WO2007058105A1 - Method for producing pancreas from es cell - Google Patents

Abstract

It is intended to produce a pancreas that fully functions from an undifferentiated cell. The inventors found that a functional pancreas can be differentiated by using activin and retinoic acid at specific concentrations and subjecting an undifferentiated cell to a specific treatment in advance, thus this object was achieved. The method of the invention comprises A) a preculture step in which an undifferentiated cell is cultured in a medium containing a leukemia inhibitory factor (LIF) to form a colony and a clump is formed; and B) an exposure step in which the undifferentiated cell is exposed to activin and retinoic acid in the absence of LIF.

Description

 Specification

 ES cell power

 Technical field

 [0001] 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.

 Background art

[0002] Several methods have been developed and reported for in vitro production of embryos from embryonic stem cells (ES cells) (for example, non-patent literature l = Loeb _e l, et al., 2003; Non-patent literature 2 = Soria, et al., 2000; Non-patent literature 3 = Shroi, et al., 2002; Non-patent literature 4 = Leon— Quinto, et al., 2004; Non-patent literature 5 = Lumelsky, et al., 2001). However, these pancreas are not perfect. This is because the beta cells (j8 cells: insulin, a hormone responsible for the suppression of glucose uptake and gluconeogenesis by body tissues, can be obtained from any mammalian undifferentiated cells, including ES cells. This is due to the fact that the pancreas has been made by focusing only on the production of endocrine cells that specifically produce and secrete these. This is because cell therapy and tissue therapy to rescue many diabetics who cannot be treated with current medical standards and technologies by transplanting β-cells with insulin secretion activity. ) Is a force aiming at regenerative medicine. Therefore, it has never been possible to create a pancreas that has the other functions and structures of the natural pancreas.

 [0003] Such natural organs and viscera are preferably

 1) Has endocrine cells (j8 cells) that secrete insulin in response to blood glucose levels (ie, blood glucose concentration)

 2) Endocrine cells that produce and secrete glucagon (alpha 1 cell, a cell: an endocrine cell that specifically produces and secretes glucagon, a hormone responsible for glycogen degradation and gluconeogenesis)

3) Has exocrine cells (acinar cells) that make digestive enzymes (eg amylase) and secrete them into the intestinal tract, 4) A pancreas with all functions and structures, such as a duct structure for exoculating digestive enzymes from the pancreas to the intestinal tract.

 [0004] In a healthy body, blood glucose levels are appropriately maintained by the competitive action of insulin and glucagon. Therefore, for the treatment of diabetes, not only a pancreas that secretes only insulin, but also a pancreas that can secrete glucagon at the same time, 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, create digestive enzymes that endure only the endocrine function, and transplant the pancreas with the ability to exoculate it. Necessary power The method to make such a pancreas in vitro was a powerful force. In addition, if digestive enzymes are secreted into the tissue, the body itself will be digested and dangerous, so a conduit for guiding digestive enzymes into the digestive tract is also necessary, but a pancreas with a conduit structure is necessary. There was no way to make this 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- 16 6, 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 Patent Document 9 (26th Information Chemistry Symposium PL-1, 2003), Non-patent Document 10 (Japan Tissue Cytochemistry General Meeting · Academic Meeting Presentation 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 (Delop. Growth Differ. 42: 593—602, 2000) [Koo! Use mouse, ί or amphibians!試 み Attempts to produce pancreas. However, pancreas produced by the methods described in these documents is not fully functional. Detailed conditions are not described. In many cases, detailed conditions are unknown during regeneration, but it has been reported that other organs can be produced with slightly different concentrations. Therefore, the present disclosure power of the present inventors is fully functional, and until the viscera can be produced.

[0006] Patent Document 3 (Japanese Patent Application Laid-Open No. 2003-9854) discloses an embryonic stem cell library comprising a step of culturing embryonic stem cells using a medium containing a specific factor. A method for forming an id body, a medium for use in the method, and an agent for inducing liquid, and using the method A method for inducing differentiated cells, the differentiation-induced cells, and use thereof are disclosed.

 [0007] In addition, an attempt to produce viscera from undifferentiated cells is described in Patent Document 4 (WO200 2/079457); Patent Document 5 (WO2002Z086107); Patent Document 6 (WO2002 / 0103 47); j¾7 (WO2002 / 059278); Patent Literature 8 (WO2002 / 092756); Non-Patent Literature 13 (Developmental Biology 264: 1—14, 2003); Non-Patent Literature 14 (Di abetes 49: February, 1-6, 2000); Patent Document 15 (Stem 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 reseach and technique 61: 448—456, 2003); Non-patent document 19 (Microscopy reseach and technique 37: 374-383, 1997); Non-patent document 20 (Stem cells 23: 656-662, 2005); Patent Document 21 (Delop. Growth Differ. 42: 593-602, 2000); Non-Patent Document 22 (Exp. Anim. 54 (3). 262, P-048, 200 5), etc. Pancreatic production has not been achieved, and nursing Production from animal cells is rarely performed. Furthermore, there has been little attempt to produce ES cells and other undifferentiated cells by dividing them, and there is no example of successful production of functional organs from mammalian ES cells. ! /

 [0008] As described above, it has not yet been achieved to produce a fully functional pancreas from various undifferentiated cells including embryonic stem cells (ES cells).

 Non-Patent Document l: Loebel, D.A., Watson, C.M., De Young, R.A., Tarn, P.P., Dev. Biol., 264, 1 (2003).

 Non-Patent Document 2: Soria, B., Roche, Berna, G., Leon—Quinto, T., Reig, J.A., Martin, F., Diabetes, 49, 157 (2000).

 Non-Patent Document 3: Shroi, A., Yoshikawa, M., Yokota, H., Fukui, H., Ishizuk a, S., Tatsumi, K., Takahashi, Y., Stem Cells, 20, 284 (2002) .

 Non-Patent Document 4: Leon-Quinto, T., Jones, J., Skoudy, A., Burcin, M., Soria, B., Diabetologia, 47, 1442 (2004).

Non-Patent Document 5: Lumelsky, N., Blondel, O., Laeng, P., Velasco, L., Ravin, R., McKay, R., Scinece, 292, 1389 (2001). Non-Patent Document 6: Experimental Medicine Vol. 19 No. 15, 159-166, 2001

 Non-Patent Document 7: History 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: Annual Meeting of the Japanese Society for Tissue Cytochemistry 'Academic Conference Lecture Program' Proceedings VOL. 44th; PAGE, 29, 2003

 Non-patent literature 11: Hyundai Kagaku VOL. 41th; PAGE, 124-131, 2002 Non-patent literature 12: Delop. Growth Differ. 42: 593-602, 2000

 Non-Patent Document 13: Bock, G., Pollard, H. B., Srivastava, M., Leapman, R. Microscopy Res. Tech. 61, 448 (1997).

 Non-Patent Document 14: Goping, B., Abdel—Moneim, M., Egerbacher, M. (2003) Patent Document 1: JP 2001-333770 A

 Patent Document 2: Japanese Patent Laid-Open No. 2001-299335

 Patent Document 3: Japanese Patent Laid-Open No. 2003-9854

 Patent Document 4: International Publication 2002Z079457 Pamphlet

 Patent Document 5: International Publication 2002Z086107 Pamphlet

 Patent Document 6: International Publication 2002Z010347 Pamphlet

 Patent Document 7: International Publication 2002Z059278 Pamphlet

 Patent Document 8: International Publication 2002Z092756 Pamphlet

 Disclosure of the invention

 Problems to be solved by the invention

[0009] An object of the present invention is to produce a fully functioning pancreas from undifferentiated cells.

[0010] 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) Has beta cells that secrete insulin in response to glucose load,

 2) As endocrine cells, not only β cells but also ex cells,

3) Has exocrine cells (acinar cells) that make digestive enzymes (amylase, etc.) 4) It is equipped with all functions and structures such as a conduit structure!

[0011] If such a pancreas can be made from undifferentiated cells of mammals, it is possible to provide a useful material for treating many patients with diabetes and soot removal. There is a potential. In addition, if the pancreas has the same function and structure as the natural pancreas, the biological and medical research related to the pancreas, research on the cause of the pancreatic disease, and the treatment and treatment of the visceral disease, It can also be expected to be a useful model and evaluation system (Atsusei system) in the development of drugs for the treatment of visceral cancer.

 Means for solving the problem

 [0012] The above problem is that the present inventors can differentiate into a functional pancreas by using a specific concentration of activin and retinoic acid and subjecting undifferentiated cells to a specific treatment in advance. It was solved by finding what I could do.

 [0013] The present invention comprises a combination of mammalian "undifferentiated cells" and "activin and retinoic acid treatment". As illustrated in Fig. 1, first, undifferentiated cells are cultured in a serum medium in the presence of leukemia in hibitory factor (LIF) on a feeder cell to form colonies of ES cells. Agglomerates (embryoid body-like spheres, EBS) are formed. Then, in the absence of feeder cells and LIF, activin and retinoic acid are simultaneously treated in a serum-free medium, and the formed aggregate is also induced by in vitro differentiation of the pancreas.

 FIG. 1 shows a schematic representative diagram. Schematic diagram of pancreatic differentiation from undifferentiated cells (ES cells). LIF, Leukemia Inhibitory Factor; EBS, Embryoia Doay—ΗΚΘ 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 serum medium (fetal bovine serum, FBS medium), and then ES cells are cultured in the presence of LIF (leukemia inhibitory factor). A colony that is an aggregate of, and further, its agglomerates. Next, the agglomerates thus obtained were subjected to induction medium (10% serum replacement, knockout serum replacement, supplemented with activin and retinoic acid under the conditions of suspension culture using a low-contact coated culture plate. KSR; Gibco) for 2 days The Furthermore, the pancreas can be made by culturing the aggregate in KSR medium in a gelatin-coated culture plate (Fig. 1).

 [0016] The spleen produced by inducing differentiation in this way includes ex cells and j8 cells that are endocrine cells of the spleen, and insulin secretion increases in response to glucose load. In addition to endocrine cells, pancreatic exocrine cells and ductal structures are also included: light microscope, electron microscope, anti-insulin antibody (anti-insulin C peptide antibody), anti-glucagon antibody, anti-amylase antibody, This was confirmed by the identification of genetic marker groups that specifically increased and decreased as the pancreas developed.

 [0017] Accordingly, the present invention provides the following.

 (1) A method for producing pancreas with unbroken cell strength, and 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) A method comprising exposing the undifferentiated cells to activin and retinoic acid in the absence of LIF.

 (2) The method according to the above item, wherein the undivided sputum cells are cultured on a feeder cell.

(3) The method according to the above item, wherein the aggregate is an embryoid body-like sphere.

 (4) The method according to the above item, wherein the pre-culture step is 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 to 4 days.

 (7) The method according to the above item, wherein the exposure step is 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 suspension culture is carried out on a low adhesion coating culture vessel. The method described.

 (13) The method according to the above item, wherein the undifferentiated sputum cell is an embryonic stem (ES) cell.

 (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 ngZml.

 (16) The method according to the above item, wherein the activin is less than lngZml to less than 50 ngZml

(17) The method according to the above item, wherein the activin is 1 ngZml to 25 ngZml.

(18) The method according to the above item, wherein the activin is 10 ngZml to 25 ngZml.

(19) The method according to the above item, wherein the retinoic acid is more than 0.01 μΜ and less than 1Μ.

 (20) The method according to the above item, wherein the retinoic acid is more than 0.01 μΜ and not more than 0.1 μΜ.

 (21) The method according to the above item, wherein the retinoic acid is 0.1. Or more and less than 1 μΜ.

(22) The method according to the above item, further comprising C) a differentiation step of culturing the differentiated aggregate in a medium on a 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 separating step is a KSR medium.

 (25) The method according to the above item, wherein the separating step is a period sufficient to produce a functional pancreas.

 (26) The method according to the above item, wherein the separating step is performed for 7 to 21 days.

 (27) The method according to the above item, wherein the separating step is performed for 10 to 14 days.

 (28) The above-described separation 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. The method described in 1.

(29) In the separation step, the cells are first cultured in a medium containing activin and retinoic acid for about 1 to 4 days, and then cultured in a medium not containing activin and retinoic acid for 1 to 3 weeks. The method according to the above item, comprising a step of nourishing.

 (30) In the separation step, the cells are first cultured in a medium containing activin and retinoic acid for about 2 days, and then cultured in a medium not containing activin and retinoic acid for about 10 days to about 2 weeks. The method according to the above item, comprising the step of:

 (31) The method according to the above item, wherein the concentration of activin and retinoic acid used is a condition selected from the group consisting of the above items 15-21.

 (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 duct structure ability. .

 (33) The functional pancreas described above is characterized in that it expresses the pancreatic marker Ipfl / pdx-Ι and does not express the marker Shh whose expression level decreases when the pancreas is formed. The method described.

 (34) The method according to the above item, wherein the functional soot /, the viscera expresses at least one pancreatic marker selected from the group consisting of insulin, glucagon and amylase 2 force.

(35) The method according to the above item, wherein the functional organ comprises oc 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 duct structure ability. .

 (37) A soot produced by the method described above.

 (38) A method for treating a patient in need of 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) Functional! / Use of activin and retinoic acid for the manufacture of a medicament for the treatment of a patient in need of a viscera.

 (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 by stem cell force.

 (45) The use according to the above item, wherein the medicament is produced from embryonic stem cells.

 [0018] Preferred embodiments of the present invention will be described below. However, those skilled in the art can appropriately implement the embodiments of the present invention and the well-known conventional technical capabilities in the field. It should be recognized that the effect is easily understood.

 [0019] Accordingly, 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. It is done.

 The invention's effect

 [0020] According to the present method, from the undeveloped mammalian sputum cells that have not been realized so far,

 1) Have β-cells that synthesize and secrete insulin in response to glucose load,

2) As an endocrine cell, it has an ex cell that synthesizes and secretes glucagon only in β cells,

 3) Has exocrine cells (acinar cells) that make digestive enzymes (such as amylase) and secrete them into the intestinal tract,

 4) Pancreatic power The world's first pancreas with all functions and structures, such as a conduit structure for exoculating digestive enzymes into the intestinal tract, has been made.

 [0021] Human ES cell power [beta] cells induced 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 is able to produce a complete viscera (functional, viscera) as shown by the present inventors, in which only β cells are present from human undifferentiated cells, Providing unprecedented useful materials to treat many diabetics and patients with visceral dysfunction, as well as patients who have had to remove the viscera for visceral cancer, etc. there's a possibility that. In addition, useful models and evaluation systems in biological and medical research related to pancreas, research on the elucidation of the cause of pancreatic diseases, and the development of drugs for the treatment and treatment of visceral diseases. It can also be expected to become (Atsusei). In this way, the practical application of this method and the industrial outlook are very bright.

Brief Description of Drawings FIG. 1 is a schematic diagram of pancreatic differentiation induction 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 unbroken cells. First, feeder cells are cultured in a gelatin-coated culture vessel in serum medium (fetal bovine serum, FBS medium), and then ES cells are cultured in the presence of LIF (leukemia inhibitory factor). The colony which is an aggregate, and the aggregate are made. Next, the agglomerates thus obtained were added to induction medium (10% serum replacement (knockout serum replacement) supplemented with activin and retinoic acid under the conditions of suspension culture using a low-contact coated culture plate. , KSR; Gi bco) for 2 days, and the pancreas can be made by culturing the aggregate in KSR medium in a gelatin-coated culture plate.

[Fig. 2] Fig. 2. A, colony aggregate (EBS) force of mouse ES cells Intestinal tract-like structure induced by differentiation between activin and retinoic acid, and a duct-like structure developed alongside it and a structure containing black spots (arrows) ). The gut-like structure in the center performs a slow peristaltic movement. This black spotted tissue is characteristic of secretory tissue. B, Colony aggregate (EBS) of mouse ES cells cultured in the same manner without conditions of activin and retinoic acid. Intestinal-like structures, duct-like structures, and tissues with black spots found in A are not observed. The scale bar is 100 mm.

[FIG. 2C] Phase-contrast micrographs of EBS including differentiated organs and gene expression after differentiation induction. (a, b) 0 .: Intestinal tubular structure (G) and visceral tubular structure (D) shown 13 days after the treatment of LM with retinoic acid and lOngZmlactivin. In the vicinity there are exocrine cells, which are observed as numerous black spots (b, arrowheads). (C) EBS treated with retinoic acid and activin and expression of pancreatic marker genes examined by RT-PCR in treated EBS. Expression of insulin II, gl ucagons, somatostatin and Ipfl / pdx-1 is enhanced on the second day after treatment! On the other hand, amylase and pancreatic polypeptide began to increase on the 6th and 8th days, respectively. Shh expression decreased after treatment [bar = 50 M (a and b)]. (D) The most efficient gene expression of all pancreatic markers in EBS is 0. retino Observed when inacid was used with lOngZmlactivin 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. 3] Fig. 3. A, Electron micrograph of a tissue (see Fig. 1) containing a black spot that is induced to differentiate with activin and retinoic acid in colony aggregates (EBS) of mouse ES cells. B, Electron microscopic image of exocrine cells of pancreas of rat fetus in late pregnancy (Bock, et al., 1997). Both cells contain a large number of granule-like structures with high electron density, and have very similar structures, such as having developed endoplasmic reticulum. ER, endoplasmic reticulum; N, nucleus.

 [Figure 3C] Figure 3. C, Electron microscopic image of a duct-like structure (see Figure 1) induced by differentiation of colony aggregates (EBS) of mouse ES cells with activin and retinoic acid. Natural! Very similar to the epidermis structure of the visceral duct. D, Electron micrograph of cells with secretory granule-like structure induced by differentiation of activin and retinoic acid from colony aggregates (EBS) of the same mouse ES cells (see Fig. 1). An enlarged image of a portion surrounded by a dotted line in the photograph is also shown. This image shows the black central part and the same as the endocrine granule image (Goping, et al., 2003; indicated by the arrow) in the β cells of the pancreas of the mouse body shown on the right (E). There is a gap between the bags that wrap the bag (indicated by arrows). Ν, nuclear.

 [Fig. 3Ε] EBS morphology including differentiated pancreas. 0 .: Section of EBS treated with L レ retinoic acid and 10 ng Zmlactivin became spherical tissue 11 days after treatment, and the area around the tubular cavity was strongly stained with toluidine blue (a, arrowhead), near the spherical tissue 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). The untreated EBS sections showed neither spherical nor tubular tissue structure (b). N is the nucleus and the scale bar is 50 m (ac) or 5 m (d).

[Fig. 4A] Fig. 4-1. Immunohistological observation image of colony aggregates (EBS) derived from mouse ES cells treated with lOngZmL activin and 0.1 M retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ′, 6-diamidino— 2-phenylindole; nuclear stain), D, A + B + C. The scale bar is 50 μΜ. Insulin C peptide and Amilla The case is made by another cell. Under these conditions, amylase is preferentially synthesized over insulin.

[FIG. 4B] FIG. 4—2. Immunohistological observation image of colony aggregate (EBS) derived from mouse ES cells treated with 25 ngZmL of activin and 0.1 M retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino-2-phenylindole; nuclear dye), D, A + B + C ₀ scale bar 50 M. Similar to Figure 4-1, the force that insulin C-peptide and amylase are made by different cells Under this condition, insulin is preferentially synthesized over amylase.

[4C] Structural analysis of EBS including isolated pancreas. In electron microscopy, cells containing large amounts of zymogen particles became apparent (a, b), which were highly developed endoplasmic reticulum and showed a dark homogeneous staining. This is a characteristic of acinar cells (a). Some cells were morphologically similar to pancreatic endocrine cells (c, d). However, they were less powerful 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 granule of pancreatic j8 cells, which was also observed in (d). (ER: endoplasmic reticulum, M: mitochondrial, N: nucleus, Z: zymogen granule). Scale bars are 5 m (a—c) and 1 m (d).

[Fig. 5] Immunohistochemical detection of marker protein. 0.: Immunostaining of EBS treated with LM retinoic acid and 1 On gZmlactivin revealed the presence of cells containing amylase positive zymogen condyles (c) and a few cells containing insulin C peptide positive granules (d) became. The pancreas of an 8 week old mouse was stained and used as a positive control (a, b). The cells were counterstained with DAPI (4, 6-diamidino-2-phenolindole) (ad). (ER: endoplasmic reticulum, M: mitochondria, N: nucleus, Z: zymogen condyle). The scale bar is 20 m.

[FIG. 6] Expression of soot /, visceral marker gene and shh in EBS. Total RNA was collected from EBS treated with retinoic acid (0, 0 .: M and: M) and activin (0, lOngZml, and 25ngZ ml), and the expression levels of the following markers were measured using real-time PCR Amylase 2 (a), insulin 11 (b), ptflaZp48 (c) and S hh (d) ₀ expression levels were normalized to those in untreated EBS. Shh expression was decreased in all combinations of treatment with retinoic acid alone, activin alone and retinoic acid and activin combination (c). Increased amounts of insulinll and amylase 2 showed no effect on insulin expression compared to untreated EBS with forceactin alone seen in EBS treated with retinoic acid alone. However, activin alone decreased amylase2 expression (a, b). When EBS is treated with a constant concentration of retinoic acid (0.1 μMia, b middle) and with a relatively low concentration of activin (lOngZml), the expression of amylase2 is expressed in retinoic acid and On the other hand, the expression of insulin II was unaltered in the two groups, while it was much higher in the group treated with both tibins than in the untreated group. In contrast, when retinoic acid was combined with a high concentration of activin (25 ngZml), 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 ptflaZp48 was strongly correlated with amylase2 mono. Murine expression profile obtained by RT-PCR of the visceral structure of mouse ES cell colony aggregate (EBS) induced by differentiation with activin and retinoic acid! Insulin 2, glucagon, amylase 2 and Ipfl Zpdx— 1 are marker genes that are specifically expressed in pancreas development (positive markers), while Shh is a gene that decreases in expression with pancreas development (negative). Marker ~~). GAPDHAgiyceraidehyde 3-phosphoate dehydrogenase; is a constitutive protein (enzyme) expressed at a high level in all cells, so it was used for the purpose of assuring that the performed assay was appropriate.

[Fig. 7] Effect of stimulus concentration on the appearance of exocrine and endocrine cells. 8-week-old mice, viscera (a), treated! /, Na! /, EBS (b) and 0.1 μΜ retinoic acid and 10 ηg / ml activin (c) or 25 ng Zml activin ( EBS treated in d and e) was stained with anti-insulin C peptide antibody, anti-amylase antibody and DAPI 13 days after treatment (19 days after starting EBS formation). Neither insulin-positive nor amylase-positive cells were observed in untreated BSs (b). In EBS treated with 0.1 M retinoic acid and lOngZmlactivin, insulin-positive cells were less potent than amylase-positive cells (c). In contrast, EBS treated with 0.1 μΜ retinoic acid and 25 ng Zmlactivin In (d), there was a marked increase in insulin-producing cells. Furthermore, 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 (&-(1) Chobi 20 111 ^).

Description of sequence listing

SEQ ID NO: 1 is the nucleic acid sequence of human inhibin 13A accession number NM002192. In the present invention, it can be used as activin.

SEQ ID NO: 2 is the amino acid sequence of accession number NM002192 of human inhibin 13A. It can be used as an activin in the present invention.

SEQ ID NO: 3 is the nucleic acid sequence of human inhibin j8 B accession number NM002193. It can be used as activin in the present invention.

SEQ ID NO: 4 is the amino acid sequence of human inhibin j8 B accession number NM002193. It can be used as an activin in the present invention.

SEQ ID NO: 5 is the nucleic acid sequence of human inhibin j8 C accession number NM005538. In the present invention, it can be used as activin.

SEQ ID NO: 6 is the amino acid sequence of human inhibin j8 C accession number NM005538. It can be used as an activin in the present invention.

SEQ ID NO: 7 is the nucleic acid sequence of human inhibin at accession number NM002191. It can be used as an activin in the present invention.

SEQ ID NO: 8 is the amino acid sequence of human inhibin at accession number NM002191. In the present invention, it can be used as activin.

SEQ ID NO: 9 is the nucleic acid sequence of Accession Number: X68250 (African Megalactivin A). It can be used as an activin in the present invention.

SEQ ID NO: 10 is the amino acid sequence of Accession Number: X68250 (African Megalactivin A). It can be used as an activin in the present invention.

SEQ ID NO: 11 is the inhibin j8 B for Xenopus laevis (accession number: S61773

) Nucleic acid sequence. In the present invention, it can be used as activin.

SEQ ID NO: 12 is the inhibin j8 B for Xenopus laevis (accession number: S61773

) Amino acid sequence. It can be used as an 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 Ipfl / pdx-1 forward primer.

SEQ ID NO: 24 is an Ipfl / 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 foam primer.

SEQ ID NO: 28 is a pancreatic polypeptide glucagon reparse 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 Ipfl / pdx-1 forward RT-PCR primer.

 SEQ ID NO: 40 is an Ipfl / 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.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0024] Hereinafter, the present invention will be described. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. Therefore, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc. in English) also include the plural concept unless otherwise stated. It is. 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.

 [0025] (Definition)

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

[0026] In this specification, "pluripotency" or "probably pluripotency" is used interchangeably and refers to the nature of a cell, and is divided into one or more, preferably two or more various tissues or organs. The ability to be jealous. Therefore, “pluripotency” and “multipotency” are used interchangeably with “undifferentiated” in this specification unless otherwise specified. Normally, the pluripotency of a cell is limited as it develops, and in adults, the cells of one tissue or organ rarely change to another. 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 tend to undergo metaplasia instead of other mesenchymal cells with a relatively simple stimulus. Embryonic stem cells are pluripotent. Tissue stem cells are pluripotent. In this specification, of pluripotency, live as a fertilized egg. The ability to differentiate into all types of cells that make up the body is totipotency, and pluripotency can encompass the concept of totipotency. Whether a cell has pluripotency includes, but is not limited to, formation of an embryoid body in an in vitro culture system, culture under differentiation-inducing conditions, and the like. In addition, assessing 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, Examples include, but are not limited to, transplantation and proliferation by injection into ascites.

 In the present specification, the “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 cells” have the ability to self-replicate and are pluripotent (ie pluripotent).

("Pluripot _e ncy"). Stem cells are usually able to regenerate the tissue when it is damaged. As used herein, stem cells can be, but are not limited to, embryonic stem (ES) cells or tissue stem cells (both tissue stem cells, tissue-specific stem cells or somatic stem cells). Also, as long as it has the above-mentioned ability, an artificially produced cell (for example, a fusion cell, a reprogrammed cell, etc. described herein) can also be a stem cell. Embryonic stem cells are 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 that have a limited direction of differentiation. They are located at specific locations in the tissue and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a poor nuclear organelle with a high nuclear Z cytoplasm ratio. Tissue stem cells generally have pluripotency and maintain proliferative capacity over the life of an individual with a slow cell cycle. As used herein, stem cells are preferably embryonic stem cells, although tissue stem cells can also be used depending on the situation.

[0029] As used herein, "feeder layer" or "feeder cell" is used interchangeably and is a cell that is provided in a culture substrate and cannot be maintained alone. To allow species growth and Z or phenotypic expression It refers to a feeder cell layer of another cell type. Tissue cells are said to have many cell types that cannot be propagated under normal cell culture conditions and cannot be propagated. Examples of such cells include certain types of stem cells (especially epithelial cells). Stem cells, embryonic stem cells, hematopoietic stem cells, etc.), cornea, epidermis and the like. These cell types are generally considered to require auxotrophic and specific growth factors and differentiation inducing factors. Even in such cell types, by utilizing a layer of specific cells formed by supporting cells of the cell type in vivo or cells similar thereto as a culture substrate, factors required by the cell type to be cultured and Z or It is said that it will proliferate and differentiate by supplying nutrients. The cell type used as a feeder cell is selected depending on the cell type to be used, but is often used by inhibiting cell growth by methods such as antibiotic administration (eg, mitomycin c) and uv irradiation. The fact that germ cells, early embryonic cells, and hematopoietic stem cells can be cultured is said to depend largely on the use of feeders. As feeder cells, in addition to fibroblasts, adipose-derived stem cells, embryonic stem cells, bone marrow stem cells, etc. can be used. It is preferable to use it after culturing according to the culture conditions to differentiate into fibroblasts!

 [0030] As used herein, "primary cultured cells" refers to cells that have been in a state of culture until cells, tissues, organs, etc. separated from the body have been implanted and the first passage has been performed.

[0031] As the cells of the present invention, any culture solution can be used 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), ascorbeto 2-phosphate, ascorbic acid and its derivatives, glyceport phosphate, Estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, aFG F, bFGF, EGF, IGF, TGF β, ECGF, BMP, PDGF and other growth factors, pituitary extract, pineal extract, retinoic acid, Vitamin D, thyroid hormone, urine fetal serum, Demethylation of serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (such as sodium selenite), linolenic acid, 3-isobutyl 1-methylxanthine, 5-azancytidine Agent, histone deacetylating agent such as trichostatin, activin, cytodynamic force such as LIF'IL-2'IL-6, hexamethylenebisacetamide (HMBA), dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMSO), ododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside (AraC), mitomycin C (MMC), sodium butyrate (NaBu ), Polyprene, selenium, cholera toxin and the like may be included as one or a combination thereof.

 [0032] As used herein, "differentiation" or "cell differentiation" has morphological and Z or functional qualitative differences among daughter cell populations resulting from the division of one cell. A phenomenon in which two or more types of cells occur. Therefore, the process by which a cell population (cell lineage) derived from cells that cannot originally detect special characteristics shows distinct characteristics such as production of specific proteins 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. By searching for intracellular or extracellular factors or conditions that cause such a gene expression state, cell differentiation is considered.匕 can be identified. The results of cell sorting are in principle stable, especially in animal cells, sorting into other types of cells can only occur exceptionally. In the present specification, the division to “pancreas” is based on the expression or disappearance of a specific marker, or the function (eg, determination of expression or secretion of insulin, glucagon, or digestive enzyme (such as amylase), specific The pancreatic marker that can be used in the present specification includes, for example, pancreatic markers ~~ 7 ~~ Ipfl / pdx— 1 , Msulm, glucagon ^ amylase 2, Isl——1, Pax——4, Pax—6, Glut—2, CK—19, etc. Another way to check for pancreas is A means for confirming a decrease in the marker Sh h whose expression level decreases at the time when the pancreas is formed can be mentioned.

[0033] In the present specification, among pluripotency, the ability to divide into all types of cells constituting a living body like fertilized eggs is called "totipotency", and pluripotency is the concept of totipotency. Can be included. However However, when distinguishing clearly, totipotency and pluripotency can be distinguished, the former refers to the ability to divide into any cell, the latter has multiple directions, but the organism is It has the ability to not be divided in all possible directions. The ability to divide only in one direction is also called monopotency.

 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 reaches the distal end of the cylinder across the ectoderm. The original stripe is an area where cell movement occurs, and as a result, the future endoderm and mesoderm are formed. By E7.5, a head process will appear in front of the nodule, and a notochord will be formed in this area, the future endoderm surrounding it, and a neural plate in the upper layer. The nodules appear around E6.5 and move backwards, forming the shaft structure from front to back. E8. By the 5th day, the embryo is somewhat longer, and a large head fold is formed at the front end, mostly with anterior neural plate strength. The body segment is E8 daily force 1. The forward force begins to form backward at a rate of 1 per 5 hours. Cells that have passed this period will no longer exhibit totipotency and will not form individuals, even if they are returned to the placenta, unless they are departed. Before this point, 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 for ES cells to establish later embryonic power, and the embryonic power after this is usually established as cells called EG (germ cell-derived) cells. It can be said that it is a turning point.

In this specification, the “pancreas” or “spleen” is a large glandular organ next to the liver associated with the digestive tract in vertebrates. Located close to the liver, the shape and the manner of opening to the intestinal tract vary depending on the type of animal.Exocrine part that secretes spleen as digestive juice, and endocrine part that secretes insulin and glucagon involved in the adjustment of blood glucose level The splenic duct (ductus pancreaticus) that opens to the duodenum has a common opening that fuses with the bile duct that begins in the liver. Developmentally, usually two to three endodermal processes from the beginning of the intestine are the primordial group, and many As the development of these animals progresses, they combine to form a pancreas, but in many teleost's, the pancreas disperses as a small tissue mass in one organ. The Langernos island tissue is often separated from the spleen secretory tissue, which is called the Blockman body, and the primordial base forms the spleen. In many mammals, two remain, and the ventral one forms the main splenic duct (Wir sung—s duct) and the dorsal one forms the accessory splenic duct (Suntorini's duct). Adult, each open separately into the duodenum.

 [0036] As used herein, "functional pancreas" or "natural pancreas" is at least selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability, and functional conduit structure ability It refers to a pancreas having one characteristic and preferably having all of it. Therefore, preferably, the functional pancreas is 1) endocrine cells that secrete insulin in response to blood glucose levels (ie, blood glucose concentration) (β cells), and 2) endocrine cells that make and secrete glucagon. (Alpha one cell, a cell: endocrine cells that specifically make and secrete glucagon, a hormone responsible for glycogen degradation and gluconeogenesis), and 3) digestive enzymes (eg amylase) It has exocrine cells (acinar cells) that are secreted into the intestinal tract, and 4) has all functions and structures such as a duct structure for exoculating digestive enzymes from the pancreas into the intestinal tract. means.

 [0037] The cells used in the present invention may be any cell derived from any organism (eg, any type of multicellular organism (eg, animal (eg, vertebrate, invertebrate), plant (eg, monocotyledonous plant, It may be a dicotyledonous plant)))). Preferably, cells derived from vertebrates (for example, metaraunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) are used, and more preferably mammals (eg, single pores). , Marsupials, rodents, wings, wings, carnivores, carnivores, long noses, odd-hoofed animals, even-hoofed animals, rodents, scales, marine cattle, cetaceans, Cells derived from primates, rodents, maggots, etc.) are used. More preferably, mammals (eg, rodents (mouse, rat, etc.), primates (monkey, human, etc.)) are used.

[0038] As used herein, "tissue" refers to a cell population having substantially the same function and Z or morphology in a multicellular organism. Usually "tissue" has the same origin Forces Cell populations with different origins can be called tissues if they have the same function and Z- or morphology. Therefore, when a tissue is regenerated using the stem cells of the present invention, cell populations having two or more different origins can constitute one tissue. Usually, the tissue forms part of an organ. Animal tissues are classified into epithelial tissue, connective tissue, muscle tissue, nerve tissue, etc. based on morphological, functional or developmental basis.

As used herein, “activin” refers to a protein hormone that also secretes force such as granulosa cells of the ovary and promotes secretion of follicle stimulating hormone (FSH) in the anterior pituitary gland. Typical activins include, for example, activin A, activin B, activin C, and inhibin. These are well conserved among animals, and humans act on African frogs and vice versa. Is expected. SEQ ID NO: 1 and SEQ ID NO: 9 (accession number: NM002192 (SEQ ID NO: human inhibin 13 A) and accession number: X68250 (African megalactivin A) = nucleic acid sequence) and SEQ ID NO: 2 and sequence Corresponding to factors having the sequence as shown in No. 10 (SEQ ID NO: human inhibin β お よ び and accession No. X68250 (African megalactivin A) = amino acid sequence, respectively) and other species of animals. Examples include but are not limited to factors (orthologs). In addition, Xenopus laevis has registered inhibin j8B (accession number: S61773) (SEQ ID NOs: 11 and 12). A molecular weight of 25000. A dimer in which the subunits of the inhibin chain are S—S bonded, and three types are known: activin Α (βΑβΑ), activin ΑΒ (βΑβΒ), and activin Β (βΒβΒ)! / Α is also called the erythroblast differentiation factor (EDF). The inhibin j8 chain has about 40% homology with the transforming growth factor TGF-β, and the position of cysteine residues in the primary structure. Is well conserved, so it may be placed in the TGF-j8 family. Activin is a protein hormone that is secreted by ovarian granulosa cells and promotes the secretion of follicle-stimulating hormone (FSH) in the anterior pituitary gland. Isolated from follicular fluid in the process of purifying inhibin (1986). Molecular weight 25000. Inhibin chain subunits are S—S-linked dimers, activin Α (βΑβ), activin ΑΒ (| 8Α | 8Β), akutibin (| 88 | 88) are known. Akutibin Α is also called ervthroblast differentiation factor (EDF). Since 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 is placed in TGF-j8 family. The expression of the activin gene in various vertebrate organs has been reported, which promotes the synthesis of FSH receptor in ovarian granulosa cells, suppresses the proliferation of erythroid progenitor cells in friend cells and bone marrow. It has many physiological activities such as induction of hemoglobin synthesis, promotion of insulin secretion from the viscera, and induction of mesoderm in amphibian embryos. For activin, reference can be made to Nakamura et al., Is oiation ana characterization of native activm B. J Biol. Chem. 1992, 267, 16385— 9; Uchiyama and Asashima, Specific erythroid differing en tiation of mouse erythroleukemia cells by activins and its enhance ment by retinoic acids.Biochem Biophys.Res. Commun. 1992, 187, 347—52; Fukui et al. 131— 9; Fukui et al., Identification of activins A, AB, and B and follistatin proteins in Xen opus embryos. Dev. Biol. 1994, 163, 279— 81; Nakano et al., Compar ison of mesoderm— inducing activity with monomeric and dimeric i nhiom alpha and beta—A subunits on Xenopus ectoderm. Horm. Re s. 1995, 44, Suppl. 2, 15—22.

In addition to mammals including humans, rats, mice, Xenopus laevis, etc., the homologues of activin are also known in Drosophila. Therefore, in the present specification, activin generally refers to activin that exists not only in mammals but also in living organisms in general. Activin 1 A is a dimer of inhibin 13 A (human inhibin 13 A accession number NM002192; SEQ ID NOs: 1 and 2 (nucleic acids and amino acids)). Activin AB is a dimer of inhibin 13 A and inhibin 13 B (human inhibin 13 B accession number NM002193; SEQ ID NOs: 3 and 4 (nucleic acids and amino acids)). Activin-B is a dimer of inhibin β Β. Activin C is a dimer of inhibin 13 C (human inhibin j8 C accession number ΝΜ005538; SEQ ID NOs: 5 and 6 (nucleic acids and amino acids)). Inhibin is a dimer of inhibin a (accession number NM0 for human inhibin a) 02191; SEQ ID NOs: 7 and 8 (nucleic acids and amino acids)).

 As such an activin gene, for example,

 (A) (a) a polynucleotide having the base sequence described 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) In the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, one or more amino acids have at least one mutation selected from the group consisting of substitution, addition, and deletion ability. A polynucleotide that 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 the polypeptide having the amino acid sequence ability as set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12;

 (f) any force of (a) to (e), a polynucleotide that hybridizes to one polynucleotide under stringent conditions and encodes a polypeptide having biological activity; or

 (g) Any one of (a) to (e) Encoding a polypeptide having a base sequence having at least 70% identity to one polynucleotide or its complementary sequence and having biological activity Polynucleotides,

including,

A nucleic acid molecule; or

 (B) (a) a polypeptide comprising the amino acid sequence set forth in 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, at least one amino acid has at least one mutation selected from the group consisting of substitution, addition, and deletion ability And a polypeptide having biological activity;

(c) a splice variant of the base sequence described in SEQ ID NO: 1, 3, 5, 7, 9, or 11 or A polypeptide encoded by an allelic variant;

 (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) a polypeptide having an amino acid sequence with at least 70% identity to any one of the polypeptides of (a) to (d) and having biological activity,

 including,

 Examples include, but are not limited to, a nucleic acid molecule encoding a polypeptide, or a polypeptide encoded by it.

 [0042] In this specification, "retinoic acid" is an important metabolite of vitamin A (retinol), which has no role in vision but is used for normal growth of animals, epithelial tissue and cartilage differentiation. It is essential. It has a morphogen-like action during vertebrate development. It exerts its effects by regulating gene expression through the retinoic acid receptor. The all-trans form is the most important isomer, but there are also 9-cis and 13-cis forms.

 [0043] [Chemical 1]

[0044] The retinoic acid receptor (RAR) is a receptor present in the nucleus necessary for retinoic acid, which is considered to be an active form of vitamin A, to exert its action. RAR — α, — j8 and mono-γ, 9-cis monoretinoic acid to which all-trans retinoic acid binds RXR— α,-β and γ are known. It is a ligand-dependent nuclear receptor similar in structure to a steroid receptor, and after binding to the ligand retinoic acid, it binds to a specific position of the gene and regulates the transcription of that gene. Involved in differentiation.

As used herein, “leukemia inhibitory factor (LIF)” refers to SEQ ID NO: 13 or 15 (nucleic acid sequence). Columns) and 14 or 16 (amino acid sequence) and the corresponding factors (orthologs) in other animals.

 [0046] LIF was purified and isolated as a factor that induces differentiation of the monocyte cell line Ml into mature macrophages (Gearing DP, Gough NM, King JA, EMBO J. 6: 3995-4002 (1987); Nough and Gough NM, Gearing DP, King JA, Proc. Natl. Acad. Sci. USA 85: 2623— 2627 (1988. Named as a leukemia inhibitory factor because of Ml cell proliferation and clonogenicity. In addition to the above reports, various group forces were discovered from various viewpoints, and differentiation-inducing factors (factor D or DIF) (Tomida et al, J Biol Chem; 259 (17): 10978-82 (1984) ; Yama moto et al, Cancer Res; 40: 4804—9 (1980)); Macrophage Z granulocyte-inducing factor type 2 (MGI—2); MN inhibitory activity (DIA); MN delay factor (DRF) Human interleukin (HILDA) against DA-la cells; growth-stimulating activity against TS-1 cells (GATS); hepatocyte-stimulating factor type I and type IV (HSF-II and H SF—III); cholinergic neuronal differentiation factor (CDF); melanocyte-derived lipoprotein lipase inhibitor (MLPLI); osteoclast activation factor (OAF) (Lass A, Weiser W, Munafo A, Loumaye E Fertil Steril; 76: 1091-6 (2001); see Knight D. Pulm Pharmacol Ther; 14: 169-76 (2001)).

[0047] LIF is found in humans and mice, and in the case of human and mouse types, is a 180-residue glycoprotein of monomers with disulfide bonds and a molecular weight of about 20 kDa. LI F is naturally glycosylated 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, Gough NM and Metcalf D. (1988) Anal. Biochem. 173: 359-367), the presence or absence of glycosyl residues depends on the activity of LIF Not relevant (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 site force-in. This site force in has a long helix, a long head A—B and CD loop, and an additional fifth helix A / rape (Purvis DH and Mabbutt BC (1997) Biochemistr y 36 (33): 10146—10154).

 [0048] Polypeptides used in the present invention also include "LIF-like factors" having 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 gpl30 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 with the LIF assay. As such a LIF assembly, for example, a mechanism for measuring the downstream signal (JAK-STAT transmission system!) Or the activity of any one molecule (eg phosphorylation) can be used. Examples include the complex of gpl30 and LIF receptor, or the use of tyrosine phosphorylation of STAT-3.

 [0049] Preferably, it binds to the LIF force receptor and induces intracellular signal transduction via gp130. Therefore, in the present invention, gpl30 ligand or LIF-like factor is used to exert an effect by the same mechanism. Can do.

 [0050] 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 has a high affinity receptor with a Kd value of 20-100 pM and a high affinity receptor with a Kd value of 1-2 ηΜ (Allan EH, Hilton DJ, Brown Μ. AJ Cell Physiol. 1 45: 110-119 (1990); Godard A., Heymann D., Raher SJ Biol. Chem. 267: 3214-3222 (1992); Hendry LA Murphy M., Hilton DJ, Nicola NA and Bartlett P. FJ. Neurosci. 12: 3427— 3434 (1992); Hil ton DJ, Nicola NA and Metcalf D. Ciba Found. Symp. 167: 227-239 (1992); Rodan SB, Wesolowski G., Hilton DJ, Nicola N. A and Rodan GA Endocrinology 127: 1602-1608 (1990); Williams

RL, Hilton DJ, Pease S. Nature 336: 684—687 (1988); and Ya mamoto—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 with gp130 and exhibits high affinity for LIF. A soluble LIF receptor has been discovered in mice, and its role has attracted attention. Yes. It has been shown that when a soluble LIF receptor-LIF receptor complex is formed, the complex can react with gpl30 and stimulate signal transduction (Heyma nn D., Goddard A., Raher S (1996) Cytokine 8 (3): 197—205). Therefore, the effects of the present invention can also be achieved by using a signal transduction mechanism using such a receptor conversion model. The LIF receptor and gpl30 are also known as components of other site force-in receptors. Examples of such site force-in include IL-6 superfamily members such as IL-6, IL 11, Examples include, but are not limited to, CNTF and OSM.

 [0051] As used herein, "ligand" refers to a substance that specifically binds to a certain protein. Examples of ligands include lectins, antigens, antibodies, hormones, and neurotransmitters that specifically bind to various receptor protein molecules present on cell membranes. If activin or retinoic acid is the ligand, ligands with similar (equivalent) action are intended to be within the scope of the present invention.

 [0052] Regarding the polypeptides such as activin and LIF used in the present invention, the moieties to which a sugar chain can be added include N-acetylyl-D-darcosamine-binding moieties, and N-darcoside-bondable moieties. Examples include moieties that form O-glycosides of acetyl-D-galactosamine (parts where serine or threonine residues frequently appear). The activin used in this specification is not particularly affected by the presence or absence of a sugar chain, but the protein to which these sugar chains are added is usually stable against degradation in vivo. Yes, it can have strong physiological activity. Accordingly, polypeptides to which these sugar chains are added are also within the scope of the present invention.

[0053] As used herein, the terms "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein and can be any length of amino acid. The polymer. This polymer may be linear or branched or cyclic. The amino acid may be a modified amino acid, whether natural or unnatural. The term can also include those assembled into a complex of multiple polypeptide chains. The term also encompasses amino acid polymers that have been modified naturally or artificially. Such modifications include, for example, disulfide bond formation, Glycosylation, lipidation, acetylation, phosphate 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 those known in the art! Other modifications are included. 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.

 [0054] As used herein, the terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably herein and refer to nucleotide polymers of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”.

[0055] As used herein, the term "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or has an unusual linkage between nucleotides, Used interchangeably. Specific examples of such oligonucleotides include, for example, 2,1-O-methyl-ribonucleotides, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide is converted to a phosphoroate bond, phosphorous in an oligonucleotide. Oligonucleotide derivative in which acid diester bond is converted to N3, P5, phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, in oligonucleotide Oligonucleotide derivatives in which uracil is replaced with C-5-propyluracil, oligonucleotide derivatives in which uracil in oligonucleotide is replaced with C 5 thiazoleuracil, and cytosine in oligonucleotide is replaced with C 5 propylcytosine Is Oligonucleotide derivatives, oligonucleotide derivatives in which the cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the DNA is replaced with 2, 1 O-propyl ribose, and in the oligonucleotide An example is an oligonucleotide derivative in which the ribose is substituted with 2, -methoxyethoxyribose. Unless otherwise indicated, a particular nucleic acid sequence may also be expressed in the same manner as an explicitly indicated sequence. Of conservatively modified variants (eg, degenerate codon substitutes) and complementary sequences are 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 a Z or deoxyinosin residue. (Batzer et al., Nucleic Acids Res. 19: 5081 (1991); Ohtsuka et al., Biol. Chem. 260: 2605-2608 (1 985); Rossolini et al., Mol. Cell. Probes 8: 91—98 (1994)). The gene of the present invention usually takes this polynucleotide form.

[0056] 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 acid and nucleic acid molecules may be included in the term “gene” concept. Nucleic acid molecules that encode a gene sequence also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid includes 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 a different (another) nucleic acid splice product encodes a different polypeptide. The mechanism of splice variant production involves alternative splicing of altering force exons. 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.

[0057] As used herein, "gene" refers to a factor that defines a genetic trait. Usually arranged in a certain order on a chromosome. Those that define the primary structure of a protein are called structural genes, and those that affect their 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 Z or translational regulatory sequence such as its promoter. In the present invention, in addition to structural genes, transcription and Z or translation, etc. It is understood that regulatory sequences are also useful for nerve regeneration, diagnosis, treatment, prevention, prognosis, etc. of neurological diseases. As used herein, “gene” refers to “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” and Z or “protein”, “polypeptide”, “oligopeptide” and “peptide”. May point. As used herein, a “gene product” also refers to a “polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule” expressed by a gene and Z or “protein”, “polypeptide”, “ Includes "oligopeptide" 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 respect to each other. In the present specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of the same sequence (individual nucleic acids, amino acids, etc.) of two or more comparable sequences. 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% identical. If%, 90%, 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In this specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when conservative substitutions are considered positive (identical) in the above homology. , Refers to the degree of identity to 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.

[0059] In the present specification, comparison of similarity, identity and homology between amino acid sequences and nucleotide sequences is performed by searching for identity, for example, NCBI BLAST 2.2.9 (issued on May 12, 2004) Can be used. The identity value in this specification is a value obtained by aligning under the default conditions usually using the above BLAST. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. Identity in multiple areas In the case of evaluation, the highest value among them shall be 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. An “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.

 [0061] The term "natural amino acid" refers to the L isomer of a natural amino acid. Natural amino acids are glycine, alanine, norine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, orthine, and lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms. Forms using D-form amino acids are also within the scope of the present invention.

 [0062] As used herein, the term "unnatural amino acid" means an amino acid that is not normally found in proteins. Examples of non-natural amino acids are norleucine, para-trophellalanine, homophenylalanine, para-fluoroferranalanin, 3-amino- 2-benzylbenzylpropionic acid, homoarginine D-form or L-form and D-phenol Lanin is mentioned.

 [0063] As used herein, "amino acid analog" refers to a molecule that is not an amino acid but is similar to the physical properties and function 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 an activin in the present specification, those containing natural amino acids, those containing such amino acid analogs or amino acid derivatives can be used.

[0064] As used herein, "nucleotide" may be natural or non-natural. “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. Such induction Examples of body nucleotides and nucleotide analogs include, but are not limited to, phosphoroates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-0-methyl ribonucleotides, peptide nucleic acids (PNA).

[0065] Amino acids may be referred to herein by either their commonly known three letter symbol power or by the one letter symbol recommended by the IUPAC — IUB Biochemica 1 Nomenclature Commission. Nucleotides can also be referred to by their generally recognized single letter codes.

 [0066] As used herein, a "corresponding" amino acid or nucleic acid has the same action as a given amino acid or nucleotide in a polypeptide or polynucleotide used as a reference for comparison in a certain polypeptide molecule or polynucleotide molecule. Amino acids or nucleotides that have or are predicted to have, especially in the case of enzyme molecules, amino acids that are present at the same position in the active site and contribute to the catalytic activity!ヽ ぅ. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Therefore, in the present 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. Accordingly, such cases are referred to herein as “corresponding” regions or domains.

[0067] As used herein, a "corresponding" gene (eg, a polypeptide molecule or a polynucleotide molecule) refers to an action similar to that of a given gene in a species as a reference for comparison. A gene that has or is predicted to have (for example, a polypeptide molecule or a polynucleotide molecule), and when there are multiple genes that have such an action, those that have the same evolutionary origin Say. Thus, the gene corresponding to a gene can be an ortholog of that gene. Therefore, genes corresponding to genes such as Xenopus laevis can also be found in other animals (human, rat, pig, ushi, etc.). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal is the reference gene for the corresponding gene (for example, It can be found by searching a sequence database of the animal (eg, human, rat) using the sequence of a gene such as tibin as a query sequence.

 [0068] As used herein, "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). Say. The length of the fragment can be changed as appropriate according to its purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2, 0, 25, 30, 40, 50 and more, and lengths expressed in integers not specifically listed here (for example, 11 etc.) are also suitable as lower limits. It can be. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. !!, NA! /, An integer length (eg 11) 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, but the above numbers are not absolute, as long as they have the same function. The above numbers as upper or lower limits are intended to include those above and below that number (or 10% above and below for example). 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. The length of a fragment useful in the present specification is retained by at least one of the functions of the full-length protein that serves as a reference for the fragment (eg, differentiation-regulating action, specific interaction with other molecules). Can be determined by whether or not.

[0069] As used herein, a first substance or factor "specifically interacts" with a second substance or factor means that the first substance or factor is relative to the second substance or factor, To interact with a higher affinity than a substance or factor other than a second substance or factor (especially another substance or factor present in the sample containing the second substance or factor). Specific interactions for a substance or factor include both nucleic acids and proteins, for example, hybridization in nucleic acids, antigen-antibody reactions in proteins, ligand-receptor reactions, enzyme-substrate reactions, etc. Protein-lipid interactions, nucleic acids—such as reactions between transcription factors and their binding sites Examples include, but are not limited to, lipid interactions. Thus, when both a substance or factor is a nucleic acid, the first substance or factor can “specifically interact” with the second substance or factor if the first substance or factor is the second substance. Alternatively, it includes at least a part of the complementarity to the factor. Also, 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, an antigen-antibody reaction interaction, Examples include, but are not limited to, interaction by septa-one ligand reaction, enzyme-substrate interaction, and the like. When two substances or factors include proteins and nucleic acids, the first substance or factor “interacts specifically” with the second substance or factor so that the transcription factor and the transcription factor Interactions with the binding region of the nucleic acid molecule of interest are included.

 Therefore, in the present specification, “an agent that specifically interacts” with a biological agent such as a polynucleotide or a polypeptide means an affinity for a biological agent such as that polynucleotide or its polypeptide. Gender is typically the same or higher than, or preferably significantly more significant than, for example, an affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. Includes those that are scientifically significant). Such affinity can be measured by, for example, hybridization assay, binding assay, or the like.

[0071] As used herein, "agent" refers to any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) as long as the intended purpose can be achieved. There may be. Examples of such substances include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, and RNA such as mRNA), Polysaccharides, oligosaccharides, lipids, small organic molecules (e.g. hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g. small molecule ligands), etc. ), And these complex molecules, but are not limited thereto. A factor specific for a polynucleotide typically has a certain sequence homology to the polynucleotide sequence (e.g., 70% or more sequence identity). Examples thereof include, but are not limited to, a polynucleotide having a complementarity and a polypeptide such as a transcription factor that binds to a 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), a polypeptide thereof. Examples include, but are not limited to, a specific ligand or receptor when the peptide is a receptor or a ligand, and a substrate when the polypeptide is an enzyme. As used herein, activin can be used for stem cell sorting, 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 common general knowledge in the art based on the disclosure herein, and are understood to be within the scope of well-known 'conventional techniques. .

 [0072] As used herein, "compound" means any distinguishable chemical or molecule, including small molecules, peptides, proteins, sugars, nucleotides, or nucleic acids. Without being limited thereto, and 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 small organic molecule has a molecular weight of about 1000 or less, but may have a higher molecular weight. Small organic molecules can be synthesized by using methods known in the art or by combining them. Such small organic molecules may be produced by living organisms. Examples of small organic molecules include hormones, ligands, signal transmitters, organic small molecules, molecules synthesized by combinatorial chemistry, and small molecules that can be used as pharmaceuticals (for example, small molecule ligands). Not limited to.

[0074] As used herein, "contacting" refers to a compound, either directly or indirectly, such as an activin of the present invention (eg, a polypeptide or a polynucleotide). It means close physical proximity. The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact refers to placing a compound in a beaker, microtiter plate, cell culture flask or microarray (e.g., a gene chip) containing a nucleic acid molecule or a polypeptide encoding the fragment. Is mentioned. Typically, contact can be achieved by placing a contact target (eg, a cell) in a solution (eg, medium) containing a factor (eg, activin) to be contacted.

 As used herein, the term “complex molecule” refers to a molecule formed by linking a plurality of types 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. As used herein, activin can also be used in the form of such complex molecules.

 [0076] As used herein, an "isolated" biological agent (eg, a nucleic acid or protein, etc.) refers to other biological cells within the cells of the organism in which the biological agent is naturally present. Factor (e.g., if it is a nucleic acid, a nucleic acid containing a non-nucleic acid and a nucleic acid sequence other than the target nucleic acid; if it is a protein, it contains an amino acid sequence other than the non-protein and the target protein) A substance substantially separated or purified from a protein or the like. “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.

 [0077] As used herein, a "purified" biological agent (eg, nucleic acid or protein) is one in which at least some of the factors that naturally accompany the biological agent have been removed. Say. Thus, the purity of a biological agent in a purified biological agent is usually higher (ie, enriched) than the state in which the biological agent is normally present.

 [0078] The terms "purified" and "isolated" as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, and even more preferably at least 95% by weight. % And most preferably at least 98% by weight of the same type of biological agent is present.

[0079] As used herein, "biological activity" refers to an activity that a certain factor (eg, polynucleotide, protein, etc.) can have in vivo, and has various functions (eg, transcription). Activity that exhibits (promoting activity) is included. For example, if two factors interact (eg, activin and its specific factor bind), its biological activity is Binding between two molecules and the resulting biological change, for example, when one molecule is precipitated with an antibody, when another molecule co-precipitates, the two molecules are considered bound It is done. Therefore, seeing such coprecipitation is one method of judgment.

 [0080] Thus, “activity” is a force that indicates or reveals binding (either direct or indirect); a measurement that affects the response (ie, responds to some exposure or stimulus). Refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide used in the present invention, or, for example, some post-stimulation or event Subsequent upstream or downstream protein quantities or other similar measures of function may be mentioned. Such activity can be measured by assays such as competitive inhibition of the binding of specific factors to activin.

 [0081] In this specification, "interaction" refers to two substances, and forces (for example, intermolecular force (van der Waals force), hydrogen bonds, and the like between one substance and the other substance. Hydrophobic interaction). Usually, two interacting substances are in an associated or bound state.

 [0082] As used herein, the term “binding” refers to a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Means. 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 due to 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 is not accompanied by other substantial chemical intermediates.

 [0083] As used herein, the terms "modulate" or "modify" mean an increase or decrease or maintenance in the amount, quality or effect of a particular activity or protein. To do.

As used herein, “polynucleotide that hybridizes under stringent conditions” refers to a polynucleotide obtained under well-known conditions commonly used in the art. The present invention Using the selected polynucleotide as a probe, the hybridization method, plaque hybridization method, Southern blot hybridization method, etc. are used. Thus, such a polynucleotide can be obtained. Specifically, using a filter immobilized with colony or plaque-derived DNA, hybridization was performed at 65 ° C in the presence of 0.7 to 1.OM of NaCl, and then 0.1 to 2 times the concentration. SSC (saline-sodium citrate) solution (1x concentrated SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) and can be identified by washing the filter under 65 ° C conditions Means polynucleotide. Hybridization is Molecular Cloning 2nd ed., Current

Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), etc. Here, preferably, sequences containing only the A sequence or only the T sequence are excluded from the sequences that are hybridized under stringent conditions. The term “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above-mentioned hybridization conditions. Specifically, the polynucleotide that can be hybridized specifically has a homology of at least 60% or more with the DNA base sequence encoding a polypeptide (for example, activin) having the amino acid sequence specifically shown in the present invention. And a polynucleotide having a homology of 80% or more, more preferably a polynucleotide having a homology of 95% or more.

As used herein, “highly stringent conditions” allow for hybridization of DNA strands that have a high degree of complementarity in nucleic acid sequences, and DNA hybridization that has significant mismatches. Exclude conditions designed to be excluded! The stringency of a hybridization is mainly determined by the temperature, ionic strength, and conditions of the denaturing agent such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015M sodium chloride, 0.0015M sodium quenate, 65-68. C, or 0.005M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42 ° C. Such a high For more stringent conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual ^ Brother 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al., Nucleic Acid Hy See bridization: a Practical approach ^ IV, IRL Press Limited (Oxford, Engl and). Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other drugs include non-specific drugs, hybridization and

For the purpose of reducing Z or background hybridization, it may be included in the hybridization buffer and wash buffer. Examples of such other drugs include

0.1% ushi serum albumin, 0.1% polybulurpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO or SDS), Ficoll, Denhardt solution,

 Four

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 carried out at pH 6.8 to 7.4; however, under typical ionic strength conditions, the speed of hybridization is almost exclusively at pH. See Anderson et al., Nucleic Acid Hybridization: a Practical Ap proach, Chapter 4, IRL Press Limited (Oxford, England).

 Factors that affect the stability of DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those skilled in the art to apply these variables and 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 / NO.72 (% formamide)

Where N is the length of the double chain formed, [Na +] is the molar concentration of sodium ions in the noble, hybridization solution or wash solution, and% G + C is The percentage of (guanine + cytosine) bases in the. Incompletely matched Neubri For a lid, the melting temperature decreases by about c for each 1% mismatch.

 [0087] As used herein, "moderately stringent conditions" refers to the formation of DNA duplexes having a higher degree of base pair mismatch than can occur under "highly stringent conditions". The condition to obtain. Typical examples of `` moderately stringent conditions '' are 0.005M salt sodium silicate, 0.0015M sodium quenate, 50-65 ° C, or 0.015M sodium chloride, 0.005M salt sodium. 0015M sodium citrate, and 20% formamide, 37-50. C. As an example, a “moderately stringent” condition of 50 ° C in 0.015M sodium ion will allow about 21% discrepancy.

 [0088] 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, in 0.015M sodium ion (no formamide), the melting temperature of a 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 skilled in the art can simply decrease the temperature or increase the ionic strength.

 [0089] For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is

 Tm = (2 ° C per A—T base) + (4 ° C per G—C base pair)

 Provided by. The sodium ion concentration in 6 X citrate sodium salt (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

[0090] A natural nucleic acid encoding a protein such as activin or a variant or fragment thereof includes, for example, a PCR primer and a hybridization probe containing a part of a nucleic acid sequence such as SEQ ID NO: 1 or a variant thereof. Easily separated from cDNA library Preferably, the nucleic acid encoding activin or a variant or fragment thereof is essentially 1% sushi serum albumin (BSA); 500 mM sodium phosphate (NaPO 4); lmM EDTA; temperature of 42 ° C. Defined by a hybridization buffer containing 7% SDS and essentially 2 X SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C Low stringency conditions More preferably, essentially 1% ushi serum albumin (BSA) at a temperature of 50 ° C; 500 mM sodium phosphate (NaPO); 15% formamide; ImM EDTA; 7% SDS

 Four

 Most preferred under low stringent conditions as defined by the hybridization buffer and essentially 1 ° SSC at 50 ° C (300 mM NaCl; 30 mM sodium citrate); 1% SDS Is essentially 1% ushi serum albumin (BSA) at a temperature of 50 ° C; 200 mM sodium phosphate (NaPO); 15% formamide; ImM EDTA

 Four

 Defined by hybridization buffer containing 7% SDS and essentially 0.5 ° S SS C (150 mM NaCl; 15 mM sodium citrate) at 65 ° C; wash buffer containing 0.1% SDS Can hybridize with one or a portion of the sequence shown in SEQ ID NO: 1 or 3 under low stringency conditions.

[0091] As used herein, "search" refers to other nuclei having a specific function and Z or property using a certain nucleobase sequence electronically or biologically or by other methods. This refers to finding an acid-base sequence. Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403—410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444). — 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195—197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol 48: 443—453 (1970)), but not limited thereto. Biological searches include stringent hybridization, macroarrays with genomic DNA attached to nylon membranes or microarrays (microarray assembly) attached to glass plates, PCR and in situ hybridizers. Examples include, but are 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.

[0092] As used herein, the percentages of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) are the comparison of two sequences that are optimally aligned in a comparison window. Sought by. Here, the portion of the polynucleotide or polypeptide sequence within the comparison window contains a criterion for the optimal alignment of the two sequences. The ability to create gaps if other sequences contain anchors. When compared to a sequence, the reference sequence here shall have no additions or deletions. (Gap) may be included. 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. Multiply by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one of various sequence comparison algorithms and programs well known in the art. Such algorithms and programs include: TBLA STN: BLASTP ゝ 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., 199 4, Nucleic Acids Res. 22 (2): 4673— 4680, Higgins et al., 1996, Met hods Enzymol. 266: 383-402, Altschul et al., 1990, J. Mol. Biol. 21 5 (3): 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272). It is not limited to this. In a particularly preferred embodiment, the Basic Local Alignment Search Tool (BLAST) known in the prior art (eg Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:22 67-2268, Altschul et al., 1990 , 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) Is used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following tasks using five dedicated BLAST programs.

 (1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;

 (2) Compare nucleotide query sequence with nucleotide sequence database in BLASTN;

(3) Comparison of the conceptual translation product of the nucleotide query sequence (both strands) converted by BLASTX in six reading frames with the protein sequence database;

(4) Use TBLASTN to change the protein query sequence in all six reading frames (both strands). Compare with converted nucleotide sequence database;

 (5) Compare the nucleotide query sequence converted by TBLASTX in 6 reading frames with the nucleotide sequence database converted in 6 reading frames.

[0094] The BLAST program is an analogy called a “high-score segment pair” between an amino acid query sequence or nucleic acid query sequence and, preferably, a protein sequence database or nucleic acid sequence database capability test sequence. The homologous sequence is identified by specifying the segment. High score segment pairs are preferred, many of which are identified (ie, aligned) by a scoring matrix well known in the art.

, 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61). Although less preferred than this matrix, PAM or PAM250 matrices can also be used (eg Schwartz and Dayhoff, ed s., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation) checking). The BLAST program evaluates the statistical significance of all identified high-scoring segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as user-specific homology . It is preferable to evaluate the statistical significance of high-scoring segment pairs using Karlin's formula for statistical significance (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267— 2268). See

[0095] (Modification of gene, protein molecule, nucleic acid molecule, etc.)

In certain protein molecules (eg, activin, etc.), certain amino acids contained in the sequence can form protein structures such as, for example, cationic regions or substrate molecule binding sites without any apparent loss or loss of interaction binding capacity. In can be substituted with other amino acids. It is the ability and nature of protein interactions that define 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. Therefore, without any apparent loss of biological utility, Modifications can be made to the peptide disclosed herein or the corresponding DNA encoding this peptide.

[0096] In designing such modifications, the hydrophobicity index of amino acids can be taken into account. 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); ferulalanin (+2.8); cystine Z cystine (+2.5); methionine (+ 1. 9); alanine (+1. 8); glycine (one 0.4); threonine (one 0.7); serine (one 0.8); tryptophan (one 0.9); tyrosine (one 1 3); Proline (— 1. 6); Histidine (1 3.2); Gnoretamic acid (1 3.5); Gnoretamine (1 3.5); Aspartic acid (1 3.5); Asparagine (1) 3.5); lysine (one 3.9); and arginine (one 4.5)).

 [0097] 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, a protein equivalent in enzyme activity). Are well known in the art. In such amino acid substitutions, 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 substitution of amino acids based on hydrophobicity is efficient.

[0098] The hydrophilicity index is also useful for modifying the amino acid sequences 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); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) Threonine (one 0.4); proline (one 0.5 ± 1); alanine (one 0.5); histidine (-0.5); cystine (one 1.0); methionine (one 1.3) Valine (one 1.5); leucine (one 1.8); isoleucine (one 1.8); tyrosine (one 2.3); ferulanine (one 2.5); To Juan (13.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and still can 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, the term “conservative substitution” refers to the amino acid substitution and the hydrophilicity index or Z and hydrophobicity index with the amino acid replaced with the original amino acid as described above. A substitution similar to. Examples of conservative substitutions include those having a hydrophilicity index or hydrophobicity index within ± 2, preferably within ± 1, and more preferably between ± 0.5. Is 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; Examples include, but are not limited to, leucine and isoleucine.

[0100] As used herein, the term "variant" refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide has been 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 Z or deletions, or one or more substitutions, additions and Z or deletions to the reference nucleic acid molecule or polypeptide. But are not limited thereto. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Thus, an “allelic variant” refers to a variant that is in an allelic relationship with a gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles and usually have nearly the same biological activity, but rarely have different biological activities. May be included. “Species homologue or homolog” means homology (preferably 60% or more, more preferably 80% or more) with a gene at the 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” means orthologue A gas gene (orthologous gene) is a gene derived from speciation from a common ancestor with two genes! /,,. For example, in the case of a hemoglobin gene family with multiple gene structures, the human and mouse α-hemoglobin genes are orthologs. . Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of the present invention can also be useful in the present invention, since orthologs can usually perform similar functions as the original species in another species.

As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified with respect to a particular nucleic acid sequence refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and is 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 field, each codon in nuclear acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) 1S produces functionally identical molecules It is thus understood that this 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 significantly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment, ligation, etc., site-specific base substitution using synthetic oligonucleotides, etc. (Specific site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) Modifications can also be made by methods commonly used in the field of molecular biology.

 [0102] In this specification, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution refers to substitution of the original peptide with one or more, for example, 1 to: LO, 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 refers to 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, and acylation (eg, acetylyl). Force including, but not limited to. The amino acid substituted or added may be a natural amino acid or an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.

 [0103] As used herein, the term "peptide analog" or "peptide derivative" is a compound that is a compound that is different from a peptide and is equivalent to at least one chemical or biological function. Say. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives have been added or replaced with respect to the original peptide. Peptide analogs have the same peptide function (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, similar water solubility). Such peptide analogs can be prepared using techniques well known in the field. Thus, a peptide analog can be a polymer containing an amino acid analog.

[0104] Chemically modified polypeptide compositions in which the polypeptide used in the present invention is bound 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). A suitable aqueous polymer may be selected from the group consisting of, for example, the following forces: polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or others Polymers based on these carbohydrates, poly (N-butylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide Z ethylene oxide copolymer, polyoxyethylated polyol (eg, glycerol) and polybut alcohol. This selected polymer is usually modified to produce a single reactive group (e.g.

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 when determined for therapeutic use.

 [0105] 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 (the propionaldehyde is water soluble) or mono-C1-C10 alkoxy derivatives or aryloxy derivatives (eg, US Pat. No. 5,252,52). 714, which is incorporated herein by reference in its entirety).

[0106] The pegylation of the polypeptide used in the present invention is carried out by any pegich reaction known in the art as described in the following references, for example. Can be: Focus on Growth Factors 3, 4-10 (1992); EP 0 154 316; and EP 0 401 384, each of which is hereby incorporated by reference in its entirety. Preferably, this veggie is carried out via an acyl or 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-lZRae28, 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 may be other proteins (eg, mono (C1-C10). ) Used to derivatize alkoxy polyethylene glycols or mono (C1-C10) aryloxy polyethylene glycols). [0107] Chemical derivatives of polypeptides used in the present invention may be performed under suitable conditions used to react biologically active substances with activated polymer molecules. . A method for preparing a polypeptide for use in a pegylated invention generally comprises the following steps: (a) Under conditions such that activin is attached to one or more PEG groups. Reacting the polypeptide with polyethylene glycol (eg, a reactive ester or aldehyde derivative of PEG) and (b) obtaining the reaction product. Based on the known parameters and the desired result, it is easy for the person skilled in the art to select the optimal reaction conditions or the acylation reaction.

 [0108] 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. However, as disclosed herein, chemically derivatized polypeptide molecules used in the present invention have additional activity, increased biological activity or increased activity compared to their non-derivative molecules. It may have reduced biological activity, or other characteristics (eg, increased half-life or reduced 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 cytodynamics, growth factors, antigens, anti-inflammatory agents and Z or chemotherapeutic agents are appropriate to treat symptoms

[0109] Similarly, a "polynucleotide analog" or "nucleic acid analog" is a compound that is a different compound from a polynucleotide or nucleic acid. The polynucleotide or nucleic acid is equivalent to at least one chemical function or biological function. Say things. 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.

[0110] As described above, a nucleic acid molecule used in the present specification has a portion of the nucleic acid sequence deleted as long as the expressed polypeptide has substantially the same activity as the native polypeptide. Alternatively, it may be substituted with another base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 ′ end and the Z or 3 ′ end. In addition, the gene encoding the polypeptide can be detected under stringent conditions. It may be a nucleic acid molecule that encodes a polypeptide that has been hybridized and has substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

 [0111] Such a nucleic acid can be obtained by a well-known PCR method or chemically synthesized. For example, a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.

 [0112] As used herein, "substitution, addition or deletion" of a polypeptide or polynucleotide refers to an amino acid or an alternative thereof, or a nucleotide or an alternative thereof, respectively, relative to the original polypeptide or polynucleotide. Physical power Replace, add, or remove. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. Any number of substitutions, additions or deletions may be used as long as it is one or more. Such numbers may be used in the variant having the substitutions, additions or deletions (eg, hormones, As long as the information transmission function is maintained). For example, such a number can be 1 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.

 [0113] (General technology)

 Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, for example, Sambrook J. et al. (, 1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and "Wiley—Intelscience; Ausubel, FM (1989) Short Protocols in Molecular Biolog y: A ompendium of Methods from Current Protocols in Molecular

Biology, Greene Pub. Associates and Wiley— Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Mol ecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Inn is, MA et al. (1995). PCR Strategies , Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, a separate volume "Experimental Methods for Recruitment and Expression Analysis", Yodosha, 1997, etc., and these are the relevant parts of this document. Obtained) is incorporated by reference.

[0114] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRLPress; Gait, MJ (1990) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F., 991) .Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, RL etal. (1992). Barova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, etc., which are incorporated herein by reference.

[0115] (Genetic Engineering)

 In the present invention, activin 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 into a target cell. Such vectors can replicate autonomously in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, individual animals, and individual plants. Another example is one that can be inserted into a chromosome and contains a promoter at a position suitable for transcription of the polynucleotide used in the present invention. Among the vectors, a vector suitable for cloning is referred to as a “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, virus 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, box virus, adeno-associated virus, Semliki Forest virus, vaccinia virus and retrovirus.

[0117] 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, a particular vector may direct the expression of the genes to which they are operably linked. Such a vector is referred to herein as an “expression vector”.

[0118] Therefore, in the present specification, an "expression vector" refers to a structural gene and a promoter that regulates its expression, in which various regulatory elements are linked in a state in which they can operate in a host cell. Refers to a nucleic acid sequence. The regulatory element may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. The type of expression vector of the organism (eg animal) and the type of regulatory elements used It is well known to those skilled in the art that it can vary depending on the cell.

[0119] "Recombinant vectors" for prokaryotic cells that can be used in the present invention include pc DNA3 (+), pBluescript—SK (+ Z), pGEM—T, pEF—BOS, pEGFP, pHAT, pUC18, pFT — Examples include DEST ^™ 42GATEWAY (Invitrogen).

 [0120] "Recombinant vectors" for animal cells that can be used in the present invention include pc DNAI / Amp, pcDNAI, pCDM8 (all sold by Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 Q [. Biochem., 101, 1307 (1987)], ρΑΜo, pAMoAQ [. Biol. Chem., 268, 22782—22787 (1993)], based on Murine Stem Cell Virus (MSCV) Examples include retrovirus-type expression vectors, pEF-BOS, and pEGFP.

 [0121] As used herein, "terminator 1" is located downstream of a gene-coding region of a gene, and is a sequence involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is. Terminators are known to affect gene stability by affecting mRNA stability.

 [0122] As used herein, the term "promoter" refers to a region on DNA that determines the initiation site of gene transcription and directly regulates its frequency, and is usually the base where RNA polymerase binds and initiates transcription. Is an array. Therefore, in this specification, a portion having a promoter function of a gene is referred to as a “promoter portion”. Since the promoter region is usually within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, the promoter region The region can be estimated. The putative promoter region varies from structural gene to structural gene, but is usually upstream of the structural gene, but is not limited to this and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream of the first exon translation initiation force.

[0123] 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. If the vector is integrated into the host cell chromosome, the latter is sufficient It can be. Alternatively, rather than using a vector containing a viral origin of replication, one skilled in the art can transform mammalian cells by a method of cotransforming a selectable marker with 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).

 [0124] 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, the mouse hox promoter (Kessel and Gruss (1990) Science 249, 374—379)) and α-feet. Toprotein promoters (Campes and Tilghman (1989) Genes De v. 3, 537-546)), albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymph specific promoter ( Calame and Eaton (1988) Adv. Immunol. 43, 235-275), especially T cell receptors (Winoto and Baltimo re (1989) EMBO J. 8, 729-733) and immunoglobulins (Banerji et al. (1983) Cell

33, 729—740; Queen and Baltimore (1983) Cell 33, 741—748) promoters, neuron specific promoters (eg, nerve fiber promoters; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86 , 5473-5477), pancreas specific promoters (Edlund et al. (1985) Science 230, 912-916), and mammary gland specific promoters (eg, whey promoter; US Pat. No. 4,873,316 and European Application Publication No. 264, 166), but is not limited thereto.

 [0125] As used herein, "enenser" refers to a sequence used to increase the expression efficiency of a target gene. Such Jenno, sensors are well known in the art. You can use multiple Hansa, but you can use one! /, Or not! /.

[0126] As used herein, "operably linked" refers to a transcriptional translational regulatory sequence (eg, promoter, henno, sensor, etc.) that has the expression (operation) of a desired sequence or It means being placed under the control of the translation arrangement. In order for a promoter to be operably linked to a gene, it is usually not necessary to place the promoter immediately adjacent to the gene. [0127] In the present specification, any technique may be used for introducing a nucleic acid molecule into a cell. Examples thereof include transformation, transduction, and transformation. Such nucleic acid molecule introduction techniques are well known and commonly used in the art, for example, Ausubel FA et al. (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 Listed in Tsuchiya, 1997. Gene transfer can be confirmed using the methods described herein, such as Northern plots, Western plot analysis, or other well-known conventional techniques.

 [0128] In addition, as a method for introducing a vector, any of the above-described methods for introducing DNA into a cell can be used. For example, transfection, transduction, transformation, etc. (for example, Calcium phosphate method, ribosome method, DEAE dextran method, electroboration method, method using particle gun (gene gun), etc.).

 [0129] As used herein, "transformant" refers to all or part of a living organism such as a cell produced by transformation. Examples of transformants include prokaryotic cells, yeast, animal cells, plant cells, insect cells and the like. A transformant is also referred to as a transformed cell, a transformed tissue, a transformed host or the like depending on the subject. The cell used in the present invention may be a transformant.

 [0130] When prokaryotic cells are used in the present invention for genetic manipulation or the like in the present invention, the prokaryotic mycelium belongs to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, 禺, Microbacterium 晨, Pseudomonas 厲, etc. Prokaryotic cells such as Escherichia coli XL1-Blue® Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

[0131] As used herein, animal cells include mouse 'myeloma cells, rat.myeloma cells, mouse's cells, hybridoma cells, Chinese' cells, CHO cells that are Muster cells, BHK cells. And African green monkey kidney cells, human leukemia cells, HBT563 7 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse 'myeloma Examples of cells include ps20 and NSO, YB2Z0 for rat myeloma cells, HEK293 (ATCC: CRL-1573) for human fetal kidney cells, BALL-1 for human leukemia cells, and African green monkey kidney cells COS-1, COS-7, human colon cancer cell lines include HCT-15, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. .

 [0132] 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)], Lipofusion method, Spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], Lithium acetate method Q [. Bacteriol., 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 7 5, 1929 (1978).

 [0133] (Polypeptide production method)

 Transformants derived from microorganisms, animal cells, etc. that possess a recombinant vector incorporating a DNA encoding the polypeptide used in the present invention (eg, activin, LIF or a variant or fragment thereof) are usually used. The polypeptide used in the present invention is produced and accumulated, and the polypeptide used in the present invention is collected from the culture of the present invention. A peptide can be produced.

 [0134] 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. However, any medium that can efficiently cultivate transformants can use natural media and synthetic media! /.

 [0135] As the carbon source, any microorganism that can be assimilated by each microorganism is acceptable. Examples of the carbon source include glucose, slatose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid and the like. Alcohols such as organic acids, ethanol and propanol can be used.

[0136] Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate. Ammonium salts of various inorganic and organic acids such as humic and ammonium phosphate, other nitrogenous substances, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, large Soybean koji and soybean koji hydrolyzate, various fermented cells and digested products thereof can be used.

 [0137] As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. may be used. it can. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

 [0138] The culture temperature is 15 to 40 ° C, and the culture time is usually 5 hours to 7 days. During the cultivation, the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. Further, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed during the culture.

 [0139] When a microorganism transformed with an expression vector using an inducible promoter as a promoter is cultured, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using the lac promoter, cultivate a microorganism transformed with an expression vector using trp promoter, such as isopropyl β-D-thiogalatatopyranoside. When doing so, indoleacrylic 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 mentor.

 [0140] For example, when animal cells are used, the culture medium for culturing the cells of the present invention is RPMI 1640 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 these media Usushi medium supplemented with fetal serum is used.

 [0141] Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C, and the presence of 5% CO.

 2

In addition, antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture. [0142] 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. Conventional enzyme isolation or purification methods commonly used in the art can be used. For example, when the polypeptide of the present invention is secreted out of the cells of the transformant for producing the polypeptide of the present invention, the culture is treated by a technique such as centrifugation, Obtain the soluble fraction. From the soluble fraction, solvent extraction method, salting-out method using ammonium sulfate, desalting method, precipitation method using organic solvent, anion using a resin such as Jetylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (Mitsubishi Chemical) Ion exchange chromatography, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose and ferrule sepharose, molecular sieve The purified sample can be obtained by using a method such as gel filtration method, final fibrity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing.

 [0143] The polypeptide used in the present invention (for example, activin, LIF, or a variant or fragment thereof) accumulates in a cell in a soluble state in the transformant for producing the polypeptide used in the present invention. In this case, the cells in the culture are collected by centrifuging the culture, and after washing the cells, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. Obtain a cell 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, jetylaminoethyl (DEAE) -Sepharose, DIAION HPA — Anion-exchange chromatography using 75 (Mitsubishi Chemical), etc., cation exchange chromatography, S-Sepharose FF (Pharmacia), butyl sepharose, ferrule Use methods such as hydrophobic chromatography using sepharose such as sepharose, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing By this, a purified sample can be obtained.

[0144] When the polypeptide used in the present invention is expressed in the form of an insoluble substance in the cells, the cells are similarly collected, disrupted and centrifuged from the precipitate fraction obtained by After recovering the polypeptide used in the present invention by an ordinary method, the insoluble substance of the polypeptide is dissolved with a polypeptide denaturant. This soluble solution is diluted or dialyzed into a dilute solution that does not contain a polypeptide denaturing agent, or the polypeptide denaturing agent does not denature the polypeptide, and is used in the present invention. After constructing the peptide into a normal three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above.

 [0145] Alternatively, the protein can be purified according to a conventional protein purification method V. 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, according to the method described in Lowe et al. [Proc. Natl. Acad. Sci., USA, 86, 8227-8231 (1989), GenesDevelop., 4, 1288 (1990)] The resulting polypeptide can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

[0146] 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 Dev elop., 4, 1288 (1990)]. In such fusion proteins, the proteolytic cleavage site in the expression vector allows the separation of the fusion protein with the recombinant protein to allow separation of the recombinant protein following fusion protein purification. Introduced at the junction. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Typical 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). [0147] Furthermore, it can be purified by affinity chromatography using an antibody against the polypeptide itself used in the present invention. The polypeptide used in the present invention is in accordance with a known method Q [. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)]. Can be produced using in vitro transcription and translation systems.

[0148] The polypeptide used in the present invention is based on the amino acid information such as Fmoc method (fluoromethyloxycarbon method), tBoc method (tbutyloxycarbon method), etc. It can also be produced by chemical synthesis. In addition, chemical synthesis can be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems ^ Pharmacia Biotech ^ Protein, echnology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

 [0149] Structural analysis of the purified polypeptide used in the present invention is performed by a method commonly used in protein chemistry, for example, protein structural analysis for gene cloning (Hirano Hisashi, published by Tokyo Chemical Dojin, (1993). The physiological activity of the polypeptide used in the present invention can be measured according to a known measurement method.

[0150] Deletion, substitution, or addition (including fusion) of amino acids of a polypeptide (for example, activin, LIF, etc.) used in the present invention can be performed by a site-directed mutagenesis method that is a well-known technique. it can. One or a few amino acids are deleted, substituted, or appended.Carlo is Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley. & Sons (1987—1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., US A, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research , 1 3, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci., USA, 81, 5662 (1984), Science, 224, 1431 (1984) ), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985), etc. [Methods described herein can be prepared according to this method. [0151] (Cell culture)

 The cell culture properties of stem cell fractions of a wide range of media formulations known in the art are dramatically improved by adjusting the amount of activin, retinoic acid, LIF or its equivalents disclosed herein. Was found to change.

 [0152] Many media formulations have been developed over the years to maximize cell growth, cell viability, and Z 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 especially cells in suspension. If grown on, include one or more anti-foarming 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) is typically part of a host that has been 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.

 [0153] Activin, retinoic acid, LIF or equivalents disclosed herein are expected to have effects on various media formulations as well as in standard formulations. The

 [0154] For example, all serum-free media formulations contain essential ingredients that allow cell growth,

(1) An energy source, typically glucose or glutamine, or other sugars, such as factose, galactose, mannose; (2) Nitrogen sources (typically by inclusion of one or more amino acids) Obtained); and (3) vitamins (complements in enzyme reactions). Also a wide range of inorganic salts including Na +, K +, Ca ²⁺ , Mg ²⁺ , CI ", HPO ^2_ etc.

 Three

And a wide range of fat and fat-soluble ingredients including fatty acids (preferably conjugates), cholesterol, phospholipids, and their precursors are also essential. The components of a standard serum-free medium formulation are listed in Table 1. This can be found in “DMEMZF-12” from media manufacturers such as Grandlsland Biological Co. (GIBCO), Grandlsland, NY. Ingredients to be included. For a review of animal cell culture media, see Mizmhi and Lazar, Cytotechnology, 1: 199 214 (1988). Media considerations for insect cell culture are disclosed in Goodwin, RH (1990) Nature 347: 209-210. An example of a defined serum-free medium that has been personalized for the culture of wildbredoma is described in Kovar, J. (1987) FoliaBiologia33: 377-384. Imagawa et al. (1989) PNAS86: 4122-4126 describes a medium developed to maximize cell proliferation in mouse mammary epithelial cells and Miyazaki et al. (1991) Res. Ex p. Med. 191: 77. —83 describes the medium to enhance the viability of rat hepatocytes in vitro.

 [0155] 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 proliferate including self, Any medium can be used as long as it is used for differentiation, maturation or preservation and is usually used for cell culture. Examples include Steinberg erg medium, (X MEM medium, RPMI-1640 medium or MEM basal medium, etc. The basic ingredients are sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, It may contain hormones, antibiotics, fatty acids, sugars or other chemical components or biological components such as serum depending on the purpose.

 [0156] Media components can typically be added in any order, and the final combination can be sterilized by standard methods, such as filter sterilization.

 [0157] Using the culture medium of the present invention, a wide variety of 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.

 [0158] In the case of animals, serum-free media such as Iskov media, RPMI media, Dulbecco MEM media, MEM media, and F12 media, which are commonly used for animal cell culture, 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, darcocorticoid, DNA and RNA synthesis nucleosides, etc. are added according to known literature. You may be careful.

[0159] As used herein, the term "culture vessel" refers to a proliferation of desired cells, for example, undifferentiated cells. It is a container used when the feeder cells such as stromal cells can maintain and survive as needed, and undifferentiated cells can maintain and survive `` differentiation '' and `` maturation ''. Any material and shape may be used. Specific examples of the culture vessel material include glass, synthetic resin, natural fat, metal, and plastic. Specific examples of the shape include triangular prisms, cubes, rectangular parallelepipeds, triangular pyramids, quadrangular pyramids, etc. Polygonal pyramids, arbitrary shapes such as gourds, spheres, hemispheres, cylinders (including round bottom, oval or semicircle), etc. The shape may be changed as necessary. Culture may be under open conditions or under closed (sealed) conditions.

 [0160] The cell culture method in the present invention is 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 matrix coating, a tertiary gel using a soft gel such as Matrijeel and a collagen sponge. Power that includes the original culture method or a method using them together Preferably, it is a two-dimensional culture method using a culture dish having a different matrix coating or a culture dish having different matrix coating, for example, gelatin. This is a two-dimensional culture method using two of a coated culture dish, type I collagen-coated culture dish, or laminin-coated culture dish. In addition, when human-derived cells are used as pluripotent cells, it is preferable to use type I collagen-coated culture dishes.

[0161] The culture conditions of the method of the present invention are not limited to specific conditions such as Examples, and may be generally acceptable conditions. For example, the cell number of minutes spoon induction ^{start, 5. Oxl0 3 ~5. OxlO} 6 can be exemplified range of cell / dish. The differentiation induction period is, for example, 2 to: LO days (preferably 5 days), or 1 to 4 days (preferably 2 days), and 2 to 5 days (preferably 3 days) in the pre-culture step. . In the case of human mesenchymal cells, the induction period is 12 to 21 days (preferably 14 days).

[0162] 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 include undifferentiated cells before killing and post-differentiation cells. Any environmental condition that prevents cells from maintaining 'surviving and undifferentiated cells from maintaining' surviving 'differentiation' maturation 'self-replication! Prefer ヽ conditions It is shown below.

 [0163] 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.

 [0164] The light may be as dark as a dark room, or as bright as the outside brightness in fine weather.

 Specifically, as for the oxygen concentration, the culture system is in contact with the gas phase having a 10% oxygen concentration in the gas phase, and the dissolved oxygen concentration in the state of V to the gas phase having a 30% oxygen concentration in the gas phase. The oxygen concentration in the contact state may be the oxygen concentration in the gas phase, preferably in contact with the gas phase having a 20% oxygen concentration in the gas phase, and in contact with the gas phase of the dissolved oxygen concentration in the state, It is the oxygen concentration in the state.

[0165] Specifically, the pH for controlling pH in a culture system is specifically pH 6.

 0 to pH 8.0, preferably a pH equivalent to physiological conditions. In order to control the pH, carbon dioxide or carbon dioxide or any other buffer may be used. Specifically, the concentration of carbon dioxide is the concentration of dissolved carbon dioxide when the culture system is in contact with the 5% gas phase.

 [0166] As used herein, a "colony" refers to a visible clump formed from a single cell in a solid medium.

[0167] 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 preferably be mixed and used immediately prior to use. Such a kit is advantageously provided with instructions describing how to treat the provided parts (eg reagents, particles, etc.). Such instructions 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 etanet, 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. [0168] (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 using a specific operation Z evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. The screening may be performed using a library generated using an in silico (computer system) system that may use a system using a real substance such as in vitro or in vivo. In the present invention, it is understood that compounds obtained by screening having the desired activity are also included within the scope of the present invention. In addition, the present invention contemplates providing a drug by computer modeling based on the disclosure of the present invention.

 [0169] In one embodiment, the present invention screens candidate or test compounds that bind to or modulate the activity of the protein of the present invention (activin), or a biologically active portion thereof. Provide an assembly for. The test compounds of the present invention can be obtained using any of a number of approaches in a combinatorial library method known in the art, including: biological libraries; spatially Accessible parallel solid phase or solution phase libraries; synthetic library methods that require back-folding; “one-bead one-compound” library methods; and synthetic library methods that use affinity chromatography selection . Biological library approaches are 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).

 [0170] 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. ) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann¾ (1994) J. M ed. Chem 37: 2678; Cho¾ (1993) Science 261: 1303; Carrell¾ (1994) An gew Chem. Int. Ed. Engl 33: 2059; Carell¾ (1994) Angew Chem. Int. E d. Engl. 33: 2061; and Gallop et al. (1994) Med. Chem 37: 1233.

[0171] A library of compounds is available in solution (eg, Houghten (1992) BioTechniques 13: 412-421) or on beads (Lam (1991) Nature 354: 82-84), on chips (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5, 22 23, 409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (3.1 01 311-11 (1990) 3 ₍ ^ nce 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (199 0) Proc. Natl. Acad. Sci. USA 87: 6378-6382; Felici (1991) J Mol B iol 222 : 301-310; Ladner supra)!

[0172] (Disease)

 In one aspect, the present invention relates to any disease, disorder or abnormal condition relating to a pancreatic disorder, such as splenitis (acute splenitis, chronic splenitis), pancreatic cancer, cystic spleen disease, diabetes, digestive organs. Methods for treating related diseases, disorders or abnormal conditions, such as diseases of the system, are provided.

 [0173] Typical pancreatic 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. In these patients, blood insulin levels are not always stable despite regular insulin infusions, resulting in longevity with many complications such as stroke, blindness, amputation, and renal dysfunction. Is shortened. In addition, there are cases where insulin resistance is caused by lifestyle habits such as lack of exercise, excessive intake of fat, and obesity, and there are cases where it becomes a force S insulin action disorder (resistance factor) such as drugs and endocrine diseases.

 [0174] As used herein, "preventing" refers to reducing the likelihood that an organism will develop a contracting force or abnormal condition.

[0175] As used herein, "treating" refers to having a therapeutic effect and the ability to or at least partially alleviate an abnormal condition in an organism. [0176] As used herein, "therapeutic effect" refers to a force that causes an abnormal condition or an inhibitory factor or an active factor that contributes to the force. The therapeutic effect will alleviate one or more of the symptoms of the abnormal condition to some extent. For the treatment of abnormal conditions, a therapeutic effect is one or more of the following: (a) increase in cell proliferation, growth, and Z or differentiation; (b) cells Inhibition of death (ie slowing or stopping); (c) inhibition of alteration; (d) some relief of one or more of the symptoms associated with the abnormal condition; and (e) of the affected cell population Strengthen functions. Compounds that exhibit efficacy against abnormal conditions can be identified as described herein.

 [0177] As used herein, "abnormal state" refers to a function in a cell or tissue of an organism that also deviates from its normal functional capabilities in the organism. The abnormal condition can be associated with cell proliferation, cell differentiation, cell signaling, or cell survival. Abnormal conditions can 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.

 [0178] Examples of abnormal cell proliferation include cancer, neoplasm, tumor and inflammation.

 [0179] Examples of the abnormal separation state include malformation and cancer.

 [0180] Examples of abnormal cell signal transduction include abnormal cell sorting.

 [0181] Abnormal cell viability is also associated with the ability to activate or arrest the apoptotic (programmed cell death) pathway. A number of protein kinases are associated with the apoptotic pathway. Abnormalities in any one function of protein kinases can result in cell immortality or premature cell death.

[0182] In another aspect, the present invention relates to a prophylactic method for treating a subject having a pancreatic-related disease, disorder or abnormal condition (affected or at risk) or having the disorder. Provide both therapeutic methods.

[0183] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the medicament of the present invention. Such containers are optionally accompanied by a notice in the form prescribed by government agencies that regulate the manufacture, use or sale of pharmaceuticals or biological products. This notice may represent governmental approval for manufacturing, use or sale for human administration.

 [0184] (Gene therapy)

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

 [0185] Any method for gene therapy available in the art can be used in accordance with the present invention. An exemplary method is as follows.

 [0186] For a general review of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12: 488—505 (1993); Wu and Wu, Biotherapy 3: 87—95 (1991); Tol stoshev, 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): See 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

 [0187] Therefore, in the present invention, gene therapy using a nucleic acid molecule that codes for activin or a variant or fragment thereof may be useful.

[0188] As used herein and as understood in the art, the term "synthes is" or "synthesize" is in contrast to enzymatic methods, A chemical substance (eg, polynucleotide, polypeptide, etc.) produced purely chemically. Thus, “globally” synthesized chemicals (eg, polynucleotides, polypeptides, etc.) are produced entirely by chemical means, and “partially” synthesized chemistry. Substances (eg, polynucleotides, polypeptides, etc.) are chemical substances (eg, polynucleotides, polypeptides, etc.) that are only partially produced by chemical means (eg, polynucleotides, polypeptides, etc.). , Polynucleotides, polypeptides, etc.).

 [0189] By the term "region" as used herein is meant a physically continuous 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 a known or suspected function of the biomolecule. A domain can have the same extent as a region or part thereof; a domain can also incorporate all or part of the region, as well as part of a biomolecule that is distinct from a particular region. Examples of the domain of activin of the present invention include, but are not limited to, signal peptides, extracellular (ie, N-terminal) domains, and leucine-rich repeat domains.

 [0190] (Regenerative Z treatment Z prophylaxis and thread composition)

 The present invention provides 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 a method. In a preferred aspect, the compound can be substantially purified (eg, in a state substantially free of substances that produce a force or an undesirable side effect that limits its effect).

 [0191] As used herein, the term "effective amount for diagnosis, prevention, treatment or prognosis" refers to an amount that is recognized as medically effective in diagnosis, prevention, treatment (or treatment) or prognosis, respectively. Say. Such amounts can be determined by one skilled in the art using techniques well known in the art, taking into account various parameters.

[0192] 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 (e.g., metaraunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (e.g., single holes, marsupials). , Rodents, crustaceans, wings, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, moths Teeth, maggots, etc.). Exemplary cover Examples of specimens include, but are not limited to, animals such as rabbits, pigs, horses, -birds, cats, and dogs. More preferably, cells derived from primates (for example, chimpanzees, monkeys, humans) are used. Most preferably, human-derived cells are used.

 [0193] When the nucleic acid molecule or polypeptide of the present invention is used as a medicine, such a composition may further contain 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.

 [0194] Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, fillers. Examples include, but are not limited to, dosages, buffers, 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, in one or more physiologically acceptable carriers, excipients. Administered in the form of a composition comprising an agent or 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. is there.

 [0195] 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, kenate, or other organic acids; ascorbic acid, a-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, poly Amino acids (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-forming counterion (Eg, sodium); and Z or non-ionic surfactants (eg, Tween, pluronic or polyethylene glycol (PEG)), and the like.

[0196] Exemplary suitable carriers include neutral buffered saline or serum albumin. Mixed saline can be mentioned. 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 Tris buffer at pH 7.0—8.5 or acetate buffer at pH 4.0—5.5, which are sardine, sorbitol, or suitable substitutes thereof. Can be included.

 [0197] A general method for preparing the pharmaceutical composition of the present invention is described below. In addition, veterinary drug compositions, quasi-drugs, marine drug compositions, food compositions, babies cosmetic compositions, and the like can also be produced by known preparation methods.

 [0198] Polypeptides, polynucleotides and the like used in the present invention are mixed with a pharmaceutically acceptable carrier, and are solid preparations such as tablets, capsules, granules, powders, powders, suppositories, or syrups. It can be administered orally or parenterally as liquid preparations such as pills, injections, suspensions, solutions and sprays. Pharmaceutically acceptable carriers include, as described above, excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, adsorbents, humectants, solubilizers in solid preparations. , Stabilizers, solvents in liquid preparations, solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. In addition, substances other than the polynucleotides and polypeptides used in the present invention can be added to the composition of the present invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.

 [0199] Examples of excipients in solid preparations include glucose, latatose, sucrose, D-mannitol, crystalline cellulose, starch, calcium carbonate, light anhydrous caustic acid, sodium chloride, kaolin and urea.

 [0200] Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, boric acid powder, colloidal key acid, talc, and polyethylene glycol.

[0201] Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mannitol, crystalline cellulose, dextrin, methylcellulose, hydroxypropenoresenorelose, hydroxypropinoremethinoresenorelose, and canoleboximethinolol. Cenorelose, de Open solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate, potassium phosphate, shellac and the like.

 [0202] Disintegrants in solid preparations include, for example, starch, carboxymethylcellulose, carboxymethylcellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium bicarbonate, calcium carbonate, Examples thereof include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, starch, monoglyceride stearate, latatose and calcium calcium glycolate.

 [0203] 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.

 [0204] Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.

 [0205] Examples of the adsorbent in the solid preparation include, but are not limited to, starch, ratatoose, kaolin, bentonite, and colloidal caustic acid.

 [0206] Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.

 [0207] Examples of solubilizing agents in solid preparations include, but are not limited to, arginine, glutamic acid, aspartic acid, and the like.

 [0208] Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin and ratatose.

[0209] When preparing tablets, pills, etc. as solid preparations, they may be coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Also good. 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, in addition to the additives listed above, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides and the like can be added, but are not limited thereto. [0210] Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene dallicol, macrogol, sesame oil, corn oil and the like.

 [0211] Preferable examples of solubilizers in liquid preparations include polyethylene glycol, propylene glycol, D-manntol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and Examples include, but are not limited to, sodium quenate.

 [0212] Preferable examples of the suspending agent in the liquid preparation include stearyl triethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, salt benzethonium, monostearic acid Surfactant such as glycerin, for example, polybulle alcohol, polybulurpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethinoresenorelose, hydroxyethinoresenorelose, hydroxyethinocellulose, hydroxypropylcellulose, etc. Examples include but are not limited to molecules.

 [0213] Preferable examples of the isotonic agent in the liquid preparation include sodium chloride salt, glycerin, D-manntol and the like, but are not limited thereto.

 [0214] Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphates, acetates, carbonates and citrates.

 [0215] Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, and pro-hydrochloric acid hydrochloride.

 [0216] Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoates, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like. .

 [0217] Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, a-tocopherol, cysteine and the like.

[0218] When preparing as an injection, the liquid and suspension are preferably sterilized and isotonic with blood. Usually, these are sterilized by filtration using a nocteria retention filter, etc., blending with a disinfectant or irradiation. Further, after these treatments, solids are obtained by freeze-drying or the like, and immediately before use, sterile water or a sterile diluent for injection (lidocaine hydrochloride) is used. Add an aqueous solution, physiological saline, aqueous glucose solution, ethanol or a mixture of these).

 [0219] Furthermore, if necessary, the pharmaceutical composition may contain coloring agents, preservatives, fragrances, flavoring agents, sweeteners, and the like, as well as other agents.

 [0220] The medicament of the present invention may 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 (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal 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 preparation form include liquids, injections, and sustained release agents.

 [0221] The medicament of the present invention may be a physiologically acceptable carrier, excipient, or stabilizer as necessary (Japanese Pharmacopoeia 14th edition, its supplement or the latest edition, Remington's Pharm aceutical Sciences , 18th Edition, AR Lrennaro, ed., Mack Publisnm g Company, 1990, etc.) and a glycan composition having the desired degree of purity to form a lyophilized cake or aqueous solution. Can be prepared and stored.

[0222] Various delivery systems are known and can be used to administer a compound of the invention (eg, ribosomes, 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 It can be administered with other biologically active agents. Administration can be systemic or local. Furthermore, the pharmaceutical compounds or compositions of the invention include any suitable route (including intraventricular injection and intrathecal injection; the intraventricular injection is attached to a reservoir such as, for example, an Ommaya reservoir). It may be desirable to introduce it into the central nervous system by an intraventricular catheter). Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

 [0223] 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 nerve, brain, etc.); For example, without limitation, local injection during surgery, topical application (eg in combination with post-surgical wound dressings), by injection, by catheter, by suppository, or by an implant 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 the protein! / ヽ material when it is not absorbed! / ヽ.

 [0224] In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly ribosomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes m tne fherapy of Infectious Disease and ancer, Lo pez— Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lo pez—Berestein, ibid., pages 317-327; see broadly )

[0225] 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); Seft on, 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 (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen And Ball (Ed.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (19 83); Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); See also Howard ^, J. Neurosurg. 71: 105 (1989)) [0226] In yet another embodiment, a controlled sustained release system can be placed near the therapeutic target, ie, the brain, and therefore requires only a portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release, (supra), Vol. 2, pages 115-138 (1984)).

 [0227] Other controlled sustained release systems are discussed in a review by Langer (Science 249

 : 1527-1533 (1990)).

 [0228] The amount of the composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. In view of this, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment history, 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 monitoring the course.

 [0229] The dosage 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. Olmg ~: LOg, preferably 0.1mg ~: Lg, lmg ~: L00mg, 0.1mg ~: LOmg, etc. For parenteral administration, it is 0. Olmg to: Lg, 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.

[0230] In the present specification, "administering" refers to the polypeptide, polynucleotide, factor and the like of the present invention or a pharmaceutical composition containing the same alone or in combination with other therapeutic agents. Or it means to be taken into the organization. The combination can be administered, for example, as a mixture simultaneously, separately but simultaneously or concurrently; or sequentially. This also includes a presentation where the combined drugs are administered together as a therapeutic mixture, and the combined drugs are administered separately but simultaneously (for example, through separate intravenous lines to the same individual). Also includes. “Combination” administration is given first, followed by administration of one of the compounds or drugs given second To further include.

 [0231] 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.

 [0232] In the present specification, "instruction" refers to a person who administers or diagnoses a method of administering or diagnosing the medicine of the present invention, etc. It is described. This instruction manual includes a word indicating 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). It will be clearly stated that it has been approved. Instructions are so-called package inserts, and are not normally limited to paper-delivered forces, such as electronic media (eg, home pages (websites) provided on the Internet, emails, etc. ) Can also be provided.

 [0233] Judgment of termination of treatment by the method of the present invention can be attributed to diseases (eg, hematological disorders) associated with standard clinical laboratory results or activin using commercially available tools or instruments. Can be supported by the disappearance of characteristic clinical symptoms. Treatment can be resumed by recurrence of a disease related to activin etc. (eg blood system disease)

[0234] 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 notification in the form of a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products may optionally be attached to such containers, which may be manufactured, used or sold for human administration. Represents approval by a government agency.

[0235] 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 used to identify drug-treated kinetics. The position 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.

 [0236] 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) the compound is administered to mice (untreated control mice should also be used); (2) The force of one mouse in each treatment group also obtains blood samples periodically via the tail vein; and (3) The samples are obtained from the number of red blood cells and white blood cells, composition, and lymphocytes and polymorphonuclear cells. Analyze percentages. Comparison of the results for each dosing regimen with controls shows whether toxicity is present.

 [0237] Further studies can be performed by sacrificing animals at the end of each toxicity study (preferably ii, American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Eutha nasia, (1993) J. Am. Vet. Med. Assoc. 202: 229-249. Then, representative animals from each treatment group can 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 as desirable as compounds that have adverse effects on major organs. In general, the greater the adverse effect, the less preferred the compound.

 [0238] (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.

[0239] In one aspect, the present invention provides a method for producing pancreas from undifferentiated cells. This method consists of the following steps: A) medium with leukemia inhibitory factor (LIF) Culturing in to form colonies and a pre-culture step to form aggregates (typically embryoid body-like spheres); and B) in the absence of LIF The method includes an exposure step in which the cells are exposed to activin and retinoic acid. Any undifferentiated cells used in the method of the present invention can be used as long as the ability to be separated remains.

 [0240] 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.

 [0241] In the method of the present invention, undifferentiated cells can be cultured by any culture method. It is advantageous that the undifferentiated cells are preferably cultured on a feeder cell.

 [0242] In the method of the present invention, the pre-culture step can be carried out for any period, but preferably about 1 to 10 days, usually 1 to 4 days, typically about 2 days or about It is advantageous to perform for 4 days. However, even if it is less than 1 day or more than 10 days, it is considered that there is no problem as long as it is suitable for separation.

 [0243] In the present invention, the undifferentiated cell used in the present invention may be any cell as long as it is a cell that can be sorted into the viscera. There has been no example in which mammalian cells have been differentiated into functional pancreas so far. Therefore, the present invention should be remarkably recognized in that mammalian cells are made functional pancreas.

 [0244] The medium used in the present invention advantageously contains serum. However, any serum that is commonly used for cell maintenance / proliferation can be used as the serum contained, for example, rabbit serum, rabbit fetal serum, horse serum, human serum, etc. But is not limited to them. The medium used may be any medium, but typically a knockout serum replacement (KSR) medium is used.

[0245] The exposure step in the method of the present invention may be performed under any culture conditions, but is preferably performed under conditions of suspension culture. In suspension culture, cells that proliferate in suspension in the living body, such as blood cells and various ascites tumor cells, can be suspended in culture without shaking and rotation. Or shake the whole culture bottle (shaking culture), or rotate the incubator (rotating culture) There is a need to. 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.

 [0246] Preferably, in an embodiment, the suspension culture is preferably performed on a low adhesion coated culture vessel (eg, available from Nunc). I don't want to be bound by theory! ヽ If you use such a low-adhesion-coated culture vessel, it seems that differentiation can be done efficiently.

 [0247] The undifferentiated cell used in the method of the present invention may be any undifferentiated cell force, preferably a stem cell, more preferably an embryonic stem cell. When embryonic stem cells are used, it is preferable to culture with feeder cells. In this case, for example, mouse embryo fibroblasts are preferably used as the feeder cells.

 [0248] Activin used in the method of the present invention may be used at a concentration of less than 50 ng Zml. For example, it can be used at concentrations of lng / ml to less than 50 ng / ml, lngZml to 25 ngZml, 10 ng / ml to 25 ngZml, and the like. It should be noted that either activin can be used depending on the purpose and the ability to vary the required amount of retinoic acid between when used in lOngZml and when used at a concentration of 25 ngZml.

 [0249] The retinoic acid used in the method of the present invention can be used in an amount of more than 0.01 μΜ and less than 1 μΜ, and usually more than 0.01 μΜ and less than or equal to 0.1 μΜ, Can be greater than or equal to Ι ^ Μ and less than 1 μ Μ.

 [0250] The method of the present invention may further include a step of C) culturing the differentiated aggregate in a medium on a coated culture vessel. By using such a process, the state of the separation can be improved. When used in the present invention, the coating can be any coating, but can preferably be a gelatin coating.

 [0251] Although any medium may be used, the medium used in the separating step is preferably a KSR medium. The separation step can be a period sufficient for the production of a functional pancreas, usually 7 to 21 days, preferably 10 to 14 days, but is not limited thereto.

[0252] In one embodiment, the separating step in the method of the present invention is performed by first performing an It includes a step of culturing in a medium containing nooic 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.

 [0253] In one preferred embodiment, the separating step is first cultured in a medium containing activin and retinoic acid for about 1 to 4 days, and then in a medium free of activin and retinoic acid. Including the step of culturing for 1 to 3 weeks.

 [0254] In one preferred embodiment, the separating step is first cultured in a medium containing activin and retinoic acid for about 2 days, and then about 10 in a medium free of activin and retinoic acid. A step of culturing for about 2 days to about 2 weeks.

 [0255] The functional organ 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 Ipf 1 / pdx-l 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, 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. This can be determined by checking.

 [0256] For example, the functional stem is characterized in that it contains all ex cells, β cells, exocrine cells, and ductal structures. Such a functional pancreas has all the characteristics selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability and functional conduit structure. The pancreas with all of these functions has never been able to be produced. In particular, ES cell-derived and mammalian ones have not been reported. There are no reports of functional conduit structures, ie, secretions that can be made in the same way as natural ones. Therefore, the present invention shows a remarkable effect in that it provides a truly “functional” pancreas.

[0257] In one aspect, the present invention provides a pancreas produced by the method of the present invention. [0258] 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.

[0259] 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 can be a mammal. Furthermore, in a preferred embodiment, the medicament is a stem cell.

(Eg, ES cells).

[0260] Hereinafter, the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the examples, but is limited only by the claims.

 Example

 [0261] Hereinafter, the present invention will be described in more detail with reference to Examples. However, 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.

 [0262] (Example 1: ES cell differentiation induction)

 (ES cell culture method and differentiation induction)

 Mouse ES cells (eg, E 14 (ATCC-CRL-1821; available from ATCC) are cultured according to the usual culture method described below: 10 μgZ mL of mitomycin C (Sigma) ~ 2.5 hours treated with embryonic fibroblasts coated with mouse embryonic fibroblasts that have stopped cell division for 5 hours, and used as feeder cells Maintenance medium for ES cells The serum medium used was 15% urine fetal serum (FBS; ES cell qualified, MEM non-essential amino acid (GIBC 0), 0.001% 13-mercaptoethanol (Sigma), and 1, 500 U / High glucose DMEM medium (Lib glutamate and pyruvate, Gibco 1 1995-065) containing mL LIF (Chemicon).

[0263] Three days after the above culture, the cells are treated with 1 mgZmL collagenase Z dispase (Roche), and the ES cells cultured in this manner are detached from the feeder cell to obtain an ES cell outlet. The colonies thus obtained are cultured in DMEM containing 15% KSR in an untreated culture dish (Iwaki). Replace this culture every 2 days Continue for 4 days. After 4 days, ES cells begin to form EBS. This EBS is coated with a low-cell multi-dish (non-biological component; Nunc). this 2曰[¾, 15 _^0/0 of KSR, Kuchihi ,, down (0, 10, 25, 50 ng / m L), and retinoic acid (0, 0.001, 0.01, 0.1, 1 / z M Sigma) in DMEM.

 [0264] (Immunostaining)

 EBS was fixed in 0.1 M phosphate buffer (pH 7.4) with 4% paraformaldehyde for 40 minutes at room temperature 13 days after the end of treatment (19 days after EBS formation). Fixed cells were detached from the dish wall, embedded in LR Gold Resin System (structural probe), an acrylic resin, and excised into thin sections of 600 nm. These sections were treated at room temperature for 40 minutes with 3% ushi serum albumin (BSA) and phosphate buffered saline (PBS = phosphate-buffered saline, 800 ml of sterile water, 8 g of NaCl, 0.2 g of KC1, Na2HP04 1 44 g, KH2P04 0.24 g, manufactured) Blocked in). They were then exposed to a pancreas-specific primary antibody for 12 hours at 4 ° C. After washing with PBS, the sections were exposed to labeled secondary antibody for 8 hours at 4 ° C. Primary antibodies used were rabbit anti-a-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); secondary antibody Were: Alexa—Fluor 488—conjugated antibody and Alexa—Fiuor 594—conjugated antibody (Molecular Probes). These sections were observed with a fluorescence microscope and connected to an ORCA-3 CCD camera AquaCosmos (H % "True" was taken by amamatsu Photonics, Hamamatsu, Japan. As a control, the pancreas of an 8-week-old mouse was fixed and processed as described above.

 [0265] (Electron microscope)

11 days after treatment (17 days after EBS formation) EBS was pre-fixed in fixative solution of 4% paraformaldehyde, 3% glutaraldehyde and 0.1M strength codylate buffer (pH 7.4) for 2 hours at room temperature did. Then, after washing with 0.1 M strength codylate buffer, these were fixed with 1% osmium tetroxide for 30 minutes at room temperature and then treated. Samples are washed with 0.1M strength codylate buffer, then dehydrated with ethanol and acetone and wrapped in epoxy resin. Buried. Very thin sections (80-90 nm) were prepared, 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 using 1 g of total RNA and Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). The forward and reverse primers used and the length of the PCR product 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 CAC T (SEQ ID NO: 19) and A AT 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) (2 34 bp);

 Ipfl / pdx-1, AGC AGT CTG AGG GTG AGC GGG TCT (SEQ ID NO: 2 3) 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 b P);

 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: No. 31) and CAT GTA GGC CCA TGA GGT CCA CCA C (SEQ ID NO:

32).

 [0267] First denaturation of cDNA at 95 ° C for 15 minutes, 45 cycles of heating at 94 ° C (20 seconds), 54 ° C (20 seconds) and 72 ° C (40 seconds), and finally Propagation was carried out by carrying out an extension process at 72 ° C for 7/7. The experiment was performed in triplicate.

[0268] (Real-time PCR)

 Real-time PCR was performed using QuantiTect ™ SYBR Green PCR Master Mix (QIA GEN) and ABI PRISM ™ 7700 Sequence Detector (Applied Biosys terns). The lengths of the forward and reverse primers used and their CRC products 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) ( 13 5 bp);

 insulin II, AGA AGC GTG GCA TTG TAG ATC AGT (SEQ ID NO: 3 7) and CAG AGG GGT AGG CTG GGT AGT G (SEQ ID NO: 38) (102 bp);

 Ipfl / 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).

[0269] cDNA was denatured at 95 ° C for the first 15 minutes, heated to 94 ° C for 45 cycles (20 seconds), 54 ° C (20 seconds) and 72 ° C (40 seconds), and finally Propagation was carried out at 72 ° C for 7/7. The experiment was performed in triplicate. (Total amount of intracellular insulin and secreted insulin Quantitative determination)

 13 days after induction (19 days from the start of EBS formation), EBS was washed with DMEM without glucose (GIBCO) and 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 lysed 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). Mouse Insulin ELI SA (TMB) Kit (SHIBAYAGI, Japan) was used to quantify the total amount of insulin in the conditioned medium or the amount of insulin in the cell lysate. Each experiment was performed with 8 EBS, and the experiment was performed in triplicate.

[0270] (Induction of pancreas)

 0. Seed EBS obtained as described above in an adhesive culture vessel (eg, 24-well well plate) coated with 1% gelatin, etc., and culture in DMEM containing 10% KSR. Adhere to the culture plate. Then, in KSR medium, treat with 25 ngZmL activin and 0.1 M retinoic acid simultaneously for 2 days in a low-adhesion-coated culture vessel. Next, the cells are cultured in the same manner in a gelatin-coated culture vessel for 10 days to 2 weeks in the absence of activin and retinoic acid. Change the culture every 3 days

[0271] (ES cell force intestinal tubular and organs, differentiation of visceral tissue)

First, ES cell colonies were isolated 3 days after treatment with collagenase Z dispase, and then cultured in DMEM supplemented with 15% KSR to form embryoid body-like aggregates (EBS). This EBS can be shared. Four days after EBS formation, the cells were transferred to a medium containing retinoic acid (0, 0.001, 0.01, 0.1 or: M) and activin (lOngZml), and then suspended in suspension for 2 days. And seeded on gelatin-coated plates. Six to eight days after treatment with retinoic acid and activin (12 to 14 days after EBS formation), the ratio of treated EBS became spherical or intestinal tubular structures, and the movement was similar to smooth muscle peristalsis . Over 20% 9-12 days after treatment (15-18 days after EBS formation) EBS treated with both retinoic acid and activin formed a tissue with black spots. This was rarely seen with unfavorable EBS or EBS treated with activin alone (Figure 2Ca, b).

[0272] Figure 2. A, colonic aggregates (EBS) of mouse ES cells. Intestinal tract-like structure induced by differentiation between activin and retinoic acid and a duct-like structure developed alongside it. Arrow). The gut-like structure in the center performs a slow peristaltic movement. This black spotted tissue is characterized by secretory tissue. B, Colony aggregates (EBS) of mouse ES cells cultured in the same manner under the conditions of V, activin and retinoic acid. The intestinal tract-like structure, duct-like structure, and tissue containing black spots are not seen in A. The scale bar is 100

[0273] (Identification of pancreas)

 The structure shown in Figure 2—— has an intestinal tract-like structure with slow peristaltic movement in the central part, and the tissue with the characteristics of secretory cells connected to it by a duct-like structure is characteristic of the natural pancreas. Show. This structure was observed with an electron microscope (Fig. 3).

 [0274] (Morphological identification of ES cell-derived pancreatic thread and weave)

Thread and histological examination of EBS sections after treatment with 0. retinoic acid and 10 ng / ml activin 11 days after the final treatment (17 days after EBS formation) showed spherical threads and weaves. It was shown that this tissue was strongly stained with toluidine blue in the region adjacent to the tubular lumen (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 (Figure 3Eb). Analysis by electron micrographs showed that the cells had well-developed endoplasmic reticulum and contained large amounts of zymogen particles (Fig. 4Ca, b). This is the same phenotype as pancreatic exocrine cells (acinar cells). The tissue formed from the treated EBS was also similar to 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 that resembled the pancreatic duct, surrounded by a low columnar epithelium (Figure 4Cd). In addition, cells that were morphologically similar to pancreatic endocrine cells (Fig. 4Cc, d) were also observed, but these cells were similar to exocrine cells. It was less powerful than the ones. These endocrine-like cells contained granules (Fig. C4d), which were morphologically different from the granules found in the secretory cells (Fig. C4b). A clear space between the dark central region and the lateral membrane was also found, which is a typical feature of j8 cell granules in the pancreas. To ascertain whether they contain these granulogenic S insulin or amylase, 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 (Figure 5c). A relatively small number of cells were aggregates with c-peptide positive granules (Fig. 5d). These morphological findings indicate that visceral tissue, including pancreatic ducts, endocrine cells and exocrine cells, was formed from EBS treated with both retinoic acid and activin.

 [0275] Figure 3. A, Colony aggregate (EBS) force of mouse ES cells An electron micrograph of a tissue (see Figure 1) that contains black spots that have been induced to differentiate with activin and retinoic acid. B, Electron micrograph of exocrine cells of the rat fetus pancreas in late pregnancy (Bock, et al., 1997). Both cells contain a large number of granule-like structures with high electron density, and have very similar structures, such as having developed vesicles. ER, endoplasmic reticulum; N, nucleus.

 [0276] Figure 3. C, Mouse ES cell colony aggregate (EBS) force Electron microscopic image of a duct-like structure (Fig. 1) induced by differentiation with activin and retinoic acid. Natural! Very similar to the epithelial structure of the visceral duct. D, Electron micrograph of a cell with a secretory granule-like structure induced by differentiation with activin and retinoic acid (see Fig. 1). An enlarged image of a portion surrounded by a dotted line in the photograph is also shown. This image is the same as the endocrine granule in the mouse β-cell (Goping, et al., 2003; indicated by the arrow) shown on the right (E). There is a gap between it and the bag that wraps it (indicated by an arrow). Ν, nuclear.

[0277] The organ-like tissue shown in Fig. 2 has a high electron density !, (black), a large number of uniform granule-like structures, and a highly developed endoplasmic reticulum (ER). ) Were observed (see Figure 3-A). These cells are the external force of the rat fetus pancreas in late gestation, akinar cysts (Bock, et al., Microscopy reseach and technique). 37: 374-383, 1997) (see Figure 3-B). On the other hand, in the same pancreas-like organization, there are also cells that contain many structures that have gaps between the contents that have a somewhat lower electron density than that shown in Fig. 3-A and the bag structure that surrounds them. (See Figure 3-D). This cell shows the typical endocrine cell morphology of the pancreas (Fig. 3-E; Goping, et al., Micros copy reseach and technique 61: 448-456, 2003). It strongly suggested that a trap occurred. In this way, both the cells with the characteristic of exocrine cells and the cells with the characteristics of endocrine cells were observed in the pancreas-like tissue formed by simultaneous treatment of activin and retinoic acid by electron microscope observation. .

[0278] As described above, typically from mouse ES cell-derived colony aggregates (EBS) treated with 10—25 ngZmL of activin and 0.1 M retinoic acid simultaneously for 2 days in vitro. It was possible to induce organs with the following structure. 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, the endocrine cells of the pancreas), glucagon (special to ex cells, the endocrine cells of the viscera), amylase 2 (su, in the viscera) Ipfl / pdx-1, pancreatic and duodenal homeoboxf actor— 1, a gene that dominates the development of pancreas). And Shh (soni c hedgehog, a gene that negatively regulates the development of the viscera and has a component)

[0279] (Pancreatic marker gene expression test)

The expression of genes that regulate visceral development and some visceral marker genes were investigated using RT-PCR (FIG. 2c). EBS treated with both retinoic acid and activin are insulin II (beta cell marker), glucagons (a cell), pancreatic polypeptide (gamma cell), somatostatin (delta cell) and amylase 2 (marker of exocrine pancreatic cell) In addition, the expression of Ipf lZpdx-1 and ptf laZp4 was markedly increased. Ipfl / pdx-1 and ptflaZp4 are genes that regulate pancreatic development. In contrast, untreated EBS had almost no expression of these genes. amylase and pancreatic polypeptide were processed on day 6 Strength increased over time (this is the 12th day of EBS formation ability). On the other hand, increased gene expression of insulin II, glucagon, somatostatin, Ipfl / pdx-1 and ptfla / p48 started immediately after the end of treatment. In untreated EBS, Shh force was found to be expressed during the S observation period. On the other hand, in EBS treated with both retinoic acid and activin, Shh gene expression was repressed after treatment.

 [0280] When the concentration of activin was kept constant (lOngZml) and the concentration of retinoic acid was varied, the gene expression of the pancreatic marker in EBS was as follows when 0.1 M retinoic acid was treated with 10 ng / ml of activin A The most efficient ones were divided (Fig. 2d). When these concentrations of retinoic acid and activin were used, a treatment EBS power of 47.6 ± 7.3% was described, exceeding all of the visceral genetic markers! If the concentration of retinoic acid is 0.001 μΜ, 0.01 μΜ, or 1 μΜ, it will be EBS [4.3! 2.5%, 12.8 ± 9. 6% and 25. 1 ± 6.6% gene expression.

 [0281] Fig. 4. Colony aggregate (EBS) strength of mouse ES cells. MRNA expression profile obtained by RT-PCR of visceral structures induced by differentiation with activin and retinoic acid. Insulin 2, glucagon, amylase 2 and Ipfl / pdx-1 are marker genes that are specifically expressed in pancreas development (positive markers), while Shh is a gene that decreases in expression with pancreas development (negative). Marker). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a constitutive protein (enzyme) that is expressed at a high level in all cells, so it was used for the purpose of assuring that the attached assay method was appropriate.

[0282] As a result, when treated with 10 ngZmL of activin A and 0.1 μM retinoic acid, it was confirmed that all positive marker groups also newly developed on the second day after treatment. In addition, these positive marker groups were not detected in the untreated group, or very few were detected. 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, not only endocrine cells (ex cells and β cells) but also exocrine cells can be prepared from colony aggregates (EBS) derived from mouse ES cells. The gene expression level is that the organ can be induced to differentiate in vitro. I understood with the bell.

 [0283] Next, the effect on the expression of insulin 2 and amylase 2 in the marker group with positive concentration of activin and retinoic acid was examined.

 [0284] Figure 5. Differentiation of mouse ES cell colony aggregates (EBS) by treatment with different activin concentrations (0, 10, 25 ngZmL) and retinoic acid concentrations (0, 0.1, 1.0 M) Difference in mRNA expression levels of insulin 2 and amylase 2 in the pancreas structure. The vertical axis shows 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 μΜ repulsive, and that it was the optimum concentration for the expression of mRNA of insulin 2 and amylase 2. Next, the insulin 2 gene is expressed most efficiently when treated with 25 ngZmL concentration of activin, also treated with 0.1 M retinoic acid, while when the activin concentration is 10 ngZmL. It was found that the amylase gene was most efficiently expressed. This result indicates that endocrine cells and exocrine cells are preferentially induced to differentiate from embryoid bodies derived from mouse ES cells, depending on the treatment concentration of activin.

[0286] Next, in these positive marker groups, it was examined histoimmunologically whether or not the insulin and amylase genes were actually transcribed and expressed as proteins. 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 genetically translated as preinsulin, a precursor, and immediately becomes proinsulin with the pre-part cleaved. This proinsulin is secreted out of the cell as active insulin which is cleaved again to remove the C peptide. That is, toヽvisceral created from a colony aggregates (EBS) in in V it _ro is to make sure that you secreting newly synthesized insulin and C peptide Nag is an antibody that binds to insulin alone Tissue immunology was performed using a binding antibody (goat anti-C peptide antibody, Linco Research). For amylase !, the usual antibody (Sigma) was used.

[0287] Figure 4-1. Immunohistological observation image of aggregates (EBS) of colonies derived from mouse ES cells treated with 10 ngZmL of activin and 0.1 M of retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino- 2-phenylindole; nuclear staining Agent), D, A + B + C. The scale bar is 50 μΜ. Insulin C peptide and amylase are made by different cells. Under these conditions, amylase is preferentially synthesized over insulin.

 [0288] Figure 4-2. Immunohistochemical image of colony aggregates (EBS) derived from mouse ES cells treated with 25 ngZmL of activin and 0.1 M retinoic acid. A, insulin C peptide; B, amylase; C, DAPI (4 ', 6-diamidino- 2-phenylindole; nuclear dye), D, A + B + C. The scale bar is 50 M. Similar to Figure 4-1, the force that insulin C peptide and amylase are made by different cells. Under this condition, insulin is preferentially synthesized over amylase.

 [0289] From the results of Fig. 4 and Fig. 3, it was found that 0.1 ^ Μ retinoic acid was the best in inducing differentiation of both endocrine and exocrine cells, and the concentration of activin was 25 ng / It was found that endocrine cells were preferentially induced when ml, but exocrine cells were preferentially induced when activin concentration was 10 ng / mL.

 [0290] (Concentration dependence)

 Ability to change the concentration of activin Changes the ratio of exocrine cells to endocrine cells.

[0291] The present inventors further tested that the effect of inducing pancreatic differentiation on EBS by activin and retinoic acid was concentration-dependent. To answer this question, total RNA was extracted from EBS 13 days after treatment with retinoic acid (0, 0.1 M or 1 μΜ) and activin (OngZml, lOngZm or 25 ngZml), and Real-time PCR was performed to compare the expression level of untreated EBS (normalized to 1) to measure the transcriptional expression levels of the following genes: amylase 2, insulin II, ptfla / p48 and Shh (Figure 6a— 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 non-treated EBS in EBS treated with retinoic acid alone than in untreated EBS. Expression was decreasing (Fig. 6a, b). Interesting results were observed when the concentration of retinoic acid was kept constant (0.1 μΜ) and the concentration of activin was varied. At relatively low concentrations of lactivin (1 OngZml), amylase2 and ptf laZp48 (markers for exocrine cells) The expression level of 1) was much higher than that of untreated EBS in the army treated with retinoic acid and akutibin, while insulin II expression did not vary between these groups. In contrast, at higher activin concentrations (25 ngZml), insulin expression was significantly increased in EBS treated with retinoic acid and activin compared to the untreated group, while the expression level of amylase 2 was 2 One group was low.

[0292] Real-time PCR results are supported by staining with both anti-insulin C peptide antibody and anti-amylase antibody (Paper Figure 7a-d). Fewer insulin-positive cells were seen than amylase-positive cells in EBS treated with 0.1 M retinoic acid and low concentrations (lOngZml) of activin. However, when a high concentration of Akuchibin (25 ng / ml) with retinoic acid 0. 1 μ Μ, _^0/0 of insulin-positive cells increased markedly (Figure 7d). In untreated EBS, insulin positive cells and amylase positive cells were not 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 (Figure 7e).

 [0293] (Summary)

 A new method of simultaneous treatment of activin and retinoic acid from a colony of mouse ES cells using a suspension culture system, synthesizing and secreting β cells and glucagon that secrete insulin from undifferentiated cells. We have succeeded in making the in vitro the endocrine cells of both OC cells, the exocrine cells that synthesize and secrete amylase, and the pancreas with the conduit for releasing the secretions in vitro for the first time in the world.

 [Example 2: Glucose responsiveness]

 In this example, pancreatic force generated in vitro by treating 25 ngZmL of activin and 0.1 μΜ of retinoic acid was used to determine whether or not the ability to increase insulin production in response to external glucose. (Table 1).

 [0295] To test insulin secretion in the in vitro mouth, 25 ng / ml activin and 0.1

The pancreatic tissue, which was also analyzed for ES cell force using μΜ retinoic acid, was tested by a low-concentration (3 mM) and high-concentration (25 mM) dalcose load test (Table 1). ACTIVIN Z REC EOS treated with noic acid secreted insulin into the medium in a dalcose-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 conditioned medium per mg of protein (about 9.7% total insulin per EBS). These results indicate that EBS treated with both retinoic acid and activin secreted insulin in response to increased glucose concentration. This is similar to the physiological function that the pancreas | 8 cell force has in vivo.

 [0296]

 Table 1. The amount of insulin secreted by the spleen in response to glucose concentration, prepared by simultaneous treatment of 25 ngZmL activin and 0.1 M retinoic acid for 13 days!

[0297] [Table 1]

Insulin secretion (pg)

 + 25 m glucose + 3 mM glucose pancreas (activin + RA) 46.0 ± 2.6 36.5 ± 2.8 untreated 37.5 ± 3.4 37.0 ± 4.2

RA, retinoic acid; mean soil SD (n = 8)

[0298] Table 1 shows changes in insulin production by changes in intracellular insulin level and glucose of EBS.

 [0299] Intracellular insulin levels in EBS treated with EBS treated with 0.1 μΜ retinoic acid and 25 ngZml of activin 13 days after induction treatment! The amount of insulin secreted from EBS was measured in response to the dalcose load. The indicated amount of insulin secreted is comparable to the amount of insulin secreted 30 minutes after the start of stimulation with 25 mM glucose. Increased insulin secretion with glucose loading in the culture medium was observed in EBS treated with both retinoin and activin.

[0300] (Example 3: Transplantation method of spleen tissue induced to differentiate in culture system) It is demonstrated that the mouse pancreas produced in Examples 1 and 2 can actually be used in a diabetes model mouse. The procedure will be described below.

[0301] (Animal)

 Diabetes model mice are males of AKITA— mice (Yoshioka, et al., Diabetes 46: 887, 1997) over 10 weeks old, or streptozotocin (STZ) that selectively destroys the splenic islets of 250 mg / kg. Use nude mice whose body condition has been caused by administration of body weight. In this case, transplantation should be performed after 1 week after administration of STZ.

[0302] (Transplantation of differentiation-induced spleen tissue)

 For transplantation, spleen 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 forceps. This is washed twice with Hanks saline.

 Mice are anesthetized with Nembutal, the abdomen is dissected, and 20-30 differentiated spleen tissues are massed and transplanted under the retroperitoneum. Implanting under the kidney capsule is also effective as another transplant site. Alternatively, spleen tissue can be treated with Liberase RI (Roche), an enzyme for islet isolation, to isolate cells and inject them into the portal vein of the liver.

 [0303] The blood glucose level is measured by scratching the tip of the mouse's tail with a scalpel or force razor and collecting several μl of blood.

 [0304] In this way, it can be confirmed whether or not the pancreas produced in the present invention actually functions in a model mouse.

 [0305] As described above, it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of the specific preferred embodiments of the present invention based on the description of the present invention and common general technical knowledge. Patents, patent applications and documents cited in this specification should be incorporated by reference in their entirety, as if the contents themselves were specifically described in this specification. Understood.

Industrial applicability [0306] By this method, from unsatisfactory mammalian cells that have not been realized so far,

 1) Have β-cells that synthesize and secrete insulin in response to glucose load,

2) As an endocrine cell, it has an ex cell that synthesizes and secretes glucagon only in β cells,

 3) Has exocrine cells (acinar cells) that make digestive enzymes (such as amylase) and secrete them into the intestinal tract,

 4) Pancreatic power The world's first pancreas with all functions and structures, such as a conduit structure for exoculating digestive enzymes into the intestinal tract, has been made.

 [0307] Human ES cell power [0307] Cells derived 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 a complete pancreas can be made as shown by the present inventors using only β cells from human undifferentiated cells by the method developed by the present inventors, visceral dysfunction likely to occur in many diabetic patients. In addition to patients, it is necessary to remove the spleen for visceral cancer, etc. ヽ It may provide unprecedented useful materials for treating patients. In addition, biological models related to pancreas' medical research, research on the cause of pancreatic diseases, and useful models and evaluation systems in the development of drugs for the treatment of pancreatic diseases and pancreatic cancer (Atsusei system) It can also be expected. Thus, the practical application of this method and the industrial prospects are judged to be extremely bright!

Claims

The scope of the claims

 [I] A method for producing pancreatic pancreas with unbroken 匕 cell force, 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) An exposure step of exposing the undifferentiated cells to activin and retinoic acid in the absence of LIF.

 [2] The method according to claim 1, wherein the undifferentiated cells are cultured on a feeder cell.

[3] The method according to claim 1, wherein the aggregate is an embryoid body-like sphere.

 [4] The method according to claim 1, wherein the pre-culture step is performed for 1 to 10 days.

[5] The method according to claim 1, wherein the exposing step is performed for 1 to 4 days.

6. The method according to claim 1, wherein the undifferentiated cell is a mammalian cell.

7. The method according to claim 1, wherein the culture medium contains serum.

 [8] The method of claim 1, wherein the medium is a knockout serum replacement (KSR) medium.

[9] The method according to claim 1, wherein the exposing step is performed under conditions of suspension culture.

[10] The method according to claim 9, wherein the suspension culture is performed on a low adhesion coating culture vessel.

 [I I] The method according to claim 9, wherein the undifferentiated cells are embryonic stem (ES) cells.

 12. The method according to claim 2, wherein the feeder cell is a mouse fetal fibroblast.

[13] The method of claim 1, wherein the activin is less than 50 ngZml.

[14] The method of claim 1, wherein the retinoic acid is more than 0.01 μΜ: less than LΜ.

[15] The method according to claim 1, further comprising a differentiation step of culturing the differentiated aggregate in the medium on the coated culture vessel.

16. The method of claim 15, wherein the coating is a gelatin coating.

[17] The method according to claim 15, wherein the medium used in the separating step is a KSR medium.

18. The method according to claim 15, wherein the differentiation step is a period sufficient for producing a functional pancreas.

[19] The method according to claim 15, wherein the differentiation step is performed for 7 to 21 days.

 [20] The differentiation step is first cultivated in a medium containing activin and retinoic acid, and then

And culturing in a medium free of activin and retinoic acid.

5. The method according to 5.

 [21] In the differentiation step, the cells are first cultured in a medium containing activin and retinoic acid for about 1 to 4 days, and then cultured in a medium not containing activin and retinoic acid for 1 to 3 weeks. 16. A method according to claim 15 comprising the steps.

[22] The method according to claim 15, wherein the working concentrations of activin and retinoic acid are conditions selected from the group consisting of claims 13-14.

[23] The functional stem / spleen has at least one characteristic selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability, and functional duct structure ability, The method of claim 1.

[24] The functional stem / spleen expresses a pancreatic marker Ipfl / pdx-Ι, and does not express a marker Shh that decreases its expression level when the pancreas is formed. The method according to claim 1.

 [25] The method of claim 1, wherein the functional pancreas expresses at least one pancreatic marker selected from the group consisting of insulin, glucagon and amylase 2 force.

26. The method according to claim 1, wherein the functional stem / spleen comprises ex cells, β cells, exocrine cells, and a duct structure.

[27] The functional stem / spleen has all the characteristics selected from the group consisting of insulin-producing ability, glucose-responsiveness, glucagon-producing ability, amylase-producing ability and functional conduit structure ability. Item 2. The method according to Item 1.

[28] A soot / spleen produced by the method according to claim 1.

 [29] A method for the treatment of a patient in need of a functional pancreas, characterized by using the pancreas according to claim 28.

30. The method of claim 29, wherein the patient is suffering from diabetes.

[31] The method of claim 29, wherein the patient is a mammal.

[32] activin for the manufacture of a medicament for the treatment of patients in need of a functional pancreas And the use of retinoic acid.

 [33] The use of claim 32, wherein the patient is afflicted with diabetes.

[34] The use of claim 32, wherein the patient is a mammal.

[35] The use according to claim 32, wherein the medicament is produced from stem cells.

[36] The use according to claim 32, wherein the medicament is produced by embryonic stem cell force.