JP5122973B2 - Method for producing pancreas from ES cells - Google Patents

Description

  The present invention relates to an organ regeneration technique. More specifically, the present invention relates to a technique for producing a pancreas from undifferentiated cells and the produced pancreas.

  Several methods for producing “pancreas” in vitro from embryonic stem cells (ES cells) have been developed and reported (for example, Non-Patent Document 1 = Loebel, et al., 2003; Non-Patent Document 2 = Soria, et al., 2000; Non-patent document 3 = Shroi, et al., 2002; Non-patent document 4 = Leon-Quinto, et al., 2004; Non-patent document 5 = Lumelsky, et al., 2001). But these pancreas are not perfect. This is because, from any undifferentiated cells of mammals, including ES cells, beta cells (β cells: insulin, which is a hormone that controls the uptake of sugar and the inhibition of gluconeogenesis by body tissues) This is due to the fact that the pancreas has been made by focusing only on making endocrine cells that are made and secreted. This is called cell therapy and tissue therapy to rescue many diabetic patients who cannot be fundamentally treated with current medical standards and technologies by transplanting β-cells with insulinotropic activity. It is because it aims at regenerative medicine. Therefore, it has not been possible to make a pancreas having other functions and structures that a natural pancreas has.

Such natural pancreas is preferably
1) having endocrine cells (β cells) that secrete insulin in response to blood glucose levels (ie, blood glucose concentration);
2) Endocrine cells that produce and secrete glucagon (alpha cells, α cells: endocrine cells that specifically make and secrete glucagon, a hormone that governs glycogen degradation and gluconeogenesis)
3) Having exocrine cells (acinar cells) that make digestive enzymes (eg amylase) and secrete them into the intestinal tract,
4) A pancreas having all functions and structures such as a duct structure for exoculating digestive enzymes from the pancreas to the intestinal tract.

  In a healthy body, blood glucose levels are properly maintained by the competitive action of insulin and glucagon. Therefore, in order to treat diabetes, not a pancreas that secretes only insulin but a pancreas that can simultaneously secrete glucagon is required, but there has been no method for making such a pancreas in vitro. To treat pancreatic dysfunction and patients whose pancreas must be removed because of pancreatic cancer, transplant the pancreas with not only the endocrine function, but also the ability to make digestive enzymes and exoculate it There was no way to make such a pancreas in vitro. In addition, if digestive enzymes are secreted into tissues, the body itself is digested, which is dangerous, so a conduit for introducing digestive enzymes into the digestive tract is also necessary. There was no way to make in vitro.

  In this regard, the present inventors have disclosed Patent Document 1 (JP 2001-333770), Patent Document 2 (JP 2001-299335), Non-Patent Document 6 (Experimental Medicine Vol. 19 No. 15, 159-166). , 2001), Non-Patent Document 7 (Ayumi of Medicine Vol. 195 No. 7, 488-489, 2000), Non-Patent Document 8 (Science Newspaper June 2, 2000, 2000), Non-Patent Document 9 ( The 26th Information Chemistry Conference PL-1, 2003), Non-Patent Document 10 (Japan Tissue Cytochemistry General Assembly / Academic Conference Lecture Program / Preliminary Collection VOL. 44th; PAGE, 29, 2003) Non-Patent Document 11 (Modern Chemistry Special Issue) VOL. 41th; PAGE, 124-131, 2002) and Non-Patent Document 12 (Delope. Growth Differ. 42: 593-60) , In 2000), we have done an attempt to produce a pancreas using a mouse or amphibian. However, the pancreas produced by the methods described in these documents is not fully functional. Further, detailed conditions are not described. In many cases, detailed conditions are unknown in regeneration, but it has also been reported that another organ is produced with a slight difference in concentration. Thus, from our previous disclosures, it cannot be said that a fully functional pancreas can be produced.

  Patent Document 3 (Japanese Patent Application Laid-Open No. 2003-9854) includes a step of culturing embryonic stem cells using a medium containing a specific factor, and a method of forming an embryoid body from embryonic stem cells, A medium and differentiation inducer for use in the method, a method for inducing differentiated cells using the method, the cells induced to differentiate, and use thereof are disclosed.

  In addition, attempts to produce pancreas from undifferentiated cells include Patent Document 4 (WO2002 / 079457); Patent Document 5 (WO2002 / 086107); Patent Document 6 (WO2002 / 010347); Patent Document 7 (WO2002 / 059278). ); Patent Document 8 (WO 2002/092756); Non-Patent Document 13 (Developmental Biology 264: 1-14, 2003); Non-Patent Document 14 (Diabetics 49: February, 1-6, 2000); cells 20: 284-292, 2002); Non-patent document 16 (Diabetologia 47: 1442-1451, 2004); Non-patent document 17 (Science 292: 1389-1394, 2001); Non-patent document 18 (Microscopy research and technique 61: 448-456, 2003); Non-patent document 19 (Microscopic research and technique 37: 374-383, 1997); Non-patent document 20 (Stem cells 23: 656-66; Non-Patent Document 21 (Develop. Growth Differ. 42: 593-602, 2000); Non-Patent Document 22 (Exp. Anim. 54 (3) .262, P-048, 2005), etc. No pancreatic production has been achieved, and production from mammalian cells is rare. Furthermore, production by differentiation from highly undifferentiated cells such as ES cells has hardly been attempted, and there is no example of successful production of a functional pancreas from mammalian ES cells.

As mentioned above, producing fully functional pancreas from a variety of undifferentiated cells including embryonic stem cells (ES cells) has not yet been achieved.
Loebel, D.C. A. Watson, C .; M.M. , De Young, R .; A. Tam, P .; P. Dev. Biol. , 264, 1 (2003). Soria, B .; Roche, Berna, G .; Leon-Quinto, T .; , Reig, J .; A. Martin, F .; Diabetes, 49, 157 (2000). Shroi, A .; Yoshikawa, M .; Yokota, H .; Fukui, H .; Ishizuka, S .; Tatsumi, K .; Takahashi, Y .; , Stem Cells, 20, 284 (2002). Leon-Quinto, T.W. Jones, J .; Skuddy, A .; Burcin, M .; , Soria, B .; Diabetologia, 47, 1442 (2004). Lumelsky, N .; Blondel, O .; Laeng, P .; , Velasco, L .; Ravin, R .; McKay, R .; , Sinece, 292, 1389 (2001). Experimental Medicine Vol. 19 No. 15, 159-166, 2001 History of Medicine Vol. 195 No. 7,488-489,2000 Science Newspaper June 2, 2000 issue, 2000 The 26th Symposium on Information Chemistry PL-1,2003 Annual Meeting of Japanese Tissue Cytochemistry / Academic Meetings / Preliminary Proceedings VOL. 44th; PAGE, 29, 2003 Hyundai Chemical Special Edition VOL. 41th; PAGE, 124-131, 2002 Delop. Growth Differ. 42: 593-602, 2000 Bock, G.M. Pollard, H .; B. , Srivastava, M .; Leapman, R .; Microscopy Res. Tech. 61, 448 (1997). Goping, B.M. Abdel-Money, M .; Egerbacher, M .; (2003). JP 2001-333770 A JP 2001-299335 A Japanese Patent Laid-Open No. 2003-9854 International Publication 2002/0779457 Pamphlet International Publication 2002/086107 Pamphlet International Publication 2002/010347 Pamphlet International Publication 2002/059278 Pamphlet International Publication 2002/092756 Pamphlet

  It is an object of the present invention to produce a fully functioning pancreas from undifferentiated cells.

To develop a method for in vitro production of pancreas having natural function and structure from mammalian undifferentiated cells. Specific issues are as follows.
1) have beta cells that secrete insulin in response to glucose load,
2) Has not only β cells but also α cells as endocrine cells,
3) Has exocrine cells (acinar cells) that make digestive enzymes (amylase, etc.)
4) To make a pancreas with all functions and structures, such as a conduit structure.

  If such a pancreas can be made from mammalian undifferentiated cells, it may provide a useful material for treating many diabetic patients, pancreatectomy patients and the like. If the pancreas has the same function and structure as the natural pancreas, biological and medical research related to the pancreas, research on the cause of pancreatic disease, treatment of pancreatic disease, and pancreatic cancer It can also be expected to be a useful model and evaluation system (assay system) in the development of drugs for treatment.

  The above-mentioned problem has been found that the present inventors can differentiate into a functional pancreas by performing specific treatment on undifferentiated cells in advance using specific concentrations of activin and retinoic acid. Solved by.

  The present invention comprises a combination of mammalian “undifferentiated cells” and “treatment of activin and retinoic acid”. As illustrated in FIG. 1, first, undifferentiated cells are cultured in a serum medium in the presence of leukemia inhibitory factor (LIF) on feeder cells to form colonies of ES cells. (Embryoid body-like spheres, EBS). Thereafter, activin and retinoic acid are simultaneously treated in a serum-free medium in the absence of feeder cells and LIF, and the pancreas is induced to differentiate from the formed aggregate in vitro.

  FIG. 1 shows a schematic representative diagram. Schematic of differentiation induction of pancreas from undifferentiated cells (ES cells). LIF, Leukemia Inhibitory Factor; EBS, Embryoid body-like spheres.

  More specifically, mouse-derived ES cells, which are good models for representing mammals, are used as undifferentiated cells. First, feeder cells are cultured in a gelatin-coated culture vessel in a serum medium (fetal bovine serum, FBS medium), and ES cells are cultured in the presence of LIF (leukemia inhibitory factor) to collect ES cells. A colony that is a body, and also its agglomerates. Next, the agglomerates thus obtained were subjected to induction medium supplemented with activin and retinoic acid (10% serum replacement (knockout serum replacement), under conditions of suspension culture using a low-contact coated culture plate. KSR; Gibco) for 2 days In addition, the pancreas can be made by culturing the aggregates in KSR medium in gelatin-coated culture plates (FIG. 1).

  The pancreas made by inducing differentiation in this way has α cells and β cells, which are pancreatic endocrine cells, and increases insulin secretion in response to glucose load. The presence of pancreatic exogenous cells and duct structures, specifically with the development of light microscope, electron microscope, anti-insulin antibody (anti-insulin C-peptide antibody), anti-glucagon antibody, anti-amylase antibody, pancreas This was confirmed by identifying gene markers that increased and decreased.

Accordingly, the present invention provides the following.
(1) A method for producing pancreas from undifferentiated cells, comprising the following steps:
A) a pre-culture step in which undifferentiated cells are cultured in a medium containing leukemia inhibitory factor (LIF) to form colonies and form aggregates; and B) activin is added to the undifferentiated cells in the absence of LIF. A method comprising an exposure step of exposure to retinoic acid.
(2) The method according to the above item, wherein the undifferentiated cells are cultured on feeder cells.
(3) The said aggregate is a method as described in the said item which is an embryoid body-like aggregate (embryoid body-like sphere).
(4) The said preculture process is a method as described in said item performed for 1 to 10 days.
(5) The method according to the above item, wherein the pre-culture step is performed for about 4 days.
(6) The said exposure process is a method as described in said item performed for 1-4 days.
(7) The said exposure process is a method as described in said item performed for about 2 days.
(8) The method according to the above item, wherein the undifferentiated cells are mammalian cells.
(9) The method according to the above item, wherein the medium contains serum.
(10) The method according to the above item, wherein the medium is a knockout serum replacement (KSR) medium.
(11) The method according to the above item, wherein the exposure step is performed under conditions of suspension culture.
(12) The method according to item 1, wherein the suspension culture is performed on a low adhesion coating culture vessel.
(13) The method according to the above item, wherein the undifferentiated cells are embryonic stem (ES) cells.
(14) The method according to item 2, wherein the feeder cell is a mouse fetal fibroblast.
(15) The method according to the above item, wherein the activin is less than 50 ng / ml.
(16) The method according to the above item, wherein the activin is 1 ng / ml to less than 50 ng / ml.
(17) The method according to the above item, wherein the activin is 1 ng / ml to 25 ng / ml.
(18) The method according to the above item, wherein the activin is 10 ng / ml to 25 ng / ml.
(19) The method according to the above item, wherein the retinoic acid is more than 0.01 μM and less than 1 μM.
(20) The method according to the above item, wherein the retinoic acid is more than 0.01 μM and 0.1 μM or less.
(21) The method according to the above item, wherein the retinoic acid is 0.1 μM or more and less than 1 μM.
(22) The method according to the above item, further comprising a differentiation step of culturing the differentiated aggregate in the medium on the coated culture vessel.
(23) The method according to the above item, wherein the coating is a gelatin coating.
(24) The method according to the above item, wherein the medium used in the differentiation step is a KSR medium.
(25) The method according to the above item, wherein the differentiation step is a period sufficient for producing a functional pancreas.
(26) The said differentiation process is a method as described in said item performed for 7 to 21 days.
(27) The method according to the above item, wherein the differentiation step is performed for 10 to 14 days.
(28) The method according to the above item, wherein the differentiation step includes a step of first culturing in a medium containing activin and retinoic acid and then culturing in a medium not containing activin and retinoic acid.
(29) The differentiation step is a step of first culturing in a medium containing activin and retinoic acid for about 1 to 4 days, and then culturing in a medium not containing activin and retinoic acid for 1 to 3 weeks. A method according to the above item comprising.
(30) The differentiation step is a step of first culturing in a medium containing activin and retinoic acid for about 2 days, and then culturing in a medium not containing activin and retinoic acid for about 10 days to about 2 weeks. A method according to the above item comprising.
(31) The method according to the above item, wherein the use concentration of the activin and the retinoic acid is a condition selected from the group consisting of the above items 15 to 21.
(32) The method according to the above item, wherein the functional pancreas has at least one characteristic selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability and functional conduit structure. .
(33) The method according to the above item, wherein the functional pancreas expresses a pancreatic marker Ipf1 / pdx-1 and does not express a marker Shh whose expression level decreases at a time when the pancreas is formed. .
(34) The method according to the above item, wherein the functional pancreas expresses at least one pancreatic marker selected from the group consisting of insulin, glucagon and amylase 2.
(35) The method according to the above item, wherein the functional pancreas comprises α cells, β cells, exocrine cells, and a duct structure.
(36) The method according to the above item, wherein the functional pancreas has all the characteristics selected from the group consisting of insulin production ability, glucose responsiveness, glucagon production ability, amylase production ability and functional conduit structure.
(37) A pancreas produced by the method described in the above item.
(38) A method for treating a patient requiring a functional pancreas, characterized by using the pancreas described in the above item.
(39) The method according to the above item, wherein the patient suffers from diabetes.
(40) The method according to the above item, wherein the patient is a mammal.
(41) Use of activin and retinoic acid for the manufacture of a medicament for the treatment of a patient in need of a functional pancreas.
(42) The use according to item 1 above, wherein the patient suffers from diabetes.
(43) The use according to the above item, wherein the patient is a mammal.
(44) The use according to the above item, wherein the medicament is produced from stem cells.
(45) The use according to the above item, wherein the medicament is produced from embryonic stem cells.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  Accordingly, it is understood that these and other advantages of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description, with reference where appropriate to the accompanying drawings and the like.

By this method, from undifferentiated mammalian cells that have not been realized so far,
1) having β cells that synthesize and secrete insulin in response to glucose load;
2) Not only β cells but also α cells that synthesize and secrete glucagon as endocrine cells,
3) Have exocrine cells (acinar cells) that make digestive enzymes (such as amylase) and secrete them into the intestine.
4) It was the first in the world to create a pancreas with all functions and structures such as a duct structure for exoculating digestive enzymes from the pancreas into the intestinal tract.

  Β cells derived from human ES cells by a method different from the method of the present inventors have already been experimentally applied to the treatment of human diabetes in the United States. If the method developed by the present inventors can produce not only β cells from human undifferentiated cells but also a complete pancreas (functional pancreas) as shown by the present inventors, many diabetes It may provide unprecedented useful materials for treating not only patients and patients with pancreatic dysfunction, but also patients who have to remove the pancreas due to pancreatic cancer and the like. Also, useful models and evaluation systems (assay systems) in biological and medical research related to the pancreas, research on the elucidation of the cause of pancreatic diseases, and the development of drugs for the treatment of pancreatic diseases and pancreatic cancer You can also expect to be. Thus, it is judged that the practical application and industrial prospect of this method are extremely bright.

Schematic of differentiation induction of pancreas from undifferentiated cells (ES cells). LIF, Leukemia Inhibitory Factor; EBS, Embryoid body-like spheres. All-cis retinoic acid (RA) and activin were used. Mouse-derived ES cells were used as undifferentiated cells. First, feeder cells are cultured in a gelatin-coated culture vessel in a serum medium (fetal bovine serum, FBS medium), and ES cells are cultured in the presence of LIF (leukemia inhibitory factor) to collect ES cells. A colony that is a body, and also its agglomerates. Next, the agglomerates thus obtained were subjected to induction medium supplemented with activin and retinoic acid (10% serum replacement (knockout serum replacement), under conditions of suspension culture using a low-contact coated culture plate. KSR; Gibco) for 2 days In addition, the pancreas can be made by culturing the aggregate in KSR medium in a gelatin-coated culture plate. FIG. A, an intestinal tract-like structure induced by activin and retinoic acid differentiation from colony aggregates (EBS) of mouse ES cells, and a tissue (arrow) containing a duct-like structure and black spots developed on its side. The gut-like structure in the center performs a slow peristaltic movement. The feature of the tissue with black spots is that of secretory tissue. B, Colony aggregate (EBS) of mouse ES cells cultured in the same manner in the absence of activin and retinoic acid. The intestinal tract-like structure, duct-like structure seen in A, and tissues containing black spots are not seen. Scale bar is 100 μM. It is a phase-contrast micrograph about EBS including a differentiated pancreas and gene expression after differentiation induction. (A, b) Intestinal tubular structure (G) and pancreatic tubular structure (D) shown 13 days after treatment with 0.1 μM retinoic acid and 10 ng / ml activin. In the vicinity there are exocrine cells, which are observed as numerous black spots (b, arrowheads). (C) Expression of pancreatic marker genes examined by RT-PCR in EBS treated and untreated EBS with retinoic acid and activin. Insulin II, glucagons, somatostatin and Ipf1 / pdx-1 expression began to increase on day 2 after treatment, while amylase and pancreatic polypeptide began to increase on days 6 and 8, respectively. Shh expression decreased after treatment [bar = 50 μM (a and b)]. (D) The most efficient gene expression of all pancreatic markers in EBS was observed when 0.1 μM retinoic acid was used with 10 ng / ml activin A. When retinoic acid and activin were used at this concentration, 47.6 ± 7.3% of the treated EBS expressed the pancreatic gene. At other concentrations, pancreatic gene expression was not efficiently induced. FIG. A, an electron microscopic image of a tissue (see FIG. 1) containing black spots differentiated with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. B, Electron microscopic image of pancreatic exocrine cells of rat fetus in late pregnancy (Bock, et al., 1997). Both cells are very similar in structure, including a large number of granule-like structures with high electron density and having developed endoplasmic reticulum. ER, endoplasmic reticulum; N, nucleus. FIG. C, Electron microscopic image of a duct-like structure (see FIG. 1) induced by differentiation with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. Very similar to the epithelial structure of the natural pancreatic duct. D, similarly, an electron microscopic image of a cell (see FIG. 1) having a secretory granule-like structure induced by differentiation with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. An enlarged image of a portion surrounded by a dotted line in the photograph is also shown. This image is similar to the endocrine granule image (Goping, et al., 2003; indicated by an arrow) in the pancreatic β cells of the mouse body shown on the right (E). Gaps are seen between the wrapping bags (indicated by arrows). N, nuclear. EBS morphology including differentiated pancreas. A section of EBS treated with 0.1 μM retinoic acid and 10 ng / ml activin became a spherical tissue on the 11th day after the treatment, and the vicinity of the tubular cavity was strongly stained with toluidine blue (a, arrowhead). A pancreas-like structure (D) was observed (c, d). A structural algae resembling a pancreatic duct surrounded by a low columnar epithelium was also observed (d). In non-treated EBS sections, neither spherical nor tubular tissue structures were seen (b). N is the nucleus, and the scale bar is 50 μm (ac) or 5 μm (d). FIG. 4 shows an immunohistological observation image of colony aggregates (EBS) derived from mouse ES cells treated with 1.10 ng / mL activin and 0.1 μM retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino-2-phenylindole; nuclear stain), D, A + B + C. Scale bar is 50 μM. Insulin C peptide and amylase are made by different cells. Under these conditions, amylase is preferentially synthesized over insulin. Fig. 4-2 Immunohistological observation image of colony aggregates (EBS) derived from mouse ES cells treated with 2.25 ng / mL activin and 0.1 µM retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino-2-phenylindole; nuclear stain), D, A + B + C. Scale bar is 50 μM. As in FIG. 4-1, insulin C peptide and amylase are produced by different cells, but under these conditions, insulin is synthesized preferentially over amylase. Structural analysis of EBS including differentiated pancreas. In electron microscopy, cells containing large amounts of zymogen particles became apparent (a, b), which were highly developed endoplasmic reticulum and showed dark homogeneous staining. This is a characteristic of acinar cells (a). Cells that were morphologically similar to pancreatic endocrine cells were also observed (c, d). However, they were fewer in number than cells similar to pancreatic exocrine cells. These cells contained large amounts of endocrine granules (d). This was morphologically different from the granules contained in exocrine cells (b). The large space between the dark central region and the lateral membrane is consistent with the pancreatic β-cell granules, which was also observed in (d). (ER: endoplasmic reticulum, M: mitochondria, N: nucleus, Z: zymogen granule). Scale bars are 5 μm (ac) and 1 μm (d). Immunohistochemical detection of marker proteins. Immunostaining of EBS treated with 0.1 μM retinoic acid and 10 ng / ml activin revealed the presence of cells containing amylase positive zymogen granules (c) and a few cells containing insulin C peptide positive granules (d) . The pancreas of an 8-week-old mouse was stained and used as a positive control (a, b). Cells were counterstained with DAPI (4 ', 6-diamidino-2-phenylindole) (ad). (ER: endoplasmic reticulum, M: mitochondria, N: nucleus, Z: zymogen granule). The scale bar is 20 μm. Expression of pancreatic marker gene and shh in EBS. Total RNA was collected from EBS treated with retinoic acid (0, 0.1 μM and 1 μM) and activin (0, 10 ng / ml, and 25 ng / ml), and the expression levels of the following markers were measured using real-time PCR Amylase 2 (a), insulin II (b), ptfla / p48 (c) and Shh (d). Expression levels were normalized to those in untreated EBS. Shh expression was decreased in all combinations of retinoic acid alone, activin alone and treatment with a combination of retinoic acid and activin (c). Increases in the amounts of insulin II and amylase 2 were seen in EBS treated with retinoic acid alone, but activin alone had no effect on insulin expression compared to untreated EBS. However, activin alone reduced amylase2 expression (a, b). When EBS was treated with a constant concentration of retinoic acid (0.1 μMla, b middle) and treated with a relatively low concentration of activin (10 ng / ml), the expression of amylase2 was observed in both retinoic acid and activin. The treatment group was much higher than the untreated group, while insulin II expression was unchanged in the two groups. In contrast, when retinoic acid was combined with high concentrations of activin (25 ng / ml), an approximately 150-fold increase in insulin II expression was seen, while a relatively low level of amylase 2 expression was observed. The expression pattern of ptf1a / p48 was strongly correlated with amylase2 mono. MRNA expression profile obtained by RT-PCR method of pancreatic structure induced to differentiate with activin and retinoic acid from colony aggregate (EBS) of mouse ES cells. Insulin 2, glucagon, amylase 2 and Ipf1 / pdx-1 are marker genes that are specifically expressed in pancreas development (positive markers), whereas Shh is a gene that decreases in expression with pancreas development (negative). Marker). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was a constitutive protein (enzyme) expressed at a high level in all cells, and was used for the purpose of ensuring that the performed assay method was appropriate. Effect of stimulatory concentration on the appearance of exocrine and endocrine cells. Antitumor of 8-week-old mice pancreas (a), untreated EBS (b) and EBS treated with 0.1 μM retinoic acid and 10 ng / ml activin (c) or 25 ng / ml activin (d, e) The cells were stained with insulin C peptide antibody, anti-amylase antibody and DAPI 13 days after treatment (19 days after the start of EBS formation). Neither insulin-positive cells nor amylase-positive cells were observed in untreated EBS (b). EBS treated with 0.1 μM retinoic acid and 10 ng / ml activin had fewer insulin positive cells than amylase positive cells (c). In contrast, EBS treated with 0.1 μM retinoic acid and 25 ng / ml activin significantly increased insulin producing cells (d). Further, by anti-glucagon antibody staining, a small but significant number of glucagon-producing cells (red) were observed mixed with insulin-producing cells (e). Scale bar: 50 [mu] (ad) and 20 [mu] m (e).

Description of sequence listing

SEQ ID NO: 1 is the nucleic acid sequence of human inhibin βA accession number NM002192. It can be used as activin in the present invention.
SEQ ID NO: 2 is the amino acid sequence of accession number NM002192 of human inhibin βA. It can be used as activin in the present invention.
SEQ ID NO: 3 is the nucleic acid sequence of human inhibin βB accession number NM002193. It can be used as activin in the present invention.
SEQ ID NO: 4 is the amino acid sequence of accession number NM002193 of human inhibin βB. It can be used as activin in the present invention.
SEQ ID NO: 5 is the nucleic acid sequence of human inhibin βC accession number NM005538. It can be used as activin in the present invention.
SEQ ID NO: 6 is the amino acid sequence of accession number NM005538 of human inhibin βC. It can be used as activin in the present invention.
SEQ ID NO: 7 is the nucleic acid sequence of accession number NM002191 for human inhibin α. It can be used as activin in the present invention.
SEQ ID NO: 8 is the amino acid sequence of accession number NM002191 for human inhibin α. It can be used as activin in the present invention.
SEQ ID NO: 9 is the nucleic acid sequence of accession number: X68250 (Xenopus activin A). It can be used as activin in the present invention.
SEQ ID NO: 10 is the amino acid sequence of accession number: X68250 (Xenopus activin A). It can be used as activin in the present invention.
SEQ ID NO: 11 is the nucleic acid sequence of inhibin βB (accession number: S61773) for Xenopus laevis. It can be used as activin in the present invention.
SEQ ID NO: 12 is the amino acid sequence of inhibin βB (accession number: S61773) for Xenopus laevis. It can be used as activin in the present invention.
SEQ ID NO: 13 is the nucleic acid sequence of human LIF.
SEQ ID NO: 14 is the amino acid sequence of human LIF.
SEQ ID NO: 15 is the nucleic acid sequence of mouse LIF.
SEQ ID NO: 16 is the amino acid sequence of mouse LIF.
SEQ ID NO: 17 is an amylase 2 forward primer.
SEQ ID NO: 18 is an amylase 2 reverse primer.
SEQ ID NO: 19 is a glucagon forward primer.
SEQ ID NO: 20 is a glucagon reverse primer.
SEQ ID NO: 21 is insulin II forward.
SEQ ID NO: 22 is an insulin II reverse primer.
SEQ ID NO: 23 is an Ipf1 / pdx-1 forward primer.
SEQ ID NO: 24 is an Ipf1 / pdx-1 reverse primer.
SEQ ID NO: 25 is a somatostatin forward primer.
SEQ ID NO: 26 is a somatostatin reverse primer.
SEQ ID NO: 27 is a pancreatic polypeptide glucagon forward primer.
SEQ ID NO: 28 is a pancreatic polypeptide glucagon reverse primer.
SEQ ID NO: 29 is a Shh forward primer.
SEQ ID NO: 30 is a Shh reverse primer.
SEQ ID NO: 31 is a GAPDH forward primer.
SEQ ID NO: 32 is a GAPDH reverse primer.
SEQ ID NO: 33 is an amylase 2 forward RT-PCR primer.
SEQ ID NO: 34 is an amylase 2 reverse RT-PCR primer.
SEQ ID NO: 35 is a GAPDH forward RT-PCR primer.
SEQ ID NO: 36 is a GAPDH reverse RT-PCR primer.
SEQ ID NO: 37 is an insulin II forward RT-PCR primer.
SEQ ID NO: 38 is an insulin II reverse RT-PCR primer.
SEQ ID NO: 39 is an Ipf1 / pdx-1 forward RT-PCR primer.
SEQ ID NO: 40 is an Ipf1 / pdx-1 reverse RT-PCR primer.
SEQ ID NO: 41 is a ptfla forward RT-PCR primer.
SEQ ID NO: 42 is a ptfla reverse RT-PCR primer.
SEQ ID NO: 43 is a Shh forward RT-PCR primer.
SEQ ID NO: 44 is a Shh reverse RT-PCR primer.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “pluripotency” or “pluripotency” is used interchangeably, refers to the nature of a cell, and refers to the ability to differentiate into one or more, preferably two or more, various tissues or organs. . Accordingly, “pluripotency” and “multipotency” are used interchangeably with “undifferentiated” in this specification unless otherwise specified. In general, cell pluripotency is limited as development progresses, and in adults, the constituent cells of one tissue or organ rarely change into another cell. Therefore, pluripotency is usually lost. In particular, epithelial cells are unlikely to change into other epithelial cells. When this happens, it is usually a pathological condition and is called metaplasia. However, mesenchymal cells have a high degree of pluripotency because they are likely to undergo metaplasia instead of other mesenchymal cells with relatively simple stimulation. Embryonic stem cells are pluripotent. Tissue stem cells are pluripotent. In the present specification, among pluripotency, the ability to differentiate into all types of cells constituting a living body like a fertilized egg is called totipotency, and pluripotency can encompass the concept of totipotency. Whether or not a certain cell has pluripotency includes, but is not limited to, formation of embryoid bodies and culture under differentiation-inducing conditions in an in vitro culture system. In addition, assay methods for the presence or absence of pluripotency using living organisms include the formation of teratomas by implantation into immunodeficient mice, the formation of chimeric embryos by injection into blastocysts, and transplantation into living tissues. Examples include, but are not limited to, proliferation by injection into ascites.

  As used herein, “undifferentiated cell” refers to a cell having multipotency as described above. Examples of undifferentiated cells include, but are not limited to, stem cells.

  As used herein, “stem cell” refers to a cell having a self-renewal ability and having pluripotency (ie, “pluripotency”). Stem cells are usually able to regenerate the tissue when the tissue is damaged. As used herein, stem cells can be embryonic stem (ES) cells or tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), but are not limited thereto. In addition, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.) can also be a stem cell. Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is limited, are present at specific positions in the tissue, and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. As used herein, stem cells may preferably be embryonic stem cells, although tissue stem cells can also be used depending on the circumstances.

  In the present specification, the term “feeder layer” or “feeder cell” is used interchangeably, and is used interchangeably, and is provided on a culture substrate, and is used to proliferate cell types that cannot be maintained in culture alone. It refers to a feeder cell layer with other cell types that allows differentiation expression. It is said that there are many types of tissue cells that cannot be differentiated or even proliferated under normal cell culture conditions. Examples of such cells include certain types of stem cells (particularly epithelial stem cells, embryos, etc.). Sex stem cells, hematopoietic stem cells, etc.), cornea, epidermis and the like. In general, these cell types are highly auxotrophic and require specific growth factors and differentiation-inducing factors. In such a cell type, by utilizing a layer of a specific cell formed by a supporting cell of the cell type in vivo or a similar cell as a culture substrate, factors required by the cell type to be cultured and / or It is said that it will proliferate and differentiate when supplied with nutrient sources. The cell type used as the feeder cell is selected depending on the target cell type, and is often used by inhibiting cell growth by a method such as antibiotic administration (for example, mitomycin C) or UV irradiation. It is said that the use of the feeder layer is largely responsible for the culture of germ cells, early embryonic cells, hematopoietic stem cells, etc. As feeder cells, fibroblasts, adipose-derived stem cells, embryonic stem cells, bone marrow stem cells, and the like can be used. In such a case, the feeder is removed and cultured under the most basic culture condition of DMEM + 10% FBS. Thus, it is preferably used after being differentiated into fibroblasts.

  In the present specification, the “primary cultured cell” refers to a cell in a state of being cultured until a cell, tissue, organ or the like separated from the body is implanted and the first passage is performed.

  Any culture solution can be used for the cells of the present invention as long as the cells are maintained or differentiated into desired differentiated cells. Examples of such a culture solution include, but are not limited to, DMEM, P199, MEM, HBSS, Ham's F12, KSR, BME, RPMI1640, MCDB104, MCDB153 (KGM), and mixtures thereof. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate-2-phosphate, ascorbic acid and its derivatives, glycerophosphate, estrogen and its derivatives, Progesterone and its derivatives, Androgen and its derivatives, aFGF, bFGF, EGF, IGF, TGFβ, ECGF, BMP, PDGF and other growth factors, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, fetal bovine Serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (such as sodium selenite), linolenic acid, 3-isobutyl-1-methyl chloride Demethylating agents such as Nthin, 5-Azancytidine, histone deacetylating agents such as trichostatin, cytokines such as activin, LIF, IL-2, IL-6, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA) , Dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMSO), iododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, Cholera toxin and the like may be included as one or a combination thereof.

  As used herein, “differentiation” or “cell differentiation” refers to two or more morphologically and / or functionally qualitative differences among daughter cell populations derived from the division of one cell. A phenomenon in which types of cells are generated. Therefore, the process in which a cell population (cell lineage) derived from cells that cannot originally detect special characteristics exhibits distinct characteristics such as production of a specific protein is also included in differentiation. At present, it is common to consider cell differentiation as a state in which a specific gene group in the genome is expressed, and cell differentiation by searching for intracellular or extracellular factors or conditions that cause such gene expression state. Can be identified. The result of cell differentiation is in principle stable, especially in animal cells, which differentiates into other types of cells only in exceptional cases. As used herein, differentiation to “pancreas” refers to expression or loss of a specific marker, or determination of expression or secretion of its function (eg, insulin, glucagon, or digestive enzyme (such as amylase), specific structure The pancreatic marker that can be used in the present specification includes, for example, pancreatic markers Ipf1 / pdx-1, insulin, glucagon, amylase 2, and Isl-1. , Pax-4, Pax-6, Glut-2, CK-19, etc. As another means for confirming whether or not the pancreas is present, the expression level is reduced at the time when the pancreas is formed. A means for confirming the decrease in the marker Shh can be mentioned.

  In the present specification, among pluripotency, the ability to differentiate into all types of cells constituting a living body like a fertilized egg is referred to as “totipotency”, and pluripotency can encompass the concept of totipotency. However, when clearly distinguishing, totipotency and pluripotency can be distinguished, the former refers to the ability to differentiate into any cell, the latter has multiple directions, but organisms are possible Having the ability to differentiate in all directions. The ability to differentiate in only one direction is also referred to as unipotency.

  In this specification, totipotency and pluripotency can be determined by, for example, the number of days after fertilization. For example, in the case of a mouse, it can be distinguished on the basis of about 8 days after fertilization. Without being bound by theory, it is normal for mice to follow the following course after fertilization. At 6.5 days after fertilization (also referred to as E6.5), the primitive streak (also referred to as the original streak) appears on one side of the epiblast, revealing the future longitudinal axis of the embryo. The primitive streak represents the future posterior end of the embryo and extends across the ectoderm to the distal end of the cylinder. The original stripe is an area where cell movement is performed, and as a result, future endoderm and mesoderm are formed. By E7.5, a head process appears in front of the nodule, and a notochord and a future endoderm surrounding the lower part of the nodule are formed in this part, and a neural plate is formed in the upper part. The nodules appear around E6.5 days and move backwards, forming the shaft structure from front to back. By day E8.5, the embryo is somewhat longer, with a large head fold consisting mostly of the anterior neural plate at the front end. The somites begin to form from front to back at a rate of one in 1.5 hours from day E8. Cells that have passed this period will no longer exhibit totipotency and will not form individuals unless they are dedifferentiated, even if they are returned to the placenta. Before this, it can be said that this is a branch point of totipotency because it can show totipotency without special treatment. This is because it is difficult to establish ES cells from subsequent embryos, and cells called EG (germ cell-derived) cells are usually established from later embryos. It can be said that it is a point.

  As used herein, “pancreas” or “pancreas” is the largest glandular organ next to the liver associated with the gastrointestinal tract in vertebrates. Located close to the liver, the shape and style of opening to the intestinal tract vary depending on the type of animal. A distinction is made between the exocrine part that secretes pancreatic juice as digestive fluid and the endocrine part that secretes insulin and glucagon involved in the regulation of blood glucose level (Island of Langerhans). The pancreatic duct as a duct for pancreatic juice opens to the duodenum, but often has a common opening fused with the bile duct starting from the liver. Developmentally, usually two to three endoderm processes from the beginning of the intestine are the primordium, and in many animals they combine to form a pancreas. Configure. However, in many teleosts and ostracoda, the pancreas is not dispersed in one organ but dispersed as a small tissue mass, in which case the Langerhans islet tissue is often separated from the pancreatic juice secreting tissue, which is blocked by the small blockman It is called a body (Blockman_s body, Stanius body). The basal base forms a pancreatic duct, but in many mammals, two remain, the ventral one is the main pancreatic duct (Wirsung_s duct), and the dorsal one is the accessory pancreatic duct (Santorini's duct) , Each opening separately into the duodenum.

  As used herein, “functional pancreas” or “natural pancreas” means at least one feature selected from the group consisting of insulin producing ability, glucose responsiveness, glucagon producing ability, amylase producing ability and functional conduit structure. And preferably refers to a pancreas having all of them. Therefore, preferably, the functional pancreas includes 1) endocrine cells (β cells) that secrete insulin in response to blood glucose level (ie, blood glucose concentration), and 2) endocrine cells that produce and secrete glucagon. (Alpha cells, α cells: endocrine cells that specifically produce and secrete glucagon, a hormone responsible for glycogen degradation and gluconeogenesis) 3) Digestive enzymes (eg, amylase) and the intestinal tract It means that it has exocrine cells (acinar cells) secreted inside, and 4) has all functions and structures such as a duct structure for exoculating digestive enzymes from the pancreas to the intestinal tract.

  The cells used in the present invention may be cells derived from any organism (for example, any kind of multicellular organisms (eg, animals (eg, vertebrates, invertebrates), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.)). ) Etc.)). Preferably, cells derived from vertebrates (eg, eel eels, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, more preferably mammals (eg, single pores, Marsupial, rodent, winged, winged, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed, rodent, scale, sea cattle, cetacean, primate , Rodents, rabbit eyes, etc.). More preferably, mammals (for example, rodents (mouse, rat, etc.), primates (monkey, human, etc.)) are used.

  As used herein, “tissue” refers to a population of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” usually has the same origin, but even cell populations with different origins can be referred to as a tissue if they have the same function and / or morphology. Therefore, when a tissue is regenerated using the stem cells of the present invention, a cell population having two or more different origins can constitute one tissue. Usually, tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis.

  As used herein, “activin” refers to a protein hormone that is secreted from ovarian granulosa cells and the like and promotes secretion of follicle-stimulating hormone (FSH) in the anterior pituitary gland. Representative activins include, for example, activin-A, activin-B, activin-C, and inhibin, which are well conserved among animals, and humans act on Xenopus and vice versa. Is also expected to be true. SEQ ID NO: 1 and SEQ ID NO: 9 (accession number: NM002192 (SEQ ID NO: human inhibin βA) and accession number: X68250 (Xenopus activin A) = nucleic acid sequence) and SEQ ID NO: 2 and SEQ ID NO: 10 (human inhibin βA, respectively) And the corresponding factor (ortholog) in other species of animals, including but not limited to those having the sequence shown in SEQ ID NO: and Accession No. X68250 (Xenopus activin A) = amino acid sequence). Xenopus has a registration of inhibin βB (accession number: S61773) (SEQ ID NOs: 11 and 12). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin is a protein hormone that is secreted from granulosa cells of the ovary and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Isolated from follicular fluid in the course of purifying inhibin (1986). Molecular weight 25000. Inhibin β-chain subunits are S—S-linked dimers, and three types of activin A (βAβA), activin AB (βAβB), and activin B (βBβB) are known. Activin A is also referred to as an erythroblast differentiation factor (EDF). Since inhibin β chain has about 40% homology with transforming growth factor TGF-β, and the position of cysteine residues in the primary structure is well conserved, activin may be included in the TGF-β family. . Activin gene expression has been reported in various vertebrate organs, promoting the synthesis of FSH receptors in ovarian granulosa cells, suppression of proliferation of erythroid progenitor cells in friend cells and bone marrow, and induction of hemoglobin synthesis, It has many physiological activities such as promotion of insulin secretion from the pancreas and mesoderm induction in amphibian embryos. For activin, reference can be made to: Nakamura et al. , Isolation and charactarization of native activin B. J Biol. Chem. 1992, 267, 16385-9; Uchiyama and Asashima, Specific erythrophilic differentiation of mouse erythroleukemia cells by activities and its sensitization activity. Biochem Biophys. Res. Commun. 1992, 187, 347-52; Fukui et al. , Isolation and characterization of Xenopus follistatin and activins. Dev. Biol. 1993, 159, 131-9; Fukui et al. , Identification of activins A, AB, and B and follistatin proteins in Xenopus embryos. Dev. Biol. 1994, 163, 279-81; Nakano et al. , Comparison of mesoderm-inducing activity with monometic and dimeric inhibin alpha and beta-A subunits on Xenopus ectoderm. Horm. Res. 1995, 44, Suppl. 2, 15-22.

  Activins are known to have homologues in mammals including humans, rats, mice and Xenopus, as well as in Drosophila. Therefore, in this specification, activin usually refers to activin which exists not only in mammals but also in living organisms in general. Activin-A is a dimer of inhibin βA (human inhibin βA accession number NM002192; SEQ ID NOs: 1 and 2 (nucleic acids and amino acids)). Activin-AB is a dimer of inhibin βA and inhibin βB (human inhibin βB accession number NM002193; SEQ ID NOs: 3 and 4 (nucleic acids and amino acids)). Activin-B is a dimer of inhibin βB. Activin-C is a dimer of inhibin βC (human inhibin βC accession number NM005538; SEQ ID NOs: 5 and 6 (nucleic acids and amino acids)). Inhibin is a dimer of inhibin α (human inhibin α accession number NM002191; SEQ ID NOs: 7 and 8 (nucleic acids and amino acids)).

Examples of such activin genes include:
(A) (a) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11 or a fragment sequence thereof;
(B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12 or a fragment thereof;
(C) a variant having one or more amino acids having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 A polynucleotide, which encodes a variant polypeptide having biological activity;
(D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11;
(E) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12;
(F) a polynucleotide that hybridizes to any one of the polynucleotides of (a) to (e) under a stringent condition and encodes a polypeptide having biological activity; or (g) (a) to A polynucleotide comprising a nucleotide sequence having at least 70% identity to any one polynucleotide of (e) or a complementary sequence thereof and encoding a polypeptide having biological activity;
including,
A nucleic acid molecule; or (B) (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12, or a fragment thereof;
(B) in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10 or 12, wherein at least one amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion; and A polypeptide having biological activity;
(C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, or 11;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12; or (e) against any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence with at least 70% identity and having biological activity;
including,
Examples include, but are not limited to, a nucleic acid molecule that encodes a polypeptide, or a polypeptide that it encodes.

  As used herein, “retinoic acid” is an important metabolite of vitamin A (retinol). Although it has no visual role, it is essential for the normal growth of animals, epithelial tissue and cartilage differentiation. Has a morphogen-like action during vertebrate development. It exerts its action by regulating gene expression via retinoic acid receptor. The all-trans form is the most important isomer, but there are also 9-cis and 13-cis forms.

  A retinoic acid receptor (RAR) is a receptor present in the nucleus necessary for retinoic acid, which is an active form of vitamin A, to exert its action. RAR-α, -β and -γ, 9-cis-retinoic acid to which all trans-retinoic acid binds are known. Retinoic acid X receptors RXR-α, -β and -γ to which all-trans-retinoic acid binds. All of these are ligand-dependent nuclear receptors with a similar structure to steroid receptors, and after binding to the ligand retinoic acid, it binds to a specific position of the gene and regulates transcription of that gene. Involved in development, morphogenesis, and cell differentiation.

  As used herein, “leukemia inhibitory factor (LIF)” corresponds to factors having sequences as shown in SEQ ID NOs: 13 or 15 (nucleic acid sequence) and 14 or 16 (amino acid sequence) and other species of animals. A factor (ortholog).

  LIF was purified and isolated as a factor that induces the differentiation of monocyte cell line M1 into mature macrophages (Gearing DP, Gough NM, King JA, EMBO J. 6: 3395). -4002 (1987); and Gough NM, Gearing DP, King JA, Proc. Natl. Acad. Sci. USA 85: 2623-2627 (1988)). It was named leukemia inhibitory factor because of M1 cell proliferation and clonogenicity. However, LIF is also found in various viewpoints from various groups other than the above reports, and differentiation inducing factor (factor D or DIF) (Tomida et al, J Biol Chem; 259 (17): 10978-82 (1984). Yamamoto et al, Cancer Res; 40: 4804-9 (1980)); macrophage / granulocyte-inducing factor type 2 (MGI-2); differentiation inhibitory activity (DIA); differentiation delay factor (DRF); DA-1a Human interleukin (HILDA) on cells; growth stimulating activity on TS-1 cells (GATS); hepatocyte stimulating factors type I and III (HSF-II and HSF-III); cholinergic neuronal differentiation factor (CDF); Melanocyte-derived lipoprotein lipase inhibitor (MLPLI) Osteoclast activating factor (OAF) (Lass A, Weiser W, Munafo A, Lomaye E .; Fertil Steril; 76: 1091-6 (2001); Knight D. Pulm Pharmacol Ther; 14: 169-76 (2001); ) Etc.).

  LIF is found in humans and mice and, for human and mouse types, is a monomeric 180-residue glycoprotein with disulfide bonds and a molecular weight of about 20 kDa. LIF exists in nature in a glycosylated manner, and purified LIF has an apparent molecular weight of approximately 38-67 kDa (Gascan H., Godard A., Ferenz C. (1989) J. Biol. Chem. 264: 21509-21515; Hilton DJ, Nicola NA, Gow NM and Metcalf D. (1988) Anal.Biochem.173: 359-367), presence or absence of glycosyl residues Not related to LIF activity (Hilton DJ et al., Supra; Gough NM, Hilton DJ, Gearing DP (1988) Blood Cells 14: 431-442). LIF has a helical structure and is classified as a long-chain type cytokine. This cytokine has a long helix, a long head AB and CD loop, and an additional fifth helix A loop (Purvis DH and Mabbutt BC (1997) Biochemistry 36 (33): 10146-10154).

  Polypeptides used in the present invention also include “LIF-like factors” that have the ability to bind to LIF receptors. “LIF-like factor” refers to a factor that has the ability to interact with the LIF receptor. As long as the LIF-like factor activates gp130 and causes subsequent signal transduction, the interaction may be direct binding or indirect action. The ability to interact with the LIF receptor can be measured in a LIF assay. Such LIF assays can utilize, for example, a mechanism that measures the activation (eg, phosphorylation) of any one of the downstream signals (JAK-STAT transduction system), such as gp130 and LIF receptors. Examples include assays that utilize tyrosine phosphorylation of the complex or STAT-3.

  Preferably, LIF binds to the LIF receptor and induces intracellular signal transduction via gp130. Therefore, in the present invention, effects can be obtained by the same mechanism using gp130 ligand or LIF-like factor.

  As used herein, “LIF receptor” is used in the same meaning as used in the art, and refers to a receptor that specifically binds to LIF. The LIF receptor includes a high affinity receptor having a Kd value of 20 to 100 pM and a high affinity receptor having a Kd value of 1 to 2 nM (Allan E. H., Hilton DJ, Brown MA). J. Cell Physiol.145: 110-119 (1990); Godard A., Heymann D., Raher S.J.Biol.Chem.267: 3214-3222 (1992); Hendry LA Murphy M., Hilton. D. J., Nicola NA and Bartlett P. FJ. Neurosci. 12: 3427-3434 (1992); -23 (1992); Rodan S.B., Wesolowski G., Hilton DJ, Nicola NA and Rodan GA and Endocrinology 127: 1602-1608 (1990); , Pease S. Nature 336: 684-687 (1988); and Yamamoto-Yamaguchi Y., Tomida M. and Hozumi M. Exp. Cell. Res. 164: 97-102 (1986)). LIF receptors are classified as hematopoietic factor receptors. The LIF receptor forms a receptor complex in cooperation with gp130 and exhibits high affinity for LIF. Soluble LIF receptors have been discovered in mice, and their role has attracted attention. It has been shown that when a receptor complex of soluble LIF receptor and LIF is formed, the complex can react with gp130 and stimulate signal transduction (Heymann D., Goddard A., Raher S.). (1996) Cytokine 8 (3): 197-205). Therefore, the effects of the present invention can also be achieved by utilizing a signal transmission mechanism using such a receptor conversion model. LIF receptors and gp130 are also known as components of other cytokine receptors, such cytokines include members of the IL-6 superfamily, such as IL-6, IL-11, CNTF, OSM However, it is not limited to them.

  As used herein, “ligand” refers to a substance that specifically binds to a protein. Examples of the ligand include lectins, antigens, antibodies, hormones, neurotransmitters, and the like that specifically bind to various receptor protein molecules present on the cell membrane. If activin or retinoic acid is the ligand, ligands with similar (equivalent) action are intended to be within the scope of the invention.

  In the polypeptides such as activin and LIF used in the present invention, as a moiety to which a sugar chain can be added, an N-glucoside-bondable moiety to which N-acetyl-D-glucosamine or the like binds, and N-acetyl-D- Examples include galactosamine O-glycosidic moieties (parts where serine or threonine residues frequently appear). The activin used in the present specification is not particularly affected by the presence or absence of a sugar chain, but proteins with these sugar chains are usually stable against degradation in vivo. , May have strong physiological activity. Accordingly, polypeptides to which these sugar chains are added are also within the scope of the present invention.

    As used herein, the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also encompass one assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is done. The gene product of the present invention usually takes the form of a polypeptide. The gene product of the present invention in such a polypeptide form is useful as a composition for diagnosis, prevention, treatment or prognosis of the present invention.

  As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”.

  As used herein, the term “oligonucleotide derivative” or “polynucleotide derivative” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has an unusual linkage between nucleotides, and is used interchangeably. Specific examples of such oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C− Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives wherein uracil in oligonucleotide is substituted with C-5 thiazole uracil, cytosine in oligonucleotide is C-5 propynylcytosine Substituted oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA is replaced with 2'-O-propylribose Examples thereof include oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98). (1994)). The gene of the present invention usually takes this polynucleotide form.

  As used herein, “nucleic acid molecule” is also used interchangeably with nucleic acids, oligonucleotides and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like. As used herein, nucleic acids and nucleic acid molecules can be included within the concept of the term “gene”. Nucleic acid molecules encoding certain gene sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition. Therefore, in the present specification, for example, a gene (activin gene) that can be used in the present invention may include a splice variant thereof.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that influence the expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Therefore, a gene such as activin usually includes both a structural gene of the gene of the present invention and a transcriptional and / or translational regulatory sequence such as its promoter. In the present invention, it is understood that in addition to structural genes, regulatory sequences such as transcription and / or translation are also useful for nerve regeneration, diagnosis, treatment, prevention, prognosis, etc. of nerve diseases. As used herein, “gene” refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”. Sometimes. Also herein, a “gene product” is a “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and / or “protein”, “polypeptide”, “oligo” expressed by a gene. Includes "peptide" and "peptide". A person skilled in the art can understand what a gene product is, depending on the situation.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In the present specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In the present specification, the similarity, identity and homology of amino acid sequences and nucleotide sequences are compared, and the search for identity is performed using, for example, NCBI BLAST 2.2.9 (issued 2004.12). be able to. In the present specification, the identity value usually refers to a value when the BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  In the present specification, the “amino acid” may be natural or non-natural as long as it satisfies the object of the present invention. “Amino acid derivative” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art.

  The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.

  As used herein, the term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine.

  As used herein, “amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. As activin in this specification, in addition to those containing natural amino acids, those containing such amino acid analogs or amino acid derivatives can be used.

  In the present specification, the “nucleotide” may be natural or non-natural. A “derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA). .

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by a generally recognized one letter code.

  As used herein, a “corresponding” amino acid or nucleic acid has the same action as a predetermined amino acid or nucleotide in a polypeptide or polynucleotide to be compared in a certain polypeptide molecule or polynucleotide molecule, or An amino acid or nucleotide that is predicted to have, especially in an enzyme molecule, refers to an amino acid that is present at a similar position in the active site and contributes similarly to catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Therefore, in this specification, a specific amino acid sequence in mouse activin can be associated with a specific amino acid in human activin by analysis such as alignment. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

  As used herein, a “corresponding” gene (for example, a polypeptide molecule or a polynucleotide molecule) has or has the same action as a given gene in a species to be compared in a certain species. It refers to a predicted gene (for example, a polypeptide molecule or a polynucleotide molecule), and when there are a plurality of genes having such an action, it refers to one having the same evolutionary origin. Thus, a gene corresponding to a gene can be an ortholog of that gene. Therefore, genes corresponding to genes such as Xenopus activin can also be found in other animals (human, rat, pig, cow, etc.). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in an animal is a sequence database of that animal (eg, human, rat) using the sequence of the gene that serves as a reference for the corresponding gene (eg, a gene such as Xenopus activin) as a query sequence. Can be found by searching.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. Whether the length of a fragment useful in the present specification retains at least one of the functions of a full-length protein that serves as a reference for the fragment (for example, differentiation-regulating action, specific interaction with other molecules) It can be decided by how.

  As used herein, a first substance or factor “specifically interacts” with a second substance or factor means that the first substance or factor has a second Interacting with a higher affinity than a substance or factor other than a substance or factor (especially another substance or factor present in a sample containing a second substance or factor). Specific interactions for a substance or factor include, for example, when both nucleic acid and protein are involved, such as hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Examples include, but are not limited to, protein-lipid interactions, nucleic acid-lipid interactions, and the like, such as reactions with transcription factor binding sites. Thus, when both a substance or factor is a nucleic acid, the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor. Further, for example, when both substances or factors are proteins, the fact that the first substance or factor “specifically interacts” with the second substance or factor includes, for example, interaction by antigen-antibody reaction, receptor- Examples include, but are not limited to, interaction by a ligand reaction and enzyme-substrate interaction. When two substances or factors include proteins and nucleic acids, the first substance or factor “specifically interacts” with the second substance or factor means that the transcription factor and the transcription factor Interaction between the binding region of the nucleic acid molecule to be included.

  Therefore, in the present specification, a “factor that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for the biological agent such as the polynucleotide or the polypeptide, The affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides is typically equivalent or higher, preferably significantly (eg, statistically significant) ) Includes the expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to. As used herein, activin can be used for stem cell differentiation, but it is understood that any factor having the same biological activity as activin can be used interchangeably with activin in the present invention. Such factors can be identified by screening using technical common sense in the field based on the disclosure in the present specification, and it is understood that these are within the scope of well-known and conventional techniques. .

  As used herein, “compound” means any identifiable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids, including: Without limitation, such compounds can be natural or synthetic.

  In the present specification, the “small organic molecule” means an organic molecule having a relatively small molecular weight. Usually, a low molecular weight organic material has a molecular weight of about 1000 or less, but may have a higher molecular weight. The organic small molecule can be synthesized usually by using a method known in the art or a combination thereof. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, small organic molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). It is not limited.

  As used herein, “contacting” refers to physically bringing a compound, either directly or indirectly, into an activin or the like (eg, a polypeptide or polynucleotide) of the present invention. It means close proximity. The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof. Typically, the contact can be achieved by placing a contact object (for example, a cell) in a solution (for example, a medium) containing a factor (for example, activin) to be contacted.

  As used herein, “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, sugars, and small molecules. Examples of such complex molecules include, but are not limited to, glycolipids and glycopeptides. In this specification, activin can also be used in the form of such a complex molecule.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, When it is a nucleic acid, it is substantially separated from a non-nucleic acid and a nucleic acid containing a nucleic acid sequence other than the target nucleic acid; Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  As used herein, “biological activity” refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). Activity is included. For example, when two factors interact (eg, activin and its specific factor bind), the biological activity is the binding between the two molecules and the resulting biological change, eg When one molecule is precipitated using an antibody, the other molecule is also co-precipitated and the two molecules are considered to be bound. Therefore, seeing such coprecipitation is one method of judgment.

  Thus, “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus); Refers to various measurable indicators, such as the affinity of a compound that binds directly to the polypeptide or polynucleotide used in the present invention, or the amount of protein upstream or downstream, eg, after some stimulation or event Alternatively, other similar measures of function can be mentioned. Such activity can be measured by assays such as competitive inhibition of binding of specific factors to activin.

  In this specification, the term “interaction” refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates.

  The term “modulate” or “modify” as used herein means an increase or decrease or maintenance in the amount, quality or effect of a particular activity or protein.

  As used herein, “polynucleotide that hybridizes under stringent conditions” refers to a polynucleotide obtained under well-known conditions commonly used in the art. Using such a polynucleotide selected from the polynucleotides used in the present invention as a probe, colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like is used to obtain such a polynucleotide. Can do. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the polynucleotide capable of hybridizing is a polynucleotide having at least 60% homology with the base sequence of a DNA encoding a polypeptide (for example, activin) having the amino acid sequence specifically shown in the present invention. A polynucleotide having a homology of 80% or more is preferable, and a polynucleotide having a homology of 95% or more is more preferable.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: a Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
Tm (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20 % Formamide, 37-50 ° C. By way of example, “moderately stringent” conditions at 50 ° C. in 0.015 M sodium ion tolerate a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is
Tm = (2 ° C. for one AT base) + (4 ° C. for one GC base pair)
Provided by. The sodium ion concentration in 6 × sodium citrate (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

Natural nucleic acids encoding proteins such as activin or variants or fragments thereof can be readily obtained from a cDNA library having PCR primers and hybridization probes comprising, for example, a portion of a nucleic acid sequence such as SEQ ID NO: 1 or a variant thereof. To be separated. A nucleic acid encoding a preferred activin or variant or fragment thereof is essentially a high containing 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; 7% SDS at a temperature of 42 ° C. More preferably essentially under low stringency conditions defined by a hybridization buffer and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C. Hybridization buffer containing 1% bovine serum albumin (BSA) at a temperature of 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS, and 1 × essentially 50 ° C. SSC (300 mM NaCl; 30 mM Sodium citrate); 1% bovine serum albumin (BSA) under low stringent conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS, and essentially 0.5 x SSC at 65 ° C (150 mM NaCl; 15 mM sodium citrate); wash containing 0.1% SDS It can hybridize to one or a portion of the sequence shown in SEQ ID NO: 1 or 3 under low stringency conditions as defined by the buffer.

  In the present specification, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. As an electronic search, BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that activin and the like used in the present invention should include corresponding genes identified by such electronic search and biological search.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but is not limited thereto. In particularly preferred embodiments, the Basic Local Alignment Tool (BLAST) (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990, well known in the prior art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare the amino acid query sequence with the protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) comparing a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX into six reading frames with a protein sequence database;
(4) Comparison of the protein query sequence with TBLASTN with a nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Strategic Stratecence. about). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). about).

(Modification of genes, protein molecules, nucleic acid molecules, etc.)
In certain protein molecules (eg, activin), certain amino acids included in the sequence can be found in other protein structures such as, for example, the cationic region or the binding site of a substrate molecule, without any apparent loss or loss of interaction binding capacity. Of amino acids. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity) It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  The hydrophilicity index is also useful for modifying the amino acid sequence of the present invention. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present specification, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid are similar as described above. Examples of conservative substitution include those having a hydrophilicity index or hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and Examples include, but are not limited to, isoleucine.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such variants include one or several substitutions, additions and / or deletions, or one or more substitutions, additions and / or deletions relative to a reference nucleic acid molecule or polypeptide. But are not limited thereto. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, site-specific base substitution methods using synthetic oligonucleotides (specification) Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). it can.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. The addition of amino acids means that one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids are added to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, shortening, lipidation, phosphorylation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg acetylation), etc. Including, but not limited to. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” or “peptide derivative” refers to a compound that is different from a peptide but is equivalent to at least one chemical or biological function of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  Chemically modified polypeptide compositions in which the polypeptide used in the present invention is conjugated to a polymer are included within the scope of the present invention. The polymer can be water soluble and can prevent precipitation of the protein in a water soluble environment (eg, a physiological environment). Suitable aqueous polymers can be selected, for example, from the group consisting of: polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymer, poly (N-vinyl pyrrolidone) polyethylene glycol, Polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol. The selected polymer is usually modified and has a single reactive group (eg, an active ester for acylation or an aldehyde for alkylation) so that the degree of polymerization can be controlled. The polymer can be of any molecular weight, and the polymer can be branched or unbranched, and mixtures of such polymers can also be used. The chemically modified polymer used in the present invention is selected for use by a pharmaceutically acceptable polymer as determined for therapeutic use.

  If the polymer is modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is modified by reductive alkylation, the polymer should have a single reactive aldehyde group. Preferred reactive aldehydes are polyethylene glycol, propionaldehyde (which is water-soluble) or mono-C1-C10 alkoxy or aryloxy derivatives thereof (eg, US Pat. No. 5,252,714). No., which is hereby incorporated by reference in its entirety).

  Pegylation of polypeptides used in the present invention can be performed by any pegylation reaction known in the art, for example as described in the following references: Focus on Growth Factors 3 , 4-10 (1992); EP 0 154 316; and EP 0 401 384, each of which is incorporated herein by reference in its entirety. Preferably, this pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of polypeptides (eg, activin, Mel-18, M33, Mph-1 / Rae28, etc.) used in the present invention is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any form of PEG, where the PEG is another protein (eg, a mono (C1-C10) alkoxy polyethylene. Glycol or mono (C1-C10) aryloxypolyethylene glycol).

  Chemical derivatization of the polypeptides used in the present invention can be carried out under suitable conditions used to react biologically active substances with activated polymer molecules. A method for preparing a pegylated polypeptide used in the present invention generally comprises the following steps: (a) polyethylene glycol (eg, under conditions such that activin is attached to one or more PEG groups. Reacting the polypeptide with a reactive ester or aldehyde derivative of PEG) and (b) obtaining the reaction product. It is easy for those skilled in the art to select the optimal reaction conditions or acylation reaction based on known parameters and the desired result.

  Pegylated polypeptides used in the present invention can generally be used to treat conditions that can be alleviated or modulated by administering the polypeptides described herein, but the present The polypeptide molecules used in the present invention that are chemically derivatized as disclosed herein have additional activity, increased or decreased biological activity, or others compared to their non-derivative molecules. Characteristics (eg, increased or decreased half-life). The polypeptides, their fragments, variants and derivatives used in the present invention may be used alone, in combination, or in combination with other pharmaceutical compositions. These cytokines, growth factors, antigens, anti-inflammatory agents and / or chemotherapeutic agents are suitable for treating symptoms.

  Similarly, “polynucleotide analog” or “nucleic acid analog” refers to a compound that is different from a polynucleotide or nucleic acid but is equivalent to at least one chemical or biological function of the polynucleotide or nucleic acid. . Thus, polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPpress; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. etal. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference in the relevant part.

(Genetic engineering)
Activins and the like and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

  In the present specification, when referring to a gene, “vector” or “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence to a target cell. Such vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals and individual plants, or can be integrated into chromosomes and used in the present invention. Examples include those containing a promoter at a position suitable for transcription of the polynucleotide to be produced. Among vectors, a vector suitable for cloning is called “cloning vector”. Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra). Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (ie, fragments that can be integrated into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenovirus, baculovirus, parvovirus, herpes virus, pox virus, adeno-associated virus, Semliki Forest virus, vaccinia virus and retrovirus.

  One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) can replicate autonomously in the host cell into which they are introduced. Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell, and are thereby replicated along with the host genome. Furthermore, certain vectors may direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors”.

  Therefore, in the present specification, the “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory elements used can vary depending on the host cell.

“Recombinant vectors” for prokaryotic cells that can be used in the present invention include pcDNA3 (+), pBluescript-SK (+/−), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY ( Invitrogen) and the like.

  Examples of “recombinant vectors” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], a retroviral expression vector based on murine stem cell virus (MSCV), pEF-BOS, pEGFP, and the like.

  As used herein, “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  In this specification, “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base sequence that initiates transcription upon binding of RNA polymerase. . Therefore, in this specification, a part having a promoter function of a gene is referred to as a “promoter part”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be deduced. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.

  As used herein, “replication origin” refers to a specific region on a chromosome where DNA replication begins. The origin of replication can either be provided by constructing the vector to include an endogenous origin, or it can be provided by the host cell's chromosomal replication machinery. The latter may be sufficient if the vector is integrated into the host cell chromosome. Alternatively, rather than using a vector containing a viral origin of replication, one of skill in the art can transform mammalian cells by a method that co-transforms a selectable marker and the DNA of the present invention. Examples of suitable selectable markers are dihydrofolate reductase (DHFR) or thymidine kinase (see US Pat. No. 4,399,216).

  For example, recombinant mammalian expression vectors can preferentially direct expression of a nucleic acid in a particular cell type by expressing the nucleic acid using tissue-specific regulatory elements. Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include developmentally regulated promoters (eg, mouse hox promoter (Kessel and Gruss (1990) Science 249, 374-379) and α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546)), albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymphoid specific promoter (Calame and Eaton (1988) Adv. Immunol.43, 235-275), in particular T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) and immunoglobulins. (Banerji et al. (1983) Cell 33, 729-740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (eg, nerve fiber promoter; Byrne and Ruddle (1989) Proc. Natl. Acad.Sci.USA 86, 5473-5477), pancreas specific promoter (Edrund et al. (1985) Science 230, 912-916), and mammary gland specific promoter (eg, whey promoter; US Pat. No. 4,873, 316 and European Application Publication No. 264,166), but are not limited thereto.

  As used herein, “enhancer” refers to a sequence used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject. The cell used in the present invention may be a transformant.

  When prokaryotic cells are used in genetic manipulation or the like in the present invention, prokaryotic cells include, for example, prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, animal cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, and human leukemia cells. HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1 and Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

  As used herein, any method for introducing a DNA can be used as a method for introducing a recombinant vector. For example, a calcium chloride method, an electroporation method [Methods. Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

(Method for producing polypeptide)
Transformants derived from microorganisms, animal cells, etc. that possess a recombinant vector incorporating a DNA encoding a polypeptide (for example, activin, LIF or a variant or fragment thereof) used in the present invention are usually used. The polypeptide used in the present invention is produced by accumulating the polypeptide used in the present invention, collecting the polypeptide used in the present invention from the culture of the present invention, and producing the polypeptide according to the present invention. Can do.

  The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.

  Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing substances, and peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin or tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter, and indole acrylic is used when a microorganism transformed with an expression vector using the trp promoter is cultured. An acid or the like may be added to the medium. Cells or organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention includes RPMI1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122). , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum or the like was added to these media A medium or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  In order to isolate or purify a polypeptide used in the present invention from a culture of a transformant transformed with a nucleic acid sequence encoding the polypeptide of the invention used in the present invention, it is well known in the art. Conventional conventional enzyme isolation or purification methods can be used. For example, when the polypeptide of the present invention is secreted outside the transformant for producing the polypeptide of the present invention, the culture is treated with a technique such as centrifugation to obtain a soluble fraction. Get minutes. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), etc. Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography A purified sample can be obtained by using a technique such as a chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide used in the present invention (for example, activin, LIF or a variant or fragment thereof) accumulates in a dissolved state in the cells of the transformant for producing the polypeptide used in the present invention, culture is performed. The cells in the culture are collected by centrifuging the product, and after washing the cells, the cells are disrupted by an ultrasonic disrupter, a French press, a Manton Gaurin homogenizer, a dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical) anion exchange chromatography using a resin, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose and phenyl sepharose A purified sample can be obtained by using a technique such as a chromatography method, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide used in the present invention is expressed by forming an insoluble substance in the cells, similarly, the cells are collected and then disrupted and centrifuged from the precipitate fraction obtained by a conventional method. After recovering the polypeptide used in the present invention, an insoluble substance of the polypeptide is solubilized with a polypeptide denaturant. The solubilized solution is diluted or dialyzed into a solution that does not contain the polypeptide denaturant or is so dilute that the polypeptide denaturant does not denature the polypeptide, and the polypeptide used in the present invention is converted into a normal three-dimensional solution. After making it into a structure, a purified sample can be obtained by the same isolation and purification method as described above.

  Further, a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. In addition, the polypeptide used in the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having affinity for the fused protein [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)]. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227-8231 (1989), Genes Develop. , 4, 1288 (1990)], the polypeptide used in the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

  In addition, the polypeptide used in the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)]. In such fusion proteins, in the expression vector, the proteolytic cleavage site joins the fusion moiety to the recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Introduced into the department. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include pGEX (Pharmacia Biotech; Smith and Johnson (1988) Gene 67, which fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein, respectively. , 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).

  Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself used in the present invention. Polypeptides used in the present invention can be prepared by known methods [J. Biomolecular NMR, 6,129-134, Science, 242, 1162-1164, J. Biol. Biochem. 110, 166-168 (1991)], it can be produced using an in vitro transcription / translation system.

  Polypeptides used in the present invention can be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid information. it can. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  The structural analysis of the purified polypeptide used in the present invention is performed by a method usually used in protein chemistry, for example, a method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Chemical Dojin, 1993). It can be implemented. The physiological activity of the polypeptide used in the present invention can be measured according to a known measurement method.

  Deletion, substitution or addition (including fusion) of amino acids of a polypeptide (for example, activin, LIF, etc.) used in the present invention can be carried out by site-directed mutagenesis which is a well-known technique. Such deletion, substitution or addition of one or several amino acids is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratories Press (1989), Current Protocols in BioJ. 1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5562 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.

(Cell culture)
The cell culture characteristics of stem cell differentiation of a wide variety of media formulations known in the art can be dramatically altered by adjusting the amount of activin, retinoic acid, LIF or equivalents disclosed herein. Was found.

  Many media formulations have been developed over the years to maximize cell growth, cell viability, and / or biologic production for a given cultured cell or cell line. These media compositions differ, for example, in the number, type, and concentration of growth factors, antibiotics, and amino acid supplements added to the media, and components such as protease inhibitors, and particularly cells in suspension. When grown, it contains one or more anti-foaming agents. Other personalized ingredients include additives that enhance recombinant protein production. For example, if the host cell was developed for gene amplification of a recombinant protein, the selectable marker DHFR (dihydrofolate reductase) typically constitutes part of the host transfected as a selectable marker, And methotrexate is contained in the culture medium. Insulin or other growth factors, such as factors that play a role in the energy source cascade, such as IGF, are also often included to enhance cell proliferation.

  The activins, retinoic acids, LIF or equivalents disclosed herein are expected to have an effect on various media formulations as well as in standard formulations.

For example, all serum-free media formulations contain essential ingredients that allow cell growth, and (1) an energy source, typically glucose or glutamine, or other sugars such as fructose, galactose, mannose, etc .; (2) contains a nitrogen source (typically obtained by inclusion of one or more amino acids); and (3) vitamins (cofactors in enzymatic reactions). Also includes a wide range of inorganic salts including Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ⁻ , HPO ₃ ^{2− and the} like, as well as fatty acids (preferably conjugates), cholesterol, phospholipids, and their precursors. A wide range of fat and fat-soluble ingredients are also essential. The components of a standard serum-free medium formulation are listed in Table 1. This includes GrandIsland Biological Co. (GIBCO), Grand Island, N .; Y. Ingredients found in “DMEM / F-12” obtained from media manufacturers such as For a review of animal cell culture media, see Mizrahi and Lazar, Cytotechnology, 1: 199-214 (1988). Media considerations for insect cell culture are described in Goodwin, R .; H. (1990) Nature 347: 209-210. An example of a defined serum-free medium that is individualized for hybridoma culture is described in Kovar, J. et al. (1987) Folia Biology 33: 377-384. Imagawa et al. (1989) PNAS 86: 4122-4126 describe a medium developed to maximize cell proliferation in mouse mammary epithelial cells, and Miyazaki et al. (1991) Res. Exp. Med. 191: 77-83 describes a medium to enhance the viability of rat hepatocytes in vitro.

  As used herein, “nutrient medium” includes natural medium, semi-synthetic medium, synthetic medium, solid medium, semi-solid medium, liquid medium, etc., but undifferentiated cells are allowed to grow, differentiate and mature, including self. Alternatively, any medium may be used as long as it is used for storage and is usually used for cell culture. Examples include Steinberg medium, α-MEM medium, RPMI-1640 medium, or MEM basic medium. As basic components, sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, sugars, or other chemical components or biological components such as serum may be contained depending on the purpose.

  The media components can typically be added in any order, and the final combination can be sterilized by standard methods, such as filter sterilization.

  Utilizing the media of the present invention, a wide range of cell stem cells (both vertebrates and invertebrates) can be differentiated in a desired direction. The culture medium of the present invention can be used to improve cell culture properties in many different cell culture systems.

  In the case of animals, serum-free media such as Iskov media, RPMI media, Dulbecco MEM media, MEM media, and F12 media that are usually used for culturing animal cells can be used. In addition, factors other than serum known to be effective for cell growth and maintenance, such as lipid and fatty acid sources, cholesterol, pyruvate, glucocorticoid, DNA and RNA synthetic nucleosides, etc. are added according to known literature. May be.

  As used herein, the term “culture vessel” refers to a vessel used when proliferating desired cells, for example, undifferentiated cells. Feeder cells such as stromal cells can be maintained and survived as needed, and undifferentiated. Any material or shape may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, or self-replication of cells. Specific examples of the culture vessel material include glass, synthetic resin, natural resin, metal, and plastic. Specific examples of the shape of the container include a triangular prism, a cube, a rectangular parallelepiped, a triangular pyramid, a quadrangular pyramid, and the like. Polygonal pyramids, arbitrary shapes such as gourds, spheres, hemispheres, cylinders (including bottom, circular, elliptical, semicircular, etc.) can be mentioned, and during culture, for example from hemispheres to spheres The shape may be changed as necessary. Cultivation may be under open conditions or under closed (sealed) conditions.

  The cell culture method in the present invention is a culture dish having a different matrix coating, a two-dimensional culture method using a culture dish having a different presence or absence of matrix coating, a three-dimensional culture method using a soft gel such as Matrigel, a collagen sponge, etc. Alternatively, a method using them in combination may be mentioned, but preferably a two-dimensional culture method using a culture dish having a different matrix coating or a culture dish having a different presence or absence of a matrix coating, such as a gelatin-coated culture dish, type I collagen. It is a two-dimensional culture method using two of a coated culture dish or a laminin coated culture dish. In addition, when human-derived cells are used as cells having multipotency, it is preferable to use type I collagen-coated culture dishes.

The culture conditions of the method of the present invention are not limited to specific conditions such as Examples, and generally acceptable conditions can be taken. For example, as the number of cells at the start of differentiation induction, a range of 5.0 × 10 ^{3 to} 5.0 × 10 ⁶ cells / culture dish can be exemplified. The differentiation induction period is, for example, 2 to 10 days (preferably 5 days), or 1 to 4 days (preferably 2 days), and 2 to 5 days (preferably 3 days) in the preculture step. In the case of human mesenchymal cells, the differentiation induction period is 12 to 21 days (preferably 14 days).

  In culturing, physical environmental conditions such as temperature, osmotic pressure, and light, and chemical environmental conditions such as oxygen, carbon dioxide, pH, and oxidation-reduction potential are maintained and maintained by undifferentiated cells before and after differentiation. Any environmental condition may be used as long as it does not inhibit the maintenance, survival, differentiation, maturation, and self-replication of undifferentiated cells. Preferred conditions are shown below.

  Specifically, the temperature is 30 ° C. to 40 ° C., preferably 37 ° C. The osmotic pressure is specifically an osmotic pressure under physiological conditions, and preferably an osmotic pressure equal to that of physiological saline.

The light may be as dark as a dark room, or as bright as the brightness outside in sunny weather.
Specifically, as for the oxygen concentration, the culture system is in contact with the gas phase having the oxygen concentration in the gas phase in contact with the gas phase having the oxygen concentration in the gas phase of 10% to 30% in the gas phase. The oxygen concentration may be the oxygen concentration in a state of being in contact with the gas phase, preferably in the state of being in contact with the gas phase having a concentration of oxygen in the gas phase of 20%. Concentration.

  In general, the pH for controlling the pH in the culture system is specifically pH 6.0 to pH 8.0, preferably the pH equivalent to physiological conditions. Carbon dioxide may be used to control the pH, or any other buffer may be used. Specifically, the concentration of carbon dioxide is the concentration of dissolved carbon dioxide in a state where the culture system is in contact with the 5% gas phase.

  As used herein, the term “colony” refers to a visible clump formed from a single cell in a solid medium.

  As used herein, the term “kit” refers to a unit in which parts to be provided (eg, reagents, particles, etc.) are usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such kits preferably include instructions that describe how to treat the provided parts (eg, reagents, particles, etc.). Such a manual may be any medium. Examples of such a medium include, but are not limited to, a paper medium, a transmission medium, and a recording medium. Examples of the transmission medium include, but are not limited to, the Internet, an intranet, an extranet, and a LAN. Examples of the recording medium include, but are not limited to, CD-ROM, CD-R, flexible disk, DVD-ROM, MD, mini disk, MO, and memory stick.

(screening)
As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For screening, a system using real substances such as in vitro and in vivo may be used, or a library generated using an in silico (computer system) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing a drug by computer modeling based on the disclosure of the present invention.

  In one embodiment, the present invention provides an assay for screening candidate or test compounds that bind to or modulate the protein of the present invention (activin), or a biologically active portion thereof. I will provide a. Test compounds of the invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; spatially Accessible parallel solid phase or solution phase library; synthetic library method requiring deconvolution; “one bead one compound” library method; and synthetic library method using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12). 145).

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. MoI. Med. Chem 37: 2678; Cho et al. (1993) Science 2611: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. MoI. Med. Chem 37: 1233.

  The library of compounds can be in solution (eg, Houghten (1992) BioTechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwilla et al. (1990) Proc. Natl. Acad. ci.U.S.A.87: 6378~6382; Felici (1991) J Mol Biol 222: 301~310; Ladner above) can be shown in.

(disease)
In one aspect, the present invention relates to any disease, disorder or abnormal condition relating to a pancreatic disorder, such as pancreatitis (acute pancreatitis, chronic pancreatitis), pancreatic cancer, cystic pancreatic disease, diabetes, digestive system disease and the like. A method for treating a related disease, disorder or abnormal condition is provided.

  Representative examples of pancreatic-related diseases include type 1 diabetes (or insulin-dependent diabetes (IDD), juvenile diabetes). This type 1 diabetes is an insulin deficiency due to autoimmune destruction of insulin-producing cells, and current therapies require patients with type 1 diabetes to inject insulin several times per day. Another type of diabetes, type 2 diabetes (or non-insulin dependent diabetes mellitus (NIDD), adult-onset diabetes) is an effective insulin deficiency due to insulin resistance. Like type 1 diabetes, type 2 diabetes requires additional insulin administration. In these patients, blood insulin levels are not always stable despite regular insulin infusions, resulting in a shortened lifespan with many complications such as stroke, blindness, limb amputations, and renal dysfunction Is done. In addition, there are cases where insulin resistance is caused by lifestyle habits such as lack of exercise, excessive intake of fat, obesity and the like, and cases where drugs, endocrine diseases and the like become insulin action disorders (resistance factors).

  As used herein, “preventing” refers to reducing the likelihood that an organism will contract or develop an abnormal condition.

  As used herein, “treating” refers to having a therapeutic effect and at least partially alleviating or suppressing an abnormal condition in an organism.

  As used herein, “therapeutic effect” refers to an inhibitor or activator that causes or contributes to an abnormal condition. The therapeutic effect alleviates one or more of the symptoms of the abnormal condition to some extent. With respect to the treatment of abnormal conditions, a therapeutic effect may refer to one or more of: (a) an increase in cell proliferation, growth, and / or differentiation; (b) inhibition of cell death. (I.e. delay or stop); (c) inhibition of degeneration; (d) some relief of one or more symptoms associated with an abnormal condition; and (e) enhance the function of the affected cell population. about. Compounds that exhibit efficacy against abnormal conditions can be identified as described herein.

  As used herein, an “abnormal condition” refers to a function in a cell or tissue of an organism that deviates from its normal function in the organism. The abnormal condition can be associated with cell proliferation, cell differentiation, cell signaling, or cell survival. Abnormal conditions may also include hematopoietic disorders, obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and irregularities in fatty acid uptake and metabolism.

  Examples of abnormal cell growth include cancer, neoplasm, tumor and inflammation.

  Examples of abnormal differentiation include malformation and cancer.

  An example of an abnormal cell signaling state is abnormal cell differentiation.

  Abnormal cell viability is also associated with conditions in which the apoptotic (programmed cell death) pathway is activated or arrested. A number of protein kinases are associated with the apoptotic pathway. Abnormalities in the function of any one of the protein kinases can result in cell immortality or premature cell death.

  In another aspect, the present invention provides both prophylactic and therapeutic methods for treating a subject having (or suspected of suffering from) a pancreatic-related disease, disorder or abnormal condition or a subject having the disorder. I will provide a.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the medicament of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may optionally be attached to such containers, which may be made, used or sold for administration to humans. Represents approval by government agencies.

(Gene therapy)
In certain embodiments, a nucleic acid comprising a normal gene nucleic acid sequence of the invention, a sequence encoding an antibody or functional derivative thereof, is associated with aberrant expression and / or activity of a polypeptide used in the invention. Administered for gene therapy purposes to treat, inhibit or prevent a disease or disorder. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

  Any method for gene therapy available in the art can be used in accordance with the present invention. An exemplary method is as follows.

  For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExplorMorL. , Stockton Press, NY (1990).

  Therefore, in the present invention, gene therapy using a nucleic acid molecule encoding activin or a variant or fragment thereof may be useful.

  As used herein and as understood in the art, the terms “synthesis” or “synthesize” are purely chemical as opposed to enzymatic methods. Refers to chemical substances (eg, polynucleotides, polypeptides, etc.) produced in Thus, “globally” synthesized chemicals (eg, polynucleotides, polypeptides, etc.) are generated entirely by chemical means, and “partially” synthesized chemicals (eg, Polynucleotide, polypeptide, etc. includes chemicals (eg, polynucleotides, polypeptides, etc.) in which only a portion of the resulting chemicals (eg, polynucleotides, polypeptides, etc.) are produced by chemical means. To do.

  By the term “region” as used herein is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of a protein, a region is defined by a contiguous portion of the amino acid sequence of the protein. The term “domain” is defined herein to refer to the structural portion of a biomolecule that contributes to the known or suspected function of the biomolecule. A domain can have the same extent as a region or portion thereof; a domain can also incorporate a portion of a biomolecule that is distinct from a particular region in addition to all or a portion of the region. Examples of activin domains of the present invention include, but are not limited to, signal peptides, extracellular (ie, N-terminal) domains, leucine-rich repeat domains.

(Administration and composition for regeneration / treatment / prevention)
The present invention provides a method for the treatment, inhibition and prevention of neurological diseases, disorders or abnormal conditions, or hematopoietic related diseases, disorders or abnormal conditions by administration of an effective amount of a compound or pharmaceutical composition of the present invention to a subject. provide. In a preferred aspect, the compound can be substantially purified (eg, in a state substantially free of substances that limit its effect or cause undesirable side effects).

  In the present specification, the “effective amount for diagnosis, prevention, treatment or prognosis” refers to an amount which is recognized as being medically effective in diagnosis, prevention, treatment (or treatment) or prognosis, respectively. Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

  The animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has a nervous system or a similar system. Preferably, it is a vertebrate (for example, larvae, lampreys, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (for example, single holes, marsupials, Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents , Rabbit eyes, etc.). Exemplary subjects include, but are not limited to, animals such as cows, pigs, horses, chickens, cats, dogs and the like. More preferably, cells derived from primates (eg, chimpanzee, Japanese monkey, human) are used. Most preferably, human-derived cells are used.

  When the nucleic acid molecule or polypeptide of the present invention is used as a medicament, such a composition may further comprise a pharmaceutically acceptable carrier and the like. Examples of the pharmaceutically acceptable carrier contained in the medicament of the present invention include any substance known in the art.

  Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers. Examples include, but are not limited to, agents, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. Typically, a medicament of the invention comprises a polypeptide or polynucleotide, such as activin or a variant or fragment thereof, or a variant or derivative thereof, one or more physiologically acceptable carriers, excipients or It is administered in the form of a composition containing with a diluent. For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other materials common to compositions for parenteral delivery. .

  Acceptable carriers, excipients or stabilizers used herein are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, for example Phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, polyvinylpyrrolidone); amino acid (Eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol) Salt formation vs. Io (E.g., sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycols (PEG)) but the like are not limited thereto.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. .

  The general preparation method of the pharmaceutical composition of this invention is shown below. In addition, veterinary drug compositions, quasi drugs, marine drug compositions, food compositions, cosmetic compositions and the like can also be produced by known preparation methods.

  Polypeptides, polynucleotides and the like used in the present invention are blended with a pharmaceutically acceptable carrier, solid preparations such as tablets, capsules, granules, powders, powders, suppositories, or syrups, injections , And can be administered orally or parenterally as liquid preparations such as suspensions, solutions, sprays and the like. As described above, the pharmaceutically acceptable carrier includes an excipient, a lubricant, a binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, an adsorbent, a humectant, a solubilizing agent in a solid preparation, Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, tonicity agents, buffers, soothing agents and the like can be mentioned. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than the polynucleotide used in this invention, a polypeptide, etc. with the composition of this invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

  Examples of the excipient in the solid preparation include glucose, lactose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous silicic acid, sodium chloride, kaolin and urea.

  Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal silicic acid, talc, and polyethylene glycol.

  Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, starch solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate , Potassium phosphate, shellac and the like.

  Examples of disintegrants in solid preparations include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Examples include, but are not limited to, esters, sodium lauryl sulfate, starch, stearic acid monoglyceride, lactose, and calcium calcium glycolate.

  Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter and hydrogenated oil.

  Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

  Examples of the adsorbent in the solid preparation include, but are not limited to, starch, lactose, kaolin, bentonite and colloidal silicic acid.

  Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

  Examples of the solubilizer in the solid preparation include, but are not limited to, arginine, glutamic acid, aspartic acid and the like.

  Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and lactose.

  When preparing tablets, pills and the like as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Tablets include tablets with ordinary coatings as necessary, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When forming into the form of a suppository, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added in addition to the additives listed above, but are not limited thereto.

  Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to them.

  Suitable examples of the suspending agent in the liquid preparation include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, Examples include, but are not limited to, hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate and citrate.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When preparing as an injection, it is preferable that the solution and suspension are sterilized and isotonic with blood. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericidal agent, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, if necessary, the pharmaceutical composition may contain coloring agents, preservatives, flavors, flavoring agents, sweeteners, and the like, as well as other agents.

  The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. Such a pharmaceutically acceptable composition can be easily prepared by those skilled in the art by considering pH, isotonicity, stability and the like. In this specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, intrarectal administration, intravaginal administration, local administration to the affected area, Skin administration, etc.). Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  The medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizer as necessary (Japanese Pharmacopoeia 14th edition, its supplement or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, A R. Gennaro, ed., Mack Publishing Company, 1990, etc.) and a sugar chain composition having a desired degree of purity, and prepared and stored in the form of a lyophilized cake or aqueous solution. Can be done.

  Various delivery systems are known and can be used to administer the compounds of the invention (eg, liposomes, microparticles, microcapsules, etc.). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through the epithelium or mucosal lining (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.), and others. It can be administered with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the invention may include any suitable route (including intraventricular and intrathecal injection; intraventricular injection, for example, intraventricularly attached to a reservoir, such as an Ommaya reservoir. It may be desirable to introduce into the central nervous system by a catheter). Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

  In certain embodiments, it may be desirable to administer the polypeptide, polynucleotide or composition of the invention locally to the area in need of treatment (eg, central nervous system, brain, etc.); this limits Although not intended, for example, local injection during surgery, topical application (eg in combination with a post-surgical wound dressing), by injection, by catheter, by suppository, or an implant (the implant is shearable ( a porous, non-porous, or glue-like material), including membranes or fibers such as (sialastic) membranes. Preferably, when administering a protein of the invention, including antibodies, care must be taken to use materials that do not absorb the protein.

  In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious). Disesease and Cancer, Lopez-Berstein and Fidler (eds.), Liss, New York, pages 353-365 (1989); see Lopez-Berstein, pages 317-327; see broadly the same book).

  In yet another embodiment, the compound or composition can be delivered in a controlled sustained release system. In one embodiment, a pump can be used (Langer (supra); Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Florida (1974); Controlled DrugDrivable Drug Bioavailability. And Ball (eds.), Wiley, New York (1984); see Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 1985); During et al., Ann. l.25: 351 (1989); see also Howard et al., J. Neurosurg. 71: 105 (1989)).

  In yet another embodiment, the controlled sustained release system can be placed near the therapeutic target, i.e., the brain, and therefore requires only a portion of the systemic dose (e.g., Goodson, Medical Applications of Controlled Release). , (Supra), Vol. 2, pages 115-138 (1984)).

  Other controlled controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  The amount of the composition used in the treatment method of the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. Can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  The dose of the polypeptide, polynucleotide and the like of the present invention varies depending on the age, weight, symptom or administration method of the subject, and is not particularly limited, but is usually 0.01 mg to 10 g in the case of oral administration per day for an adult. Preferably, it may be 0.1 mg to 1 g, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like. In the case of parenteral administration, it is 0.01 mg to 1 g, preferably 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 10 mg, and the like.

  As used herein, “administering” refers to a polypeptide, polynucleotide, factor or the like of the present invention or a pharmaceutical composition containing the same, alone or in combination with other therapeutic agents, to cells or tissues of an organism. It means capturing. The combinations can be administered, for example, either as a mixture simultaneously, separately but simultaneously or in parallel; or sequentially. This also includes a procedure in which the combined drugs are administered together as a therapeutic mixture, and the combined drugs are administered separately but simultaneously (eg, through separate intravenous lines to the same individual). Also includes. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

  An abnormal condition can also be prevented or treated by administering a compound (such as an agent identified by the present invention) to a group of cells having an abnormality in a signal transduction pathway to an organism. The effect of administering the compound on the biological function can then be monitored. This organism is preferably a laboratory animal such as a mouse, rat, rabbit, guinea pig, goat or monkey (monkey or ape), and most preferably a human.

  In the present specification, the “instruction” describes a method for administering or diagnosing the pharmaceutical of the present invention to a doctor, a patient or other person who performs administration, or a person to be diagnosed (which may be the patient). It is. This instruction describes a word for instructing a procedure for administering the diagnostic agent or medicine of the present invention. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert, and is usually provided as a paper medium, but is not limited thereto. For example, an electronic medium (for example, a home page (web site) or e-mail provided on the Internet) It can also be provided in such a form.

  Determination of the end of treatment according to the method of the present invention is based on clinical symptoms characteristic of diseases (eg, hematological disorders) associated with standard clinical laboratory results or activins such as commercially available assays or instrumentation. Can be supported by the disappearance of Treatment can be resumed by the recurrence of a disease (eg, a blood system disease) associated with activin.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product may optionally be attached to such containers, which may be made, used or sold for administration to humans. Represents approval by government agencies.

  Plasma half-life and biodistribution of drugs and metabolites in plasma, tumors and major organs can also be determined to facilitate selection of the most appropriate drug to inhibit the disorder. Such a measurement can be performed. For example, HPLC analysis can be performed on the plasma of a drug-treated animal, and the location of the radiolabeled compound can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that exhibit potent inhibitory activity in screening assays but lack pharmacokinetic characteristics can be optimized by chemical structure alteration and retesting. In this regard, compounds that exhibit good pharmacokinetic characteristics can be used as models.

  Toxicity studies can also be performed by testing the compositions of the present invention. For example, toxicity studies can be performed in appropriate animal models such as: (1) a compound is administered to mice (untreated control mice should also be used); (2) each Blood samples are periodically obtained from one mouse in the treatment group via the tail vein; and (3) the sample is obtained from the number of red blood cells and white blood cells, the composition and the ratio of lymphocytes to polymorphonuclear cells Analyze about. Comparison of the results for each dosing regimen with controls shows whether there is toxicity.

  Further studies can be performed by sacrificing the animals at the end of each toxicology study (preferably, the American Veterinary Medical Guidelines Report of the American Veterinary Medical Asc. E. A. 93). Vet.Med.Assoc.202: 229-249). A representative animal from each treatment group can then be tested by global examination for direct evidence of metastasis, abnormal illness or toxicity. Overall abnormalities in the tissue are described and the tissue is examined histologically. Compounds that cause weight loss or blood component loss are not preferred, as are compounds that have adverse effects on major organs. In general, the greater the adverse effect, the less preferred the compound.

(Best Mode for Carrying Out the Invention)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  In one aspect, the present invention provides a method for producing pancreas from undifferentiated cells. This method includes the following steps: A) Undifferentiated cells are cultured in a medium containing leukemia inhibitory factor (LIF) to form colonies, and aggregates (typically embryoid body-like aggregates (embryoids) body-like spheres)); and B) exposing the undifferentiated cells to activin and retinoic acid in the absence of LIF. As the undifferentiated cell used in the method of the present invention, any cell can be used as long as the ability to differentiate remains.

  In the method of the present invention, colony formation can be achieved by culturing undifferentiated cells in the presence of LIF. Colony formation can be confirmed with the naked eye, and the culture period can be appropriately set by those skilled in the art.

  In the method of the present invention, the undifferentiated cells can be cultured by any culture method, but it is advantageous that they are preferably cultured on feeder cells.

  In the method of the present invention, the pre-culture step can be performed for any period, but preferably about 1 to 10 days, usually 1 to 4 days, typically about 2 days or about 4 days. Is advantageous. However, even if it is less than 1 day or 10 days or more, it is considered that there is no problem as long as it is suitable for differentiation.

  The undifferentiated cells used in the present invention may be any cells as long as they can differentiate into pancreas. There has been no example in which mammalian cells have been differentiated into functional pancreas so far, and therefore, the present invention should be recognized in that the mammalian cells have become functional pancreas.

  The medium used in the present invention advantageously contains serum. However, any serum generally used for cell maintenance / proliferation can be used as the serum contained, such as bovine serum, fetal bovine serum, horse serum, human serum, etc. Is not limited to them. The medium used may also be any medium, but typically a knockout serum replacement (KSR) medium is used.

  The exposure step in the method of the present invention may be performed under any culture condition, but it is advantageous to perform it under the condition of suspension culture. In suspension culture, cells that proliferate in a floating state in vivo, such as blood cells and various ascites tumor cells, can be suspended in suspension without being shaken or rotated. (Stirring culture), shaking the whole culture bottle (shaking culture), or rotating the incubator (rotating culture) is required. Examples of the suspension culture include suspension culture using a low adhesion coating culture vessel, suspension culture using a spinner flask (with or without oxygen gas), and the like.

  In a preferred embodiment, the suspension culture is preferably performed on a low adhesion coated culture vessel (eg, available from Nunc). Although not wishing to be bound by theory, it is because differentiation seems to be performed efficiently when such a low adhesion coating culture vessel is used.

  The undifferentiated cell used in the method of the present invention may be any undifferentiated cell, but is preferably a stem cell, more preferably an embryonic stem cell. When embryonic stem cells are used, it is preferably cultured with feeder cells. In this case, for example, mouse embryo fibroblasts are preferably used as the feeder cells.

  The activin used in the method of the present invention may be used at a concentration of less than 50 ng / ml. For example, it can be used at a concentration of 1 ng / ml to less than 50 ng / ml, 1 ng / ml to 25 ng / ml, 10 ng / ml to 25 ng / ml, and the like. It should be noted that activin can be used depending on the purpose because the required amount of retinoic acid varies depending on whether it is used at 10 ng / ml or 25 ng / ml. is there.

  The retinoic acid used in the method of the present invention may be used at more than 0.01 μM and less than 1 μM, and usually more than 0.01 μM and 0.1 μM or less, and may be 0.1 μM or more and less than 1 μM.

  The method of the present invention may further include C) a differentiation step of culturing the differentiated aggregate in a medium on a coated culture vessel. By using such a process, the state of differentiation can be improved. When used in the present invention, the coating can be any coating, but can preferably be a gelatin coating.

  Any medium may be used, but preferably, the medium used in the differentiation step is a KSR medium. The differentiation step can be a period sufficient to produce a functional pancreas, usually 7 to 21 days, and preferably 10 to 14 days, but is not limited thereto.

  In one embodiment, the differentiation step in the method of the present invention comprises first culturing in a medium containing activin and retinoic acid and then culturing in a medium not containing activin and retinoic acid. This is because such a method has been shown to promote differentiation.

  In one preferred embodiment, the differentiation step is initially cultured in a medium containing activin and retinoic acid for about 1 to 4 days, and then in a medium not containing activin and retinoic acid for 1 to 3 weeks. Including the step of culturing.

  In one preferred embodiment, the differentiation step is first cultured in a medium containing activin and retinoic acid for about 2 days, and then in a medium free of activin and retinoic acid for about 10 days to about 2 weeks. Including the step of culturing.

  The functional pancreas obtained by the present invention has at least one characteristic selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability and functional conduit structure. Such a functional pancreas is characterized in that it expresses the pancreatic marker Ipf1 / pdx-1 and does not express the marker Shh whose expression level decreases at the time when the pancreas is formed. Markers can be detected by immunological techniques. For example, confirming that such a functional pancreas expresses at least one or more pancreatic markers selected from the group consisting of insulin, glucagon and amylase 2, preferably all such markers. Can be determined.

  For example, a functional pancreas is characterized by including all α and β cells, exocrine cells and ductal structures. Such a functional pancreas has all the characteristics selected from the group consisting of insulin production ability, glucose responsiveness, glucagon production ability, amylase production ability and functional conduit structure. A pancreas having all such functions could not be produced until now. In particular, those derived from ES cells and mammals have not been reported. There are no reports of functional conduit structures, ie those that can be secreted in the same way as the natural one. Thus, the present invention exhibits a significant effect in that it provides a truly “functional” pancreas.

  In one aspect, the present invention provides a pancreas produced by the method of the present invention.

  In another aspect, the present invention provides a method for the treatment of a patient in need of a functional pancreas, characterized by using the pancreas of the present invention.

  In a further aspect, the present invention provides the use of activin and retinoic acid for the manufacture of a medicament for the treatment of a patient in need of a functional pancreas. Here, the patient may be a mammal. Furthermore, in a preferred embodiment, the medicament can be manufactured from stem cells (eg, ES cells).

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the embodiments, but only by the claims.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Animals were handled according to the spirit of animal welfare, in compliance with the standards stipulated at the University of Tokyo Animal Experiment Facility.

(Example 1: Differentiation induction of ES cells)
(ES cell culture method and differentiation induction)
Mouse ES cells (eg, E14 (ATCC-CRL-1821; available from ATCC) are cultured according to the usual culture method as described below, ie, with 10 μg / mL mitomycin C (Sigma) in advance. Cells seeded in culture vessels coated with mouse embryonic fibroblasts treated for ~ 2.5 hours to stop cell division are used as feeder cells and used as a maintenance medium for ES cells. Serum medium consists of 15% fetal bovine serum (FBS; ES cell qualified, MEM non-essential amino acid (GIBCO), 0.001% β-mercaptoethanol (Sigma), and 1,500 U / mL LIF (Chemicon). ) High glucose DMEM medium (L-Glutamine) And pyruvic acid, Gibco 11995-065).

  Three days after the culture, the cells are treated with 1 mg / mL collagenase / dispase (Roche), and the ES cells thus cultured are detached from the feeder cells to obtain ES cell colonies. The colonies thus obtained are cultured in DMEM containing 15% KSR in an untreated culture dish (Iwaki). The culture is changed every 2 days and the culture is continued for 4 days. After 4 days, ES cells begin to form EBS, but this EBS is transferred to a low-adhesive culture vessel (low-cell multi-dish; coated with a non-living component; Nunc), and then for another 2 days. , 15% KSR, activin (0, 10, 25, 50 ng / mL), and retinoic acid (0, 0.001, 0.01, 0.1, 1 μM; Sigma) in DMEM .

(Immunostaining)
13 days after the end of the treatment (19 days after the formation of EBS), EBS was fixed in 0.1M phosphate buffer (pH 7.4) with 4% paraformaldehyde for 40 minutes at room temperature. Fixed cells were detached from the dish walls, embedded in acrylic resin LR Gold Resin System (structural probe), and excised into thin sections of 600 nm. These sections were treated at room temperature for 40 minutes with 3% bovine serum albumin (BSA) and phosphate buffered saline (PBS = phosphate-buffered saline, 800 ml of sterile water, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4, Produced by 0.24 g of KH2PO4). They were then exposed to a pancreas-specific primary antibody for 12 hours at 4 ° C. After washing with PBS, sections were exposed to labeled secondary antibody for 8 hours at 4 ° C. The primary antibodies used were rabbit anti-α-amylase antibody (1: 1000 dilution, Sigma), anti-insulin monoclonal antibody (1: 400 dilution, Sigma) and goat anti-C peptide antibody (1: 800 dilution Linco Research); It was as follows: Alexa-Fluor 488-conjugated antibody and Alexa-Fluor 594-conjugated antibody (Molecular Probes). These sections were observed with a fluorescence microscope and photographed with AquaCosmos (Hamamatsu Photonics, Hamamatsu, Japan) connected to an ORCA-3 CCD camera. As a control, the pancreas of an 8 week old mouse was fixed and processed as described above.

(electronic microscope)
Eleven days after treatment (17 days after EBS formation), EBS was pre-fixed in a fixative solution of 4% paraformaldehyde, 3% glutaraldehyde and 0.1 M cacodylate buffer (pH 7.4) for 2 hours at room temperature. Then, after washing with 0.1 M cacodylate buffer, these were fixed with 1% osmium tetroxide for 30 minutes at room temperature and then treated. Samples were washed with 0.1 M cacodylate buffer, then dehydrated with ethanol and acetone and embedded in epoxy resin. Very thin sections (80-90 nm) were prepared and stained with uranium acetate and lead citrate and observed with a transmission electron microscope (JEM-1200CX, JEOL).

(RT-PCR)
Total RNA was extracted from 16 EBSs and normalized for gene expression between EBSs using ISOGEN (Nippon Gene). DNA was synthesized from 1 μg of total RNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). The forward and reverse primers used and the PCR product lengths are as follows:
amylase 2, GCC AAG GAA TGT GAG CGA TAC TTA (SEQ ID NO: 17) and CCA GAA GGC CAG TCA GAC GA (SEQ ID NO: 18) (418 bp);
glucagon, AAT GAA GAC AAA CGC CACT (SEQ ID NO: 19) and AAT TCA TAT ACA ATC GTT GGG TTA (SEQ ID NO: 20) (554 bp);
insulin II, GGC TTC TTC TAC ACA CCC ATG TCC (SEQ ID NO: 21) and TTT ATT CAT TGC AGA GGG GTA GGC (SEQ ID NO: 22) (234 bp);
Ipf1 / pdx-1, AGC AGT CTG AGG GTG AGC GGG TCT (SEQ ID NO: 23) and AAC CTC CAA CAG CCG CCT TTC GT (SEQ ID NO: 24) (412 bp);
somatostatin, CCC AGA CTC CGT CAG TTT CT (SEQ ID NO: 25) and TCA ATT TCT AAT GCA GGG TCA AGT (SEQ ID NO: 26) (377 bp);
pancreatic polypeptide, GCC CAA CAC TCA CTA GCT CAG (SEQ ID NO: 27) and AGA GGA AAG AGC TGG ACC TGT ACT (SEQ ID NO: 28) (419 bp);
Shh, ACA TGT CCC TTG TCC TGC GTT TCA (SEQ ID NO: 29) and CTC GTG GGC TCG CTG CTA GGT (SEQ ID NO: 30);
GAPDH, TGA AGG TCG GTG TGA ACG GAT TTG GC (SEQ ID NO: 31) and CAT GTA GGC CCA TGA GGT CCA CCA C (SEQ ID NO: 32).

  The cDNA was denatured at 95 ° C for the first 15 minutes, heated for 45 cycles at 94 ° C (20 seconds), 54 ° C (20 seconds) and 72 ° C (40 seconds), and finally at 72 ° C for 7 minutes. Proliferation was performed by an extension process. The experiment was performed in triplicate.

(Real-time PCR)
Real-time PCR was performed using QuantTect ^™ SYBR Green PCR Master Mix (QIAGEN) and ABI PRISM ^™ 7700 Sequence Detector (Applied Biosystems). The forward and reverse primers used and their PCR product lengths are as follows:
amylase 2, ATA CTC TGC TTG GGA CTT TAA CGA (SEQ ID NO: 33) and CAG AAG GCC AGT CAG ACG A (SEQ ID NO: 34) (100 bp);
GAPDH, GCT ACA CTG AGG ACC AGG TTG TC (SEQ ID NO: 35) and AGC CGT ATT CAT TGT CAT ACC AGG (SEQ ID NO: 36) (135 bp);
insulin II, AGA AGC GTG GCA TTG TAG ATC AGT (SEQ ID NO: 37) and CAG AGG GGT AGG CTG GGT AGT G (SEQ ID NO: 38) (102 bp);
Ipf1 / pdx-1, GAT GAA ATC CAC CAA AGC TCA CGC (SEQ ID NO: 39) and GGG TGT AGG CAG TAC GGG TCC TC (SEQ ID NO: 40) (101 bp);
ptfla, ATG CAG TCC ATC AAC GAC GCC TTC (SEQ ID NO: 41) and GGC TTG CAC CAG CTC GCT GAG (SEQ ID NO: 42) (138 bp);
Shh, GAC TGC GGG CAT CCA CTG GTA CTC (SEQ ID NO: 43) and GTC GGG CTT CAG CTG GAC TTG AC (SEQ ID NO: 44) (114 bp).

The cDNA was denatured at 95 ° C for the first 15 minutes, heated for 45 cycles at 94 ° C (20 seconds), 54 ° C (20 seconds) and 72 ° C (40 seconds), and finally at 72 ° C for 7 minutes. Proliferation was performed by an extension process. The experiment was performed in triplicate. (Quantification of total intracellular insulin and secreted insulin)
13 days after induction (19 days from the start of EBS formation), EBS was washed with DMEM without glucose (GIBCO) and then incubated with DMEM with 3 mM glucose (Sigma) for 60 minutes. EBS was washed again with DMEM, incubated in DMEM containing 3 mM or 25 mM glucose for 30 minutes, and conditioned medium was collected. These cells were treated with lysis buffer (50 mM Tris-HCl [pH 7.0], 0.5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% CHAPS, 0.1% SDS, 1 mM PMSF) and the total amount of protein was measured with a protein assay kit (BioRad). The total amount of insulin in the conditioned medium or the amount of insulin in the cell lysate was quantified using a Mouse Insulin ELISA (TMB) Kit (SHIBAYAGI, Japan). Each experiment was performed with 8 EBS, and the experiment was performed in triplicate.

(Induction of pancreas)
An EBS obtained as described above is seeded in an adhesive culture vessel (for example, a 24-well well plate) coated with 0.1% gelatin, and cultured in DMEM containing 10% KSR. Adhere to the culture plate. Then, in a KSR medium, in a low adhesion coat culture vessel, treatment with 25 ng / mL activin and 0.1 μM retinoic acid simultaneously for 2 days. Subsequently, the cells are cultured for 10 days to 2 weeks in the same manner in a gelatin-coated culture vessel under the absence of activin and retinoic acid. The culture medium is changed every 3 days.

(Intestinal tubular and pancreatic tissue differentiation from ES cells)
First, ES cell colonies were isolated 3 days after collagenase / dispase treatment, and then cultured in DMEM supplemented with 15% KSR to form embryoid body-like aggregates (EBS). This EBS can differentiate. Four days after the formation of EBS, the cells were transferred to a medium containing retinoic acid (0, 0.001, 0.01, 0.1 or 1 μM) and activin (10 ng / ml), and suspended in culture for another 2 days. Seeded on gelatin-coated plates. Six to eight days after treatment with retinoic acid and activin (12-14 days after EBS formation), the proportion of treated EBS became spherical or intestinal tubular structures, and the movement was similar to smooth muscle peristalsis. . 9-12 days after treatment (15-18 days after EBS formation), EBS treated with more than 20% of both retinoic acid and activin formed tissues with black spots. This was rarely seen with non-conventional EBS or EBS treated with activin alone (FIG. 2Ca, b).

  FIG. A, an intestinal tract-like structure induced by activin and retinoic acid differentiation from colony aggregates (EBS) of mouse ES cells, and a tissue (arrow) containing a duct-like structure and black spots developed on its side. The gut-like structure in the center performs a slow peristaltic movement. The feature of the tissue with black spots is that of secretory tissue. B, Colony aggregate (EBS) of mouse ES cells cultured in the same manner in the absence of activin and retinoic acid. The intestinal tract-like structure, duct-like structure seen in A, and tissues containing black spots are not seen. The scale bar is 100 μM.

(Identification of pancreas)
In the structure shown in FIG. 2-A, there is an intestinal tract-like structure that performs a slow peristaltic movement in the central part, and the tissue having the characteristics of secretory cells connected to it by a duct-like structure has the characteristics of the natural pancreas. Show. This structure was observed in more detail with an electron microscope (FIG. 3).

(Morphological identification of ES cell-derived pancreatic tissue)
Histological examination of sections of EBS after treatment with 0.1 μM retinoic acid and 10 ng / ml activin 11 days after the last treatment (17 days after EBS formation) showed a globular tissue, It was shown that the region adjacent to the tubular lumen was strongly stained with toluidine blue (FIG. 3Ea). Pancreatic tube-like structures were found in the vicinity of the spherical tissue (FIGS. 3Ec, 3d). In contrast, neither spherical tissue nor tube-like structures were observed in untreated sections of EBS (FIG. 3Eb). Analysis by electron micrographs showed that the cells had well-developed endoplasmic reticulum and contained a large amount of zymogen particles (FIG. 4Ca, b). This is the same phenotype as pancreatic exocrine cells (acinar cells). Tissue formed from treated EBS also resembled pancreatic acinar cells in that a tube-like (gap region) was seen in the adjacent region of the tissue (FIG. 4Ca). There was also a structure similar to the pancreatic duct, surrounded by a low columnar epithelium (FIG. 4Cd). In addition, cells morphologically similar to pancreatic endocrine cells (FIG. 4Cc, d) were also observed, but fewer than those of cells resembling exocrine cells. These endocrine-like cells contained granules (Fig. C4d), which were morphologically different from the granules found in the secretory cells (Fig. C4b). A transparent space between the dark central region and the lateral membrane was also found, which is a typical feature of β-cell granules in the pancreas. To ascertain whether these granules contain insulin or amylase, the cells were immunostained with anti-amylase antibody or anti-insulin C peptide (proinsulin) antibody. These EBS treated with both retinoic acid and activin contained cells with amylase positive zymogen granules (FIG. 5c). Moreover, a relatively small number of cells were aggregates having c peptide positive granules (FIG. 5d). These morphological findings indicate that pancreatic tissue, including pancreatic ducts, endocrine cells and exocrine cells, was formed from EBS treated with both retinoic acid and activin.

  FIG. A, an electron microscopic image of a tissue (see FIG. 1) containing black spots differentiated with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. B, Electron microscopic image of pancreatic exocrine cells of rat fetus in late pregnancy (Bock, et al., 1997). Both cells are very similar in structure, including a large number of granule-like structures with high electron density and having developed endoplasmic reticulum. ER, endoplasmic reticulum; N, nucleus.

  FIG. C, Electron microscopic image of a duct-like structure (see FIG. 1) induced by differentiation with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. Very similar to the epithelial structure of the natural pancreatic duct. D, similarly, an electron microscopic image of a cell (see FIG. 1) having a secretory granule-like structure induced by differentiation with activin and retinoic acid from a colony aggregate (EBS) of mouse ES cells. An enlarged image of a portion surrounded by a dotted line in the photograph is also shown. This image is similar to the endocrine granule image (Goping, et al., 2003; indicated by an arrow) in the pancreatic β cells of the mouse body shown on the right (E). Gaps are seen between the wrapping bags (indicated by arrows). N, nuclear.

  In the pancreas-like tissue shown in FIG. 2, cells having many electron-dense (black) uniform granule-like structures and cells having highly developed endoplasmic reticulum (ER) were observed ( (See FIG. 3-A). This cell is very similar to the exocrine cells of the rat fetus pancreas in late pregnancy (acinar cells: Bock, et al., Microscopic research and technique 37: 374-383, 1997) (see FIG. 3-B). On the other hand, in the same pancreas-like tissue, cells containing many structures in which gaps are seen between the contents having a somewhat lower electron density than that in FIG. (See Figure 3-D). This cell shows the typical endocrine cell morphology of the pancreas (FIG. 3-E; Goping, et al., Microscopic research and technique 61: 448-456, 2003), and differentiation of insulin producing cells occurred. I strongly suggested that. As described above, both the cells having the characteristics of exocrine cells and the cells having the characteristics of endocrine cells were observed in the pancreas-like tissue formed by simultaneously treating activin and retinoic acid by electron microscope observation.

  As described above, a colony-like structure (EBS) derived from mouse ES cells, typically treated with 10-25 ng / mL activin and 0.1 μM retinoic acid simultaneously for 2 days, shows in vitro pancreatic-like structure. Differentiated organs could be induced. Next, whether or not this pancreas-like organ actually expresses various proteins (pancreatic markers) expected to be specifically expressed in the pancreas was examined at the gene expression level. The pancreatic markers examined were insulin 2 (specific to β cells, pancreatic endocrine cells), glucagon (specific to α cells, pancreatic endocrine cells), amylase 2 (specific to exogenous cells in the pancreas) , Ipf1 / pdx-1 (pancreatic and dual homeboxfactor-1, a gene known to positively control pancreatic development). And Shh (a sonic hedgehog, a gene known to negatively control pancreatic development).

(Pancreatic marker gene expression test)
The expression of genes regulating pancreatic development and several pancreatic marker genes were investigated using RT-PCR (FIG. 2c). EBS treated with both retinoic acid and activin are insulin II (β cell marker), glucogons (α cell), pancreatic polypeptide (γ cell), somatostatin (δ cell) and amylase 2 (marker of exocrine pancreatic cells) and Ipf1. It was found that the expression of / pdx-1 and ptf1a / p4 was significantly increased. Ipf1 / pdx-1 and ptf1a / p4 are genes that regulate pancreatic development. In contrast, untreated EBS had almost no expression of these genes. Amylase and pancreatic polypeptide increased over time from day 6 of treatment (this is day 12 counting from EBS formation). On the other hand, the increase in gene expression of insulin II, glucagon, somatostatin, Ipf1 / pdx-1 and ptf1a / p48 started immediately from the end of the treatment. In untreated EBS, Shh was found to be expressed during the observation period, whereas in EBS treated with both retinoic acid and activin, Shh gene expression was repressed after treatment.

  When the activin concentration was kept constant (10 ng / ml) and the retinoic acid concentration was varied, the gene expression of the pancreatic marker in EBS was most apparent when 0.1 μM retinoic acid was treated with 10 ng / ml activin A. It was found to be efficient (FIG. 2d). When these concentrations of retinoic acid and activin were used, 47.6 ± 7.3% of the treated EBS was exceeded for all of the described pancreatic genetic markers. When 0.001 μM, 0.01 μM and 1 μM were used as the concentration of retinoic acid, 4.3 ± 2.5%, 12.8 ± 9.6% and 25.1 ± 6.6% in EBS, respectively. Gene expression was seen.

  FIG. MRNA expression profile obtained by RT-PCR method of pancreatic structure induced to differentiate with activin and retinoic acid from colony aggregate (EBS) of mouse ES cells. Insulin 2, glucagon, amylase 2 and Ipf1 / pdx-1 are marker genes (positive markers) that are specifically expressed in pancreas development, while Shh is a gene (negative) that decreases in expression with pancreas development. Marker). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was a constitutive protein (enzyme) expressed at a high level in all cells, and was used for the purpose of ensuring that the performed assay method was appropriate.

  As a result, when treated with 10 ng / mL of activin A and 0.1 μM retinoic acid, it was confirmed that all positive marker groups were newly expressed from the second day after the treatment. In addition, these positive marker groups could be detected at all or only in the untreated group. On the other hand, the expression of Shh, a negative marker, was observed in the whole untreated group and on day 0 of the treated group, but disappeared completely after 2 days of treatment with activin A and retinoic acid. From these results, by simultaneously treating with activin A and retinoic acid, the pancreas with not only endocrine cells (α cells and β cells) but also exocrine cells was obtained from colony aggregates (EBS) derived from mouse ES cells. It was found at the level of gene expression that differentiation can be induced in vitro.

  Next, the effects of activin concentration and retinoic acid concentration on the expression of insulin 2 and amylase 2 in the positive marker group were examined.

  FIG. From the colony aggregate (EBS) of mouse ES cells, differentiation of pancreatic structures induced by treatment with different activin concentrations (0, 10, 25 ng / mL) and retinoic acid concentrations (0, 0.1, 1.0 μM). Difference in mRNA expression level between insulin 2 and amylase 2. The vertical axis represents the relative expression level of each gene.

  As a result, it was found that the concentration of retinoic acid to be treated first was 0.1 μM, which is the optimum concentration in terms of insulin 2 and amylase 2 mRNA expression. Next, when treated with activin at a concentration of 25 ng / mL while also treating with 0.1 μM retinoic acid, the gene for insulin 2 is most efficiently expressed, while the concentration of activin is 10 ng / mL It was found that the amylase gene was most efficiently expressed at mL. This result indicates that endocrine cells and exocrine cells are preferentially induced to differentiate from mouse ES cell-derived embryoid bodies by the treatment concentration of activin.

  Next, in these positive marker groups, whether or not the insulin and amylase genes were actually transcribed and expressed as proteins was examined from histoimmunology. However, since insulin may be contained in the culture medium, it is necessary to identify only insulin that is newly synthesized and secreted in the differentiation-induced pancreas. Insulin is first translated from the gene as a precursor, preinsulin, and immediately becomes proinsulin with the prepart cleaved. This proinsulin is secreted to the outside of the cell as active insulin which has been cleaved again to remove the C peptide portion. That is, in order to confirm that the pancreas prepared from colony aggregates (EBS) in vitro newly synthesized and secreted insulin, an antibody that binds only to the C peptide, not an antibody that binds to insulin ( Tissue immunology was performed using a goat anti-C peptide antibody, Linco Research). For amylase, an antibody (Sigma) was usually used.

  Fig. 4-1.10 An immunohistological observation image of colony aggregates (EBS) derived from mouse ES cells treated with ng / mL Activin and 0.1 µM retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino-2-phenylindole; nuclear stain), D, A + B + C. Scale bar is 50 μM. Insulin C peptide and amylase are made by different cells. Under these conditions, amylase is preferentially synthesized over insulin.

  Fig. 4-2. Immunohistological observation image of colony aggregate (EBS) derived from mouse ES cells treated with 25 ng / mL activin and 0.1 μM retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino-2-phenylindole; nuclear stain), D, A + B + C. Scale bar is 50 μM. As in FIG. 4-1, insulin C peptide and amylase are produced by different cells, but under these conditions, insulin is synthesized preferentially over amylase.

  From the results of FIG. 4 and FIG. 3, it can be seen that 0.1 μM retinoic acid is best for inducing differentiation of both endocrine cells and exocrine cells, and that endocrine cells are activated when the activin concentration is 25 ng / mL. However, it was found that exocrine cells are preferentially induced to differentiate when the activin concentration is 10 ng / mL.

(Concentration dependence)
Changes in activin concentration change the ratio of exocrine cells to endocrine cells.

  The inventors further tested that the effect of activin and retinoic acid on pancreatic differentiation on EBS was concentration-dependent. To answer this question, total RNA was extracted from EBS 13 days after treatment with retinoic acid (0, 0.1 μM or 1 μM) and activin (0 ng / ml, 10 ng / ml or 25 ng / ml), and Amylas 2, insulin II, ptf1a / p48, and Shh were measured by performing real-time PCR and comparing (normalized to 1) the expression level of untreated EBS to measure the transcriptional expression levels of the following genes: d). Shh expression was reduced by treatment with retinoic acid alone, activin alone and retinoic acid plus activin (FIG. 6c). Insulin II and amylase 2 expression was higher in untreated EBS in EBS treated with retinoic acid alone, and activin alone had no significant effect on the level of insulin expression, but amylase over untreated EBS. Expression was reduced (Figure 6a, b). Interesting results were observed when the concentration of retinoic acid was kept constant (0.1 μM) and the concentration of activin was varied. At relatively low concentrations of activin (10 ng / ml), the expression levels of amylase2 and ptf1a / p48 (exocrine cell marker) are much higher in the army treated with retinoic acid and activin than untreated EBS On the other hand, the expression of insulin II did not vary between these groups. In contrast, at higher activin concentrations (25 ng / ml), the expression of insulin is significantly increased in EBS treated with retinoic acid and activin, compared to the untreated group, whereas the expression level of amylase 2 is The two groups were low.

  Real-time PCR results are supported by staining with both anti-insulin C peptide antibody and anti-amylase antibody (Paper Figure 7a-d). In EBS treated with 0.1 μM retinoic acid and low concentrations (10 ng / ml) of activin, fewer insulin positive cells were seen than amylase positive cells. However, when high concentrations of activin (25 ng / ml) were used with 0.1 μM retinoic acid, the percentage of insulin positive cells was significantly increased (FIG. 7d). In untreated EBS, neither insulin-positive cells nor amylase-positive cells were observed (FIG. 7b). These results indicate that the ratio of endocrine cells to exocrine cells in tissues derived from EBS depends on the activin concentration. Immunocytochemical analysis also revealed that some of the cells produced glucagon, an alpha cell marker protein (FIG. 7e).

(Summary)
From a mouse ES cell colony, using a suspension culture system, a new method of simultaneous treatment of activin and retinoic acid is used to synthesize and secrete β cells and glucagon that synthesize and secrete insulin from undifferentiated cells. We have succeeded for the first time in the world to create in vitro the endocrine cells of both α cells, the exocrine cells that synthesize and secrete amylase, and the pancreas having a conduit for releasing the secretions to the outside.

(Example 2: Glucose responsiveness)
In this example, whether pancreas prepared in vitro by treating 25 ng / mL activin and 0.1 μM retinoic acid increases the amount of insulin secreted in response to external glucose. It examined (Table 1).

  To test insulin secretion in vitro, pancreatic tissue differentiated from ES cells with 25 ng / ml activin and 0.1 μM retinoic acid was subjected to low (3 mM) and high (25 mM) glucose tolerance tests. Tested (Table 1). Activin / retinoic acid treated EBS secreted insulin into the medium in a glucose dependent manner. On the other hand, insulin secretion from untreated EBS was below the detection limit under the same conditions. Individual EBS treated with both retinoic acid and activin secreted an average of 0.24 ng of insulin in the conditioned medium per mg of the protein (approximately 9.7% total insulin per EBS). These results indicate that EBS treated with both retinoic acid and activin secreted insulin in response to increasing glucose concentration. This is similar to the physiological function that pancreatic β cells have in vivo.

Table 1.25 Amount of insulin secreted by pancreas made by simultaneous treatment of ng / mL activin and 0.1 μM retinoic acid for 13 days depending on glucose concentration

  Table 1 shows changes in the amount of insulin produced by EBS intracellular insulin amount and glucose change.

  Intracellular insulin levels in EBS treated and untreated with 0.1 μM retinoic acid and 25 ng / ml activin 13 days after induction treatment. The amount of insulin secreted from EBS in response to glucose load was measured. The indicated amount of secreted insulin is comparable to the amount of insulin secreted 30 minutes after the start of stimulation with 25 mM glucose. Increased insulin secretion due to glucose loading in the culture medium was observed in EBS treated with both retinoin and activin.

(Example 3: Transplantation method of pancreatic tissue induced to differentiate in a culture system)
It demonstrates that the mouse pancreas produced in Examples 1-2 can actually be used in a diabetes model mouse. The procedure will be described below.

(animal)
Diabetes model mice can be 250 mg / kg body of AKITA-mouse (Yoshioka, et al., Diabetes 46: 887, 1997) or streptozotocin (STZ) that selectively destroys pancreatic islets of 10 weeks of age or older. Nude mice whose pathology is caused by administration of weight are used. In this case, transplantation is performed when one week has elapsed after administration of STZ.

(Transplantation of differentiation-induced pancreatic tissue)
For transplantation, pancreatic tissue that has been induced to differentiate in the culture system is collected from the culture dish so as not to damage the tissue with tweezers. This is washed twice with Hanks' saline.
Mice are anesthetized with Nembutal, the abdomen is dissected, and 20-30 differentiation-induced pancreatic tissues are massed and transplanted under the retroperitoneum. As another transplant site, it is also effective to implant under the renal capsule. There is also a method in which pancreatic tissue is previously treated with Liberase RI (Roche) RI, which is an enzyme for islet isolation, to isolate cells and inject them into the portal vein of the liver.

  The blood glucose level is measured by damaging the tip of the mouse tail with a scalpel or razor and collecting several μl of blood.

  In this way, it is possible to confirm whether the pancreas produced in the present invention actually functions in the model mouse.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

By this method, from undifferentiated mammalian cells that have not been realized so far,
1) having β cells that synthesize and secrete insulin in response to glucose load;
2) Not only β cells but also α cells that synthesize and secrete glucagon as endocrine cells,
3) Have exocrine cells (acinar cells) that make digestive enzymes (such as amylase) and secrete them into the intestine.
4) It was the first in the world to create a pancreas with all functions and structures such as a duct structure for exoculating digestive enzymes from the pancreas into the intestinal tract.

  Cells derived from human ES cells by a method different from the method of the present inventors have already been experimentally applied to the treatment of human diabetes in the United States. If the method developed by the present inventors can produce not only β cells from human undifferentiated cells but also a complete pancreas as shown by the present inventors, many patients with diabetic dysfunction are likely to be diabetic. In addition, there is a possibility of providing an unprecedented useful material for treating a patient who has to remove the pancreas due to pancreatic cancer or the like. Also, useful models and evaluation systems (assay systems) in biological and medical research related to the pancreas, research on the elucidation of the cause of pancreatic diseases, and the development of drugs for the treatment of pancreatic diseases and pancreatic cancer You can also expect to be. Thus, it is judged that the practical application and industrial prospect of this method are extremely bright.

Claims (21)

A method for producing mouse pancreas from mouse embryonic stem (ES) cells, comprising the following steps:
A) A step of culturing mouse ES cells on a feeder cell in a medium containing serum and leukemia inhibitory factor (LIF) to form a colony,
B) Thereafter, a pre-culture step of culturing the colony formed in a medium containing no LIF to form an embryoid body-like sphere;
C) then an exposure step in which the embryoid body-like aggregates are exposed simultaneously to activin and retinoic acid in the absence of feeder cells and LIF; and D) is then cultured in a medium without activin and retinoic acid. A method comprising the steps. The method according to claim 1, wherein the pre-culture step is performed for 1 to 10 days. The method according to claim 1, wherein the exposing step is performed for 1 to 4 days. The method according to claim 1, wherein the medium used in the steps C) and D) is a KnockOut Serum Replacement ™ (KSR ™ ) medium. The method according to claim 1, wherein the exposing step is performed under conditions of suspension culture. The method according to claim 5, wherein the suspension culture is performed on a low adhesion coating culture vessel. The method of claim 1, wherein the feeder cell is a mouse fetal fibroblast. The method according to claim 1, wherein the activin in the step C) is 1 ng / ml to less than 50 ng / ml. The method according to claim 1, wherein the retinoic acid in step C) is more than 0.01 μM and less than 1 μM. Furthermore, the said process D) includes the differentiation process which culture | cultivates the differentiated said aggregate in the culture medium on the coated culture container. The method of claim 10, wherein the coating is a gelatin coating. The method according to claim 10, wherein the medium used in the differentiation step is a KSR (trademark) medium. 11. The method of claim 10, wherein the differentiation step is a period sufficient to produce a functional pancreas. The method according to claim 10, wherein the differentiation step is performed for 7 to 21 days. The step C) includes a step of culturing in a medium containing activin and retinoic acid for 1 to 4 days, and the step D) is cultured in a medium not containing activin and retinoic acid for 1 week to 3 weeks. The method according to claim 10, comprising the step of: 11. The method according to claim 10 , wherein in step C), the use concentration of activin is 1 ng / ml to less than 50 ng / ml, and the use concentration of retinoic acid is more than 0.01 μM and less than 1 μM . Wherein S w organs insulin production ability, has at least one feature glucose-responsive, glucagon-producing ability is selected from the group consisting of amylase, etc. producing ability and functional channel structure, The method of claim 1. Wherein S w visceral express pancreas marker Ipf1 / pdx-1, the expression level at a time when the pancreas is formed is equal to or not expressing the marker Shh to decrease, The method of claim 1. Wherein S w organs are, insulin, express at least one pancreatic markers selected from the group consisting of glucagon and amylase 2, The method of claim 1. Wherein S w organs is characterized in that it comprises α cells and β cells and exocrine cells and the conduit structure, The method of claim 1. Wherein S w organs insulin production ability has glucose-responsive, glucagon-producing ability, all features selected from the group consisting of amylase, etc. producing ability and functional channel structure, The method of claim 1.