level is indispensable.

  To date, attempts have been made to induce adult pancreatic cells for the purpose of cell-level analysis. Differentiation ability of neonatal pancreatic cells has been shown by inducing glucagon-producing cells and insulin-producing cells from neonatal pancreatic cells (Non-patent Document 20). However, since cells are induced by long-term culture from neonatal cells that have already had islets, there is a question as to whether they reflect physiological phenomena. It is suggested that islet regeneration is due to islet cell division and that cells other than islets do not differentiate into islets (Non-patent Document 21). Therefore, regeneration is considered to be a fundamentally different mechanism from occurrence. The neonatal pancreas is thought to have created a special state of regeneration rather than development because the islet structure is already complete. Above all, no islets are formed in this culture system. Fractionation of neonatal pancreatic cells for identifying pancreatic progenitor cells has also been attempted, and fractionation is performed by the expression of a number of blood cell markers, and pancreatic progenitor cells are used as indicators of high proliferative ability. Further, the obtained fraction was cultured by the above culture method, and the expression of insulin and glucagon was confirmed (Non-patent Document 22). However, in this conventional method, since negative markers are used in addition to many blood cell markers, the localization of pancreatic progenitor cells in the tissue cannot be confirmed, and there are many problems due to the same problems as in the above culture method. The question remains.

Podocalyxin-like protein 1 (PCLP1) (also called MEP1, thrombomucin) is a cell surface antigen found to be expressed in the E11.5 mouse Aorta-Gonad-Mesoderm (AGM) region. Yes, it is a single-transmembrane glycoprotein whose extracellular region is highly glycosylated. PCLP1 is classified as the sialomucin family because the N-terminal extracellular domain undergoes a characteristic sugar chain modification, and its members include hematopoietic cells such as CD34, CD164, CD162, CD43, and endoglycan or hematopoietic microenvironment ( Those expressed in vascular endothelial cells and the like) belong. PCLP1 has already been molecularly identified in the following species, and the presence of PCLP1 molecules is also expected from other vertebrates.
Human (Non-patent Document 23)
Mouse (Non-patent Document 24)
Rat (Accession No. AB020726)
Rabbit (Non-patent Document 25)
Chicken (Non-patent Document 26)

  It is known that the PCLP1 molecule has low conservation between species of the N-terminal amino acid sequence (Non-patent Documents 23 and 25). Homologous amino acid residues in the PCLP1 molecule are found in the intracellular region. PCLP1 counterparts in chickens have also been reported to have activity as hematopoietic progenitor cells, and similar tissue localization has been reported in rats, rabbits, mice, and humans. It is considered to be a substance with sex and role. Cells expressing PCLP1 are considered to be hematopoietic progenitor cells and vascular endothelial progenitor cells (Non-patent Document 24). PCLP1 is often expressed in cancer tissues, suggesting that it is expressed in undifferentiated cells.

  Membrane protein Dlk (delta-like) is a membrane protein having six EGF-like domains and homology to Delta. In vitro, inhibition of adipocyte differentiation and hematopoietic support have been reported, and knockout mice have been reported to have delayed growth and obesity. A fetal hepatocyte differentiation induction system was constructed by Professor Atsushi Miyajima et al. At the University of Tokyo (Non-patent Document 27), and a protein expressed on the surface of the fetal liver cell membrane was identified (Non-patent Document 28). The cell population containing hepatic progenitor cells can be analyzed by fractionation of hepatocytes and induction of differentiation of the separated cells. These in vitro analysis results have been confirmed in vivo, and the differentiation potential of Dlk-positive cells is not altered by non-physiological properties due to external factors in vitro, but is normal under physiological conditions. It was shown to be a property (Non-patent Document 28). In addition, it was discovered that Dlk-positive cells have high proliferative capacity on laminin-coated plates, and these cells that can be subcultured on laminin maintain their function as hepatic progenitor cells. However, since it replicated itself, it was considered that it retains more undifferentiated stem cell-like properties (Non-patent Document 29). This passage cell was named Hepatic Progenitor cells Proliferate on Laminin (HPPL), and was also shown to include more undifferentiated pluripotent cells that can transdifferentiate into the pancreas (Non-patent Document 29). A method for detecting undifferentiated hepatocytes using dlk gene expression, and further a method for obtaining cells that can be differentiated into hepatocytes, bile duct epithelial cells and pancreatic cells by culturing the detected cells on laminin Have also been proposed (Patent Documents 1 and 2).

WO2002 / 103033 JP 2005-312303 A

Cabrera O et al. (2006) Proc Natl Acad Sci USA 103: 2334-39 Konstantinova I et al. (2007) Cell 129: 359-370 Pictet RL, Rutter WJ (1972) Development of the embryonic endocrine pancreas.In handbook of physiology.Steiner DF, Frenkel N, Eds.Washington DC, Williams and Wilkins, p.25-66 Pictet RL et al. (1972) Dev Biol 29: 436-467 Githens S (1988) J Pediatr Gastroenterol Nutr 7: 486-506 Slack JM (1995) Development 121: 1569-1580 Hao E et al. (2006) Nat Med 12: 310-316 Schwitzgebel VM et al. (2000) Development 127: 3533-3542 Gradwohl G et al. (2000) Proc Natl Acad Sci USA 97: 1607-1611 Gu G et al. (2002) Development 129: 2447-2457 Cockell M et al. (1989) Mol Cell Biol 9: 2464-2476 Krapp A et al. (1996) EMBO J 15: 4317-4329 Kawaguchi Y et al. (2002) Nat Genet 32: 128-134 Edlund H (1999) Current Opinion in Cell Biology 11: 663-668 Pedro L Herrera (2002) Int J Dev Biol 46: 97-103 Wilson ME et al. (2003) Mech Dev 120: 65-80 Zhang Y-Q et al. (2003) Diabetes Matab Res Rev 19: 363-374 Gu G et al. (2003) Mechanisms of Development 120: 35-43 Jensen J (2004) Dev Dyn 229: 176-200 Suzuki A et al. (2002) Cell Transplant 11: 451-453 Dor Y et al. (2004) Nature 429: 41-6 Suzuki A et al. (2004) Diabetes 53: 2143-2152 Kershaw DB et al. (1997) J Biol Chem 272: 15708-15714 Hara T et al. (1999) Immunity 11: 567-578 Kershaw DB et al. (1995) J Biol Chem 270: 29439-29446 Kelly M et al. (1997) J Cell Biol 138: 1395-1407 Kamiya A et al. (1999) EMBO J 18: 2127-2136 Tanimizu et al. (2003) J Cell Sci 116: 1775-1786 Tanimizu et al. (2004) J Cell Sci 117: 6425-6434

Diabetes is a serious chronic disease that causes many complications that are difficult to treat after onset. Islet transplantation has attracted attention as one of the treatments for diabetes. However, chronic donor shortage is a serious problem, and ES cells are attracting attention as a source of islets, and there are many reports that induce insulin-producing cells from ES cells. In 2006, Novocell succeeded in inducing insulin-producing cells from ES cells in a manner consistent with normal development, and it is generally thought that artificial differentiation induction of insulin-producing cells has become realistic. (D'Amour KA et al. (2006) Nat Biotechnol 24: 1392-1401). However, as described in the paper, the insulin-producing cells produced by them are immature fetal types and are only scattered in the culture system. Therefore, further maturation and purification of transplanted cells and removal of undifferentiated ES cells are necessary for practical use. Furthermore, as already revealed by islet transplantation, the three-dimensional structure of the islets is important for the function of β cells in vivo. In order to solve these problems, a culture system that forms the three-dimensional structure of islets in vitro is necessary.
The cell isolation and identification method using cell membrane antigen expression as an index is useful for elucidating organ formation and also from the clinical aspect of regenerative medicine. However, a technique for fractionating pancreatic cells by the expression of cell membrane antigen has not been developed.

In the inventor's laboratory, a method for in vitro analysis of liver development was developed, and by using this method, progenitor cells of the liver were identified. Furthermore, the results of analysis performed in vitro were confirmed in vivo. Since it was observed that hepatic progenitor cells were differentiated into the pancreas, it was inferred that the same technique as the liver could be used in the pancreas, leading to the present invention.
Therefore, in the present invention, first, a database search was performed for the expression of cell surface antigens during pancreatic development, and Dlk and PCLP1 were found. In the pancreas, it was revealed that islet and progenitor cells of the pancreatic duct can be separated using the expression of cell surface antigen as an index, as in the liver.

  Furthermore, in the present invention, a novel culture system has been developed that constructs a three-dimensional structure of adult islets and pancreatic ducts from fetal pancreatic cells. The first established culture system (hereinafter referred to as the old culture system) formed islets and pancreatic ducts on planar fibroblast-like cells and exocrine areas, but the improved culture system (hereinafter referred to as the new culture system) mainly The fibroblast-like cell layer has a thickness and becomes a solid rather than a flat surface, in which a three-dimensional structure of islets and pancreatic ducts is built. Therefore, the ratio of fibroblast-like cells is higher in the new culture system than in the old culture system, and the number of islets is higher in the old culture system despite the increased expression of insulin and the like. It is considered that these expressions did not increase in the new culture system. Observing the cross-section of the culture with frozen sections, it is assumed that fibroblast-like cells have increased more than 10 times in the new culture system. The number of islets increased 5 times compared to the old culture system, but if the total number of cells was thought to be 10 times, the ratio of islets to the whole cells was halved, which is almost the same as the result by RT-PCR . Furthermore, since the expression of genes expressed in progenitor cells disappears after culture in the new culture system, the cells are considered to be differentiated.

  In addition, from the gene expression data, it is considered that cells that have built a pancreatic three-dimensional structure by culture are producing these proteins themselves. In particular, since the center of the islet is C-peptide positive, Is not adsorbed (Rajagopal J et al. (2003) Science 299: 363). In this culture system, there is no doubt that the three-dimensional structure of the pancreatic islets has been constructed, but in reality not all the spherical structures have constructed the pancreatic islet structure. In many spherical structures, CK19-positive cyst (cyst / cyst) -like structures were constructed. Considering this, the number of islets formed by culture becomes almost equal to the number of adult islets. It has been suggested that the cells that make up cyst-like structures in the liver are undifferentiated (Tanimizu N et al. (2007) Mol Biol Cell 18: 1472-1479), so the pancreas is also composed of undifferentiated cells. There is a high possibility. Actually, a part of the cyst-like structure has differentiated into insulin-producing cells, and it has been observed that a mass of insulin-producing cells sprouting from the cyst-like structure, and these are considered to be differentiated into islets.

  In this culture system, a three-dimensional structure of the acinus and blood vessels was not constructed. The acinar has already built a 3D structure with E16.5, and it may have lost the ability to build 3D structures. However, even though E14.5 has not yet formed a three-dimensional structure of the acinus, the three-dimensional structure was not constructed even in the culture of E14.5. The results of immunostaining confirmed that the acinar structure has not yet been constructed in E14.5. However, there is a report that non-patent literature 19; Cano DA et al (2007) Gastroenterology 132: 745-762), and E14.5 already has a three-dimensional structure of the acinus. It is possible that the decisive stage has been completed. Blood vessels are thought to enter from outside the pancreas. In contrast, the pancreatic duct was constructed with E16.5 culture, although it was already constructed with E16.5. Unlike the acini and pancreatic islets, the pancreatic duct builds a three-dimensional structure at the developmental stage and then extends the duct structure as the pancreas enlarges, complicating the branching structure. This suggests that pancreatic duct-forming cells still have the ability to be reconstructed after the construction of the three-dimensional structure. Thus, it was suggested that the three-dimensional structure construction mechanism of islets and pancreatic ducts is different.

  In any case, conventional methods could only induce differentiation into insulin-producing cells and create aggregates of insulin-producing cells (Non-patent Documents 20 and 23). It is an epoch-making thing which can form the physiological islet which a cell surrounds. This culture system shows that pancreatic cells before the formation of islets in vivo have the ability to construct a three-dimensional islet structure, but pancreatic cells that have already had islets have no such function. This culture system is considered to reproduce the developmental process. Furthermore, the time to form pancreatic islets was almost the same as that of living organisms, suggesting that this culture system is physiologically normal.

  Furthermore, in the present invention, the construction of a three-dimensional structure of the pancreatic duct was also successful. So far, attempts have been made to construct a three-dimensional structure of the exocrine duct epithelium in other parenchymal organs such as the bile duct in the liver, but no examples have been realized. In addition, it has been suggested that stem cells and progenitor cells that produce parenchymal cells in each parenchymal organ are present in or near the duct (Non-patent Documents 3-7), but definitive evidence has not been obtained. This culture system can analyze the interaction between pancreatic duct and islet progenitor cells at the time of islet development by realizing the three-dimensional structure construction of islet and pancreatic duct under the same conditions in the same dish. In fact, contact with the pancreatic ducts and islets also provided evidence that some of the pancreatic ducts were differentiated into islets. Thus, this culture system is a very useful tool for molecular cell biological analysis of the 3D structure of ducts and the relationship between ducts and stem / progenitor cells. We also developed a technique to separate islet progenitor cells and pancreatic duct progenitor cells. It is considered that the details of the mechanism can be elucidated by this culture system.

  In this invention, it succeeded in isolate | separating the progenitor cell of an islet and a pancreatic duct. In the culture system, islet progenitor cells form islets, but cells other than islet progenitor cells do not form islets, and pancreatic duct progenitor cells form pancreatic ducts, but islet progenitor cells do not form pancreatic ducts. That is, in the main culture system, it does not differentiate in a direction that is not originally differentiated. This suggests that it is not a culture system that induces differentiation non-physiologically in a special environment in a test tube. Taken together with the fact that only cells prior to the formation of islets at the developmental stage formed islets, it was strongly suggested that the culture system followed the developmental stage normally.

  In the present invention, separation is performed using only the expression of two cell surface antigens, PCLP1 and Dlk, as an index, but it is considered that further purification of pancreatic progenitor cells can be achieved by combining the expression of other cell surface antigens. . In fact, data suggesting that further purification is possible by expression of the c-kit gene (for example, human gene: Yarden Y et al. (1987) EMBO J 6: 3341-3351).

In the present invention, it has been found that the liver and pancreas, which are similar parenchymal organs generated from the same site, follow similar differentiation processes, and thus the intestinal tract that is the origin of these is focused. Since the liver and pancreas sprouting from the intestinal tract, considering the possibility that these progenitor cells remain in the intestinal tract, when fetal intestinal epithelial cells are cultured by the islet formation culture method of the present invention, the islet-like structure is formed. When the constructed intestinal epithelial cells were analyzed by FCM, PCLP1 and Dlk-expressing cells existed. When these cells were fractionated using the expression and cultured by the islet formation culture method, PCLP1 ⁺ Dlk ⁺ cells were A similar structure was formed, but no other cells were formed. Furthermore, similar results were obtained when adult intestinal epithelial cells were cultured. This suggested that islet progenitor cells may remain in the intestinal tract even in adults.

  By developing a culture system that can construct 3D structures of islets and pancreatic ducts from fetal pancreatic cells, it is also possible to construct 3D structures of islets and pancreatic ducts from ES cells by combining with the differentiation induction system of ES cells It is considered to be. In fact, the present inventors succeeded in forming an islet-like three-dimensional structure from ES cells.

In recent years, the development of induced pluripotent stem cells (iPS cells) has cleared the problem of immune rejection (Takahashi K et al. (2006) Cell 126: 663-676; Okita K et al. (2007) Nature 448: 313-317; Meissner A. et al (2007) Nat Biotechnol 25: 1177-1181; Blelloch R et al. (2007) Cell Stem Cell 1: 245-247; Nakagawa et al (2008) Nat Biotechnol 26: 101-106; Yu J et al. (2007) Science318: 1917-1920; Takahashi K et al. (2007) Cell 131: 861-872; Park IH et al. (2007) Nature 451: 141-146 Hanna J et al. (2007) Science 318: 1920-1923), and is thought to be able to form physiological and functional islets like ES cells. However, ES cells and iPS cells may produce tumors called teratomas in the process of differentiation into specific tissues (Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, GR Martin, Proc. Natl. Acad. Sci. USA 78 (1981), pp. 7634-7638.) This has been a problem when transplanting ES cells and iPS cells into patients with specific tissues and organs. Since undifferentiated ES cells and iPS cells do not express PCLP-1 or Dlk, the progenitor cells found by the present inventors express these cell surface antigens. Antigens can be used to eliminate undifferentiated cells that can form teratomas.
For this reason, islets formed by the main culture system in the near future can be expected as a source of safe islet transplantation. Experiments conducted by the present inventors also suggested the possibility of removing undifferentiated ES cells by actually fractionating with the expression of cell surface antigen as an index when constructing islet-like constructs from ES cells. It was.

Specifically, the present invention provides the following inventions [1] to [36].
[1] A method for detecting pancreatic progenitor cells, comprising detecting the expression of Dlk gene and PCLP1 gene in cells.
[2] The method for detecting pancreatic progenitor cells according to [1], wherein the cells in which the expression of the Dlk gene and the PCLP1 gene are detected are determined as pancreatic parenchymal progenitor cells.
[3] The method for detecting pancreatic progenitor cells according to [1], further comprising detecting the expression of the c-kit gene for the cells in which the expression of the Dlk gene and the PCLP1 gene is detected.
[4] The detection method according to [1] to [3], wherein the gene expression is detected using an antibody that recognizes an expression product of each gene.
[5] A method for preparing islet progenitor cells, comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells bound with anti-Dlk antibody and anti-PCLP1 antibody as islet progenitor cells [6] A method for preparing pancreatic duct progenitor cells comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) a step of separating cells not bound to at least one of the anti-Dlk antibody and the anti-PCLP1 antibody as pancreatic duct progenitor cells [7] the step of separating cells (3) is performed using a cell sorter, [5] or [6] The preparation method according to the above.
[8] Cell samples containing pancreatic progenitor cells are fetal pancreatic cells, adult pancreatic cells, fetal intestinal epithelial cells, adult intestinal epithelial cells, fetal liver or cells derived from regenerating liver, adult liver cells, stem cells, ES cells, and iPS. The preparation method according to [5] or [6], which is selected from cells.
[9] In the step (2), an anti-KIT antibody is further added, and in addition to the anti-Dlk antibody and the anti-PCLP1 antibody in the step (3), the anti-KIT antibody-bound cells are separated as islet progenitor cells. [5] The preparation method according to [5].
[10] A method for culturing pancreatic islets including the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) A step of culturing the prepared cell sample under conditions for inducing islets [11] A method for culturing pancreatic islets comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells bound with anti-Dlk antibody and anti-PCLP1 antibody as islet progenitor cells
(4) A step of culturing the isolated islet progenitor cells under conditions for inducing islets [12] A method for culturing pancreatic ducts comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells to which at least one of the anti-Dlk antibody and the anti-PCLP1 antibody has not bound as pancreatic duct progenitor cells
(4) The step of culturing the isolated pancreatic duct progenitor cells under conditions for inducing the pancreatic duct [13] The culture according to [11] or [12], wherein the step of separating cells (3) is performed using a cell sorter Method.
[14] Cell samples containing pancreatic progenitor cells are fetal pancreatic cells, adult pancreatic cells, fetal intestinal epithelial cells, adult intestinal epithelial cells, fetal liver or cells derived from regenerating liver, adult liver cells, stem cells, ES cells, and iPS. The culture method according to [10] to [12], which is selected from cells.
[15] The culture method according to [10] to [12], wherein the culture in step (4) is performed in a DMEM / F12 medium supplemented with serum, 2Me, nicotinamide, HEPES, and gentamicin.
[16] The culture method according to [15], wherein the medium further contains insulin, EGF and GLP-1.
[17] The culture method according to [10] to [12], wherein the medium is changed during the culture in the step (4).
[18] Islet progenitor cells prepared by the method of [5].
[19] Pancreatic duct progenitor cells prepared by the method of [6].
[20] A pancreatic islet cultured by the method of [10] or [11].
[21] A pancreatic duct cultured by the method of [12].
[22] The cell, pancreatic islet or pancreatic duct according to [18] to [21], which is used for transplantation.
[23] A pharmaceutical composition comprising the cell according to [18] or the pancreatic islet according to [20].
[24] The pharmaceutical composition according to [23], which is used for treating diabetes.
[25] A kit for detecting or preparing pancreatic progenitor cells, comprising a reagent for detecting the expression of PCLP1 gene and Dlk gene.
[26] The kit according to [25], wherein the reagents for detecting the expression of the PCLP1 gene and the Dlk gene are an anti-PCLP1 antibody and an anti-Dlk antibody.
[27] The kit according to [25], further comprising a reagent for detecting the expression of the c-kit gene.
[28] The kit according to [27], wherein the reagent for detecting the expression of the c-kit gene is an anti-KIT antibody.
[29] The kit according to [25] to [28], which contains a cell culture medium in addition to a reagent for detecting gene expression.
[30] The kit according to [25] to [29], wherein the pancreatic progenitor cells to be detected or prepared are islet progenitor cells or pancreatic duct progenitor cells.
[31] A pancreatic islet or pancreatic duct culture medium comprising a DMEM / F12 medium containing serum, 2Me, nicotinamide, HEPES, and gentamicin.
[32] The medium according to [31], further comprising insulin, EGF and GLP-1.
[33] A screening method for a substance having a regulating action on insulin production / secretion, comprising the following steps.
(1) A step of culturing the islets of [20] in the presence of a test substance
(2) A step of detecting the level of insulin production / secretion from the islets
(3) identifying a test substance that alters insulin production / secretion level as a substance having an action of regulating insulin production / secretion compared to the control

Furthermore, the present invention provides the following inventions [34] to [36].
[34] A method for treating diabetes comprising the step of administering the cell according to [18] or the pancreatic islet according to [20] to an individual.
[35] Use in production of a pharmaceutical composition for the treatment of diabetes according to [18] or the islet according to [20].
[36] The cell according to [18] or the islet according to [20] for use in the treatment of diabetes.

In the present invention, islet progenitor cells could be isolated as a fraction of PCLP1 ⁺ Dlk ⁺ . Immunostaining using PCLP1 and Dlk as markers also revealed the localization of islet progenitor cells. Since the cells can be separated only by the expression of these two cell surface antigens, the separation operation is simplified. As a result, the damage to the cells due to the staining operation at the time of separation is reduced, and the progenitor cells are separated. It became possible to accurately separate in a lively state.

  To date, there has been no in vitro reproduction of not only the pancreas but also the three-dimensional structure of an adult parenchymal organ in vitro, and the culture system of the present invention is the first system to reproduce the three-dimensional structure of a parenchymal organ. As a result, it is very useful as a model system for analyzing in detail the mechanism of 3D structure construction of the real organ, which has been difficult to analyze at the cellular level.

  Differentiation into pancreas has been reported by introducing Pdx-1 gene into intestinal cells and culturing for a long time (Kojima H et al. (2002) Diabetes 51: 1398-1408; Yoshida S et al. (2002) Diabetes 51: 2505-2513), in the present invention, only wild-type cells are cultured as they are, which is close to a normal state. Most patients with type 2 diabetes are obese due to overeating. On the other hand, in Europe and the United States, as a measure against obesity caused by overeating, treatment for excising part of the intestine is being performed. If the culture method of the present invention is applied to the resected intestinal tract, there is a possibility that it can be linked to diabetes treatment.

  Moreover, it is considered that the generation of islets and pancreatic ducts can be understood in detail at the cellular level by examining in detail the process of constructing the three-dimensional structure of islets and pancreatic ducts from ES cells using the method of the present invention. The above culture system for constructing a three-dimensional structure of islets and pancreatic ducts from ES cells and iPS cells can be applied to screening for therapeutic agents for diabetes, pancreatic duct cancer, pancreatitis and the like, and research of these diseases.

It is a figure which shows the result of having investigated the expression of the cell surface antigen in a fetal pancreatic cell. (a) The expression of PCLP1 and Dlk in fetal pancreas was analyzed by flow cytometer. (b) Fetal pancreatic cells were divided into 4 fractions by the expression of PCLP1 and Dlk, and the ratio (%) of each fraction was tabulated. (c) E14.5, E16.5, E18.5 The total number of fetal pancreas cells was graphed. (d) From the results of (b) and (c), the number of cells in each fraction was converted into a graph. It is a photograph which shows the result of having investigated the expression of the cell surface antigen in E14.5 fetal pancreatic tissue by immuno-staining. E14.5 pancreas frozen sections were prepared and immunostained with a combination of PCLP1 and Dlk, amylase and insulin, and glucagon and insulin. Each cell is indicated by PCLP1 ⁺ Dlk ⁺ (arrow), PCLP1 ⁺ Dlk ⁻ (open arrow), and PCLP1 ⁻ Dlk ⁺ (arrow head). It is a photograph which shows the result of having investigated the expression of the cell surface antigen in E16.5 fetal pancreatic tissue by immuno-staining. E16.5 pancreas frozen sections were prepared and immunostained with a combination of PCLP1 and Dlk, amylase and insulin, and glucagon and insulin. Each cell is indicated by PCLP1 ⁺ Dlk ⁺ (arrow), PCLP1 ⁺ Dlk ⁻ (open arrow), and PCLP1 ⁻ Dlk ⁺ (arrow head). It is a photograph which shows the result of having investigated the expression of the cell surface antigen in E18.5 fetal pancreatic tissue by immuno-staining. E18.5 pancreas frozen sections were prepared and immunostained with a combination of PCLP1 and Dlk, amylase and insulin, and glucagon and insulin. Each cell is indicated by PCLP1 ⁺ Dlk ⁺ (arrow), PCLP1 ⁺ Dlk ⁻ (open arrow), and PCLP1 ⁻ Dlk ⁺ (arrow head). It is a figure which shows the result of having performed the property analysis by quantitative PCR of the fractionated cell. (A) The cell lineage assumed from the information reported so far is shown. The expression of Ngn3 (b), p48 (c), and nestin (d) in vivo obtained by quantitative PCR was shown. It is a figure which shows the result of having analyzed the property of PCLP1 ⁺ Dlk ⁺ fraction of E16.5 fetus pancreas, and another fraction. (a) The colony forming ability of each fraction was shown. (b) The size of the formed colony was classified into three according to the number of cells in the colony, and the number of each colony was graphed. It is a photograph which shows the result of having immunostained the frozen section | slice of the fetal pancreas in order to pinpoint the time of 3D structure construction of an islet / acini occurring in mouse pancreas development. It is the figure and photograph which show the result of having analyzed the property of the new culture system of fetal pancreatic cells. (a) The number of E16.5 and E18.5-derived islet-like structures obtained by a new culture system of fetal pancreatic cells is shown. (b) The expression of pancreatic genes before and after culture of E16.5 pancreatic cells was compared. It is a figure and a photograph which show the result of having carried out the immuno-staining of the cell obtained by the fetal pancreatic cell culture system. Production of amylase was observed in the flat area (lower left), and in the islet-like structure, insulin was observed in the central part and glucagon was produced in the peripheral part (lower right). Similar to adult mouse islet structure (top right). It is the figure and photograph which show the differentiation ability to the islet of the fractionated fetal pancreatic cell. (a) The number of islet-like structures formed by culturing was quantified and graphed. (b) The obtained culture was immunostained with insulin and amylase. It is a figure which shows the result of having investigated the raise of the blood glucose level by the dosage of STZ for preparation of the diabetes model mouse by STZ administration. It is a graph which shows the result of having measured the blood glucose level of the transplantation diabetes model mouse in order to investigate the improvement effect of hyperglycemia by cell transplantation. It is a figure which shows the result obtained about the culture condition examined in order to improve a culture system. The culture conditions are shown on the left. (A) The number of islets formed in the culture using each serum was graphed. (B) The number of islet formation by each medium condition was graphed. Cultures that have not been established are excluded. It is a phase-contrast micrograph after culture | cultivation of a novel culture system. It is the figure and photograph which show the result of having analyzed the property of the novel culture system. (A) E14.5, E16.5, E18.5 fetal pancreatic cells were cultured by a novel culture method, and the number of formed islets was graphed. (B) The number of islet formation was compared between the old culture system and the new culture system. (C) RNA of the new culture system (right 2 lanes), fetal pancreas (left), and fetal pancreatic cells (second from the left) were collected, and RT-PCR was performed on pancreas-specific genes. It is a photograph which shows the immuno-staining result about the frozen section after culture | cultivation of a novel culture system. It is a figure and a photograph which show the result of having investigated the effect by the transplant of the islet cell formed by the novel culture system. (A) The change in blood glucose due to transplantation of cultured cells was graphed. (B) Photographs of immunostained frozen sections of kidney 3 months after islet transplantation. It is a photograph showing differentiation induction of mouse iPS cells into islet structures. Stain the nucleus with blue (Dapi), stain insulin with red (Alexa594), and stain Gcg / SST / PP (glucagon, somatostatin, pancreatic polypeptide) with green.

  The inventors of the present application have determined that the cells expressing the Dlk gene and the PCLP1 gene are a cell population containing pancreatic parenchymal progenitor cells that can differentiate into pancreatic islets and acinar. Therefore, the present invention provides a method for detecting pancreatic progenitor cells by examining the expression of Dlk gene and PCLP1 gene in cells. Furthermore, in the present invention, it was revealed that pancreatic progenitor cells can be further purified by detecting the expression of the c-kit gene. Therefore, as another aspect, the present invention provides a method for detecting pancreatic progenitor cells by examining the expression of Dlk gene, PCLP1 gene and c-kit gene in cells. In the method of the present invention, the cells (Dlk, PCLP1, and c-kit positive cells) in which the expression of Dlk, PCLP1, and c-kit genes are detected are determined and determined to be pancreatic parenchymal progenitor cells.

  Although the order in which these gene expressions are confirmed in the present invention is not limited, the expression of the c-kit gene is confirmed at the same time as the expression of the Dlk gene and the PCLP1 gene or in cells that are determined to be positive for the expression of these genes. It is desirable. That is, first, the expression of the Dlk gene and the PCLP1 gene is determined, and then the expression of the c-kit gene is confirmed. Expression of the Dlk gene and the PCLP1 gene may be performed simultaneously or sequentially. In addition to these three types of gene expression, the expression of marker genes such as known pancreatic progenitor cells, islet progenitor cells, or pancreatic duct progenitor cells may be examined as necessary.

  The method for detecting pancreatic progenitor cells of the present invention can be carried out on vertebrates having a dlk protein, a PCLP1 protein, and, if necessary, a KIT protein encoded by a c-kit gene as cell surface antigens.

  In the present specification, “dlk protein” refers to a delta-like (dlk) protein and homologous proteins thereof, and “Dlk gene” refers to a nucleic acid encoding the protein. dlk protein is also called predipocyte factor 1 (pref-1), zona glomerulosa-specific factor (ZOG), fetal antigen 1 (FA1), etc. (Smas CM et al. (1993) Cell 73: 725-734; Laborda J et al. (1993) J Biol Chem 268: 3817-3820; Jensen CH et al. (1994) Eur J Biochem 225: 83-92). The sequence of the Dlk gene is known in humans (GenBank Accession No. NM_003836 and U15979), rat (AB046763 and D84336), bovine (AB009278) and the like.

  On the other hand, in the present specification, “PCLP1 protein” refers to a single-transmembrane glycoprotein podocalyxin-like protein 1 and its homologous protein present in the cell membrane, and “PCLP1 gene” means a nucleic acid encoding the protein. The sequence of PCLP1 is known in humans (NM_001018111; NM_005397; U97519), mice (NM_013723; BC054530; BC052442; AB028048), rats (AB020726), rabbits (NM_001082766; U35239), chickens (Non-patent Document 26), and the like.

  As used herein, “KIT protein” refers to receptor tyrosine kinase KIT and its homologous protein that play an important role in early hematopoiesis, and “c-kit gene” refers to a nucleic acid encoding the protein. Its sequence has been clarified in various species including humans. (For example, see the human gene: Yarden Y et al. (1987) EMBO J 6: 3341-3351).

Therefore, in the method for detecting pancreatic progenitor cells of the present invention, gene expression can be detected with reference to these known sequence information. However, the application target of the detection method of the present invention is not limited to known biological species in the sequences of these Dlk gene, PCLP1 gene, and c-kit. A gene sequence encoding a protein exhibiting a specific activity is generally considered to be conserved between species. For a gene whose sequence has been determined in a certain organism species, the gene or a partial sequence thereof is a nucleic acid probe. / hybridization using as primers (Sambrook J et al. (1989 ) Molecular Cloning 2 nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press , etc.), and the gene by a known method by gene amplification (PCR, etc.), etc. It is well known that a gene encoding a protein having a function homologous to the protein encoded by can be isolated from other species. Therefore, the above-mentioned Dlk gene, PCLP1 gene, and c-kit gene whose expression is detected in the method of the present invention are known in addition to the genes known at the time of filing of the present application, due to the conservation of genes between such species. Homologous genes of unknown sequence that can be detected and obtained by gene sequence are included.

  In the method of the present invention, gene expression can be performed by detecting expression of a gene product in a cell. For example, the presence of mRNA as a gene product can be detected. Various techniques for detecting the presence of nucleic acids of known sequence have been developed and can be utilized in the present invention. For example, detection can be performed using a nucleic acid hybridization technique using an appropriate nucleic acid probe, primer or the like prepared based on the sequence information of each gene. Known methods such as Northern hybridization, RNA protection assay, RT-PCR and the like can be mentioned.

  It is also possible to detect proteins expressed in cells as gene products. Since the proteins Dlk and PCLP1 encoded by the genes whose expression is detected in the method of the present invention are cell surface antigens, the detection method for the portion of these proteins exposed on the cell surface is directed against cells. It is considered advantageous when the purpose is to further isolate the detected cells with little damage.

  The detection of the protein can be suitably performed by using, for example, an antibody that recognizes the protein. Examples include Western blot, immunoprecipitation, immunohistochemistry, ELISA, FACS and the like.

  Various antibodies that recognize PCLP1, Dlk, and KIT proteins are known. Moreover, as long as the desired protein (PCLP1, Dlk, or KIT) can be recognized, an antibody obtained by any technique can be used in the method of the present invention regardless of its origin. Here, recognizing a protein means, but not limited to, that an antibody binds in response to an antigen on a specific protein. The antibody used in the method of the present invention may recognize any antigen of the protein serving as each marker, but preferably recognizes a site exposed on the cell surface. Antibodies that can be used in the present invention include both monoclonal and polyclonal antibodies. For example, an antigen can be sensitized to an immunized animal, and serum obtained from the animal can be used as a polyclonal antibody. In addition, hybridomas are obtained by fusing immune cells (spleen cells, etc.) obtained from antigen-sensitized immunized animals or antigen-sensitized lymphocytes with cells having permanent division ability, such as myeloma cells, Monoclonal antibodies may be obtained from hybridomas. In obtaining the antibody, any method known to those skilled in the art may be employed. The obtained antibody may be used after separation and purification as required by general protein separation and purification methods (Antibodies: A Laboratory Manual, Harlow and David Lane ed., Cold Spring Harbor Laboratory Press (1988)). )).

As used herein, “antibody” refers to an immunoglobulin molecule that binds to a specific antigen. The antibody used in the method of the present invention need not be a complete antibody molecule as long as it retains the ability to bind to an antigen, and may be a partial fragment of a normal antibody or a modified antibody. . For example, Fab fragment (also called F (ab)), Fab ′ fragment, F (ab ′) ₂ fragment, F (ab ′) fragment, Fv fragment, diabody (Holliger P et al. (1993) Proc Natl Acad Sci USA 90: 6444-6448 etc.), single chain antibody (hereinafter "scFv"; see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer Verlag, NY, pp. 269-315. ) Etc. are mentioned as antibody fragments. If desired, multispecific antibodies (eg, bispecific antibodies) fused with two or more types of antigen recognition sites may be used (Milstein C et al. (1983) Nature 305: 537-540; Keler T et al. (1997) Cancer Res 57: 4008-4014; Brennan M et al. (1985) Science 229: 81-83; Kostelny SA et al. (1992) J Immunol 148: 1547-53; Holliger T et al. (1993) Proc Natl Acad Sci USA 90: 6444-6448). With regard to antibody modification, for example, a technique for binding an antibody to polyethylene glycol or the like is known. Appropriate chemical modifications can be applied as necessary for the purpose of antibody stabilization. Furthermore, a method for modifying an antibody-encoding gene sequence is also known, and a genetically modified antibody is produced according to the purpose, for example, to increase the binding ability of the antibody or to increase stability, and used in the method of the present invention. Also good.

  Detection of cells with an antibody may be detected by directly or indirectly labeling the antibody to be used, or by using another labeled antibody that recognizes the detection antibody. The antibodies that bind to each antigen are preferably labeled with different labels. Methods for detecting proteins via various antibodies have been devised, and any known technique may be employed in the present invention.

  The method for detecting pancreatic progenitor cells using the expression of Dlk gene and PCLP1 gene as an index of the present invention can also be applied as a method for preparing islet progenitor cells and pancreatic duct progenitor cells. That is, the present invention provides a method for preparing pancreatic islet progenitor cells and pancreatic duct progenitor cells, the method comprising (1) preparing a cell sample containing pancreatic progenitor cells, and (2) anti-preparing the prepared cell sample. This can be achieved by adding Dlk antibody and anti-PCLP1 antibody. Here, cells that bind to both anti-Dlk antibody and anti-PCLP1 antibody are islet progenitor cells, and other cells, that is, both Dlk and PCLP1, or cells in which either Dlk or PCLP1 is not detected Can be determined as pancreatic duct progenitor cells. The cell population determined as islet progenitor cells by binding with anti-Dlk antibody and anti-PCLP1 antibody can be further purified by examining the binding property with anti-KIT antibody. If necessary, in addition to these three types of antibodies, in order to detect the expression of other known cell markers, antibodies against those cell markers may be added.

  In this specification, “preparing” islet or pancreatic duct progenitor cells means increasing the proportion of these cells in the cell population, and obtaining a cell population containing a larger number of corresponding cells than the original cell population. Means that. “Purification”, “fractionation”, “isolation”, “recovery”, “concentration” and the like are also used in the same meaning in the present specification.

  Cell samples containing pancreatic progenitor cells include, but are not limited to, fetal pancreatic cells, adult pancreatic cells, fetal intestinal epithelial cells, adult intestinal epithelial cells, fetal liver or regenerating liver-derived cells, adults Suitable examples include liver cells, various stem cells, ES cells and iPS cells. Examples of stem cells include digestive system gland tissues such as salivary glands, digestive system organs such as stomach, bone marrow, umbilical cord blood, and stem cells obtained from adipose tissue.

  A method for isolating a target cell using a specific cell surface antigen as an index is known. For example, by using a fluorescently labeled antibody, target cells can be separated by a cell sorter using a fluorescent signal as an index (FACS or the like).

In addition, a method is also known in which cells are reacted with magnetic particles to which antibodies are immobilized, target cells are captured by the magnetic particles, and then the magnetic particles are collected by magnetism to recover the target cells. In order to separate cells bound to magnetic particles, methods using magnets and devices such as magnetic cell sorters are known (BD IMag-DM Particles reagent, BD IMag-MSC Particles reagent (Becton, Dickinson &Co.); Dynabeads (Invitrogen); MACS products (Miltenyi Biotech Co., Ltd.).
In addition, the cells can be separated by a panning method in which the antibody is coated on the plate and the cells expressing the antigen are concentrated.

  In the step (2) of adding the antibody of the preparation method of the present invention, the order of adding each antibody (anti-Dlk antibody and anti-PCLP1 antibody, and optionally anti-KIT antibody and / or other antibody) is particularly determined. Absent. One type of antibody may be added, and the following antibodies may be added to cells in which binding of the antibody has been confirmed. If each type of antibody is labeled with a different label, all antibodies May be added simultaneously.

The present invention further provides islet progenitor cells and pancreatic duct progenitor cells. These progenitor cells can be prepared by the above-described cell preparation method using the binding to anti-Dlk antibody and anti-PCLP1 antibody as an index. Three-dimensional structure of pancreatic islets by culturing prepared islet progenitor cells (Dlk ⁺ PCLP1 ⁺ cells), and three-dimensional structure of pancreatic duct by culturing prepared pancreatic duct progenitor cells (other than Dlk ⁺ PCLP1 ⁺ cells) It was also revealed that can be built. Therefore, islets and pancreatic ducts obtained by culturing and differentiating islet progenitor cells and pancreatic duct progenitor cells obtained by the cell preparation method of the present invention are also an embodiment of the present invention.

  These cells and tissues help elucidate the mechanism of pancreatic development, and by examining the effects of various drug candidates on them, these cells and tissues are involved in diabetes, pancreatic cancer, pancreatitis, etc. It may be useful for screening drugs for disease. Furthermore, it was confirmed that islet progenitor cells and islets have a blood glucose improving effect by transplantation into a diabetes model mouse (see Examples 3. (4) and (7)). Therefore, the islet progenitor cells and islets of the present invention are considered to be useful for the treatment of diabetes. When used for transplantation treatment, use specific materials (e.g., using iPS cells as starting cells) to suppress rejection of transplanted cells, or genetically modify cells as needed before transplantation Can also be added.

  Thus, as another aspect, the present application provides a pharmaceutical composition comprising islet progenitor cells or islets prepared by the method of the present invention. Such a pharmaceutical composition can be used for treatment of a disease involving islet cells such as diabetes, preferably for transplantation. In addition to islet progenitor cells or islets, the pharmaceutical composition of the present invention can include a pharmaceutically acceptable carrier or solvent required for such treatment.

  When using islet progenitor cells or islets prepared by the method of the present invention as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, if desired, it can be prepared into an injectable form that can be administered parenterally by making it a sterile solution or suspension in water or any other pharmaceutically acceptable liquid. . For example, pancreatic islet progenitor cells or pancreatic islets prepared by the method of the present invention to be included in the pharmaceutical composition are allowed carriers or solvents, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspension, It can be mixed with surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders and the like to provide generally acceptable unit doses necessary for pharmaceutical use. The term “pharmaceutically acceptable” indicates that the material is inert and includes conventional materials used as diluents or vehicles for drugs. Suitable excipients and formulations thereof are described, for example, in Remington's Pharmaceutical Sciences, 16th ed. (1980) Mack Publishing Co., ed. Oslo et al.

  Other isotonic solutions including saline, glucose, and adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. They are used with suitable solubilizers such as alcohols, specifically ethanol and polyalcohols (eg propylene glycol and polyethylene glycol), and nonionic surfactants (eg Polysorbate 80 ™ or HCO-50) can do.

  Sesame oil or soybean oil can be used as an oily liquid, and benzyl benzoate or benzyl alcohol may be used as a solubilizer together with these. Buffers (such as phosphate buffer and sodium acetate buffer), analgesics (such as procaine hydrochloride), stabilizers (such as benzyl alcohol and phenol), and antioxidants can be used in the formulation. The prepared injection solution can be filled into an appropriate ampoule.

  Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The pharmaceutical composition comprises a pharmaceutically effective amount of the active ingredient (islet progenitor cells or islets prepared by the method of the present invention). A “pharmaceutically effective amount” of a compound is an amount sufficient to treat and / or prevent a disorder in which islet progenitor cells or islets that are modulated by the methods of the present invention play an important role. An example of a pharmaceutically effective amount may be the amount necessary to treat or prevent a disorder caused by diabetes, for example, when administered to an individual (patient), for example, lowering blood glucose levels.

  Assessment to determine such pharmaceutically effective amounts of islet progenitor cells or islets prepared by the methods of the present invention can be made using standard clinical protocols.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose of the pharmaceutical composition containing islet progenitor cells or islets prepared by the method of the present invention can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100000 mg / body per patient, but is not necessarily limited to these values. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

  For the first time, the present invention has made it possible to form in vitro a physiological islet in which β cells are surrounded by α, δ, and PP cells. The method for culturing pancreatic islets of the present invention includes (1) a step of preparing a cell sample containing pancreatic progenitor cells, and (2) a step of culturing the prepared cell sample under conditions for inducing islets. Alternatively, it is a culture method for differentiating cells obtained by the above-described method for preparing islet progenitor cells of the present invention into islets as a starting material. That is, another aspect of the islet culture of the present invention includes (1) a step of preparing a cell sample containing pancreatic progenitor cells, (2) a step of adding an anti-Dlk antibody and an anti-PCLP1 antibody to the prepared cell sample, 3) a step of separating cells bound with anti-Dlk antibody and anti-PCLP1 antibody as islet progenitor cells, and (4) culturing the isolated islet progenitor cells under conditions for inducing islets.

  Furthermore, the present invention also provides a method for constructing a three-dimensional structure of the pancreatic duct. The method of culturing pancreatic ducts of the present invention comprises (1) a step of preparing a cell sample containing pancreatic progenitor cells, (2) a step of adding an anti-Dlk antibody and an anti-PCLP1 antibody to the prepared cell sample, Separating a cell to which at least one of the Dlk antibody or the anti-PCLP1 antibody has not bound as a pancreatic duct progenitor cell, and (4) culturing the separated pancreatic duct progenitor cell under conditions for inducing the pancreatic duct.

  Examples of cell samples containing pancreatic progenitor cells used in the above three culture methods include cells similar to those mentioned in the method for preparing islet progenitor cells and pancreatic duct progenitor cells. As in the case, the present invention is not limited to these, and any cell sample may be used as long as it includes pancreatic progenitor cells. A person skilled in the art can easily confirm whether pancreatic progenitor cells are contained in various cell populations by applying the method for detecting a cell surface antigen of the present invention.

  For the step of separating progenitor cells using the binding to anti-Dlk antibody and anti-PCLP1 antibody as an index in the above-described culture method, the description of the above-described method for preparing pancreatic islet and pancreatic duct progenitor cells can be referred to. That is, for the purpose of further purifying the islet progenitor cells, if necessary, the cells may be further classified based on the binding property with the anti-KIT antibody, or other cell markers may be employed. Similarly, desired cells can be separated using a cell sorter using fluorescently labeled antibodies or magnetic particles.

  In the examples herein, Fetal Bovine Serum (JRH 12303-500M) was used as serum. In this case, cell detachment was suppressed by lowering the serum concentration in the medium for culturing pancreatic cells (5%). Therefore, in the culture method of the present invention, the serum concentration can be set to about 5%, for example. For example, 1-10%, preferably 3-8%, more preferably 4-6%, or 10%, 9%, 8%, 7%, 6% or 5% or less, 1%, 2%, It can be 3%, 4% or 5% or more. However, it is apparent that sera from other species can be used in the method of the present invention in addition to fetal calf serum (FCS; FBS) other than those used in the Examples. A person skilled in the art will appropriately determine a concentration suitable for the type of serum to be used based on the well-known technique or the description of the examples in the specification (especially, see 2. (4) and 3. (5)). Can do. It is also possible to culture using a serum substitute product such as a cocktail of growth factors instead of serum.

  The medium used for the culture of the present invention is a DMEM / F12 medium containing 2Me, nicotinamide, HEPES, gentamicin, insulin, EGF and GLP-1 in addition to serum. Each of these components can be added to the medium at the following concentrations. Nicotinamide: 10 mM; HEPES: 5 mM; Gentamicin: 50 μg / ml; Insulin: 10 nM; EGF: 20 ng / ml; GLP-1: 10 ng / ml. However, it should be understood that the concentrations for these components are not limited to this, and those skilled in the art will be aware of well-known techniques or descriptions of the examples herein (especially 2. (4) and 3. (5) ))) And the like, the concentration of the component to be used can be appropriately determined. It has also been confirmed that insulin, EGF and GLP-1 do not necessarily have to be added.

  In the culturing step of the present invention, the medium can be exchanged as necessary. For example, the medium can be changed every 12 to 48 hours.

  The islets and pancreatic ducts formed by such a culture method of the present invention are also included in the present invention. The formed islet and pancreatic duct can be confirmed by examining whether or not a component known to be specifically expressed in each tissue is produced. The components can be confirmed by, for example, immunostaining, and commercially available antibodies can be used for immunostaining. Components specific to adult islets include insulin and Pdx-1 (pancreas duodenum homeobox 1).

  A medium suitable for the culture of the aforementioned islets and pancreatic ducts is also included in the present invention. The medium for forming the islets and / or pancreatic ducts of the present invention is preferably a DMEM / F12 medium containing 2Me, nicotinamide, HEPES, gentamicin, insulin, EGF and GLP-1 in addition to serum.

  The present invention also relates to a kit comprising a reagent suitable for the detection of pancreatic progenitor cells described above and preparation of islet progenitor cells or pancreatic duct progenitor cells. The kit includes a reagent for detecting the expression level of the PCLP1 gene and the Dlk gene. Furthermore, the kit may contain a reagent for detecting the expression level of the c-kit gene. These reagents may be nucleic acid probes and primers for detecting nucleic acid products (eg, mRNA) transcribed from each gene and / or antibodies for detecting proteins translated from each gene. There may be. For these probes, primers and antibodies, reference can be made to the description of the method for detecting pancreatic progenitor cells.

  The kit of the present invention may contain components / apparatuses and the like required for detection / separation of cells using the above-described nucleic acid probe, primer, or antibody. For details, reference can be made to the description of the method for detecting pancreatic progenitor cells.

  The kit of the present invention may contain a medium suitable for culturing pancreatic progenitor cells, islet progenitor cells, and / or pancreatic duct progenitor cells, in addition to a reagent for detecting the expression level of each gene. Moreover, the culture medium which can induce | guide | derive the three-dimensional structure of the above-mentioned islet and pancreatic duct of this invention may be included. The medium may be included in the kit in a prepared form, or the necessary components may be included in the kit in an individually packaged form so that the medium can be prepared at the time of use.

  Furthermore, the kit of the present invention may contain, as necessary, instructions contained in a normal kit, such as instructions for handling reagents, culture media, components and devices contained in the kit, appropriate containers, control reagents and the like. .

  The islet cells obtained by the method of the present invention were confirmed to be functional islet cells whose insulin secretion is regulated by the blood glucose level in vivo over a long period of time by transplantation into a diabetes model mouse. Such islet cells are useful not only for the treatment of diabetes by transplantation but also for the screening of therapeutic drugs for diabetes in vitro. Therefore, the present invention comprises (1) a step of culturing pancreatic islets cultured by the method of the present invention in the presence of a test substance, (2) a step of detecting the level of insulin production / secretion from the pancreatic islets, and (3) a control and In comparison, the present invention provides a screening method for a substance having an action of regulating insulin production, comprising the step of identifying a test substance that changes the level of insulin production / secretion as a substance having an action of regulating insulin production / secretion. .

  The insulin production / secretion level can be evaluated and detected immunologically by using, for example, an antibody against insulin. Antibodies that recognize insulin are commercially available, and can also be produced by well-known antibody production techniques as necessary.

  As a control for comparing the level of insulin production / secretion, for example, pancreatic islets cultured under the condition where no test substance is added can be used. If necessary, a solution (for example, physiological saline) in which the test substance is dissolved can be added to the islet culture medium as a control in the same volume as when the test substance is added. On the other hand, when the pancreatic islet to which a substance known to change the insulin production / secretion level in the pancreatic islet is used as a control, the degree of activity between the test substance and the control substance can be compared and evaluated.

  Test substances that can be used in the screening method of the present invention include purified proteins (including antibodies), expression products of gene libraries, synthetic peptide libraries, RNA libraries, cell extracts, cell culture supernatants, And libraries of synthetic low molecular weight compounds, but are not limited thereto.

A substance having an action of regulating insulin production / secretion selected by the screening method of the present invention is a candidate compound for a therapeutic agent for diseases (such as diabetes) involving insulin. That is, the present invention provides a therapeutic agent for diabetes containing a substance selected by the screening of the present invention as an active ingredient. The present invention also relates to the use of a compound selected by the screening method of the present invention in the manufacture of a therapeutic agent for diabetes. When the substance isolated by the screening method of the present invention is used as a therapeutic agent, it can be formulated and used by a known pharmaceutical manufacturing method. For example, it is administered to a patient together with a pharmacologically acceptable carrier or vehicle (saline, vegetable oil, suspension, surfactant, stabilizer, etc.). Administration is percutaneous, intranasal, transbronchial, intramuscular, intravenous or oral, depending on the nature of the substance. The dosage varies depending on the age, weight, symptoms, administration method, etc. of the patient, but those skilled in the art can appropriately select an appropriate dosage.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
1. material
(1) Mouse
C57BL / 6 wild type mice were purchased from SLC. Mice were reared in a SPF environment so that food and water could be freely accessed in a light / dark cycle every 12 hours. Mice were handled in accordance with the University of Tokyo animal experimentation regulations.

(2) Cell culture Dulbecco's modified Eagle's medium (DMEM) [Gibco BRL # 12800-082]
DMEM / F12ham [Gibco BRL # 11320-033]
Fetal bovine serum [Gibco BRL # 26140-979]
Fetal bovine serum [JRH # 12303-500M]
Nicotinamide [Sigma # N0636]
2-Me [Wako # FSM1874]
V-type collagenase [Sigma # C9263]
200 mM L-glutamine [Gibco BRL # 25030-081]
Gelatin [Sigma # G-1890]
Laminin [BD # 354232]
Insulin Transferrin-Selenium X (ITS) [Gibco BRL # 51500-056]
Protease inhibitor cocktail [Roche # 1697498]
Trypsin inhibitor soybean [Sigma # T6522]
Cell strainer (70μm) [Falcon # 12350]
Cell culture dish (6 wells) [Corning # 430166]
Type IV collagen coat dish [Iwaki # 4810-014]

(3) Serum for cell culture studies
Gibco BRL 26140-979
Gibco BRL 16170-078 511116
Gibco BRL 10437-069 1333198
Gibco BRL 26140-061 1287369
JRH 12303-500M 4L0299
JRH 12303-500M 6D0973
JRH 12303-500M 6D0974
JRH 12303-500M 4G0758
Equitech-Bio SFBM30 1489
Equitech-Bio SFBM30 1847
Equitech-Bio SFBM30 1850
CosmoBio SH30397 KQM25242
SIGMA CR1211 066K3395
WAKO SH30396.03 KPJ21975
Biological Industries 04-001-1E 115203
Biological Industries 04-001-1E 115171
Biological Industries 04-001-1E 115388
Japan BioSealam Chile JBS-5094
Japan Bioseam Mexico JBS-5145
Bio west S1500 S05495
CCT CC3008-502-S 05090310

(4) Cytokines & hormones Endothelial cell growth factor (EGF) [Peprotech # 315-09]
Hepatocyte growth factor (HGF) [Peprotech # 100-39]
Glucagon like protein-1 (GLP-1) [Sigma # G3265]
Dexamethasone (Dex) [Sigma # D1756]
Insulin [Sigma # I9278]

(5) Antibody <br/> Biotinylated anti-Dlk rat polyclonal antibody [Homemade]
Anti-PCLP-1 rat polyclonal antibody [MBL # D072-3]
Anti-PCLP1 PE [MBL # D072-5]
Anti-FcR rat polyclonal antibody [Homemade]
Anti-insulin guinea pig polyclonal antibody [Dako # A-0564]
Anti-glucagon rabbit polyclonal antibody [Dako # A-0565]
Anti-somatostatin rabbit polyclonal antibody [Dako # A056601]
Anti-amylase rabbit polyclonal antibody [Sigma # A-8273]
Anti-C-peptide goat polyclonal antibody [Linco # 4023-01]
Anti-CK19 rabbit polyclonal antibody [Homemade]
Anti-guinea pig IgG [Inter-Cell # F103-1]
Anti-rabbit IgG [Vector # I-1000]
Anti-goat IgG [Vector # I-5000]
Biotinylated anti-guinea pig IgG [Vector # BA-7000]
Streptavidin Alexa 594 [Molecular Probes # S-11227]
Anti-Rabbit IgG Alexa 488 [Molecular Probes # A21206]
Anti-rat IgG Alexa 555 [Molecular Probes # A21434]
Streptavidin-conjugated APC [BD # 554067]
7AAD [BD # 51-68981E]

(6) Primer
Pdx1 sense strand: 5'-TTACAAGCTCGCTGGGATCA-3 '(SEQ ID NO: 1)
Antisense strand: 5'-GTCCCGCTACTACGTTTCTT-3 '(SEQ ID NO: 2)
PTF1a-p48 sense strand: 5'-AGGAAAGGGAGTGCCCTGCAAG-3 '(SEQ ID NO: 3)
Antisense strand: 5'-GGCCCAGAAGGTCATCATCTGC-3 '(SEQ ID NO: 4)
Neurogenin 3 sense strand: 5'-AGTGCTCAGTTCCAATTCCAC-3 '(SEQ ID NO: 5)
Antisense strand: 5'-AAGAAGTCTGAGAACACCAGT-3 '(SEQ ID NO: 6)
HNF1β sense strand: 5'-GAAAGCAACGGGAGATCCTC-3 '(SEQ ID NO: 7)
Antisense strand: 5'-CCTCCACTAAGGCCTCCCTC-3 '(SEQ ID NO: 8)
HNF6 sense strand: 5'-CAGCACCTCACGCCCACCTC-3 '(SEQ ID NO: 9)
Antisense strand: 5'-CAGCCACTTCCACATCCTCCG-3 '(SEQ ID NO: 10)
Nestin sense strand: 5'-CGACAGGCCACTGAAAAGTT-3 '(SEQ ID NO: 11)
Antisense strand: 5'-GACCCTGCTTCTCCTGCTC-3 '(SEQ ID NO: 12)
Insulin 1 sense strand: 5'-CAGAGACCATCAGCAAGCAG-3 '(SEQ ID NO: 13)
Antisense strand: 5'-CACTTGTGGGTCCTCCACTT-3 '(SEQ ID NO: 14)
Glucagon sense strand: 5'-CGACTACAGCAAATACCTCG-3 '(SEQ ID NO: 15)
Antisense strand: 5'-CAGCCAGTTGATGAAGTCC-3 '(SEQ ID NO: 16)
Amylase sense strand: 5'-CATGACAATCAGCGAGGACA-3 '(SEQ ID NO: 17)
Antisense strand: 5'-CATAAATCCAACAGCCATTTTG-3 '(SEQ ID NO: 18)
CK7 sense strand: 5'-GGAGATGGCCAACCACAG-3 '(SEQ ID NO: 19)
Antisense strand: 5'-GGCCTGGAGTGTCTCAAACTT-3 '(SEQ ID NO: 20)
GAPDH sense strand: 5'-ACCACAGTCCATGCCATCAC-3 '(SEQ ID NO: 21)
Antisense strand: 5'-TCCACCACCCTGTTGCTGTA-3 '(SEQ ID NO: 22)
As other general reagents, Wako's special grade reagents were mainly used.

2. Method
(1) Isolation of fetal pancreatic cells The fetal pancreas from embryonic day 16.5 (E16.5) was carefully separated so as not to contaminate surrounding tissues. The separated pancreas was placed in ice-cold PBS, centrifuged at 4 ° C. and 1200 rpm for 3 minutes, and the supernatant was discarded. 2 mg / ml collagenase V / PBS that had been pre-dissolved, filtered, and warmed to 37 ° C. was added, and incubated at 37 ° C. for 10 minutes. During this time, the cells were dispersed by pipetting every 3 minutes. The cell dispersion obtained here was passed through a 70 um cell strainer to remove undigested sites and aggregates, and double the amount of DMEM / F12 + 10% FCS to stop the enzyme activity of collagenase. . Thereafter, centrifugation was performed in the same manner as described above, and the pellet was dispersed again with a culture medium for islet formation culture (DMEM / F12 ham + 5% FCS + gentamicin + nicotinamide + 2Me + HEPES), and the number of cells was counted and used for the following experiment. .

(2) Separation of Dlk-positive PCLP-1-positive cells using a cell sorter The cells obtained by the above method were DMEM containing 10% FBS, 1xITS, 10 mM nicotinamide, 20 ng / ml insulin, 5 mM L-glutamine. Suspend in / F12 medium (pancreatic cell culture medium), add 1 / 50-fold amount of anti-FcR antibody for 15 minutes at 4 ° C, then add 1 / 50-fold amount of biotinylated anti-Dlk antibody at 4 ° C. Processed for 30 minutes. After washing with 10 times the amount of ice-cold PBS, the stock PE anti-PCLP1 antibody was added and suspended, and 1/50 times the amount of streptavidin-conjugated APC was added and treated at 4 ° C. for 30 minutes. Next, 1 / 50-fold amount of 7AAD was added and suspended, and treated at 4 ° C. for 15 minutes. After washing with 10 times the amount of ice-cold PBS, the cells were suspended in PBS, and dead cells were removed by FACS Vantage, followed by fractionation using Dlk and PCLP1 expression as indicators. After the fractionation for each experiment, the separation accuracy by Dlk and PCLP1 was reanalyzed by FACS. As a result, almost no contamination of other fractions was observed.

(3) Colony-forming ability Cells that have been isolated by a cell sorter are modified pancreatic cell culture media developed by Suzuki et al. (DMEM / F12 + 10% FCS + 2-Me + nicotinamide + HEPES + gentamicin + dexamethasone + ITS-x + EGF + HGF) and seeded on a gelatin-coated dish at a cell density of 100 cells / cm ² . Colonies formed after 5 days of culture were observed with a phase contrast microscope, and the number of colonies was counted. Furthermore, the size of the formed colony is classified using the number of cells in the colony as an index, and the colonies in which the number of cells of 50 or less, 51 to 100, 101 or more are recognized in the colonies are small, medium, It was big. The number of each colony was counted and digitized into a graph.

(4) Islet Formation Culture Method The cells obtained by the above cell preparation method were suspended in the islet formation culture medium and seeded at 5 × 10 ⁴ cells / cm ² at 200 μl / cm ² . After 24 hours, the medium is replaced with medium supplemented with 20 ng / ml EGF, 10 ng / ml insulin, 10 nM, 10 ng / ml GLP-1, and the medium is replaced every 48 hours thereafter. The culture becomes confluent around the 5th day of culture, and the 3D culture is seen around the 10th day of culture. These were collected at an arbitrary timing and used for the following experiments.

(5) Preparation of diabetes model mice
Streptozotocin (STZ) was administered intraperitoneally at 150 mg / kg (body weight) to 8-week-old C57BL / 6 wild-type mice, and blood glucose was measured every other week. Mice whose blood glucose level increased from 400 to 500 mg / dL were used as diabetes model mice. Moreover, since the blood glucose level of the wild type mouse was 237.7 ± 5.6 mg / dL, a mouse showing a blood glucose level almost equivalent to the wild type less than 300 mg / dL was used as a mouse with no high blood sugar.

(6) Fetal pancreatic cells transplanted under the kidney capsule or cells on the 12th day of culture are collected by collagenase treatment, double the amount of DMEM / F12 + 10% FCS is added to inhibit the enzyme activity, and then the centrifuged pellet is added to PBS. 5 × 10 ⁵ cells were suspended and then transplanted under the kidney capsule of a diabetes model mouse with a 28G needle.

(7) Blood glucose measurement A cut was made in the tail vein of the mouse with a scalpel, and after bleeding venous blood was collected with a hematocrit tube, blood glucose was immediately measured with a simple blood glucose meter (ACCU-Chek Compact).

(8) The immunostaining culture on the plate was washed with PBS and fixed with ice-cooled fresh 4% PFA / PBS for 3 hours. Thereafter, the plate was washed twice with PBS, treated with 0.02% Triton / PBS for 30 minutes at room temperature, further washed with PBS, and then blocked using 15 μl / ml goat serum or donkey serum as necessary. The blocked sample was reacted with the primary antibody at 4 ° C. overnight. The next day, the sample was washed 3 times with PBS for 30 minutes, and reacted with the secondary antibody at 4 ° C. overnight. The plate was again washed with PBS three times for 30 minutes, and reacted with the tertiary antibody at 4 ° C. overnight. After washing with PBS for 30 minutes 3 times, DAPI was stained for 10 minutes with nucleus, washed with PBS for 30 minutes 3 times, washed with pure water, and encapsulated by dropping the mounting agent (Gel / Mount) onto one side. . The mounting medium was dried at 4 ° C. overnight and then observed with an inverted microscope manufactured by Nikon.

(9) The immunostained culture of the frozen section was recovered by peeling it into a sheet by collagenase treatment, and fixed with ice-cooled fresh 4% PFA / PBS at 4 ° C. overnight. The fetal pancreas and transplanted mass were fixed overnight at 4 ° C. with fresh 4% PFA / PBS cooled with ice immediately after excision. Thereafter, 10% sucrose / PBS, 20% sucrose / PBS, and 30% sucrose / PBS were sequentially replaced, embedded with OCT Compound, and stored at -80 ° C. The embedded block was frozen with a microtome (Cryostat), attached to a slide glass, air-dried and immunostained. The air-dried frozen section was washed with PBS for 5 minutes, treated with 0.02% Triton / PBS, washed again with PBS, and blocked with 15 μl / ml goat serum or donkey serum as necessary. The blocked sample was reacted with the primary antibody overnight at 4 ° C., washed with PBS three times for 10 minutes, and then reacted with the secondary antibody for 90 minutes at room temperature. After washing with PBS for 10 minutes 3 times, react with the tertiary antibody at room temperature for 90 minutes, again wash with PBS for 10 minutes 3 times, stain 5 minutes with DAPI, and further wash with PBS for 10 minutes 3 times with pure water After washing, the mounting medium was dropped, mounted with a cover glass, and observed with a Zeiss upright microscope.

(10) Microscopic observation As for immunostaining by fluorescence, staining on a plate was observed with an inverted microscope made by Nikon, and staining of a frozen section was observed with an upright microscope made by Zeiss. In both cases, the fluorescence intensity was photographed for each color with a MATSUNAMI digital camera, and colored with pseudo color using image processing software (Aqua cosmos).

3. result
(1) Identification and characterization of fetal pancreatic progenitor cells Cell classification by cell surface antigen was established by the most advanced blood cell system research in stem / progenitor cell research. It has been clarified that this method can be applied to the liver which is one of the organs (Non-patent Document 28), and the present invention considered that the same method can be applied to the pancreas. Based on the results in the liver, we focused on the molecules expressed in the progenitor cells of the liver, and searched the database for the expression of cell surface antigens in the pancreas. As a result, it was revealed that Dlk and PCLP1 expressed in progenitor cells in the fetal liver were also significantly expressed in the pancreas. Therefore, the following experiment was performed on the assumption that these molecules are expressed in undifferentiated pancreatic cells as in the liver.

First, the expression of PCLP1 and Dlk in pancreas development was analyzed by a flow cytometer (FCM) (FIG. 1a). As a result, PCLP1 and Dlk were expressed in the fetal pancreas. PCLP1 expression decreased after birth, and Dlk expression was observed from fetal to adult. These cells were divided into four fractions, PCLP1 ⁺ Dlk ⁺ , PCLP1 ⁺ Dlk ⁻ , PCLP1 ⁻ Dlk ⁺ , and PCLP1 ⁻ Dlk ⁻ , and the ratio of each fraction was quantified (FIG. 1 b). Next, based on the number of cells recoverable from each fetal pancreas (FIG. 1c), the cells contained in the above four fractions were converted to the number of cells per individual (FIG. 1d). According to this cell preparation method, the total cell number in the pancreas per individual E14.5 in 2.6 x 10 ^5, E16.5 in 6.9X10 ^5, a 1.1 × 10 ⁶ in E18.5, from E14.5 E18.5 In the meantime, the number of cells in the pancreas grew at about twice the pace in 2 days (FIG. 1c)). From this result and the ratio of each fraction obtained by FCM, the number of cells of each fraction contained in one individual was calculated (FIG. 1d)). PCLP1 ⁺ Dlk ⁺ cells increased from E14.5 to E16.5, but hardly increased from E16.5 to E18.5. PCLP1 ⁺ Dlk ⁻ cells were a rare fraction and constantly increased from E14.5 to E18.5. PCLP1 ⁻ Dlk ⁺ cells increased about 2.5 times from E14.5 to E16.5, but hardly changed from E16.5 to E18.5. PCLP1 ⁻ Dlk ⁻ cells increased only slightly from E14.5 to E16.5, but increased approximately 5-fold from E16.5 to E18.5.

Next, immunostaining was performed to examine the localization of cells expressing PCLP1 and Dlk in the pancreas development process (FIGS. 2-4). The localization of cells expressing PCLP1 and Dlk was examined by preparing serial sections of fetal pancreas and staining adjacent sections with antibodies of proteins expressed in the pancreas. In E14.5, PCLP1 ⁺ Dlk ⁺ cells (arrow in Fig. 2) and PCLP1 ⁺ Dlk ^- cells (arrow in Fig. 2), PCLP1 ^- Dlk ⁺ cells (arrow in Fig. 2) are randomly scattered in fetal pancreas, amylase, PCLP1 and Dlk expression was not observed in cells expressing insulin, glucagon and the like. PCLP1 ⁻ Dlk ⁻ cells gathered at sites distant from PCLP1 ⁺ Dlk ⁺ cells and PCLP1 ⁺ Dlk ⁻ cells, and differentiated cells were included in this fraction (FIG. 2). From the above, in E14.5, various cells are randomly scattered, but there are independent areas where differentiated cells and undifferentiated cells exist, and there are undifferentiated cells that express PCLP1 and Dlk. It became clear that it exists in the part to be.

In E16.5, PCLP1 ⁺ Dlk ⁺ cells are scattered in the duct-like structural site (arrow in Fig. 3) and its periphery, and PCLP1 ⁺ Dlk ^- cells are placed in large and small ducts (arrow in Fig. 3), PCLP1 ^- Dlk ⁺ The cells were outside the PCLP1 ⁺ Dlk ⁺ cells around the duct (the part surrounded by the arrow in Fig. 3), and the PCLP1 ^- Dlk ^- cells were already differentiated cells that produced amylase and insulin in other areas. (Outlined arrow in Fig. 3). From these immunostaining results, differentiated and undifferentiated cells were scattered to some extent in E14.5, but in E16.5, undifferentiated cells were present in the duct and its surroundings, and differentiated cells. Was found to exist in a different region. As described later in (2), at this time, cells that form acinar, pancreatic islets, pancreatic ducts and blood vessels begin to form their own structures (Fig. 3-10). It is possible that the movement is happening.

In E18.5, PCLP1 ⁺ Dlk ⁺ cells are present in the periphery of large and small duct-like structures (Fig. 4 arrows), PCLP1 ⁺ Dlk ^- cells are in ducts (open arrows in Fig. 4), and PCLP1 ^- Dlk ⁺ cells are It was present around the PCLP1 ⁺ Dlk ⁺ cells (arrowhead in FIG. 4). On the other hand, PCLP1 ^- Dlk ^- cells existed in a different region and produced amylase and insulin. Thus, in E18.5, where the three-dimensional structure of the acinus, pancreatic islet, pancreatic duct, and blood vessel has already been completed, the area where differentiated cells existed in E16.5 and the area where undifferentiated cells exist Seems to be more clearly separated.

In summary, cells that express amylase, glucagon, and insulin already exist in E14.5, but PCLP1 and Dlk-expressing cells are different from differentiated cells, as are randomly scattered cells. Randomly present at locations. E16.5 to E18.5 are the time when pancreatic islets, acinar, blood vessels and ducts are formed, but they are present at specific sites in the pancreas and they do not express PCLP1 or Dlk. Cells expressing PCLP1 and Dlk are present at sites distinctly different from the cells that form these structures. As the development further progressed and after birth, there was no region where such differentiated cells were present, and cells expressing PCLP1 and Dlk were scattered around the duct or in a narrow space between the parenchyma and the parenchyma. Taken together, these data suggest that PCLP1 ⁺ Dlk ⁺ cells before E16.5 may be pancreatic stem / progenitor cells.

Quantify expression of pancreatic cell differentiation-specific genes (Figure 5a) to ensure that pre-E16.5 PCLP1 ⁺ Dlk ⁺ fractions contain pancreatic stem / progenitor cells It examined by PCR (FIG. 5b-d). As a result, Ptf1a-p48 is PCLP1 of E14.5 expressing the acinar progenitors ^- Dlk ⁺ cells, PCLP1 of E16.5 ^- Dlk ⁺ cells, PCLP1 ^- Dlk ^- also expressed in cells (Fig. 5c) Ngn3 expressed in islet progenitor cells was expressed in PCLP1 ^- Dlk ^- cells of E14.5 (FIG. 5b). Since PTF1a-p48 and Ngn3 are not expressed in E18.5, which forms acinars and islets, the cells that express them are immature and do not form a three-dimensional islet structure. It was suggested that the cells were fate-determined as parenchymal cells such as bunches and islets. On the other hand, PCLP1 ⁺ Dlk ⁺ cells and PCLP1 ⁺ Dlk of E16.5 from E14.5 ^- cells because it does not express Ptf1a-p48 and Ngn3, or not fate decisions in the duct, or still not determined fate at this point It was suggested that there were no cells. As a result of immunostaining, PCLP1 ⁺ Dlk ⁺ cells and PCLP1 ⁺ Dlk ⁻ cells were present in and around the duct and were not expressed in cells expressing amylase, glucagon and insulin. Nestin (Zulewski H et al. (2001) Diabetes 50, 521-533), which is said to be expressed in the pancreas stem cells existing in the duct and its surroundings, is PCLP1 ⁺ Dlk ⁺ , PCLP1 ⁺ Dlk ⁻ cells of E14.5 and E16.5 of ^{PCLP1 + Dlk +, PCLP1 + Dlk} -, PCLP1 - Dlk + since expressing (FIG. 5d) to the cells, nestin-positive cells has been considered to include pancreatic stem cells ever single It was divided into three fractions based on surface antigen expression, rather than the cell population. From the previous immunostaining results (Fig. 2~4), PCLP1 ⁺ Dlk ⁺ cells are present in and around the duct, PCLP1 ⁺ Dlk ^- since cells are present only in the duct, PCLP1 ⁺ Dlk ^- cells in the duct It is thought that PCLP1 ⁺ Dlk ⁺ cells are fetal pancreatic stem / progenitor cells.

From the above results, PCLP1 ⁺ Dlk ⁺ cells up to E16.5 contain fetal pancreatic stem / progenitor cells, and the previously proposed pancreatic stem / progenitor cells were treated with surface antigens PCLP1 and Dlk. Further fractionation by expression could be concentrated as PCLP1 ⁺ Dlk ⁺ cells.
The results of immunostaining and gene expression suggested that PCLP1 ⁺ Dlk ⁺ cells before E16.5 contain pancreatic stem / progenitor cells. Therefore, in order to investigate stem / progenitor cell activity using proliferation ability as an index, the colony-forming ability of E16.5 PCLP1 ⁺ Dlk ⁺ cells and cells other than PCLP1 ⁺ Dlk ⁺ (hereinafter, other cells) was compared (Fig. 6). Disperse E16.5 fetal pancreas into single cells by enzyme treatment, fractionate into PCLP1 ⁺ Dlk ⁺ cells and other cells using a cell sorter, and seed at low density (100 cells / cm ² ) in gelatin-coated dishes Cultured for 5 days. As a result, 43.5 ± 3.8 unfractionated cells, 74.3 ± 8.3 PCLP-1 ⁺ Dlk ⁺ cells, and 37.9 ± 2.45 other cells per 1 × 10 ³ cells in the E16.5 pancreas (Fig. 6a). That is, it was shown that the colony forming ability of PCLP1 ⁺ Dlk ⁺ cells was about 2 times higher. Further, the colony is divided into three groups: large (the number of cells in the colony is 101 or more), medium (the number of cells in the colony is 50 to 100), and small (the number of cells in the colony is less than 50). Was examined (Fig. 6b). As a result, PCLP1 ⁺ Dlk ⁺ cells formed many large colonies, but other cells only formed small colonies, so in E16.5 pancreatic cells, cells with high proliferative potential are included in PCLP1 ⁺ Dlk ⁺ cells It became clear.
From the above, PCLP1 ⁺ Dlk ⁺ cells before E16.5 contain highly proliferative pancreatic progenitor cells, but these cells are considered to be differentiated after E18.5. Other cells are considered to contain cells that are already destined for the pancreatic duct, exocrine region, and endocrine region, respectively, based on gene expression and histological analysis.

(2) Development of a new pancreatic cell culture method As described above, PCLP1 ⁺ Dlk ⁺ cells before E16.5 were considered to contain pancreatic progenitor cells, and a new fetal pancreatic culture system was used to analyze these in detail. Worked on creating.
First, in order to identify the timing of islet formation, frozen sections of fetal pancreas were prepared, and amylase, insulin, and glucagon were immunostained (FIG. 7). As a result, in E14.5, cells that produce amylase, insulin, and glucagon existed, but were scattered in the fetal pancreas. In E16.5, acinar began to form, and cells producing glucagon and insulin gathered around the site that should be present in adults, but no islets were formed. These cells began to form islet structures at E18.5 and had a three-dimensional structure similar to that of adults after birth. These results suggest that there may be progenitor cells that have the ability to construct a three-dimensional structure of the acinus and islets in the pancreas before E16.5.

  To examine the ability of E16.5 fetal pancreatic cells to differentiate into pancreatic islets, just before the formation of pancreatic islets, we created a culture system that allows E16.5 fetal pancreatic cells to be cultured to form exocrine sites, islets, and pancreatic ducts . In this culture system, cells were seeded using a medium supplemented with 10% FCS, 2Me, nicotinamide, HEPES, ITS-x, insulin, L-glutamine, and gentamicin in DMEM / F12, and EGF, 24 hours after cell seeding. The medium was changed to a medium supplemented with HGF, and thereafter the medium was changed every 48 hours. When E16.5 fetal pancreas was dispersed into single cells by enzyme treatment and seeded on a gelatin-coated dish, a single adherent cell layer was formed. Thereafter, a spherical cell mass was formed on the monolayer adherent cells around day 5, and the spherical cell mass became larger by the 12th day. As described in detail below, this cell mass expresses insulin and glucagon and is considered to be a functional islet. Furthermore, when the number of islet-like structures formed in this culture was converted per individual, it was about 120 (FIG. 8a). This is almost equal to the number of islets formed in the living body. From the above, it is considered that this fetal pancreas culture method reproduces the three-dimensional structure of islets. However, by the same culture, an islet-like structure was not formed in E18.5 pancreatic cells (FIG. 8a). In other words, in this culture method, pancreatic cells before the formation of E16.5 islets form an islet-like structure, but pancreatic islets already formed from E18.5 and later arelet-like structures. Not (Figure 8a). Therefore, it was suggested that this culture system faithfully reflects the developmental process and does not reverse the differentiation process of development.

In order to evaluate the function of the islet-like structure formed by the culture method described above, the cultured cells were collected and examined for the expression of genes that are specifically expressed in the acinar and islets of the adult pancreas (FIG. 8b). As a result, compared to before culture, expression of Pdx-1 and insulin specifically expressed in adult islets increased after culture, and amylase specifically expressed in acinar decreased. Thus, this culture method is thought to induce differentiation of undifferentiated E16.5 fetal pancreatic cells into islet cells. Furthermore, by immunostaining for amylase, insulin, and glucagon (Fig. 9), some of the single-layer adherent cells that occupy most of the cultured cells produce amylase, and the spherical cell mass is insulin in the center and the periphery. It was revealed that glucagon producing cells exist. This structure is consistent with the islet structure in the adult mouse pancreas. In addition, the gene expression data indicates that these cells do not adsorb insulin or the like contained in the medium but express each gene by themselves.
From the above results, this culture system is considered to reproduce the three-dimensional cell construction of the adult pancreas.

(3) Functional analysis of fetal pancreatic progenitor cells As described above, a culture system that reproduces the three-dimensional cell structure of adult pancreas from E16.5 fetal pancreatic cells was created. By this culture method, the differentiation ability of E16.5 fetal pancreatic PCLP1 ⁺ Dlk ⁺ cells considered to contain progenitor cells was evaluated using 3D cell construction as an index. As a result, only PCLP1 ⁺ Dlk ⁺ cells formed the islet-like structure, and other cells did not form the islet-like structure, but formed only a single fibroblast-like planar structure. When the frequency of islet-like structures is quantified (Figure 10), 19.0 ± 1.7 islets are formed per 1x10 ⁵ cells in the whole pancreas culture, whereas 1x10 ⁵ cells in the case of PCLP1 ⁺ Dlk ⁺ cells 41.3 ± 4.0 cells were formed, and other cells did not form islet-like structures at all. These results indicate that the cells forming the islets are contained only in the PCLP1 ⁺ Dlk ⁺ fraction. Furthermore, when comparing the size of the islet-like structures, the diameter was 125 to 150 μm in the culture of unfractionated cells, and the diameter was 75 to 100 μm in the culture of PCLP1 ⁺ Dlk ⁺ cells. These results suggest that PCLP1 ⁺ Dlk ⁺ cells form islets and other cells have some effect on islet growth and maturation.

In order to analyze the structure of a three-dimensional cell cluster formed from PCLP1 ⁺ Dlk ⁺ cells, immunostaining of amylase and insulin was performed (FIG. 10b). As a result, in the culture of PCLP1 ⁺ Dlk ⁺ cells, monolayer adherent cells produced amylase, and the central part of the islet-like structure produced insulin. On the other hand, amylase and insulin were hardly produced in other cell cultures. From these results, it was found that the cells constituting the acinar and pancreatic islets appeared only from PCLP1 ⁺ Dlk ⁺ cells and not from other cells. Furthermore, when other cells were cultured for a long period (1 month or longer), tubular structures appeared, but PCLP1 ⁺ Dlk ⁺ cells did not form tubular structures. Therefore, it was suggested that other cells include progenitor cells that form a tubular structure such as the pancreatic duct. Furthermore, immunostaining and gene expression results suggest that PCLP1 ⁺ Dlk ⁻ cells contain progenitor cells that form tubular structures such as the pancreatic duct.

The above in vitro experimental results showed that PCLP1 ⁺ Dlk ⁺ cells are pancreatic parenchyma acinar and islet (exocrine and endocrine) stem / progenitor cells. In addition, other cells have already differentiated to some extent, and gene expression results suggest that they are progenitor cells of the acinus, islets, and pancreatic ducts, but in vitro experiments differentiate into pancreatic ducts and mesenchymal cells. It has been suggested.

(4) In vivo functional analysis of PCLP1 ⁺ Dlk ⁺ cells by transplantation into diabetes model mice
In order to analyze the in vivo differentiation potential of E16.5 fetal pancreatic PCLP1 ⁺ Dlk ⁺ cells, these cells were transplanted into diabetic model mice. In order to investigate the function of islets in vivo, it is best to create a diabetes model mouse and transplant the islets under the kidney capsule to confirm that hyperglycemia is improved. Therefore, in order to create a diabetes model mouse, streptozotocin (streptozocin; STZ) was administered to wild type mice. As a result, it was found that STZ had no effect at 100 mg / kg body weight or less, but at higher doses, the rate of hyperglycemia increased in a dose-dependent manner (FIG. 11). However, there were large individual differences, for example, there were mice that were ineffective and those that died even when 150 mg / kg body weight was administered. Furthermore, it is not easy to create a diabetes model mouse because the responsiveness to STZ varies depending on the season. Therefore, STZ was administered to a large number of mice, and mice with blood glucose levels of 400 to 500 mg / dL were designated as hyperglycemic mice and mice with blood glucose levels of 300 mg / dL or less were subjected to the following experiments. .

The fetal pancreatic cells separated by the cell sorter were transplanted under the renal capsule of a diabetes model mouse prepared by STZ administration, and the blood glucose improvement effect was examined (FIG. 12). As a result, unfractionated pancreatic cells and PCLP1 ⁺ Dlk ⁺ cells showed hypoglycemic ability in hyperglycemic mice, but other cells did not have such a function. On the other hand, when PCLP1 ⁺ Dlk ⁺ cells were transplanted into normoglycemic mice, hypoglycemia did not occur, indicating that insulin production was regulated by blood glucose. That is, PCLP1 ⁺ Dlk ⁺ cells are considered to have differentiated into functional islet cells in vivo. However, the hypoglycemic effect of cell transplantation was maintained only for about 1 week, and it returned to the original blood glucose after 2 weeks from the transplantation. From this, it is understood that hyperglycemia was improved although it was transient. This is because although fetal-derived PCLP1 ⁺ Dlk ⁺ cells differentiated into islet cells in vivo, they did not form a three-dimensional structure of islets in the environment of the organism, so that the function as islets could not be maintained for a long time. It is thought to be the cause. In fact, the Edmonton protocol also shows that hypoglycemic ability is not maintained for a long time even when β cells that do not form islet structures are transplanted.
Thus, PCLP1 ⁺ Dlk ⁺ cells were shown to differentiate into islet cells that function normally in vivo.

(5) Improvement of pancreatic cell culture method The above culture system required a specific lot of serum. When the lot was changed, the medium immediately became yellow, and at the same time as the islet formation, the cells detached from the dish into a sheet. Thus, as many serum samples as possible were collected and fetal pancreatic cells were cultured, but the cells were detached in any of the 24 types collected. Based on this result, it was judged that the serum used above was special, and it was difficult to construct a culture system similar to the previous one only by changing the serum. It is thought that cells are directly or indirectly affected by excess or deficiency of some component contained in serum. Considering that the subject of culture is the pancreas, we focused on the digestive enzymes secreted by the pancreas. Since it was already known that fetal pancreatic cells differentiate into exocrine cells of the acinus, considering the possibility of self-digestion by the digestive enzymes they release, a cocktail of protease inhibitors was added to the medium, Cell detachment was significantly suppressed. Therefore, trypsin inhibitor was added, but since the culture was detached as before, the cells may have been detached by self-digestion by proteases other than trypsin secreted by cells differentiated into exocrine cells. Was considered. Protease activity can be inhibited to some extent by the addition of serum, so increasing the serum concentration unexpectedly increases cell detachment when the serum concentration is increased (15%, 20%). Lowering (5%) suppressed cell detachment. From this result, it is possible that the serum used previously had few factors that induce differentiation of fetal pancreatic cells into exocrine cells or secrete and stimulate proteases other than trypsin to exocrine cells.

In order to investigate whether the obtained culture system has serum lot dependency, and to further improve the culture system, the culture system was improved using cell detachment and islet formation frequency as indices. First, the serum concentration in the culture system was changed to 5%, and fetal pancreatic cells were cultured using the collected serum samples. As a result, exfoliation of the cells was suppressed except for some serums, and it was found that the frequency of islet formation varies depending on the serum (FIG. 13a). Among the sera with relatively high islet formation efficiency, the following experiments were conducted using sera stocked in large quantities by the present inventors (FIG. 13a, Ser20). Next, when the serum was fixed at 5% and the components to be added to the medium were examined again, efficient conditions without cell detachment were narrowed down to three (FIG. 13b, Med-4, 7, 8). Examining these in more detail, Med-4 and 7 showed some peeling at the edge of the dish, so the optimal condition was Med-8 (DMEM / F12 + 5% FCS + 2Me + nicotinamide + HEPES + gentamicin + insulin + EGF + GLP-1).
Unlike the previous culture system, this culture system had three-dimensional fibroblast-like cells that covered the entire dish, and there was an islet-like structure (Fig. 14).

(6) Characterization of improved culture system In order to analyze the properties of cells obtained under the above culture conditions, E14.5, E16.5, E18.5 fetal pancreatic cells were cultured and I counted the number. As a result, the islets were formed from the E14.5 and E16.5 pancreatic cells before the formation of the islets in the living body, but the islets were formed from the E18.5 pancreatic cells that had already been formed in the living body. There was no (Figure 15a). From this, it is considered that the culture system reproduces the normal development process as in the previous culture method. Compared with the E16.5 culture (FIG. 15b), this culture system obtained an islet formation rate more than 5 times that of the previous method, which is more frequent than the number of islets in the living body. This suggests that this improved culture system is more suitable for differentiating E16.5 pancreatic cells into islets. On the other hand, even when E14.5 pancreatic cells were cultured, the frequency of islet formation was lower than when E16.5 pancreatic cells were cultured. It begins to acquire gradually from before .5, and the ability is considered to reach its peak between E14.5 and E16.5.

  Next, in order to confirm differentiation by the main culture system, the expression of genes specifically expressed at each stage of pancreas development was confirmed by RT-PCR (FIG. 15c). As a result, the expression of insulin, lipase, and CK7 hardly changed before and after the culture, but the expression of glucagon and amylase was decreased. On the other hand, the expression of Ngn3 and PTF1a-p48 specifically expressed in endocrine cells and progenitor cells of exocrine cells was significantly reduced. From this, it is considered that the progenitor cells are differentiated and lost in this culture system. Also, unlike the previous culture system, this culture system is dominated by fibroblast-like cells, and the total number of cells increases with the number of islets, so the apparent expression level has not increased. it is conceivable that. Next, immunostaining was performed to examine the structure of the three-dimensional cell cluster obtained by this method (FIG. 16). In this culture system, since a three-dimensional islet-like structure is formed in the three-dimensional fibroblast-like cells, it was difficult to observe on the dish as it was in the previous culture. However, taking advantage of the fact that the cells are easy to peel off, the cultured cells were peeled off into a sheet by pipetting, and a frozen section was prepared to allow immunostaining and photography. Therefore, the cultured cells were collected into a sheet form by pipetting after 12 days, and frozen sections were prepared and evaluated by immunostaining with an antibody of a protein specific to the adult pancreas. As a result, this culture system not only builds a three-dimensional structure of islets from fetal pancreatic cells in the same way as the previous culture system, but also reproduces the duct structure that was confirmed in long-term culture in the previous culture system this time. It was also confirmed that it built the three-dimensional structure of the pancreatic duct. These results suggest that this culture system is a culture system that reproduces the three-dimensional structure of islets and pancreatic ducts from fetal pancreatic cells.

(7) Functional analysis in vivo of islets formed by culture In order to confirm that the islets constructed with a three-dimensional structure in the above culture system are equivalent to adult islets, the function in vivo was analyzed.
Pancreatic islets formed by culturing under the renal capsule or cells before culturing were transplanted into a diabetes model mouse and a mouse with low blood sugar prepared by administration of STZ (FIG. 17a). As a result, when transplanted islets formed by culture significantly decreased blood glucose over a long period of time, cells before culture had little effect on lowering blood glucose and were temporary. Furthermore, since the islets formed by culture do not lower the blood glucose level of mice that exhibit normal blood glucose levels, it is thought that they correctly perform the physiological function of releasing insulin in response to the blood glucose level of the living body. . At 12 weeks after transplantation, kidneys were collected, frozen sections were prepared, and the transplanted site was immunostained (Fig. 17b). Under the renal capsule, many 3D structures of islets were retained. It is thought that it was functioning as a pancreatic islet. From the above, it has been clarified that islets formed from fetal pancreatic cells by this culture system function normally in vivo.

(8) Differentiation induction of mouse iPS cells 1x10 ⁶ iPS cells were cultured in collagen type4 coated ⁶ well plate with DMEM + 10% KSR + 55uM 2-Me + 50ng / ml Activin for 4 days, then DMEM + 10% KSR + 55uM 2 -Me + 1uM Retinoic acid was cultured for 4 days. Then, DMEM / F12 + 55uM 2-Me + 1x ITS-x + 2mg / ml BSA + 10ng / ml FGF basic for 3 days, DMEM / F12 + 55uM 2-Me + 1x ITS-x + 2mg / ml BSA The cells were cultured for 5 to 7 days in +10 ng / ml FGF basic + 10 mM Nicotinamide (FIG. 18 was cultured for 5 days). The cells obtained here were peeled off with collagenase and re-plated on a gelatin-coated plate. When 1 well was spread to 1 well and 2 well was spread to 1 well, it appeared that the frequency of islet formation increased when 2 well was spread to 1 well (no data). This is the result of culturing for 14 days using the islet formation culture method developed by us.
The medium was changed every day in the whole process so far. The immunostaining was performed in the same manner as in the above-described “2. Method (8) Immunostaining on the plate” in which the pancreatic islets formed from fetal pancreatic cells were stained on the plate. iPS cells were maintained in the same manner as described in Takahashi K et al. (2007) Nat Protocols 2: 3081-3089.
FIG. 18 shows the result of imaging the same site during culture. The Merge image is a composite of blue, green and red signals on a PC. The staining method was performed in the same manner as the method of staining the islets formed from fetal pancreatic cells on the plate in “2. Method (8) Immunostaining on plate” described above. Gcg / SST / PP, which is stained with Dapi and stained with red (Alexa594) and with insulin (green), stands for glucagon, somatostatin, pancreatic polypeptide. These three are rat antibodies, so they are seen together in one color (Alexa 488). The interpretation of the results is that the β-cells that produce insulin are surrounded by α-, δ-, and PP-cells that produce glucagon, somatostatin, and pancreatic polypeptide. . Although FIG. 18 is a representative photograph, it can be said that a plurality of such structures were observed during the culture, and that the islet was successfully formed from the iPS cells by this culture system.

Claims (33)

A method for detecting pancreatic progenitor cells, comprising detecting expression of a Dlk gene and a PCLP1 gene in a cell. 2. The method for detecting pancreatic progenitor cells according to claim 1, wherein the cells in which expression of the Dlk gene and the PCLP1 gene are detected are determined as pancreatic parenchymal progenitor cells. 2. The method for detecting pancreatic progenitor cells according to claim 1, further comprising detecting c-kit gene expression for cells in which expression of the Dlk gene and PCLP1 gene is detected. 4. The detection method according to claim 1, wherein the gene expression is detected using an antibody that recognizes an expression product of each gene. A method for preparing islet progenitor cells, comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells bound with anti-Dlk antibody and anti-PCLP1 antibody as islet progenitor cells A method for preparing pancreatic duct progenitor cells, comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells to which at least one of the anti-Dlk antibody and the anti-PCLP1 antibody has not bound as pancreatic duct progenitor cells 7. The preparation method according to claim 5 or 6, wherein the step (3) of separating cells is performed using a cell sorter. Cell samples containing pancreatic progenitor cells are selected from fetal pancreatic cells, adult pancreatic cells, fetal intestinal epithelial cells, adult intestinal epithelial cells, fetal liver or cells derived from regenerating liver, adult liver cells, stem cells, ES cells and iPS cells The preparation method according to claim 5 or 6. The method further comprises adding an anti-KIT antibody in step (2), and in addition to the anti-Dlk antibody and the anti-PCLP1 antibody in step (3), separating the cells bound with the anti-KIT antibody as islet progenitor cells. The preparation method described. A method for culturing pancreatic islets comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) A step of culturing the prepared cell sample under conditions for inducing islets A method for culturing pancreatic islets comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells bound with anti-Dlk antibody and anti-PCLP1 antibody as islet progenitor cells
(4) A step of culturing the isolated islet progenitor cells under conditions for inducing islets A method for culturing pancreatic ducts comprising the following steps.
(1) A step of preparing a cell sample containing pancreatic progenitor cells
(2) adding anti-Dlk antibody and anti-PCLP1 antibody to the prepared cell sample
(3) A step of separating cells to which at least one of the anti-Dlk antibody and the anti-PCLP1 antibody has not bound as pancreatic duct progenitor cells
(4) culturing the isolated pancreatic duct progenitor cells under conditions that induce the pancreatic duct 13. The culture method according to claim 11 or 12, wherein the step (3) of separating cells is performed using a cell sorter. Cell samples containing pancreatic progenitor cells are selected from fetal pancreatic cells, adult pancreatic cells, fetal intestinal epithelial cells, adult intestinal epithelial cells, fetal liver or cells derived from regenerating liver, adult liver cells, stem cells, ES cells and iPS cells 13. The culture method according to claim 10 to 12, wherein 13. The culture method according to claim 10, wherein the culture in step (4) is performed in a DMEM / F12 medium supplemented with serum, 2Me, nicotinamide, HEPES, and gentamicin. 16. The culture method according to claim 15, wherein the medium further contains insulin, EGF and GLP-1. 13. The culture method according to claim 10, wherein the medium is exchanged during the culture in the step (4). 6. Islet progenitor cells prepared by the method of claim 5. 7. Pancreatic duct progenitor cells prepared by the method of claim 6. 12. A pancreatic islet cultured by the method of claim 10 or 11. 13. A pancreatic duct cultured by the method of claim 12. 22. The cell, islet or pancreatic duct according to claim 18 to 21, which is used for transplantation. 21. A pharmaceutical composition comprising the cell of claim 18 or the islet of claim 20. 24. The pharmaceutical composition according to claim 23, which is used for treating diabetes. A kit for detecting or preparing pancreatic progenitor cells, comprising a reagent for detecting the expression of PCLP1 gene and Dlk gene. 26. The kit according to claim 25, wherein the reagents for detecting the expression of the PCLP1 gene and the Dlk gene are an anti-PCLP1 antibody and an anti-Dlk antibody. 26. The kit according to claim 25, further comprising a reagent for detecting the expression of the c-kit gene. 28. The kit according to claim 27, wherein the reagent for detecting the expression of the c-kit gene is an anti-KIT antibody. 29. The kit according to any one of claims 25 to 28, which comprises a cell culture medium in addition to a reagent for detecting gene expression. 30. The kit according to claim 25 to 29, wherein the pancreatic progenitor cells to be detected or prepared are islet progenitor cells or pancreatic duct progenitor cells. A pancreatic islet or pancreatic duct culture medium comprising a DMEM / F12 medium containing serum, 2Me, nicotinamide, HEPES, and gentamicin. 32. The medium of claim 31, further comprising insulin, EGF and GLP-1. A screening method for a substance having a regulating action on insulin production / secretion, comprising the following steps.
(1) A step of culturing the islet of claim 20 in the presence of a test substance
(2) A step of detecting the level of insulin production / secretion from the islets
(3) identifying a test substance that alters insulin production / secretion level as a substance having an action of regulating insulin production / secretion compared to the control