JP2006238835A - Marker for adult hematopoietic stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify a factor for efficiently distinguish adult hematopoietic stem cells. <P>SOLUTION: The problem is solved by finding that endomucin itself unpredictably has a marker function of efficiently distinguishing adult hematopoietic stem cells from fetal hematopoietic stem cells. Consequently, a composition for detecting adult hematopoietic stem cells comprises a factor specific to a nucleic acid molecule containing a nucleic acid sequence encoding endomucin or its part is obtained. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to regenerative medicine and stem cells. More particularly, the present invention relates to markers associated with adult hematopoietic stem cells, determination methods, kits and systems associated therewith, and treatment and prevention utilizing the same.

  Disease treatment by regenerative medicine (regenerative medicine) has recently attracted attention. However, it has not yet been routinely applied to many patients with organ or tissue dysfunction. To date, treatment of such patients has been only applied to patients with limited use of assistive systems in medical devices in addition to organ transplantation. However, these therapies have problems such as donor shortage, rejection, infection, and service life. In particular, the shortage of donors is a serious problem, and in the case of bone marrow transplantation, it is difficult to provide a limited sample to many patients even though the bone marrow or cord blood bank is gradually enriched in Japan and overseas. Therefore, in order to overcome these problems, there is an increasing expectation for regenerative medicine centered on stem cell therapy and its application.

  The body of an organism loses a part of its organ or suffers a major injury during a lifetime due to trauma or disease. In that case, whether or not a damaged organ can be regenerated depends on the organ (or animal species). Regenerative medicine is to regenerate organs (or tissues) that cannot be regenerated naturally and restore their functions. Whether an organization has regenerated can be determined by whether its function has improved. Mammals have a certain level of power to regenerate tissues and organs (eg, regeneration of skin, liver and blood). However, it has been considered that organs such as the heart, lungs, and brain have poor regenerative ability, and that once they are damaged, their functions cannot be regenerated. Therefore, conventionally, for example, when an organ is damaged, there is almost no effective measure except treatment by organ transplantation.

  It has long been assumed that stem cells are present in organs with high regeneration ability. The correctness of this concept has been proved by experimental bone marrow transplantation using animal models. Subsequent studies revealed that stem cells in the bone marrow are the source of all blood cell regeneration. It has also been clarified that stem cells are present in organs with high regeneration ability such as bone marrow and skin. Furthermore, it has become clear that stem cells exist even in the brain, which has long been thought not to be regenerated. That is, it has been found that there are stem cells in every organ in the body, and more or less is responsible for the regeneration of each organ. In addition, stem cells present in each tissue are more plastic than expected, and it has been pointed out that stem cells in a certain organ can be used for regeneration of other organs.

  Stem cells may also be used to regenerate organs that were previously impossible. Regenerative medicine is gaining increasing attention, especially in stem cell therapy. In recent years, as the need for research on regeneration from the medical / medical field has increased, knowledge about stem cells or organogenesis has been increasing every year. For example, the establishment of embryonic stem cells (ES cells) having totipotency and the production of cloned individuals from adult somatic cells have attracted particular attention. Because development and regeneration technologies can be used for stem cell therapy, regenerative medicine using stem cells is in preparation for clinical application in various fields. There are also fields that are already in practical use.

  In regenerative medicine, organ-organ reconstruction is the most important. In this case, there are broadly divided into a method for constructing an organ in vitro and using it as an artificial organ, and a method for reconstructing the organ in vivo. In either case, stem cells are required for organ construction. Important properties for the stem cells used here include pluripotency and self-renewal ability.

  Stem cells are roughly classified into embryonic stem cells and somatic (tissue) stem cells. Among somatic stem cells, hematopoietic stem cells have attracted attention for a long time. In order to maintain a short-lived mature blood cell for the lifetime of a human, hematopoietic stem cells must have the ability of self-renewal and pluripotency, which was already proposed in 1961 (Non-patent Document 1). ). Since a bone marrow transplantation method using a mouse model was established in 1972 (Non-patent Document 2) and hematopoietic stem cells can be detected by examining hematopoietic system remodeling, research on hematopoietic stem cells has been a breakthrough. Made progress. Subsequent research has shown the presence of hematopoietic stem cells having both self-renewal ability and pluripotency (Non-patent Document 3). However, hematopoietic stem cells are present at a low frequency of about several in 100,000 bone marrow cells even in the case of mice, so that they need to be concentrated and purified for actual research or clinical application.

  Conventionally, hematopoietic stem cell transplantation treatment has been performed, but there are various side effects because natural cells are concentrated. For example, side effects (RRT) due to pre-transplantation treatment with high dose anticancer drugs or radiation were present. Bacterial / fungal infections during bone marrow suppression by pretreatment, bleeding; in the case of transplantation from another person, when the donor's white blood cells have engrafted and increased, the patient's organ is regarded as a foreign body (other person) and attacks ( Graft-versus-host disease = GVHD); various pulmonary complications centering on cytomegalovirus (CMV) pneumonia; various visceral disorders due to damage of vascular endothelial cells (cells lining the blood vessels); Various infectious diseases affected during prolonged immunosuppressive state (at least 1 to 2 years); chronic GVHD with prolonged and various symptoms; side effects such as secondary carcinogenesis, gonadal dysfunction, late effects such as infertility was there.

  Due to the above complications, it is often the case that the transplantation is performed, but the general condition temporarily worsens. In addition, about 10 to 20% of patients who die due to complications are found in autologous transplants, and about 20 to 40% in transplants from others. In addition, even if these complications are overcome, the original disease may recur, and the current transplantation treatment has been limited.

  In order to prevent early death due to complications after bone marrow transplantation, treatment methods have been developed that isolate and purify stem cells, produce large amounts of progenitor cells from stem cells in vitro, and transplant them together with stem cells. Clinical trials are already being attempted.

  Since the introduction of FACS (fluorescence activated cell sorter) in the 1980s, methods using FACS have been frequently used for enrichment and purification of hematopoietic stem cells. It has been clarified that high-purity hematopoietic stem cells can be obtained by separating CD34-KSL cells from multiple stained bone marrow cells (Non-patent Document 4). As mentioned above, pluripotency and self-renewal ability are the essence of stem cells. In order to fully exploit its ability, it is important to concentrate and purify stem cells and amplify them by in vitro culture. However, the enrichment necessary for clinical application requires the use of various markers, and the selection by the markers cannot be said to correlate with 100% transplant compatibility or stem cell undifferentiation. It has also been pointed out that the therapeutic effect is not always sufficient.

  Various proteins play an important role as a mechanism for regulating stem cell differentiation. For example, stem cell factor (steel cell factor or steel factor; also referred to as SCF) is a factor attracting attention in hematopoietic stem cells.

  SCF is produced by bone marrow stromal cells and acts on pluripotent stem cells, myeloid cells such as CFU-GM and CFU-Meg of CFU-GM, and lymphoid stem cells to support their differentiation. That is, it acts on the differentiation stage cells of almost all lineages from hematopoietic stem cells to differentiated cells, and helps to induce differentiation to the final differentiation stage by other cytokines (Non-patent Document 5). However, such differentiation factors cannot be markers.

  Cell surface markers such as CD34, Lin (lineage marker), c-Kit and Sca-1 are commonly used to identify stem cells. However, these markers are disadvantageous in that they are weak in specificity and are insufficient to determine transplant compatibility and stem cell undifferentiation alone. Also, there is a question as to whether adult stem cells can be separated by nearly 100% even when these markers are combined.

Hematopoietic cells include primary hematopoiesis that originates from hemangioblast (a common progenitor of vascular endothelial cells and primary hematopoietic cells) in the yolk sac, and the back of the paraaortic reproductive midrenal region (AGM region). It is derived from vascular endothelial cells of the aorta such as the lateral aorta, and is classified into secondary hematopoiesis that is responsible for fetal liver hematopoiesis and further adult bone marrow hematopoiesis (Non-patent Documents 6 to 8). At present, it is said that adult hematopoietic stem cells capable of reconstructing adult bone marrow hematopoiesis appear for the first time in the para-aortic reproductive midrenal region around the 11th day of gestation (Non-Patent Documents 8 to 9). On the other hand, hematopoietic stem cells found in yolk sac cells at this time can be engrafted only when injected into the neonatal liver, and it is assumed that there is a problem in the expression of homing molecules essential for engrafting directly into the bone marrow. (Non-Patent Document 10).
In any case, the generation of hematopoietic stem cells is closely related to vascular endothelial cells, and hematopoietic stem cells and vascular endothelial cells express many common molecules. Therefore, many membrane surface antigens specific to vascular endothelial cells are useful for identification of hematopoietic stem cells, and several molecules have been studied so far. For example, Tie-2, PCLP-1, and Sca-1 identify hemogenic endogenium having the ability to produce adult hematopoietic stem cells among the vascular endothelial cells of the dorsal aorta, and identification of adult hematopoietic stem cells derived from hemogenic endothelium. (Non-patent Documents 8, 11 to 12). On the other hand, it has been clarified that fetal hematopoiesis and adult hematopoiesis can be induced from ES cells, and Flk-1 positive VE-cadherin positive cells are regarded as hematopoietic endothelium (Non-patent Document 13). In an induction system using an embryoid body, it has been proved using a bone marrow transplantation system that hematopoietic stem cells that can reconstruct adult bone marrow hematopoiesis for a long time are generated (Non-Patent Documents 14 to 15). Currently, c-Kit and CD41 are used as markers for undifferentiated hematopoietic cells including adult (secondary hematopoietic) hematopoietic stem cells generated from ES cells or generated in the fetus (Non-Patent Documents 15 to 18). . However, they are expressed also in fetal type (primary hematopoietic) hematopoietic progenitor cells, and the purity of hematopoietic stem cells by these markers is insufficient, and more specific markers for hematopoietic stem cells are required.

On the other hand, a molecule called endomucin that exists in the cell membrane has been reported (Non-patent Documents 19 to 23). Endomucin is a sialomucin membrane protein expressed specifically in vascular endothelial cells (Non-patent Document 19). Applicants have identified that endmucin is also specifically expressed in adult hematopoietic stem cells, and expression of endmucin is expressed in CD34 ⁻ KSL hematopoietic stem cells and CD34 ⁺ KSL hematopoietic progenitor cells using a monoclonal antibody that recognizes endmucin. It was confirmed that all hematopoietic stem cells with long-term bone marrow remodeling ability expressed endomucin from analysis using bone marrow transplantation. Application 2003-391514, unpublished).

However, it is not known whether this molecule can distinguish adult hematopoietic stem cells.
Till, J .; E. , Et al. Radiat. Res. 14: 213-222, 1961 Micklem, H.M. S. et al. : J. Cell Physiol. 79: 293-298, 1972. Dick, J .; E. , Et al. Cell 42; 71-79, 1985; Osawa, M .; et al. , Science, 273: 242-245, 1996. S. Kitamura, Frontline of Cytokines, Yodosha, Toshio Hirano, pp. 174-187, 2000 Palis J, Yoder MC. (2001). Exp Hematol. 29, 927-936 Dzierzak E.M. (2003) Curr Opin Hematol. 10, 229-34. de Bruijn, M.M. F. , Ma, X. Robin, C .; , Ottersbach, K .; Sanchez, M .; J. et al. , And Dzierzak, E .; (2002). Immunity 16, 673-83 Muller, A.M. M.M. Medvinsky, A .; Stroboulis, J. et al. Grosveld, F .; , And Dzierzak, E .; (1994). Immunity 1, 291-301. Yoder, M.M. C. and Hiatt, K.K. (1997). Blood 89, 2176-2183 Hamaguchi, I. et al. Huang, X .; L. Takakura, N .; Tada, J .; Yamaguchi, Y .; , Kodama, H .; , And Suda, T .; (1999) Blood 93, 1549-1556. Hara, T .; Nakano, Y .; Tanaka, M .; , Tamura, K .; , Sekiguchi, T .; Minehata, K .; Copelland, N .; G. Jenkins, N .; A. Okabe, M .; , Kogo, H .; , Mukouyama, Y .; , And Miyajima, A .; (1999) Immunity 11, 567-78. Nishikawa SI, Nishikawa S, Hiroshima M, Matsushima N, Kodama H. (1998) Development. 125, 1747-1757. Kyba, M.M. , And Daley, G .; Q. (2003) Exp. Hematol. 31, 994-1006 Burt RK, Verda L, Kim DA, Oyama Y, Luo K, Link C.I. (2004) J Exp Med. 199, 895-904. Sanchez MJ, Holmes A, Miles C, Dzierzak E .; (1996) Immunity. 5, 513-525. Ferkowicz, M.M. J. et al. , Starr, M .; , Xie, X. Li, W .; Johnson, S .; A. Shelley, W .; C. Morrison, P .; R. , And Yoder, M.M. C. (2003) Development 130, 4393-4403. Mikcola, H.M. K. A. , Fijiwara, Y .; , Schlaeger, T .; M.M. , Traveler, D.A. , And Orkin, S .; H. (2003) Blood 101, 508-516. Morgan S.M. M.M. , Et al. , Blood 93, p165-175 (1999) Liu C.I. , Et al. , BBRC 288, 129-136 (2001) Kinoshita M. et al. et al. , FEBS Lett. 499.121-126 (2001) Ueno et al. , BBRC 287, 501-506 (2001) Samulowitz U. et al. , Am. J. et al. of Pathol. , 160, 1669-181, 2002)

  An object of the present invention is to identify a factor that can efficiently identify adult hematopoietic stem cells.

  The present invention, in part, as a result of intensive studies by the present inventors, endomucin itself unexpectedly has a marker function that can efficiently distinguish adult hematopoietic stem cells, particularly from fetal-derived types. As a result, the above-mentioned problems have been solved.

Endomucin is a sialomucin membrane protein expressed specifically in vascular endothelial cells. In the present invention, in the present invention, the expression of endomucin in the process of generating adult hematopoietic cells from ES cells is expressed by the pan-hematopoietic cell marker CD45 and the undifferentiated hematopoietic cell marker CD41 that is recognized early in the development of hematopoiesis. A detailed discussion was given in the context of this. As a result, most of the embryoid bodies 4-7 days after culture by forming embryoid bodies from ES cells are CD45-negative cells, CD41 + Endomucin +, CD41 ⁺ Endomucin ^-, CD41 ^- Endomucin ^+, CD41 ^- Endomucin ^- of being fractionated into four groups of cells, highly multipotent progenitor cells CD41 ⁺ Endomucin ⁺ cell fraction was revealed that they are concentrated. In addition, more mature blood cells positive for CD45 appeared in the embryoid bodies after the 7th day, but most of them were endmucin positive. Similar findings embryonic 10 corresponding to embryoid bodies 5 day ES occurrence site of adult hematopoiesis in 5 days fetuses are confirmed in the AGM region, CD41 + Endomucin in CD45 negative cells +, CD41 ⁺ Endomucin ^-., CD41 ^- Endomucin ^+, CD41 ^- Endomucin ^- in 4 fractions include CD41 ⁺ Endomucin ⁺ highly multipotent progenitor cells cell fraction is concentrated, further long-term permits reconstruction hematopoietic stem cells adult bone marrow hematopoiesis It was confirmed. Endomucin is a specific marker of adult hematopoietic stem cells, unlike C-Kit and CD41, because the expression of endomucin is not observed at all in the yolk sac of embryonic day 8.5, which is the place of fetal hematopoiesis. It was judged that there was. Endomucin is a novel marker specific to adult hematopoietic stem cells based on the results of ES cell hematopoiesis and fetal hematopoiesis, and is a useful molecule that can enrich adult hematopoietic stem cells more highly than known markers Became clear. If these findings are applied to human ES cells in the future, the industrial utility value will be high.

Accordingly, the present invention provides the following.
(1) A composition for detecting adult hematopoietic stem cells, comprising means for identifying endomucin.
(2) The composition according to the above item, wherein the adult hematopoietic stem cell comprises a cell having a long-term bone marrow remodeling ability.
(3) The composition according to the above item, wherein the identification means comprises a specific factor for a nucleic acid molecule comprising a nucleic acid sequence encoding endomucin or a part thereof.
(4) The above endmucin is
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof;
(B) in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, at least one amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and an organism A polypeptide having a biological activity;
(C) a polypeptide encoded by an allelic variant or splice variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10; or (e) identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is at least 70% and having biological activity;
The composition according to the above item, comprising:
(5) The composition according to the above item, wherein the endomucin comprises the sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10.
(6) The composition according to the above item, wherein the nucleic acid molecule comprises a sequence represented by SEQ ID NO: 1, 3, 5, 7 or 9.
(7) The composition according to the above item, wherein the means for identifying endomucin is an anti-endomucin antibody.
(8) The composition according to the above item, wherein the means for identifying endmucin is a nucleic acid molecule comprising a nucleic acid sequence encoding endmucin or a sequence complementary to a part thereof.
(9) The composition according to the above item, wherein the identification means is labeled or labelable.
(10) The composition according to the above item, wherein the label is selected from the group consisting of a radioactive substance, a chemiluminescent substance, a fluorescent substance, a phosphorescent substance, a color former, a ligand, a hapten, and raw materials thereof.
(11) The composition according to the above item, further comprising means for identifying a marker.
(12) The composition according to the above item, wherein the additional marker comprises at least one marker selected from the group consisting of CD34, Lin (Lineage marker), c-Kit, CD41 and CD45.
(13) The composition according to the above item, wherein the additional marker comprises at least one marker selected from the group consisting of CD41 and CD45.
(14) The composition according to the above item, wherein the additional marker comprises CD41 and CD45.
(15) The composition according to the above item, wherein the marker has an ability to discriminate between fetal hematopoietic progenitor cells and adult hematopoietic stem cells.
(16) The composition according to the above item, wherein the adult hematopoietic stem cell is a cell having pluripotency.
(17) The composition according to the above item, wherein the adult hematopoietic stem cell is a cell derived from an embryonic stem cell.
(18) The composition according to the above item, wherein the adult hematopoietic stem cell is a cell used for bone marrow transplantation.
(19) The composition according to the above item, wherein the adult hematopoietic stem cell is a cell produced by forming an embryoid body from an embryonic stem cell.
(20) The composition according to the above item, wherein the label contains at least one dye selected from the group consisting of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin.
(21) A method for detecting adult hematopoietic stem cells in a sample,
A) A method comprising the step of bringing the sample into contact with a means for identifying endmucin.
(22) Furthermore,
B) determining that adult hematopoietic stem cells are present when the above-mentioned endmucin-positive cells are found,
The method according to the above item, comprising:
(23) C) The method further comprises contacting the sample with a marker factor identification means selected from the group consisting of CD41 and CD45.
The method described in the above item.
(24) It is determined that adult hematopoietic stem cells are present when the CD41-positive cells are found and at least one selected from the group consisting of when the CD45-positive cells are found The method described in the above item.
(25) The method according to the above item, wherein the sample contains or is expected to contain embryonic stem cells.
(26) A kit for detecting adult hematopoietic stem cells in a sample, the kit comprising:
A) A kit comprising means for identifying endomucin.
(27) A method for producing a composition in which adult hematopoietic stem cells are isolated or concentrated,
A) providing a source comprising or expected to contain adult hematopoietic stem cells;
B) contacting the source with endumin identification means;
C) separating or concentrating endomucin positive cells,
Including the method.
(28) The method according to the above item, wherein the identification means is an anti-endomucin antibody, and the concentration is performed by flow cytometry.
(29) The method according to the above item, further comprising a step of concentrating CD41 positive cells.
(30) The method according to the above item, further comprising a step of concentrating CD45 negative cells.
(31) determining the reactivity of the sample to a further marker selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit, and determining said CD34, Lin (Lineage marker), CD34, CD41 and The c-Kit gene expression indicates that the candidate cell is a stem cell, and the negative expression of the Lin (Lineage marker) gene indicates that the candidate cell is a stem cell. The method according to the above item, further comprising a step of concentrating.
(32) The method according to the above item, wherein the separation or concentration is performed using panning by an antibody-immobilized dish, flow cytometry, or magnetic beads.
(33) The method according to the above item, wherein the separation or concentration is performed by combining FACS and magnetic beads.
(34) A kit for producing a composition in which adult hematopoietic stem cells are isolated or concentrated,
A) means for identifying endomucin;
B) means for contacting said identifying means with a source comprising or expected to contain adult hematopoietic stem cells; and C) means for isolating or concentrating endomucin positive cells;
A kit comprising:
(35) An adult hematopoietic stem cell composition prepared by the method described in the above item.
(36) An adult hematopoietic stem cell composition in which cells expressing endomucin and CD41 are concentrated.
(37) The adult hematopoietic stem cell composition according to the above item, wherein the adult hematopoietic stem cell composition is enriched with cells that do not express CD45.
(38) A method for determining whether a candidate cell is an adult hematopoietic stem cell,
(I) providing a candidate cell to be determined;
(II) contacting the candidate cell with a nucleic acid molecule encoding endomucin or a part thereof, or an agent that specifically reacts with endomucin polypeptide or a part thereof; and (III) the factor and the endmucin. A step of confirming whether the gene for the endmutin is expressed in the candidate cell by detecting a specific reaction with the encoding nucleic acid molecule or a portion thereof, or an endmucin polypeptide or a portion thereof,
Wherein the endomucin gene expression in the candidate cell indicates that the candidate cell is an adult hematopoietic stem cell.
(39) The method according to the above item, wherein the expression is confirmed by flow cytometry.
(40) further comprising contacting the candidate cell with at least one factor specific for a marker selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit, ,
The gene expression of CD34, CD41 and c-Kit in the candidate cell indicates that the candidate cell is a stem cell, and the negative expression of the Lin (Lineage marker) gene indicates that the candidate cell is a stem cell The method according to the above item, which indicates that
(41) The method according to the above item, further comprising a step of depleting a lineage positive cell.
(42) The said depletion process is a method as described in said item performed by MACS.
(43) The method according to the above item, wherein the depletion step is performed using at least one LIN (Lineage) marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220. .
(44) A kit for determining whether a candidate cell is an adult hematopoietic stem cell,
(I) means for identifying endomucin;
(II) A kit comprising means for detecting the expression of endomucin in a candidate cell by the identification means, wherein the gene expression of the endomucin in the candidate cell indicates that the candidate cell is an adult hematopoietic stem cell.
(45) A method for treating, preventing or prognosing a subject requiring transplantation of adult hematopoietic stem cells,
(I) a step of confirming whether the gene for endomucin is expressed in the cells to be transplanted;
(II) a step of isolating or concentrating cells expressing the endomucin gene; and (III) a step of transplanting the isolated or enriched cells into the subject.
Including the method.
(46) A system for treating, preventing or prognosing a subject requiring transplantation of adult hematopoietic stem cells,
(I) means for identifying endomucin;
(II) means for isolating or concentrating cells expressing the endomucin gene; and (III) means for transplanting the separated or enriched cells into the subject,
A system comprising:
(47) A medicament for treatment, prevention or prognosis of a subject requiring transplantation of adult hematopoietic stem cells,
A) a cell population enriched with stem cells expressing endomucin,
Containing a medicament.
(48) Use of endomucin for the determination of adult hematopoietic stem cells.
(49) Use of stem cells expressing endomucin for producing a medicament for transplantation of adult hematopoietic stem cells.

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

  According to the present invention, adult hematopoietic stem cells can be easily identified, separated and concentrated. In addition, the present invention has made it possible to determine transplantation compatibility of adult hematopoietic stem cells easily and efficiently.

  As a transplant used in the method of the present invention, for example, transplantation of adult hematopoietic stem cells derived from embryonic stem cells (ES cells) can be mentioned as a representative example. Here, among the stem cells, examples of the purpose and target diseases for transplantation of adult hematopoietic stem cells include the following. 1) Diseases in which qualitative and / or quantitatively abnormal stem cells need to be replaced with normal stem cells: severe aplastic anemia, myelodysplastic syndrome, thalassemia, congenital immune deficiency syndrome, etc .; Diseases that need to avoid irreversible lymphoid or hematopoietic tissue damage due to chemotherapy or radiation: leukemia (excluding chronic lymphocytic leukemia (CLL)), malignant lymphoma, multiple myeloma, solid (For example, breast cancer, neuroblastoma, small cell lung cancer, ovarian cancer, etc.); 3) diseases requiring permanent enzyme supplementation: inborn errors of metabolism.

  With the use of the method of the present invention, adult hematopoietic stem cells can be efficiently selected, separated and concentrated, so that more effective treatment and reduction of side effects can be achieved in the transplantation of adult hematopoietic stem cells as described above. Can now do. In addition, if an amplification factor or the like is used, it is possible to prepare an amount necessary for treatment after efficiently removing a small amount of adult hematopoietic stem cells. After preparing the required amount, appropriate cells may be further concentrated by the method for determining adult hematopoietic stem cells of the present invention. In such cases, rejection (eg, graft-versus-host disease) is reduced or impossible.

  Endomucin is a novel marker specific to adult hematopoietic stem cells, and it has become clear that it is a useful molecule that can enrich adult hematopoietic stem cells to a higher degree than known markers. it is conceivable that.

  Hereinafter, preferred embodiments of the present invention will be described. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, singular articles (for example, “a”, “an”, “the” in the case of English, “ein”, “der”, “das”, “die”, etc. in the case of German and their case changes) Corresponding articles in other languages such as "un", "une", "le", "la" in Spanish, "un", "una", "el", "la", etc. It should be understood that the adjectives, etc. also include the plural concept thereof unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

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

(Endomucin)
As used herein, “endomucin” is a sequence as shown in SEQ ID NO: 1, 3, 5, 7 or 9 (nucleic acid sequence) and SEQ ID NO: 2, 4, 6, 8 or 10 (amino acid sequence). And the corresponding factor (ortholog) in other species of animals. Endomucin is a sialomucin protein identified as a membrane surface molecule that is specifically expressed in vascular endothelial cells (Morgan SM, et al., Blood 93, p165-175 (1999); Liu C., et al. , BBRC 288, 129-136 (2001)). It is said to be a type 248 amino acid (mouse) type I membrane protein with a large number of threonine and serine residues (35%) outside the cell and is also known to be susceptible to degradation by O-sialoglycoprotein endopeptidase. (Morgan et al., Ibid.). In humans, it is known as a molecule having 261 amino acids (Liu et al., Ibid.). This molecule is said to have an apparent molecular weight of 45 kDa by sialidase and O-glycosidase. Furthermore, in 2001, it was reported that endomucin 1 and endomucin 2 exist in humans (Kinoshita M. et al., FEBS Lett. 499. 121-126 (2001)). These molecules were identified as molecules of approximately 25 kDa type I membrane protein with multiple glycosylation motifs that are susceptible to O-sialoglycoprotein endopeptidase digestion. It has been suggested that its properties negatively regulate cell adhesion to the extracellular matrix.

  Endomucin has been reported to be strongly expressed in the dorsal aorta of mouse embryos, especially in endothelial cells, in which four splice variants (full length = 1a = 1-261 (identical to Kinoshita et al. ) 1b = 130-142 (same as reported by Morgan et al.), 1c = 92-129 is missing, 1d = 92-142 is reported) (Ueno et al., BBRC 287) , 501-506 (2001)) (SEQ ID NO: 1-2, SEQ ID NO: 3-4, 5-6, 7-8 (odd numbers are nucleic acid sequences, even numbers are amino acid numbers), respectively). In the present specification, it is understood that 1a is a full-length sequence and has the sequence shown in SEQ ID NOs: 1 and 2 (odd numbers are nucleic acid sequences and even numbers are amino acid numbers). In the present invention it is understood that all these variants can be used as markers. Endomucin is also present in humans and has been shown to have the sequences shown in SEQ ID NOs: 9 and 10 (odd numbers are nucleic acid sequences and even numbers are amino acid numbers), and the above splice variants exist It is understood that

  Endomucin has also recently been reported as a potential L-selectin ligand (Samlouitz U. et al., Am. J. of Pathol., 160, 1669-181, 2002).

  However, these reports only show that endmucin is specifically expressed on vascular endothelium (especially venous endothelium, capillaries; less expressed on arterial endothelium) and its function is not known. Although it has been suggested to be used in the diagnosis of cancer and in relation to extracellular matrix, expression analysis in stem cells has not been performed, and it was not known to be a marker for transplantation compatibility. Et al., Japanese Patent Application No. 2003-391514 confirmed that it becomes a stem cell and transplantation compatibility marker.

  Endomucin is known not only in humans but also in mammals including rats and mice, and its homologue is known. Therefore, in this specification, endmucin usually refers to endmucin present in mammals in addition to humans. Endomucin is also known for various splice variants (or isoforms), but it is understood that all of these splice variants can be used as markers.

  Endomucin used in the present invention can be produced artificially or synthetically. Examples of such an artificially produced endmucin include endometin into which a mutation has been introduced.

  To determine whether it is endmucin, it is sufficient that a significant activity can be determined by at least one of the endmutin activities. Examples of such endomucin activity include molecules that are recognized by endomucin-specific monoclonal antibodies, rich in addition of o-glycoside-linked sugar chains, and highly expressed in specific cell lines in vitro. Examples include, but are not limited to, the property of inhibiting adhesion. Examples of methods for confirming such activity include, but are not limited to, sugar chain identification assay, cell transformation assay for adhesion inhibition, etc. It is understood that a method for determining activity can be constructed as appropriate.

  In the endmucin used in the present invention, as a moiety to which a sugar chain can be added, an N-glucoside-bondable moiety (asauparagine-X-serine / threonine) to which N-acetyl-D-glucosamine and the like bind, and N-acetyl -The part (part which a serine or threonine residue appears frequently) which carries out O- glycosidic bonds, such as -D-galactosamine and sialic acid, is mentioned. As used herein, endomucin or endomucin is not particularly affected by the presence or absence of sugar chains, but proteins with these sugar chains are usually stable against degradation in vivo. And may have strong physiological activity. Therefore, polypeptides to which these sugar chains are added are also within the scope of the present invention.

(cell)
In the present invention, cells that are intended to determine the undifferentiated state and / or transplantation compatibility are targeted, and usually stem cells can be used, but cells that can become desired cells by treatment with the factor of the present invention Any cell can be used as long as it is.

  In the present specification, the “stem cell” is defined as a cell having self-replicating ability and pluripotency, and actually refers to a cell that can regenerate the tissue when it is damaged. Stem cells used in the present invention can be embryonic stem cells (ES) or tissue stem cells (also referred to as tissue-specific stem cells or somatic stem cells). Embryonic stem cells refer to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Thus, in one preferred embodiment of the present invention, embryonic stem cells (Embryonic stem cells and Embryonic germ cells) can be used as the cells. In another preferred embodiment, tissue stem cells (eg, bone marrow cells (eg, hematopoietic stem cells)) can be used.

  As used herein, “progenitor cell” refers to a cell having the ability to differentiate into a cell when referring to the cell. Thus, it is understood that hematopoietic progenitor cells can become hematopoietic cells upon differentiation. In the present specification, “stem cell” and “progenitor cell” are used interchangeably.

  As used herein, “hemangioblast” refers to a cell derived from mesoderm that is a source of blood vessels and blood cells. Initially, it forms an isolated cell population, also called a blood island. Things around it become flat and differentiate into endothelial cells. The cells in the center become hematopoietic stem cells from which various blood cells differentiate. Blood islands eventually coalesce into continuous blood vessels. Accordingly, hemangioblasts can be referred to as blood cell and vascular progenitor cells.

  As used herein, “adult hematopoietic stem cell” is used interchangeably with “adult hematopoietic stem cell” or “adult hematopoietic progenitor cell”, such as the dorsal aorta of the paraaortic reproductive midrenal region (AGM region) This refers to stem cells derived from aortic vascular endothelial cells and responsible for fetal liver hematopoiesis, and also for secondary bone hematopoiesis (Non-Patent Documents 6 to 8). At present, adult hematopoietic stem cells capable of reconstructing adult bone marrow hematopoiesis are said to appear for the first time in the para-aortic reproductive midrenal region around the 11th day of embryogenesis (Non-Patent Documents 8 to 9). On the other hand, hematopoietic stem cells found in yolk sac cells at this time can be engrafted only when injected into the neonatal liver, and it is assumed that there is a problem in the expression of homing molecules essential for engrafting directly into the bone marrow. (Non-Patent Document 10). It became clear that fetal hematopoiesis and adult hematopoiesis can also be induced from ES cells, and Flk-1-positive VE-cadherin-positive cells are regarded as hematopoietic endothelium (Non-patent Document 13). In an induction system using an embryoid body, it has been proved using a bone marrow transplantation system that hematopoietic stem cells that can reconstruct adult bone marrow hematopoiesis for a long time are generated (Non-Patent Documents 14 to 15).

  In this specification, “fetal hematopoietic stem cell” refers to a stem cell responsible for primary hematopoiesis that is generated from hemangioblast (a common progenitor cell of vascular endothelial cells and primary hematopoietic cells) in the yolk sac and is responsible for hematopoiesis in the mid-fetal period. Say. Different from adult type, fetal hematopoietic stem cells cannot fully take on the functions of adult type, so for those who need adult type hematopoietic stem cells, we provide adult type of hematopoietic stem cells It is necessary. Fetal hematopoietic stem cells are often produced in the yolk sac of day 8.5.

  As a method for measuring stem cells such as adult stem cells used in the present specification, for example, a colony assay method (eg, stem cell culture method), a flow cytometry (FCM) method using CD34 as an index (eg, fluorescence histogram) Display method = FL display conventional method, fluorescent one side scattered light display method = FL-SSC method) and the like. In the colony assay method, proliferation ability and number can be evaluated by counting the number of colonies obtained by culturing stem cells on a medium. In the fluorescence histogram display method, after staining with a fluorescently labeled anti-CD34 antibody, the cells are counted by FCM, and all cells showing FL values higher than the control are counted as positive cells. Therefore, it can be said that the fluorescence histogram display method is a numerical evaluation method. However, this display method is calculated including some monocytes, so it may be extremely high. In the FL-SSC method, after staining with a fluorescently labeled anti-CD34 antibody, counting is performed by FCM, and only a stem cell region clearly separated from others on a two-dimensional graph drawn by FL value and SSC value is regarded as a positive cell. Count. This can also be said to be a number evaluation method. Furthermore, since the FL-SSC method is hardly affected by monocytes, it has a feature that the correlation with the colony assay is extremely good.

  As an index of stem cells, for example, CD41 (SEQ ID NOs: 33 to 36), CD45 (SEQ ID NOs: 37 to 40), CD34 (SEQ ID NOs: 13 to 16), Lin (Lineage marker), c-Kit (SEQ ID NOs: 25 to 26). ), Sca-1 (SEQ ID NO: 27-28) and Flt3 / Flk2 (SEQ ID NO: 11-12), but are not limited thereto.

  In the present specification, “Lin (marker)”, “Lineage (marker)”, and “differentiation antigen (marker)” are used interchangeably and refer to a marker of differentiation state. This means something that has not been differentiated. Examples of the Lin marker include Gr-1, Mac-1 (SEQ ID NO: 17-18), TER119, CD4 (SEQ ID NO: 19-20), CD8 (SEQ ID NO: 21-22), and B220 (SEQ ID NO: 23-24). However, it is not limited to them.

  Unlike embryonic stem cells, tissue stem cells are cells in which the direction of differentiation is relatively limited, are present in tissues, and have an undifferentiated intracellular structure. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. Thus, in one preferred embodiment of the invention, tissue stem cells directed to blood cells as cells can be used.

  Tissue stem cells can be classified into ectoderm, mesoderm, and endoderm-derived stem cells according to origin. Tissue stem cells derived from ectoderm include neural stem cells that are present in the brain, epidermal stem cells that are present in the skin, hair follicle stem cells, and pigment stem cells. Tissue stem cells derived from mesoderm include hemangioblasts, hematopoietic stem cells and mesenchymal stem cells found in bone marrow and blood. Endoderm-derived tissue stem cells are mainly present in organs, and include liver stem cells, pancreatic stem cells, and intestinal epithelial stem cells. In addition, germ line stem cells exist in the sperm and egg layers. In a preferred embodiment of the present invention, mesoderm-derived stem cells can be used. In a more preferred embodiment of the invention, bone marrow cells (eg, hematopoietic stem cells) can be used.

  When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, and nervous system. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells. In one embodiment of the invention, it has been unexpectedly found that bone marrow cells are preferred.

  In a preferred embodiment, in the present invention, bone marrow cells can be used as a source as they are, or a specific cell population (eg, tissue stem cells) that has been concentrated or purified can be used as a cell source.

  In this specification, “regeneration” means that a damaged tissue or organ is restored to its original state, and is also called pathological regeneration. The body of an organism loses a part of its organ or suffers a major injury during a lifetime due to trauma or disease. In that case, whether or not a damaged organ can be regenerated depends on the organ (or animal species). Regenerative medicine is to regenerate organs (or tissues) that cannot be regenerated naturally and restore their functions. Whether an organization has regenerated can be determined by whether its function has improved. Mammals have a certain level of power to regenerate tissues and organs (eg, regeneration of skin, liver and blood). However, it has been considered that organs such as the heart, lungs, and brain have poor regenerative ability, and that once they are damaged, their functions cannot be regenerated. Therefore, conventionally, for example, when the heart is damaged, only a treatment by heart transplantation has been effective.

  It has long been assumed that stem cells are present in organs with high regeneration ability. The correctness of this concept has been proved by experimental bone marrow transplantation using animal models. Subsequent studies revealed that stem cells in the bone marrow are the source of all blood cell regeneration. It has also been clarified that stem cells are present in organs with high regeneration ability such as bone marrow and skin. Furthermore, it has become clear that stem cells also exist in the brain, heart, etc., which have long been thought to be unregenerated. That is, it has been found that there are stem cells in every organ in the body, and more or less is responsible for the regeneration of each organ. In addition, stem cells present in each tissue are more plastic than expected, and it has been pointed out that stem cells in a certain organ can be used for regeneration of other organs. Therefore, the amplification action found in the present invention is effective in such various tissue stem cells, and its uses are infinite.

  As used herein, “differentiation” or “cell differentiation” refers to two or more types of cells that have morphologically and functionally qualitative differences among daughter cell populations derived from the division of one cell. This is a phenomenon that occurs. Therefore, the process in which a cell population (cell lineage) derived from cells that cannot originally detect special characteristics exhibits distinct characteristics such as production of a specific protein is also included in differentiation. At present, it is common to consider cell differentiation as a state in which a specific gene group in the genome is expressed, and cell differentiation by searching for intracellular or extracellular factors or conditions that bring about such gene expression state. Can be identified. The result of cell differentiation is in principle stable, and in animal cells in particular, differentiation to another type of cell occurs only exceptionally. Therefore, “undifferentiated” refers to the state of cells that have no morphological and functional qualitative differences.

  As used herein, “maintenance” in an undifferentiated state refers to maintaining pluripotency. Therefore, whether or not the undifferentiated state is maintained can be determined by determining whether or not pluripotency is maintained.

  In the present specification, “expansion” of a stem cell means that the number of cells increases while maintaining the self-amplification ability and pluripotency (multipotency) of the stem cell.

  In the present specification, “differentiated cells” refer to cells (for example, skeletal muscle cells, nerve cells, cardiomyocytes, blood cells, etc.) that have specialized functions and morphologies. Unlike stem cells, they are pluripotent. There is little or no. Examples of differentiated cells include epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, bile duct cells, blood cells (eg, erythrocytes, platelets, T cells, B cells, etc.), cardiomyocytes, skeletal muscle cells, osteoblasts. Examples include cells, skeletal myoblasts, nerve cells, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells, chondrocytes and the like. Accordingly, in one embodiment of the present invention, when a certain source cell can be differentiated into a differentiated cell such as leukocyte by treating with a factor of the present invention (for example, endomucin polypeptide or nucleic acid), Such differentiated cells are also within the scope of the present invention.

  The cells used as the source of the present invention can be differentiated into hematopoietic cells by treatment with the polypeptide, nucleic acid, composition, kit and / or medicament used in the present invention.

  As used herein, “pluripotency” or “pluripotency” is used interchangeably, refers to the nature of a cell, and refers to the ability to differentiate into cells belonging to various tissues or organs. Cell pluripotency is usually limited as development progresses, and in adults, differentiated cells that make up one tissue or organ rarely change into cells of another tissue or organ. Therefore, pluripotency is usually lost. When this happens, it is usually a pathological condition and is called metaplasia. However, mesenchymal cells have a high degree of pluripotency because they are likely to undergo metaplasia instead of other mesenchymal cells with relatively simple stimulation. Therefore, the source cell of the present invention may preferably be a pluripotent cell itself, but this is not always necessary.

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

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

(Biochemistry / Molecular biology)
As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acid may be natural or non-natural and may be a modified amino acid. The term can also be assembled into a complex of multiple polypeptide chains. The term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is included. When specifically mentioned, the “polypeptide” of the present invention may refer to endomucin or endomucin.

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

  As used herein, “nucleic acid” is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. Particular nucleic acid sequences also include “splice variants”. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. As the name suggests, a “splice variant” is the product of alternative splicing of a gene. After transcription, the initial nucleic acid transcript can be spliced such that different (another) nucleic acid splice products encode different polypeptides. The production mechanism of splice variants varies, but includes exon alternative splicing. Other polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any product of a splicing reaction (including recombinant forms of splice products) is included in this definition. When used in the present invention, endomucin can also take this nucleic acid form. In particular, endomucin has been shown to undergo various splice mutations, and it is assumed that all of them function as markers, and therefore all of them are of the undifferentiated state of stem cells and / or transplantation compatibility. It is understood that it is used as a marker.

  As used herein, “gene” refers to a factor that defines a genetic trait. Usually arranged in a certain order on the chromosome. A gene that defines the primary structure of a protein is called a structural gene, and a gene that affects its expression is called a regulatory gene. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein” “polypeptide”, “oligopeptide” and “peptide”. As used herein, “homology” of genes refers to the degree of identity of two or more gene sequences with respect to each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous.

  In the present specification, comparison of similarity, homology and identity of amino acid and base sequences is calculated using BLAST, which is a tool for sequence analysis, using default parameters. Where specifically mentioned, identity searches can be performed, for example, using NCBI's BLAST 2.2.9 (issued 2004.5.12). In the present specification, the identity value usually refers to a value when the above BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value.

  As used herein, “ligand” refers to a substance that specifically binds to a protein. Examples of the ligand include lectins, antigens, antibodies, hormones, cytokines, and extracellular matrices that specifically bind to various receptor protein molecules present on the cell membrane. Therefore, it is understood that the ligand for the end mucin of the present invention can be used as a factor for detecting the marker of the present invention.

  As used herein, “polynucleotide that hybridizes under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization is performed according to methods described in experimental documents such as 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). It can be done according to this. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. Therefore, a polypeptide (eg, endmucin 1 or a splice variant thereof) used in the present invention is hybridized under stringent conditions to a nucleic acid molecule encoding a polypeptide particularly described in the present invention. Also included are polypeptides encoded by soy nucleic acid molecules.

  In the present specification, the “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide has at least 60% or more homology with the base sequence of the DNA encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8 or 10. A polynucleotide, preferably a polynucleotide having 80% or more homology, and more preferably a polynucleotide having 95% or more homology. Nucleic acid sequence homology is shown in score by using, for example, a search program BLAST using an algorithm developed by Altschul et al. (J. Mol. Biol. 215, 403-410 (1990)).

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y.1989), 3rd edition (2001) And Anderson et al., Nucleic Acid Hybridization: A Practical approach, IV, IRL Press Limited (Oxford, England). Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. See Anderson et al., Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

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

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

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of 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 of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

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

  As used herein, an “isolated” biological factor (eg, a cell, nucleic acid or protein, or factor thereto) is defined as any other factor in the cell of the organism in which the biological factor naturally occurs. Biological factors (for example, in the case of a nucleic acid, a nucleic acid that includes a non-nucleic acid factor and a nucleic acid sequence other than the target nucleic acid; in the case of a protein, a protein that includes a non-protein factor and an amino acid sequence other than the target protein. Etc.) is substantially separated or purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins. In the case of a cell, it means that a cell having a specific property (for example, an adult hematopoietic stem cell) is separated and isolated from a cell having another property (for example, a differentiated cell, a fetal stem cell, etc.). .

  As used herein, a “purified” or “purified” biological agent (eg, a cell, nucleic acid or protein, or a factor thereto) is at least an agent that naturally accompanies the biological agent. This refers to a part that has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present. In the case of cells, cells with certain properties (eg, adult hematopoietic stem cells) are separated from cells with other properties (eg, differentiated cells, fetal stem cells, etc.) and purified / purified. Say.

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

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA can also be an aspect of expression. More preferably, such a polypeptide form may be post-translationally processed. In a preferred embodiment, such an expressed polypeptide can be glycosylated endmucin. In the present invention, expression is understood to include both transcription and translation. Therefore, it should be understood that when confirming the expression of the gene for endomucin of the present invention, it is contemplated to confirm the presence of the transcript and / or the translation.

  As used herein, “expression” is “negative”, “weakly positive”, “positive” or “strongly positive” as observed by protein expression when the primary antibody (eg, biotinylated anti-endomucin antibody) and It is shown by fluorescence intensity (FI) when a secondary antibody (for example, PE-labeled streptavidin) is used. Where A: Strongly positive (High (++)): FI> 500; B: Weakly positive (dull (+)): 100 <FI <500; C: Negative (negative (-)): FI <100 A + B is collectively referred to as positive. In the case of endmucin, it is called endmucin positive. “Expression” as “negative”, “weakly positive”, “positive” or “strongly positive” may alternatively be expressed as −, ±, +, ++ herein.

  For the expression intensity at the mRNA level, RT-PCR or expression analysis using a microarray can be used. When RT-PCR is used, it can be quantified relatively using a densitometer, and when quantified in more detail, less expression relative to the housekeeping gene HRPT in the microarray Negative, equivalent can be weak positive, and stronger (statistically strong) levels can be expressed as strong positive.

  In this specification, the term “interaction” refers to two substances. Force (for example, intermolecular force (van der Waals force), hydrogen bond, hydrophobic interaction between one substance and the other substance. Etc.). Usually, two interacting substances are in an associated or bound state. Therefore, when a factor specific to the marker is used to confirm the presence of the marker, the presence of the marker can be confirmed by confirming the interaction between the factor and the marker.

  The term “binding” as used herein means a physical or chemical interaction between two proteins or compounds or related proteins or compounds, or a combination thereof. Bonds include ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. A physical interaction (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effects of another protein or compound and does not involve other substantial chemical intermediates. Therefore, when confirming the expression of endmucin of the present invention, it is understood that the expression of endmucin has been confirmed by confirming the binding using a factor specific thereto.

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

Natural nucleic acids encoding proteins such as endomucin or variants or fragments thereof include, for example, PCR primers that contain all or part of a nucleic acid sequence such as SEQ ID NO: 1, 3, 5, 7 or 9, or variants thereof. Easily separated from a cDNA library with hybridization probes. Nucleic acids encoding preferred endomucins or variants or fragments thereof are essentially 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; high containing 7% SDS at a temperature of 42 ° C. More preferably essentially under low stringency conditions defined by a hybridization buffer, and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C. Hybridization buffer containing 1% bovine serum albumin (BSA) at a temperature of 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS, and 1 × essentially 50 ° C. SSC (300 mM NaCl; 30 mM Sodium citrate); 1% bovine serum albumin (BSA) under low stringent conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS and essentially 0.5x SSC at 65 ° C (150 mM NaCl; 15 mM sodium citrate); wash containing 0.1% SDS It can hybridize to one or a portion of the sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9 under low stringency conditions as defined by the buffer.

  In the present specification, the “identification means” of endmucin refers to any means capable of identifying endmucin (eg, endmucin polypeptide, endmucin nucleic acid, etc.). As used herein, it is understood that any means known in the art can be used as long as endmucin expression can be identified. For example, a probe, a primer, an antibody, other having specific binding, and the like. Can be mentioned. For example, a nucleic acid molecule that can hybridize under stringent conditions with a nucleic acid encoding the endmucin of the present invention or a part thereof can be used as a probe. It can be understood that such a nucleic acid molecule is useful as a probe because it can bind a nucleic acid encoding endomucin even when cross-linked to other molecules. Such identification means are usually labeled or labelable, either directly or indirectly.

  In the present specification, the “marker” refers to an index that can confirm the presence of a certain molecule (eg, end mucin), and examples thereof include CD antigens (eg, CD34, CD41, CD45, etc.). It is not limited to.

  In the present specification, the “detection composition” refers to a composition for detecting the presence of a certain molecule (for example, endomucin).

  As used herein, the term “probe” refers to a substance to be searched for in biological experiments such as screening in vitro and / or in vivo. For example, a nucleic acid molecule containing a specific base sequence or a specific Examples include, but are not limited to, peptides containing amino acid sequences.

  Examples of nucleic acid molecules that are usually used as probes include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is homologous or complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotide lengths, at least 14 contiguous nucleotide lengths, at least 15 contiguous nucleotide lengths, at least 20 contiguous nucleotide lengths, at least 25 contiguous nucleotide lengths, at least 30 It can be at least a nucleic acid sequence of at least 40 contiguous nucleotides, at least 50 contiguous nucleotides, of contiguous nucleotides. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included. Preferably, the probe can be labeled with a label.

  Examples of proteins that are usually used as probes include, but are not limited to, antibodies or derivatives thereof. When a protein is used as a probe, it can be directly or indirectly labeled with a label.

  As used herein, “search” refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods. Say. Electronic search includes BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)), the Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and the Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Bio3. 48: -453 (1970)), but is not limited thereto. Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, endomucin or other marker (CD41, CD45, CD34, etc.) used as a marker in the present invention includes a corresponding gene identified by such an electronic search or biological search. It is intended to be.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. Is also possible. Here, a portion of the polynucleotide or polypeptide sequence within the comparison window is a reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequence contains additions, Reference sequences herein should be free of additions or deletions) and may include additions or deletions (ie gaps). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol.Biol.215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res.22 (2): 4673-4680, Higgins et al., 1996, Methods Enzymol.266: 383-402. , Altschul et al., 1990, J. Mol. Biol. 215 (3): 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272), but is not limited thereto. In particularly preferred embodiments, the Basic Local Alignment Tool (BLAST) (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 90, well known in the art. J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res. 25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

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

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

  In the present specification, the “primer” refers to a substance necessary for initiation of a reaction of a polymer compound to be synthesized in a polymer synthase reaction. In the synthesis reaction of a nucleic acid molecule, a nucleic acid molecule (for example, DNA or RNA) complementary to a partial sequence of a polymer compound to be synthesized can be used.

  Examples of nucleic acid molecules that are usually used as primers include those having a nucleic acid sequence of at least 8 consecutive nucleotides that is complementary to the nucleic acid sequence of the target gene. Such a nucleic acid sequence is preferably at least 12 contiguous nucleotides long, at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, and even more preferably at least 11 contiguous nucleotides. At least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 At least 19 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 Nucleotides long that connection, at least 50 contiguous nucleotides in length, may be a nucleic acid sequence. Nucleic acid sequences used as probes are nucleic acid sequences that are at least 70% homologous, more preferably at least 80% homologous, more preferably at least 90% homologous, at least 95% homologous to the sequences described above. Is included. A sequence suitable as a primer may vary depending on the nature of the sequence intended for synthesis (amplification), but those skilled in the art can appropriately design a primer according to the intended sequence. Such primer design is well known in the art, and may be performed manually or using a computer program (eg, LASERGENE, PrimerSelect, DNAStar).

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” 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 derivative amino acids and amino acid analogs are well known in the art.

  As used herein, “natural amino acid” means an L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to in this specification are in L form.

  As used herein, “unnatural amino acid” means an amino acid that is not normally found naturally in proteins. Examples of non-natural amino acids include the aforementioned D-form amino acids, norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine D-form or L-form, and D-phenylalanine Is mentioned.

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

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

The character codes are as follows.
3 letter code of amino acid 1 letter code Meaning Ala A alanine Cys C cysteine Asp D aspartic acid Glu E glutamic acid Phe F phenylalanine Gly G glycine His H histidine Ile I isoleucine Lys K lysine Leu L leucine Met M methionine Asl G Glutamine Arg R Arginine Ser S Serine Thr T Threonine Val V Valine Trp W Tryptophan Tyr Y Tyrosine Asx Asparagine or Aspartate Glx Glutamine or Glutamate Xaa Unknown or other amino acids.
Base symbol Meaning
a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine / uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine / uracil keto group s guanine or cytosine w adenine or thymine / uracil b guanine or cytosine Thymine / uracil d adenine or guanine or thymine / uracil h adenine or cytosine or thymine / uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine / uracil, unknown or other base.

  As used herein, a “corresponding” amino acid or nucleic acid has or has the same action as a predetermined amino acid or nucleotide in a reference polypeptide or polynucleotide in a polypeptide molecule or polynucleotide molecule. In particular, in the case of an enzyme molecule, it means an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity. For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. In the endomucin of the present invention, the corresponding amino acid can be, for example, a site that is phosphorylated. In another embodiment, in the endomucin of the present invention, the corresponding amino acid may be an amino acid responsible for glycosylation. Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain.

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

  In the present specification, the “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. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-type nucleic acids (PNA). Not.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit.

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

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

  As used herein, an “agent” can be any substance or other element (eg, energy such as light, radioactivity, heat, electricity, etc.) that can achieve the intended purpose. Good. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

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

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

  As used herein, “contacting” means bringing a compound into physical proximity, either directly or indirectly, to a polypeptide or polynucleotide of the present invention. . The polypeptide or polynucleotide can be present in many buffers, salts, solutions, and the like. Contact includes placing the compound in, for example, a beaker, microtiter plate, cell culture flask or microarray (eg, gene chip) containing a polypeptide encoding a nucleic acid molecule or fragment thereof.

  As used herein, “biological activity” refers to an activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes an activity that exhibits various functions. For example, when an agent is a ligand, the biological activity includes the activity of the ligand binding to the corresponding receptor. In the case of the endomucin of the present invention, the biological activity is at least one of the activities possessed by endomucin (such as the ability to negatively regulate cell adhesion to the extracellular matrix, the ability to bind to endmutin-specific antibodies, etc.). Include.

  As used herein, a method for assaying biological activity includes, but is not limited to, an assay using (for example, the above-described assay for determining endomucin).

  As a method for producing the polypeptide used in the present invention, for example, primary cultured cells or established cells that produce the polypeptide are cultured, and the polypeptide is isolated or purified from the culture supernatant or the like. The method of obtaining is mentioned. Alternatively, using a genetic manipulation technique, a gene encoding the polypeptide is incorporated into an appropriate expression vector, and an expression host is transformed using the gene, and a recombinant polypeptide is obtained from the culture supernatant of the transformed cell. Obtainable. The host cell is not particularly limited as long as it expresses a polypeptide having physiological activity, and various host cells conventionally used in genetic manipulation (for example, E. coli, yeast, animal cells, etc.) are used. It is possible. As long as the polypeptide derived from the cells thus obtained has substantially the same action as the native polypeptide, one or more amino acids in the amino acid sequence are substituted, added and / or deleted. The sugar chain may be substituted, added and / or deleted.

  Certain amino acids can be substituted for other amino acids in a protein structure, such as, for example, a cationic region or a binding site of a substrate molecule, without an apparent reduction or loss of interaction binding capacity. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

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

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

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

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. 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; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine; However, it is not limited to these.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, splice variants and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, the orthologs of the endomucins of the present invention may also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

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

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

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

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

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

  Chemical derivatization of the polypeptides of the present invention may be performed under suitable conditions used to react biologically active substances with activated polymer molecules. A method for preparing a PEGylated polypeptide of the invention generally comprises the following steps: (a) conditions such that endomucin or an active form thereof or an activator thereof 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. It is easy for those skilled in the art to select the optimal reaction conditions or acylation reaction based on known parameters and the desired result.

  PEGylated polypeptides of the invention can generally be used to treat conditions that can be alleviated or modulated by administering the polypeptides described herein, but herein The chemically derivatized polypeptide molecules of the present invention disclosed in (1) may have additional activity, increased or decreased biological activity, or other characteristics (eg, increased) compared to their non-derivatized molecules. Reduced half-life). The polypeptides, fragments, variants and derivatives thereof of the invention can be used alone, in combination, or in combination with other pharmaceutical compositions.

  In the present invention, a nucleic acid form (nucleic acid molecule) can also be used as long as the expressed substance generates endmucin constitutively or transiently. As long as the expressed polypeptide has substantially the same activity as that of the native endomucin, a part of the nucleic acid sequence is deleted or substituted with other bases as described above. Or a part of other nucleic acid sequence may be inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

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

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

  The polypeptide used in the present invention may be derived from any organism. Preferably, the organism is a vertebrate (eg, mammal, reptile, amphibian, fish, bird, etc.), more preferably a mammal (eg, rodent (mouse, rat, etc.), primate (human). Etc.). The polypeptide used in the present invention may be synthesized as long as the desired effect is exhibited. Such polypeptides can be synthesized by synthetic methods well known in the art. For example, a synthesis method using an automatic solid-phase peptide synthesizer is described in Stewart, JM et al. (1984). Solid Phase Peptide Synthesis, Pierce Chemical Co .; Grant, GA (1992). Synthetic Peptides: A User's Guide, WH Freeman. Bodanszky, M. (1993). Principlesof Peptide Synthesis, Springer-Verlag; Bodanszky, M. et al. (1994). The Practiceof Peptide Synthesis, Springer-Verlag; Fields, GB (1997). Phase PeptideSynthesis, Academic Press; Pennington, MW et al. (1994). Peptide Synthesis Protocols, Humana Press; Fields, GB (1997). Solid-Phase Peptide Synthesis, Academic Press.

  The polypeptide used in the present invention (for example, an agent specific for an endmucin polypeptide or polynucleotide) expresses a fusion protein with only the hinge region portion of the antibody, and dimers via disulfide bonds. Higher than a dimer containing a fusion protein made to be expressed at the C-terminus, N-terminus, or elsewhere, in a form that forms a disulfide bond in a way that forms a disulfide bond in a way that does not affect activity Multimeric polypeptides having specific activity can also be utilized. In addition, a method of arranging a sequence in series to give a multimeric structure can also be used in the present invention. Accordingly, any dimer or more form produced by genetic engineering techniques is within the scope of the present invention. Alternatively, the monomers contained in the dimer of the present invention or in a form exceeding the dimer may be the same or different.

(Immunology)
In the present specification, the term “antibody” is used in the meaning normally used in the art, and in the present specification, the whole antibody and fragments, derivatives, conjugates and the like thereof are also included. An antibody that recognizes the polypeptide used in the present invention is preferred, and an antibody that specifically recognizes the polypeptide used in the present invention is more preferred. Such an antibody may be either a polyclonal antibody or a monoclonal antibody. In one embodiment of the invention, such antibodies are also encompassed within the scope of the invention.

  As used herein, the term “antigen” refers to a substance that binds to an antibody or binds to a specific receptor such as B lymphocyte or T lymphocyte to cause an immune reaction such as antibody production and / or cell damage (for example, Protein, lipid, sugar and the like, but not limited thereto). Binding to an antibody or lymphocyte receptor is referred to as “antigenicity”. Properties that induce an immune response, such as antibody production, are referred to as “immunogenicity”. The substance used as the antigen includes, for example, at least one target substance (for example, protein). The substance to be contained is preferably full length, but may be a partial sequence as long as it contains at least one epitope capable of inducing immunity. As used herein, “epitope” or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided.

  Epitopes can be used even if their exact location and structure are not known. Thus, an epitope is required for a set of amino acid residues involved in recognition by a particular immunoglobulin, or in the case of T cells, for recognition by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors. A set of amino acid residues is included. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. In the immune system field, in vivo or in vitro, an epitope is a molecular feature (eg, primary peptide structure, secondary peptide structure or tertiary peptide structure and charge) and is recognized by an immunoglobulin, T cell receptor or HLA molecule. Form a site. An epitope comprising a peptide can comprise more than two amino acids in a spatial conformation unique to the epitope. In general, an epitope consists of at least 5 such amino acids, typically consisting of at least 6, 7, 8, 9, or 10 such amino acids. Longer epitope lengths are generally preferred because they are more similar to the antigenicity of the original peptide, but may not necessarily be so when considering conformation. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. Furthermore, identification of epitopes in a given protein is readily accomplished using techniques well known in the art. See, for example, Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998 (a general method for rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); US Pat. No. 4,708,871 (identifying epitopes of antigen and See Procedure for Chemical Synthesis); and Geysen et al. (1986) Molecular Immunology 23: 709 (a technique for identifying peptides with high affinity for a given antibody). Antibodies that recognize the same epitope can be identified in a simple immunoassay. Thus, methods for determining epitopes comprising peptides are well known in the art, and once such epitopes are provided with the primary sequence of a nucleic acid or amino acid, those skilled in the art will use such well known techniques. Can be determined.

  Thus, for use as an epitope comprising a peptide, a sequence of at least 3 amino acids in length is required, preferably this sequence is at least 4 amino acids, more preferably at least 5 amino acids, at least 6 amino acids, at least 7 amino acids. A sequence of amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids in length may be required. Epitopes may be linear or conformational.

  Macromolecular structures (eg, polypeptide structures) can be described in terms of various levels of configuration. For a general discussion of this configuration, see, for example, Alberts et al. , Molecular Biology of the Cell (3rd edition, 1994) and Canter and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally arranged three-dimensional structures within a polypeptide. These structures are generally known as domains. A domain is a portion of a polypeptide that forms a compact unit of the polypeptide and is typically 50-350 amino acids long. Exemplary domains are made up of parts such as β-sheets (such as β-strands) and α-helix stretches. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to a three-dimensional structure formed by non-covalent association of independent tertiary units. Terms relating to anisotropy are used in the same way as terms known in the energy field. Therefore, the polypeptide used in the present invention can include a polypeptide having any amino acid sequence as long as it has a higher-order structure having an ability equivalent to that of endomucin.

  In the present specification, a gene is "specifically expressed" means that the gene is expressed at a level (preferably high) that is different from (preferably high) at least one other site or time in a specific site or time in the living body. That means. With specific expression, it may be expressed only at a certain site (specific site) or may be expressed at other sites. The expression specifically preferably means that it is expressed only at a certain site. Therefore, the gene encoding the endmucin of the present invention can be manipulated so that it is expressed specifically at the site or time when expression is desired. Such techniques are well known in the art and are described in the literature incorporated herein.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in the present specification 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-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocolsin Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCRProtocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). ShortProtocols in Molecular Biology: A Compendium of Methods from Current Protocolsin Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995) PCRStrategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, andannual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics , Academic Press, separate experimental medicine “Gene Transfer & Expression Analysis Experimental Method”, Yodosha, 1997, etc., and related parts (which may be all) are incorporated herein by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). TheBiochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM etal. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Technologies, Academic Press, etc. These are incorporated herein by reference in the relevant part.

(Genetic engineering)
Endomucin and the like and fragments and variants thereof used in the present invention and factors (for example, antibodies and the like) thereto can be produced using genetic engineering techniques.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Such a vector can be autonomously replicated in a host cell such as an individual animal, or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. Are illustrated. As used herein, a vector can be a plasmid.

  As used herein, “virus vector” refers to a virus-derived vector. As used herein, the term “virus” refers to an infectious microstructure that has either DNA or RNA as a genome and that grows only in infected cells. Viruses include retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, poxviridae, herpesviridae, baculoviridae and hepa A virus belonging to a family selected from the group consisting of the family Donnaviridae. As used herein, “retrovirus” refers to a virus having genetic information in the form of RNA and synthesizing DNA from the RNA information by reverse transcriptase.

  In the present specification, the “retroviral vector” refers to a form using a retrovirus as a gene carrier (vector). Examples of the “retroviral vector” used in the present invention include, but are not limited to, a retrovirus type expression vector based on Moloney Murine Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV), and the like. Preferably, retroviral vectors include, but are not limited to, pGen-, pMSCV, and the like.

  As used herein, “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory element used can vary depending on the host cell. In the case of humans, the expression vector used in the present invention may further contain pCAGGS (Niwa H et al, Gene; 108: 193-9 (1991)).

  As used herein, “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence into a target cell. Examples of such vectors include those that can autonomously replicate in a host cell such as an individual animal or can be integrated into a chromosome and contain a promoter at a position suitable for transcription of the polynucleotide of the present invention. Is done.

  In this specification, “recombinant vectors” for animal cells include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 (Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3,133 (1990)), pREP4 (Invitrogen) ), PAGE103 (J. Biochem., 101, 1307 (1987)), pAMo, pAMoA (J. Biol. Chem., 268, 22882-22787 (1993)), pCAGGS (NiwaH et al, Gene; 108: 193- 9 (1991)).

  In the present specification, the terminator is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene. Examples of the terminator include, but are not limited to, a CaMV35S terminator, a nopaline synthase gene terminator (Tnos), and a tobacco PR1a gene terminator in addition to mammalian terminators.

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

  In the present specification, when used for expression of a gene, in general, “site specificity” means the expression of the gene in a part of an organism (eg, animal) (eg, in the case of an animal, heart, cardiomyocyte, etc.). Specificity. “Time specificity” refers to the specificity of expression of a gene according to a specific stage (eg, at the time of an attack) of an organism (eg, an animal). Such specificity can be introduced into a desired organism by selecting an appropriate promoter.

  In the present specification, an “enhancer” can be used to increase the expression efficiency of a target gene. When used in plants, the enhancer is preferably an enhancer region containing a sequence upstream of a human cytomegalovirus immediate-early enhancer. A plurality of enhancers may be used, but one may be used or may not be used.

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

  The present invention can be utilized in any animal. Techniques for use in such animals are well known and commonly used in the art, such as Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J. et al. (1987) Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, separate volume of experimental medicine "Gene transfer & expression analysis experiment method" Yodosha, 1997, etc. It is described in.

  As used herein, “transformant” refers to all or part of a living organism such as a cell produced by transformation. Examples of the transformant include animal cells. A transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject, and encompasses all of these forms herein, but may refer to a particular form in a particular context. .

  As used herein, “animal” is used in the broadest sense in the art and includes vertebrates and invertebrates. Examples of animals include, but are not limited to, mammals, birds, reptiles, amphibians, fishes, insects, and worms. Preferably, the animal includes a mammal.

  It is contemplated that the polypeptides, nucleic acids, kits, systems, compositions and methods used in the present invention will function in whole animals, including other animals as well as mammals. This is because endomucin is expected to exist in organisms other than mammals.

  A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism, surrounded by a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism.

  In the present specification, the “tissue” of an organism refers to a group of cells having a certain similar action in the group. Thus, the tissue can be part of an organ (organ). In an organ (organ), it often has cells having the same function, but those having slightly different functions may be mixed, so in the present specification, as long as the tissue shares certain characteristics, Various cells may be mixed.

  In this specification, an “organ” refers to a structure having one independent form and a structure that performs a specific function by combining one or more tissues. Examples of animals include stomach, liver, intestine, pancreas, lung, trachea, nose, heart, artery, vein, lymph node (lymphatic system), thymus, egg layer, eye, ear, tongue, skin, etc. It is not limited.

  In the present specification, “treatment with endomucin or an endomucin-activating factor” means that cells are exposed to the endmucin or the factor that activates endmucin of the present invention to be amplified. Therefore, such treatment includes not only direct contact but also techniques such as transforming cells to be transiently expressed and inducing their expression.

  As used herein, the term “transgenic” refers to an organism (including an animal (such as a mouse) or a plant) that is incorporated into an organism in which a specific gene is present.

  When a transgenic organism is used in the present invention, such a transgenic organism can be obtained by microinjection method (microinjection method), viral vector method, ES cell method (embryonic stem cell method), sperm vector method, chromosomal fragment It can be produced using a technique for producing a transgenic animal utilizing a method of introduction (transzomic method), an episome method or the like. Techniques for producing such transgenic animals are well known in the art.

(screening)
The present invention also contemplates providing a computer modeled drug or marker based on the disclosure of the present invention.

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

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

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. Org. Med. Chem 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. Org. Med. Chem. 37: 1233.

  Compound libraries can be in solution (eg, Houghten, BioTechniques 13: 412-421, 1992), or on beads (Lam, Nature 354: 82-84, 1991), on a chip (Fodor, Nature 364: 555). 556, 1993), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869, 1992). Or on phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (19 0) Proc.Natl.Acad.Sci.U.S.A.87: 6378~6382; Felici (1991) J.Mol.Biol.222: 301~310; Ladner above) can be shown in.

The present invention, in other embodiments, is a computer-aided quantitative structure-activity relationship as a tool for screening agents that are equally effective as the active ingredient of the present invention (eg, an endmucin polypeptide or nucleic acid or means for identifying the same). Also included in the present invention are compounds obtained using (quantitative structure activity relationship = QSAR) modeling techniques. Here, the computer technique includes creation of a substrate template, a pharmacophore, and a homology model of an active site created by several computers. In general, from data obtained in vitro, a method for modeling the normal characteristic groups of an interacting substance for a substance is described by the CATALYST ^™ pharmacophore method (Ekins et al., Pharmacogenetics, 9: 477-489, Ekins et al., J. Pharmacol. & Exp. Ther., 288: 21-29, 1999; Ekins et al., J. Pharmacol. & Exp. Ther., 290: 429-438, 1999; al., J. Pharmacol. & Exp. Ther., 291: 424-433, 1999) and comparative molecular field analysis (CoMFA). (Jones et al., Drug Metabolism & Disposition, 24: 1-6, 1996) and the like. In the present invention, computer modeling may be performed using molecular modeling software such as CATALYST ^™ version 4 (Molecular Simulations, Inc., San Diego, Calif.).

  For example, fitting a compound to the active site of endomucin can be performed using any of a variety of computer modeling techniques known in the art. Visual inspection and manual manipulation of compounds for active sites are described in QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. Louis, MO, 1992). et al., J. Am. Chem. Soc., 106: 765-784, 1984), CHARMM (Brooks et al., J. Comp. Chem., 4: 187-217, 1983) and the like. Can be done using. In addition, energy can be minimized using standard force fields such as CHARMM, AMBER, and the like. Other more specialized computer modeling are GRID (Goodford et al., J. Med. Chem., 28: 849-857, 1985), MCSS (Miranker and Karplus, Function and Genetics, 11: 29-34, 1991), AUTODOCK (Goodsell and Olsen, Proteins: Structure, Function and Genetics, 8: 195-202, 1990), DOCK (Kuntz et al., J. Mol. Biol., 161: 269-288). Etc. Further structural compounds include LUDI (Bohm, J. Comp. Aid. Molec. Design, 6: 61-78, 1992), LEGEND (Nishibata and Itai), in blank active sites, active sites in known small molecule compounds, and the like. , Tetrahedron, 47: 8985, 1991), LeapFrog (Tripos Associates, St. Louis, MO), and the like, can be newly constructed. Such modeling is well known and commonly used in the art, and a person skilled in the art will appropriately design a compound that falls within the scope of the present invention (for example, endmucin or an equivalent of a factor specific thereto) according to the disclosure of the present specification. can do.

(Administration / Infusion / Medicine)
A cell (eg, an adult hematopoietic stem cell, a transplant containing it, a cell differentiated therefrom (eg, a hematopoietic cell)) or cell composition prepared according to the present invention may be in a form suitable for transfer to an organism. For example, it can be provided in any formulation form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent.

  Administration methods include oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration to affected areas, skin administration, etc.) Examples include direct administration to the affected area. Formulations for such administration can be provided in any dosage form. Examples of such a pharmaceutical form include a liquid, an injection, and a sustained release agent. The compositions and medicaments of the present invention may be in the form of an orally acceptable aqueous solution that is pyrogen-free when administered systemically. The preparation of such pharmaceutically acceptable protein solutions is within the skill of the artisan, provided that considerable attention is paid to pH, isotonicity, stability, and the like.

  The solvent used for pharmaceutical formulation in the present invention may have either aqueous or non-aqueous properties. In addition, the vehicle can be used to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, isotonicity, disintegration rate, or odor of the formulation. Material may be included. Similarly, the compositions of the present invention may include other formulation materials to modify or maintain the release rate of the active ingredient or to promote absorption or permeation of the active ingredient.

  The present invention, when formulated as a medicament or pharmaceutical composition, is optionally a physiologically acceptable carrier, excipient or stabilizer (Japanese Pharmacopoeia 14th Amendment and Supplement, Remington's Pharmaceutical Sciences 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990) and a selected composition having the desired degree of purity in the form of a lyophilized cake or aqueous solution. Can be prepared for storage.

  Such suitable pharmaceutically acceptable factors include, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents. , Fillers, bulking agents, buffering agents, delivery vehicles, diluents, excipients and / or agricultural or pharmaceutical adjuvants. Typically, the medicament of the present invention comprises the active ingredient of the present invention (eg, a polypeptide or nucleic acid) in the form of a composition together with one or more physiologically acceptable carriers, excipients or diluents. Can be administered. 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. . Such acceptable carriers, excipients or stabilizers are non-toxic to the recipient and are preferably inert at the dosages and concentrations used, and include the following: Phosphate, citrate, or other organic acids; antioxidants (eg, ascorbic acid); low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymers (eg, polyvinylpyrrolidone) 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 formation Ion (e.g., sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)).

  The composition or kit of the present invention may also further comprise a biocompatible material. This biocompatible material is, for example, a group consisting of silicone, collagen, gelatin, glycolic acid / lactic acid copolymer, ethylene vinyl acetic acid copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyacrylate, and polymethacrylate. It may include at least one more selected. Silicone is preferred because it is easy to mold. Examples of biodegradable polymers include collagen, gelatin, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.) and hydroxytricarboxylic acids (eg, , Citric acid, etc.) from one or more selected from the group consisting of polymers, copolymers or mixtures thereof, poly-α-cyanoacrylates, polyamino acids (for example, Poly-γ-benzyl-L-glutamic acid and the like), and polyanhydrides of maleic anhydride copolymers (for example, styrene-maleic acid copolymer and the like). The form of polymerization may be random, block, or graft. When α-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, D-form, L-form, and DL-form Any of these can be used. Preferably, a copolymer of glycolic acid / lactic acid can be used.

  Injections containing cells and the like can be prepared by methods well known in the art. For example, after dissolving in an appropriate solvent (physiological saline, buffer solution such as PBS, sterilized water, etc.), sterilized by filtration with a filter, and then filled into a sterile container (eg, ampoule) Can be prepared. This injection may contain a conventional pharmaceutical carrier, if necessary. Administration methods using non-invasive catheters can also be used. Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the medicament of the present invention is formulated as a lyophilization agent using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. . The pH of the solution should also be selected based on the relative solubility of the active ingredient of the invention (such as a polypeptide or nucleic acid) at various pHs.

  The formulation procedure of the preparation of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, the pharmacopoeia of other countries, and the like. Accordingly, those skilled in the art can determine the amount of polypeptide and cell to be administered without undue experimentation as described herein.

  In one embodiment, the compositions and medicaments of the present invention can be provided in sustained release form. When administered in a sustained release form, the active ingredient (eg, nucleic acid or polypeptide) is released gradually, which is effective when long-term efficacy is expected. The sustained-release form can be any form known in the art as long as it can be used in the present invention. As such a form, for example, it may be a preparation such as a rod form (pellet form, cylinder form, needle form, etc.), a tablet form, a disc form, a spherical form, or a sheet form. Methods for preparing sustained release forms are known in the art and are described, for example, in the Japanese Pharmacopeia, the US Pharmacopeia and pharmacopoeia in other countries. Examples of the method for producing a sustained-release agent (sustained administration agent) include a method utilizing dissociation of a drug from a complex, a method of making an aqueous suspension injection, a method of making an oily injection or an oily suspension injection. And an emulsion injection solution (o / w type, w / o type emulsion injection solution, etc.).

  In another embodiment, the present invention also contemplates administration of additional agents. Such a drug can be any drug known in the art, for example, such a drug can be any drug known in pharmacy (eg, antibiotics, etc.). Of course, such agents can be more than one other agent. Examples of such drugs include those listed in the latest version of the Japanese Pharmacopeia, the latest version of the US Pharmacopeia, and the latest version of the pharmacopoeia in other countries.

  In other embodiments, the cells prepared by the methods of the present invention can comprise two or more types of cells. When using two or more types of cells, cells of similar nature or origin may be used, or cells of different nature or origin may be used.

  The amount of polypeptide, nucleic acid, composition, medicament and cell used in the method of the present invention is the purpose of use, the age, size, sex, history of the subject, polypeptide, nucleic acid, composition, medicament or cell. It can be easily determined by those skilled in the art in view of the form or type, the form or type of cells, and the like.

  The frequency with which the composition of the present invention is applied to an object can also be easily determined by those skilled in the art taking into consideration the purpose of use, the age, size, sex, medical history, treatment history, etc. of the object. . Examples of the frequency include administration once to several times a day, once every day to several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  In the present invention, gene therapy such as inducing adult hematopoietic stem cells using a nucleic acid molecule encoding endomucin or a variant or fragment thereof may be useful.

  When administering a composition of the invention comprising a nucleic acid molecule, the nucleic acid molecule can be administered, such as by administration in a non-viral vector form or viral vector form, or by direct administration with naked DNA. Such administration forms are well known in the art. For example, separate experimental medicine “Basic Technology of Gene Therapy” Yodosha, 1996; Separate Experimental Medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, etc. It is explained in detail.

  In certain embodiments, a nucleic acid comprising a normal gene nucleic acid sequence of the invention, a sequence encoding an antibody or functional derivative thereof, is a disease or disorder associated with abnormal expression and / or activity of a polypeptide of the invention. Is administered for the purpose of gene therapy. Gene therapy refers to a therapy performed by administering to a subject a nucleic acid that is expressed or expressible. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

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

  For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, 90, described in StockNon.

  In the case of a non-viral vector form, a method for introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofectin method, lipofectamine method, etc.), microinjection method, gene gun (Gene Gun) ) And a method of transferring nucleic acid molecules into cells together with carriers (metal particles). Examples of expression vectors include pCAGGS (Gene 108: 193-9, Niwa H, Yamamura K, Miyazaki J (1991)), pBJ-CMV, pcDNA3.1, pZeoSV (available from Invitrogen, Stratagene, etc.) Is mentioned.

  The HVJ-liposome method involves encapsulating a nucleic acid molecule in a liposome made of a lipid bilayer and fusing the liposome with an inactivated Sendai virus (Hemagglutinating virus of Japan, HVJ). This HVJ-liposome method is characterized in that it has a much higher fusion activity with the cell membrane than the conventional liposome method. The HVJ-liposome preparation method is described in detail in a separate volume of experimental medicine “Basic technology of gene therapy” Yodosha, 1996; a separate volume of experimental medicine “Gene transfer & expression analysis experimental method” Yodosha, 1997. As HVJ, any strain can be used (for example, ATCC VR-907, ATCC VR-105, etc.), and Z strain is preferable.

  When the composition of the present invention is provided in the form of a nucleic acid of a viral vector, a viral vector such as a recombinant adenovirus or retrovirus is used. DNA viruses or RNA viruses such as detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, SV40, human immunodeficiency virus (HIV), A gene can be introduced into a cell or tissue by introducing a nucleic acid encoding endomucin or a nucleic acid encoding endomucin and infecting the cell or tissue with the recombinant virus. In these viral vectors, the adenoviral vector system is preferably used because the infection efficiency of adenovirus is much higher than that of other viral vectors.

  In the case of the Naked DNA method, the above-described non-viral vector expression plasmid is dissolved in physiological saline and administered as it is. For example, Tsurumi Y, Kearney M, Chen D, Silver M, Takeshita S, Yang J, Symes JF, Isner JM. , Circulation 98 (Suppl. II), 382-388 (1997), can be directly injected into tissues of living organisms.

  Therefore, the amount of the polypeptide (eg, endmucin) as an active ingredient contained in the compositions and kits of the present invention is about 1 μg to about 1000 mg, preferably about 5 μg It can be about 100 mg. The lower limit of the amount range of this polypeptide is, for example, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, etc. It can be any number between about 1 μg and about 1 mg. The upper limit of the amount range of this polypeptide is, for example, about 1000 mg, about 900 mg, about 800 mg, about 700 mg, about 600 mg, about 500 mg, about 400 mg, about 300 mg, about 200 mg, about 100 mg, about 75 mg, about 50 mg, about It can be any numerical value from about 1000 mg to about 1 mg, such as 25 mg, about 10 mg, about 5 mg, and the like.

  When the active ingredient of the present invention is in the form of a nucleic acid (for example, a nucleic acid encoding endomucin or a nucleic acid encoding endomucin), for an adult (body weight of about 60 kg), about 1 μg to about 10 mg, preferably about 1 μg to about 1000 μg More preferably from about 5 μg to about 400 μg. The lower limit of the range of the amount of nucleic acid is, for example, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, etc. It can be any number between 1 μg and about 20 μg. The upper limit of the amount range of this nucleic acid is, for example, about 10 mg, about 9 mg, about 8 mg, about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, about 1 mg, about 750 μg, about 500 μg, about 250 μg. Any numerical value from about 10 mg to about 10 μg, such as about 100 μg. Even when two or more types of cell physiologically active substances (SCF, etc.) are contained, the above amounts are applied individually. When administered as a viral vector or a non-viral vector, the dose is usually 0.0001 to 100 mg, preferably 0.001 to 10 mg, more preferably 0.01 to 1 mg. Examples of the administration frequency include daily administration-once every several months (for example, once a week-once a month).

The amount of cells contained in the composition comprising cells prepared in the present invention is, for example, from about 1 × 10 ³ cells to about 1 × 10 ¹¹ cells, preferably from about 1 × 10 ⁴ cells to about 1 × 10 ¹⁰ cells. More preferably about 1 × 10 ⁵ cells to about 1 × 10 ⁹ cells. These cells may be present as solutions such as, for example, about 0.1 ml, 0.2 ml, 0.5 ml, 1 ml saline. As the upper limit of the range of the amount of cells, for example, about 1 × 10 ¹¹ cells, about 5 × 10 ¹⁰ cells, about 2 × 10 ¹⁰ cells, about 1 × 10 ¹⁰ cells, about 5 × 10 ⁹ cells, about 2 × 10 ⁹ cells, about 1 × 10 ⁹ cells, about 5 × 10 ⁸ cells, about 2 × 10 ⁸ cells, about 1 × 10 ⁸ cells, about 5 × 10 ⁷ cells, about 2 × 10 ⁷ cells, about 1 × 10 ⁷ cells and the like. As the lower limit of the amount of cells, for example, about 1 × 10 ³ cells, about 2 × 10 ³ cells, about 5 × 10 ³ cells, about 1 × 10 ⁴ cells, about 2 × 10 ⁴ cells, about 5 × 10 ⁴ cells. Examples include cells, about 1 × 10 ⁵ cells, about 2 × 10 ⁵ cells, about 5 × 10 ⁵ cells, and about 1 × 10 ⁶ cells.

  The amount of stem cells contained in a composition (transplant, medicine, etc.) containing cells concentrated or separated in the present invention is, for example, usually at least about 10%, preferably at least about 20% of the total proportion, At least about 30%, at least about 40%, at least about 50%, more preferably at least about 60%, at least about 70%, at least about 80%, more preferably at least about 90%, at least about 95%, and the like. The composition of such cells can be assayed by detecting or quantifying the expression level of the endomucin of the present invention.

  As used herein, “detection” or “quantification” of polypeptide expression can be accomplished using any suitable method, including, for example, measuring mRNA and immunological methods. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of immunological measurement methods include ELISA methods using microtiter plates, RIA methods, fluorescent antibody methods including FACS, Western blotting methods, immunohistochemical staining methods, and the like. Examples of the quantitative method include an ELISA method and an RIA method.

  As used herein, “expression level” refers to the amount of polypeptide or mRNA expressed in a target cell or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method using the antibody of the present invention. The amount of expression of the polypeptide of the present invention at the protein level, or the polypeptide used in the present invention evaluated by any suitable method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. The expression level of the peptide at the mRNA level can be mentioned. “Change in expression level” means expression at the protein level or mRNA level of the polypeptide used in the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Means that the amount increases or decreases.

  In the present specification, “instruction” describes the method for administering the cell medicine of the present invention to doctors, patients and other persons who administer it. This instruction describes a word indicating an administration method of the medicine or the like of the present invention. In addition, the instructions may include a word indicating the location and method of administration (for example, by injection) of skeletal muscle or the like as the administration site. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction is a so-called package insert and is usually provided as a paper medium, but is not limited thereto. For example, a form such as an electronic medium (for example, a homepage or e-mail provided on the Internet) is used. But it can be provided.

  The “disease” targeted by the present invention may be any disease as long as it requires a certain amount of adult hematopoietic stem cells or differentiated cells, tissues or organs derived therefrom. The disease or disorder that can be treated according to the present invention can be a disease or disorder associated with a disorder of differentiated cells, tissues or organs from which the adult hematopoietic stem cells of the present invention can differentiate.

  In a typical embodiment of such a thing, treatment can be performed using conventional bone marrow cells or purified hematopoietic stem cells, for obstructive arteriosclerosis, Buerger's disease, angina pectoris, myocardial infarction, various arteriosclerosis. A typical example of the subject is an accompanying ischemic disease. This is because administration of bone marrow cells or purified hematopoietic stem cells creates collateral blood vessels. Without wishing to be bound by theory, this neoplasia involves a mechanism in which hematopoietic cells become blood vessels and / or are stimulated by factors emanating from hematopoietic cells, or both. It is thought that there is.

  In one embodiment, the differentiated cell, tissue or organ may be a circulatory system (such as a blood cell). Such diseases or disorders include, for example, but are not limited to: anemia (eg, aplastic anemia (particularly severe aplastic anemia), renal anemia, cancerous anemia, secondary anemia) , Refractory anemia, etc.), cancer or tumor (eg, leukemia) and hematopoietic failure after chemotherapeutic treatment, thrombocytopenia, acute myeloid leukemia (especially first remission phase (High-risk group), second remission) Remission period and later), acute lymphoblastic leukemia (especially the first remission period, remission period after the second remission period), chronic myelogenous leukemia (especially the chronic phase, transition phase), malignant lymphoma (especially the first remission phase) Remission period (High-risk group), remission period after the second remission period, multiple myeloma (especially early after onset), and the like.

  The cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates). Preferably, cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. Most preferably, human-derived cells are used.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In this specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene or factor is introduced in vitro, and then returned to the same subject again. . The amplification factor of the present invention is also useful for ex vivo treatment.

  As used herein, “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

  As used herein, a “recipient” (recipient) is an individual that receives a graft or transplant and is also referred to as a “host”. In contrast, an individual that provides a graft or transplant is referred to as a “donor”.

  As used herein, “self” or “self” refers to a transplant derived from an individual to be transplanted. In this specification, the term “autograft” may include a graft from another genetically identical individual (for example, an identical twin).

  As used herein, “same species” (same allogeneic) refers to transplantation from another individual that is genetically different even if it is the same species. Due to genetic differences, allogeneic grafts can elicit an immune response in the transplanted individual (recipient). Examples of such grafts include, but are not limited to, parent-derived grafts for children.

  As used herein, “heterologous” refers to being transplanted from a heterogeneous individual. Thus, for example, when a human is a recipient, a graft from a pig is referred to as a xenograft.

(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.

  The present invention provides a technique for simply and / or efficiently determining the state of a stem cell (for example, an adult hematopoietic stem cell). It was clear that the adult hematopoietic stem cells could be easily separated and concentrated, so that the present invention greatly contributes to the treatment (for example, transplantation) using the adult hematopoietic stem cells, and has a surprising effect in this field. provide.

(Adult hematopoietic stem cell detection composition using endomucin identification means)
In one aspect, the present invention provides a composition for detecting adult hematopoietic stem cells, comprising means for identifying endomucin. The present invention was completed by finding the ability of endomucin to distinguish adult hematopoietic stem cells from fetal hematopoietic stem cells.

  In one embodiment, the adult hematopoietic stem cell used in the present invention may be a cell having long-term bone marrow remodeling ability.

  In one embodiment, the identification means used in the present invention can be a specific factor for a polypeptide comprising endomucin or a portion thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding endomucin or a portion thereof. Examples of such factors include an anti-endomucin antibody, a nucleic acid encoding endomucin, a part thereof, or a nucleic acid that can hybridize under stringent hybridization conditions (for example, a nucleic acid molecule encoding a complementary strand, etc. Can be mentioned.

  The identification means used in this specification is labeled or labelable, and the label is a radioactive substance, a chemiluminescent substance, a fluorescent substance, a phosphorescent substance, a color former, a ligand, a hapten and their raw materials, etc. obtain. Such a label can be at least one dye selected from the group consisting of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin.

  In preferred embodiments, the present invention may include additional marker identification means. Such a marker may be any marker as long as it assists in identification of stem cells, and examples thereof include CD34, Lin (Lineage marker), c-Kit, Sca-1, Flt3 / Flk2, CD41 and CD45. But not limited to them. For the fetal type, CD34, CD41, Lin (Lineage marker), c-Kit and the like are preferable, where the gene expression of CD34, CD41 and c-Kit indicates that the candidate cell is a stem cell, and Lin (Lineage marker) ) Negative expression of the gene indicates that the candidate cell is a stem cell. These combinations can be used in fetal molds. Preferably, the additional marker may be at least one marker selected from the group consisting of CD41 and CD45, more preferably both. This is because CD41 positive and CD45 negative are preferred indicators of stem cells or pluripotency. However, since CD41 and CD45 cannot clearly distinguish between fetal hematopoietic progenitor cells and adult hematopoietic stem cells, endomucin is important for clearly distinguishing fetal hematopoietic progenitor cells from adult hematopoietic stem cells.

  In one embodiment, the adult hematopoietic stem cell targeted by the present invention may be a cell that retains pluripotency.

  In one embodiment, the adult hematopoietic stem cell targeted by the present invention may be an embryonic stem cell-derived cell or a fetus-derived cell. Cells derived from embryonic stem cells are preferred. This is because the established embryonic stem cell-derived cell has few ethical problems. Making it possible to distinguish hematopoietic stem cells from cells derived from embryonic stem cells is important when the use of fetal stem cells is restricted or should be restricted. In such an aspect, the present invention plays an important role. Is expected to fulfill Here, adult hematopoietic stem cells from embryonic stem cells can be produced by forming embryoid bodies from embryonic stem cells. Specifically, for example, a bacterium in which ES cells loosened with Trypsin / EDTA are suspended at a concentration of 100 cells in a 50 μl culture solution and inverted in a 50 μl culture drop by a hanging drop method (hanging drop). An embryoid body is formed by culturing in a state suspended in a petri dish. After 24 to 48 hours, embryoid bodies are collected from each culture drop and further cultured in a petri dish for bacteria to generate hematopoietic stem cells in the embryoid body. Alternatively, hematopoietic stem cells are generated by seeding ES cells loosened with Trypsin / EDTA directly on stromal cells and repeating co-culture with stromal cells. Such specific procedures are also illustrated and demonstrated in the examples.

(Adult hematopoietic stem cell marker-factor for nucleic acid morphology)
In one embodiment, the present invention provides an adult hematopoietic stem cell detection composition comprising an agent specific for a nucleic acid molecule comprising a nucleic acid sequence encoding endomucin or a portion thereof. Therefore, any factor can be used in the present invention as long as it can confirm the expression (eg, mRNA) of endomucin in stem cells. Here, an amount effective for diagnosis, prevention, treatment or prognosis can be determined by a person skilled in the art using techniques well known in the art in consideration of various parameters, in order to determine such an amount. Can be easily determined by those skilled in the art in consideration of, for example, the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. . In the present invention, since it has been clarified that the expression of endomucin corresponds to adult hematopoietic stem cells, it is efficiently used to identify such a state and property.

  In a preferred embodiment, the factor of the present invention may be a factor selected from the group consisting of nucleic acid molecules, polypeptides, lipids, sugar chains, small organic molecules, and complex molecules thereof. It can be understood that any such factor may be used as long as it specifically binds to the nucleic acid molecule of the present invention.

  In preferred embodiments, it may be advantageous that the agent of the invention is labeled or capable of binding to the label. When so labeled, various conditions that can be measured by the agents of the present invention can be measured directly and / or easily. Such a label may be any label as long as it is distinguishably labeled, such as fluorescence, phosphorescence, chemiluminescence, radioactivity, color former, ligand, hapten, enzyme substrate reaction and antigen-antibody reaction. Techniques, but not limited to. Alternatively, when the factor interacts using an immune reaction such as an antibody, a system often used in an immune reaction such as biotin-streptavidin may be used.

  In a preferred embodiment, the agent of the present invention is a nucleic acid molecule. Where the agent of the invention is a nucleic acid molecule, such a nucleic acid molecule can be at least 8 contiguous nucleotides in length, preferably the nucleic acid sequence of endomucin (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 11) and the like. The appropriate nucleotide length of the nucleic acid molecule of the present invention can vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides long, more preferably at least 15 contiguous nucleotides long, and even more preferably at least 20 contiguous nucleotides long. The lower limit of these nucleotide lengths is, in addition to the numbers specifically mentioned, numbers between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more numbers (for example, 21, 22, ... 30, etc.). As long as the nucleic acid molecule of the present invention can be used for the intended use (for example, antisense, RNAi, marker, primer, probe) or can interact with a predetermined factor, its upper limit length May be the full length of the sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, etc., or a complement thereof, or may be longer than that. Alternatively, when used as a primer, it can usually be at least about 8 nucleotides in length, preferably about 10 nucleotides in length, more preferably about 15 nucleotides in length. When used as a probe, it can usually be at least about 15 nucleotides in length, preferably about 17 nucleotides in length.

  Thus, in one exemplary embodiment, an agent of the invention comprises a nucleic acid molecule having a sequence that is complementary to or at least 70% identical to the nucleic acid sequence of a polynucleotide encoding endomucin. It can be.

  In another exemplary embodiment, the agent of the present invention comprises (a) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or a complement thereof, or a fragment sequence thereof; A polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10; or a fragment thereof; (c) one or more in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 A polynucleotide that encodes a variant polypeptide having at least one mutation selected from the group consisting of substitutions, additions and deletions, and having biological activity; (d ) A polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (c) and has biological activity. A polynucleotide encoding a peptide; or (e) a nucleotide sequence having at least 70% identity to a polynucleotide of any one of (a) to (c) or a complementary sequence thereof, and having biological activity A nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid sequence of any of the endomucin genes of the polynucleotide encoding the polypeptide it has. The stringency may be high, medium or low, and the degree of stringency can be appropriately determined by a person skilled in the art depending on the situation.

  Alternatively, the factor of the present invention is preferably a factor specific for a nucleic acid molecule comprising the sequence represented by SEQ ID NO: 1, 3, 5, 7 or 9 or a complement thereof. Such sequences are particularly useful for examining the degree of differentiation of hematopoietic tissues or individuals because they can identify the undifferentiated nature of hematopoietic stem cell lines, but are not limited thereto.

  In one embodiment, the stem cells found by the marker of the present invention are tissue stem cells, preferably hematopoietic stem cells, but are not limited thereto, and other representative examples include vascular endothelial cells and vascular endothelial cells. It is understood that hemangioblast, which is a common progenitor cell of blood cells, and the like can also be included as a subject.

  In one embodiment, the endomucin of the present invention comprises (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof; (b) SEQ ID NO: 2, 4, 6 A polypeptide having one or more amino acids selected from the group consisting of substitutions, additions and deletions and having biological activity in the amino acid sequence according to claim 8, 8 or 10; c) a polypeptide encoded by an allelic variant or splice variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9; (d) in SEQ ID NO: 2, 4, 6, 8 or 10 A polypeptide that is a species homologue of the described amino acid sequence; or (e) an amino acid that is at least 70% identical to any one polypeptide of (a)-(d) It has an acid sequences, and includes a biologically active polypeptide, a.

  More preferably, the endomucin of the present invention may comprise the sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10. Alternatively, a nucleic acid molecule comprising a nucleic acid sequence encoding endmucin of the present invention can comprise the sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9.

  In one preferred embodiment, the number of substitutions, additions and deletions in (c) above is limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less. 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably similar to or substantially similar to endomucin comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 As long as they have the same activity).

  In another preferred embodiment, the biological activity of the variant polypeptide is specific to, for example, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof. Include, but are not limited to, interaction with specific antibodies, maintenance of undifferentiation, interaction with extracellular matrix, and the like. Preferably, such biological activity includes undifferentiated maintenance. Endomucin is thought to play an important role in maintaining undifferentiated cells. In order to measure this activity, gene introduction experiments, gene deletion experiments, RNAi experiments, protein function inhibition experiments with antibodies, and the like can be considered.

  In another preferred embodiment, the allelic variant preferably has at least 90% homology with the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9. For example, such allelic variants preferably have at least 99% homology, such as within the same strain.

  When the species homologue database of the species exists, the species homologue is defined as the entire endomucin polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 of the present invention. Or by identifying as a query sequence all or part of a nucleic acid molecule encoding endomucin, including a part of the amino acid sequence or the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9 can do. Alternatively, it can be identified by screening such a gene library using all or part of the nucleic acid sequence of the endomucin of the present invention as a probe or primer. Such identification methods are well known in the art and are also described in the literature described herein. The species homologue preferably has at least about 30% homology with, for example, the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9. Species homology preferably has at least about 50% homology with the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9.

  In a preferred embodiment, the identity to any one of the polynucleotides (a) to (e) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%, even more preferably May be at least about 98%, and most preferably at least about 99%.

  In preferred embodiments, the nucleic acid molecules of the invention can be at least 8 contiguous nucleotides. The appropriate nucleotide length of the nucleic acid molecule of the present invention can vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides long, more preferably at least 15 contiguous nucleotides long, and even more preferably at least 20 contiguous nucleotides long. The lower limit of these nucleotide lengths is, in addition to the numbers specifically mentioned, numbers between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more numbers (for example, 21, 22, ... 30, etc.). As long as the nucleic acid molecule of the present invention can be used for the intended use (for example, a marker), the upper limit length is the full length of the sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9. The length may be longer than that. Alternatively, when used as a primer, it can usually be at least about 8 nucleotides in length, preferably about 10 nucleotides in length. When used as a probe, it can usually be at least about 15 nucleotides in length, preferably about 17 nucleotides in length.

  In a more preferred embodiment, the present invention provides (a) a polynucleotide having the base sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9 or a fragment sequence thereof; or (b) SEQ ID NO: 2, 4, 6, It may be a polynucleotide encoding a polypeptide consisting of the amino acid sequence described in 8 or 10 or a fragment thereof.

  In certain preferred embodiments, the nucleic acid molecules of the invention have a structure in which the extracellular domain has multiple Ser and Thr residues in addition to the two N-glycosylation sites (Asn-X-Ser / Thr). There are 25 potential 0-glycosylation sites. In the intracellular domain, there are three sites phosphorylated by protein kinase C (Ser / Thr-X-Lys). It is preferred in one embodiment that the corresponding positions of these characteristic domains are preserved, but not limited thereto.

  Here, in a more preferred embodiment, the identity to any one of the polynucleotides (a) to (b) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%. More preferably at least about 98%, most preferably at least about 99%.

  In a preferred embodiment, the nucleic acid molecule encoding endmucin of the present invention or fragments and variants thereof may be at least 8 contiguous nucleotides in length. The appropriate nucleotide length of the nucleic acid molecule of the present invention can vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides long, more preferably at least 15 contiguous nucleotides long, and even more preferably at least 20 contiguous nucleotides long. The lower limit of these nucleotide lengths is, in addition to the numbers specifically mentioned, numbers between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more numbers (for example, 21, 22, ... 30, etc.). As long as the nucleic acid molecule of the present invention can be used for an intended use (for example, antisense, RNAi, marker, primer, probe, capable of interacting with a predetermined factor), the upper limit length thereof is It may be the full length of the sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9, or may be longer than that. Alternatively, when used as a primer, it can usually be at least about 8 nucleotides in length, preferably about 10 nucleotides in length. When used as a probe, it can usually be at least about 15 nucleotides in length, preferably about 17 nucleotides in length.

  In one preferred embodiment, the composition of the invention comprises at least one nucleic acid molecule specific for a nucleic acid molecule encoding a polypeptide selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit. A factor may further be included. More preferably, such additional factors include at least two, more preferably three, even more preferably four, and even more preferably five. Here, since Lin is a combination of a plurality of markers, it is understood that these markers can vary. Examples of the Lin marker include, but are not limited to, Gr-1, Mac-1, TER119, CD4, CD8, and B220.

  More preferably, CD34 used in the present invention includes the nucleic acid sequence shown in SEQ ID NO: 13 or 15, or a part thereof or a modification thereof, and Lin used in the present invention includes SEQ ID NOs: 17, 19, 21, and / or Or c-Kit used in the present invention includes the nucleic acid sequence shown in SEQ ID NO: 25 or a part thereof or a variant thereof, and includes the nucleic acid sequence shown in FIG. Sca-1 used includes the nucleic acid sequence shown in SEQ ID NO: 27 or a part thereof, or a modification thereof, and Flt3 / Flk2 used in the present invention is the nucleic acid sequence shown in SEQ ID NO: 11, a part thereof or a part thereof. Including variants.

  As used herein, Gr-1 (granulocyte-differentiation antigen) recognizes Ly-6G (21 to 25 kDa glyco-phospho-inositol (GPI) -binding membrane protein) (TJ Fleming, ML Fleming and TR). (See Malek, The Journal of Immunology, Vol 151, Issue 5 2399-2408, 1993). Therefore, in this specification, this Gr-1 is used as an indicator of the differentiation state.

  B220 is recognized by the antibody TER119 (Kina T, Ikuta K, Takayama E, Wada K, Majudar AS, Weissman IL, Katsura Y, Br J Haematol. 2000 May; 109 (2): 280-7. Therefore, in this specification, this Gr-1 is used as an indicator of the differentiation state.

  These additional factors can also be labeled with a label. In such a case, the label is preferably varied depending on each factor. When a plurality of labels are used, labels such as Texas-Red, PE, FITC, APC, and biotin can be used. Hoechst Blue, Hoechst 33342, and Hoechst Red can be used as DNA staining dyes.

(Stem cell marker-factor for polypeptide form)
In another embodiment, the present invention provides an adult hematopoietic stem cell detection composition comprising an agent that specifically binds to an endomucin polypeptide or a portion thereof. Such factors include, but are not limited to, polypeptides (eg, antibodies, single chain antibodies), polynucleotides, sugar chains, lipids, and complex molecules thereof. It can be understood that such factors may be any as long as they specifically bind to the polypeptide of the present invention. More preferably, the agent of the present invention is an antibody or a derivative thereof (eg, a single chain antibody). Thus, the agents of the present invention can be used as probes and / or inhibitors.

  In preferred embodiments, it may be advantageous that the agent of the invention is labeled or capable of binding to the label. When so labeled, various conditions that can be measured by the agents of the present invention can be measured directly and / or easily. Such a label may be any label as long as it is distinguishably labeled, and examples thereof include techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction, and antigen-antibody reaction. It is not limited. Alternatively, when the factor interacts using an immune reaction such as an antibody, a system often used in an immune reaction such as biotin-streptavidin may be used. The label used in the present invention may be fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Texas Red, biotin. Hoechst Blue, Hoechst 33342, and Hoechst Red can be used as DNA staining dyes.

  In a preferred embodiment, the agent of the present invention may be an antibody. The antibodies may be, for example, monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof, such as F (ab ′) 2 and Fab fragments, and others It may be a conjugate produced by recombination. Such an antibody can be used as a tool for determining the expression of the gene of the present invention, and thus can be used for screening.

  In a preferred embodiment, the polypeptide of endmucin of the present invention comprises (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof; (b) SEQ ID NO: 2, 4 A polypeptide having at least one mutation selected from the group consisting of substitutions, additions and deletions, and having biological activity in the amino acid sequence according to claim 6, 8, 8 or 10 (C) a polypeptide encoded by an allelic variant or splice variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9; (d) SEQ ID NO: 2, 4, 6, 8 or A polypeptide that is a species homologue of the amino acid sequence of claim 10; or (e) at least identity to any one polypeptide of (a)-(d) It has an amino acid sequence that is at 0%, and has a biological activity, polypeptides,.

  More preferably, the endomucin of the present invention may comprise the sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10.

  In one preferred embodiment, the number of substitutions, additions and deletions in (b) above may be limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less. 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably similar or substantially similar to endomucin having the sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 As long as they have the same activity).

  In another preferred embodiment, it is preferred that the allelic variant in (c) above has at least about 90% homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10. Preferably, the allelic variant in (c) has at least about 99% homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10.

  In another preferred embodiment, the species homologue can be identified as described hereinabove and has at least about 30% homology with the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10. It is preferable to have. Species homology preferably has at least about 50% homology with the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9.

  In another preferred embodiment, the biological activity of the variant polypeptide in (e) above is, for example, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, or a polypeptide thereof Examples include, but are not limited to, interaction with an antibody specific to a fragment, interaction with an extracellular matrix, and the like. Preferably, such biological activity includes undifferentiated maintenance. Endomucin is thought to play an important role in maintaining undifferentiated cells. In order to measure this activity, gene introduction experiments, gene deletion experiments, RNAi experiments, protein function inhibition experiments with antibodies, and the like can be considered.

  In preferred embodiments, the identity to any one of the polypeptides (a)-(d) above may be at least about 80%, more preferably at least about 90%, and even more preferably at least about 98. %, Most preferably at least about 99%.

  The polypeptide of the present invention usually has at least 3 consecutive amino acid sequences. The amino acid length of the polypeptide of the present invention can be any length as long as it suits the intended use, but preferably a longer sequence can be used. Therefore, it may be preferably at least 4 amino acids long, more preferably 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, 9 amino acids long, 10 amino acids long. More preferably it may be at least 15 amino acids long, and still more preferably at least 20 amino acids long. The lower limit of these amino acid lengths is a number between them (for example, 11, 12, 13, 14, 16, etc.) or more (for example, 21, 22, ..., Etc.). As long as the polypeptide of the present invention can be used for the intended use (for example, immunogen, marker), the upper limit is the full length of the sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10. It may be the same as or longer than that.

  In a preferred embodiment, the polypeptide of the present invention has a structure in which the extracellular domain has multiple Ser and Thr residues in addition to the two N-glycosylation sites (Asn-X-Ser / Thr). There are potential 0-glycosylation sites. The intracellular domain has three phosphorylation sites by protein kinase C (Ser / Thr-X-Lys). Thus, it is preferred in one embodiment that the polypeptides of the invention are conserved in at least one, preferably all of the corresponding positions of these characteristic domains, but are not limited thereto. In the polypeptide sequence, the above region corresponds to the corresponding sequence in the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8 or 10, or a polypeptide corresponding thereto (for example, SEQ ID NO: 2, 4, 6, 8 or The gene corresponding to the amino acid sequence shown in Fig. 10), but is not limited thereto. Such sequences can be identified by those skilled in the art using well-known techniques.

  Here, in a further preferred embodiment, the identity to any one of the polypeptides (a) to (b) above may be at least about 80%, more preferably at least about 90%, even more preferably May be at least about 98%, and most preferably at least about 99%.

  In one preferred embodiment, the composition comprising a polypeptide of the invention comprises at least one factor specific for a polypeptide selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit. May further be included. More preferably, such additional factors include at least two, more preferably three, and even more preferably four. Here, since Lin is a combination of a plurality of markers, it is understood that these markers can vary.

  More preferably, CD34 used in the present invention includes the amino acid sequence shown in SEQ ID NO: 14 or 16, or a part thereof or a variant thereof, and Lin used in the present invention includes SEQ ID NOs: 18, 20, 22, and 24. C-Kit used in the present invention is the amino acid sequence shown in SEQ ID NO: 26 or a part thereof or a modification thereof. Sca-1 used in the present invention including a modified form includes the amino acid sequence shown in SEQ ID NO: 28 or a part thereof or a modified form thereof, and Flt3 / Flk2 used in the present invention is shown in SEQ ID NO: 12. The amino acid sequence or a part thereof or a modification thereof is included.

  In one preferred embodiment, the composition of the invention comprises at least one nucleic acid molecule specific for a nucleic acid molecule encoding a polypeptide selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit. A factor may further be included. More preferably, such additional factors include at least two, more preferably three, and even more preferably four. Here, since Lin is a combination of a plurality of markers, it is understood that these markers can vary.

  Preferably, the composition comprises a factor such that it can be determined that CD34 expression is negative or weakly positive. Alternatively, the composition comprises a factor that can determine that c-Kit and Sca-1 expression is positive, particularly strong positive. Alternatively, the composition of the present invention comprises a factor that can determine the negative expression of Lin. Such negative, weak positive, positive and strong positive can preferably be based on the expression intensity employed in quantitative PCR. In such cases, the expression intensity is determined by a level that is relatively determined by PCR. Alternatively, the expression intensity can be determined using a microarray or the like based on the expression intensity of a housekeeping gene such as HRPT. Therefore, a factor capable of sensitively determining the degree of positiveness is preferable. Examples of such a factor include a nucleic acid molecule containing a perfect match complementary sequence, a nucleic acid molecule containing a complementary sequence of a base mismatch, and a completely random sequence. Examples include, but are not limited to, sequences and combinations thereof.

  More preferably, CD34 used in the present invention includes the nucleic acid sequence shown in SEQ ID NO: 13 or 15, or a part thereof or a modification thereof, and Lin used in the present invention includes SEQ ID NOs: 17, 19, 21, and / or Or c-Kit used in the present invention includes the nucleic acid sequence shown in SEQ ID NO: 25 or a part thereof or a variant thereof, and includes the nucleic acid sequence shown in FIG. Sca-1 used includes the nucleic acid sequence shown in SEQ ID NO: 27 or a part thereof, or a modification thereof, and Flt3 / Flk2 used in the present invention is the nucleic acid sequence shown in SEQ ID NO: 11, a part thereof or a part thereof. Including variants.

  These additional factors can also be labeled with a label. In such a case, the label is preferably varied depending on each factor. When a plurality of labels are used, labels such as Texas-Red, PE, FITC, APC, and biotin can be used.

  In another aspect, the present invention provides a composition for determining the transplant suitability of a transplant, comprising a specific factor for an endomucin polypeptide or a part thereof. Cells that were judged to be positive for expression of the polypeptide of the endmucin of the present invention were shown to be transplantable. It was also shown that cells that were not judged positive were not transplantable. Therefore, it has been shown for the first time by the present invention that judgment of positive / negative of endmucin can be used as it is for the suitability of transplantation. Therefore, an agent specific for the endomucin polypeptide of the present invention or a part thereof has utility that it can be used to determine the transplant suitability of a transplant (eg, a preparation containing stem cells).

  In a preferred embodiment, factors specific for the endomucin polypeptides of the invention or portions thereof can determine the transplant suitability of a transplant containing stem cells. More preferably, the stem cell comprises a hematopoietic stem cell.

  Factors specific for the endomucin polypeptide of the present invention or a part thereof used in the composition for determining the transplant suitability of the transplant of the present invention are the above-mentioned (stem cell marker-polypeptide forms). Any form of the nucleic acid molecules and factors indicated in Factors for) can be used.

  In one preferred embodiment, the composition of the present invention further comprises at least one factor specific for a polypeptide selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit. May be. More preferably, such additional factors include at least two, more preferably three, and even more preferably four. Here, since Lin is a combination of a plurality of markers, it is understood that these markers can vary.

  Preferably, the composition comprises a factor such that it can be determined that CD34 expression is negative or weakly positive. Alternatively, the composition comprises a factor that can determine that c-Kit and Sca-1 expression is positive, particularly strong positive. Alternatively, the composition of the present invention comprises a factor that can determine the negative expression of Lin. Such negative, weak positive, positive and strong positive may preferably be based on expression intensity employed in techniques using FACS or magnetic beads. FACS or magnetic beads can be performed using procedures as exemplified herein, based on techniques well known in the art. Therefore, a factor that can judge the degree of positivity with high sensitivity is preferable. Examples of such a factor include, but are not limited to, a monoclonal antibody with high specificity, a specific ligand, or a variant or fragment thereof. Not.

  More preferably, CD34 used in the present invention includes the amino acid sequence shown in SEQ ID NO: 14 or 16, or a part thereof or a modification thereof, and Lin used in the present invention includes SEQ ID NO: 18, 20, 22, and / or Or c-Kit used in the present invention includes the amino acid sequence shown in SEQ ID NO: 26 or a part thereof or a variant thereof. Sca-1 used includes the amino acid sequence shown in SEQ ID NO: 28 or a part thereof, or a modification thereof, and Flt3 / Flk2 used in the present invention is the amino acid sequence shown in SEQ ID NO: 12, a part thereof or a part thereof. Including variants.

  These additional factors can also be labeled with a label. In such a case, the label is preferably varied depending on each factor. When a plurality of labels are used, labels such as Texas-Red, PE, APC, FITC, and biotin can be used. Hoechst Blue, Hoechst 33342, and Hoechst Red can be used as DNA staining dyes.

  Specific procedures for analysis by FACS using such specific factors include, but are not limited to, the following.

  (1) Sample cells are collected from, for example, bone marrow (femur, tibia, etc.), suspended in a buffer solution such as PBS, and passed through a filter (eg, 70 μm) such as a nylon filter to collect cell mass, muscle, etc. Remove contaminated tissue.

(2) Count the number of cells. Usually 1 × 10 ⁷ to 1 × 10 ⁸ sample cells are obtained.

(3) Adjust the cell concentration to 1 × 10 ⁷ / ml with PBS (dissolve NaCl 9 g in 1000 ml of 0.1 molar phosphate buffer) and use it as a medium for density gradient centrifugation such as 5 ml Ficoll. Layer.

  (4) Centrifuge at 400 × g for 20 minutes at room temperature.

(5) Cells at the interface are collected and suspended in SM (staining medium = PBS containing 5% FBS, 0.05% NaN ₃ ).

  (6) Count the number of cells. In the case of Ficoll separation, about half of the number of cells is recovered.

  (7) Appropriate amounts of biotinylated lineage antibody (Gr-1 antibody (eg RB6-8C5), Mac-1 antibody (eg M1 / 70), Ter119 antibody, B220 antibody (eg RA3-6B2), CD4 antibody ( For example, RM4-5) and a CD8 antibody (for example, a mixture of 53.6.7) are added and allowed to react on ice for 30 minutes.

  (8) Wash twice with SM and resuspend in 100 μl SM.

  (9) Take the required amount of streptavidinized Dynabeads (M-280, Dynal, Norway) (5-6 beads / cell) and wash with SM using a magnet stand. After washing, Dynabeads are resuspended in SM.

  (10) React on ice for 30 minutes.

  (11) After adding 7 ml of SM, negative selection (removing lineage positive cells bound to beads) is performed using a magnetic stand.

  (12) Count the number of cells. About 5-10% of cells remain after line depletion.

  (13) After centrifugation (4 ° C., 400 × g, 5 minutes), the following antibodies are added in a cell pellet state.

  (14) Add biotinylated lineage antibody again. (Here, staining with biotinylated lineage antibody needs to be performed twice before and after lineage depletion) (5-10% of the amount initially used).

  (15) For example, the required amount of PE-anti-Sca-1 antibody (for example, E13-161.7) and APC-c-Kit antibody (for example, ACK2 or 2B8) and FITC-CD34 antibody (RAM34) are added simultaneously.

  (16) After incubation on ice, the cells are washed and streptavidin-Texas Red is added.

(17) After incubation on ice, the cells are washed, and the suspension of cells is adjusted with 5 × 10 ⁵ / ml as a guide.

  (18) Analysis and / or sorting by FACS (for example, using flow cytometry equipped with two lasers such as FACS Vantage (Becton Dickinson)).

  Whether the adult hematopoietic stem cells of the present invention are suitable for transplantation can be confirmed by judging suitability after transplanting the sample. Specific procedures of such a method are exemplified below.

(1) A small amount of 0.1 M EDTA · Na ₂ / H ₂ O is taken into a capillary tube (for example, 100 μl) using capillary action.

  (2) Anesthetize the target organism as necessary.

  (3) Collect blood from the orbital vein until the capillary tube is full.

  (4) Transfer to another tube (Eppendorf tube etc.) and stir well.

  (5) Blood is similarly collected from another organism.

  (6) For example, the blood is sedimented by centrifugation at 2,000 rpm for several seconds.

  (7) For example, 350 μl of distilled water is added and left at room temperature for about 30 seconds.

  (8) Add 350 equivalents (for example, 350 μl) of PBS with double concentration and stir well.

  (9) Centrifuge at 2,000 rpm for 2 minutes.

  (10) Discard the supernatant containing the hemolyzed red blood cells.

(11) Suspend blood cells in SM (staining medium = PBS containing 5% FBS, 0.05% NaN ₃ ), bisect and centrifuge. At this time, for example, if transferred to a round bottom 96-well plate and then centrifuged, antibody staining of a large number of samples is easy.

  (12) For example, two types of antibody staining are performed on the cell pellet. For example, biotinylated Ly5.1 (A20) antibody, FITC-Ly5.2 antibody (104) antibody, PE-Mac-1 antibody, PE-Gr-1 antibody and APC-B220 antibody are added to one of the two halves. This staining is used to determine the reconstruction of the myeloid lineage and the B lymphocyte lineage. For example, biotinylated Ly5.1 (A20) antibody, FITC-Ly5.2 (104) antibody, PE-CD4 antibody and PE-CD8 antibody are added to the other bisected sample. With this staining, the reconstruction of the T lymphocyte system can be determined.

  (13) After incubation at 4 ° C., the cells are washed and streptavidin-Texas Red is added.

  (14) After incubation at 4 ° C., the cells are washed, resuspended in 200 μl SM, analyzed by FACS, and judged after sorting.

  (15) Chimerism, reconstruction unit (RU), and competitive reconstruction unit (CRU) can be determined as follows. RU represents the amount of hematopoietic stem cell activity, and CRU represents the number of hematopoietic stem cells.

Chimerism calculation method:
Total Chimerism (%) = (% Ly5.1 cell × 100) / (% Ly5.1 cell +% F1 cell)
B lymphocyte lineage chimerism (%) = (% B220 ⁺ Ly5.1 cells × 100) / (% B220 ⁺ Ly5.1 cells +% B220 ⁺ F1 cells)
T lymphocyte lineage chimerism (%) = (% CD4 ⁺ CD8 ⁺ Ly5.1 cells × 100) / (% CD4 ⁺ CD8 ⁺ Ly5.1 cells +% CD4 ⁺ CD8 ⁺ F1 cells)
Myeloid lineage chimerism (%) = (% Mac-1 ⁺ Gr-1 ⁺ Ly5.1 cells x 100) / (% Mac-1 ⁺ Gr-1 ⁺ Ly5.1 cells +% Mac-1 ⁺ Gr-1 ⁺ F1 cell).

  The calculation method of RU follows the definition of Harrison (Harrison DE, et al .: Exp. Hematol. 21: 206-219, 1993) and can be obtained by a calculation formula using data obtained by FACS analysis. .

RU = (% Ly5.1 cells × (number of competing cells / 10 ⁵ )) /% F1 cells.

In the above example, when 2 × 10 ⁵ bone marrow cells are used as competing cells,
RU = (% Ly5.1 cells × 2) /% F1 cells.

  Stem cells can also be represented by CRUs. The frequency of CRU can be determined by fitting the number of test cells and the ratio of negative mice to the maximum likelihood calculation formula. A positive mouse is a mouse that exhibits 1% or more of chimerism in all three strains, and a negative mouse is defined as a mouse of less than that. The calculation formula is as follows. Groth S. F. , J .; Immunol. Methods 49, R11-R23, 1982.

  The mean activity of stem cells (MAS) means the RU per hematopoietic stem cell and can be determined by the following calculation formula.

  MAS = RU / CRU (RU / cell).

  As used herein, being adapted for transplantation means that the chimerism is 1% or more, preferably 10% or more.

  Examples of the transplant used in the method of the present invention include transplanting adult hematopoietic stem cells as they are, and transplanting cells obtained by differentiating or amplifying adult hematopoietic stem cells. Here, examples of the purpose of transplantation of adult hematopoietic stem cells and target diseases include the following. 1) Diseases in which qualitative and / or quantitatively abnormal stem cells need to be replaced with normal stem cells: severe aplastic anemia, myelodysplastic syndrome, thalassemia, congenital immune deficiency syndrome, etc .; Diseases that need to avoid irreversible lymphoid or hematopoietic tissue damage due to chemotherapy or radiation: leukemia (excluding chronic lymphocytic leukemia (CLL)), malignant lymphoma, multiple myeloma, solid (For example, breast cancer, neuroblastoma, small cell lung cancer, ovarian cancer, etc.); 3) diseases requiring permanent enzyme supplementation: inborn errors of metabolism.

  The use of the method of the present invention enables efficient selection, separation and enrichment of cells suitable for transplantation, thereby enabling more efficient treatment and side effect reduction in stem cell transplantation as described above. I can do it now. In addition, since transplantable cells can be selected with high efficiency, transplantation treatment can be performed using only autologous raw materials. In addition, if an amplification factor or the like is used, it is possible to prepare a necessary amount for treatment after efficiently removing a small amount of transplant-compatible cells. After the necessary amount is prepared, appropriate cells may be further concentrated by the transplant suitability determination method of the present invention. In such cases, rejection (eg, graft-versus-host disease) is reduced or impossible. Also, when cells enriched for cells that are suitable for transplantation are used, the speed of recovery after transplantation (for example, recovery of the hematopoietic system) is faster than those for which transplantation compatibility has not been determined. Therefore, the effect that a therapeutic effect appears early is also show | played.

(Endomucin itself)
In another aspect, the present invention provides a composition for identification of adult hematopoietic stem cells, comprising a nucleic acid molecule encoding endomucin or a part thereof. Endomucin itself is also a marker for adult hematopoietic stem cells, so that itself can also be used as a composition for detecting adult hematopoietic stem cells or a composition for preparing the same. In particular, it is understood that the present invention is useful as a composition for identification and / or determination of adult hematopoietic stem cells because endmucin can be used to screen for specific factors. . Such screening methods include, for example, a step of providing a nucleic acid molecule encoding endomucin or a part thereof, a step of bringing a candidate chemical substance into contact with the nucleic acid molecule or a part thereof, and a candidate for specific binding. Including a step of selecting.

  Preferably, as the nucleic acid molecule encoding endmucin, any form of the nucleic acid molecules shown in the above (Adult hematopoietic stem cell marker-factor for nucleic acid form) can be used. More preferably, the nucleic acid molecule encoding endomucin of the present invention comprises the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9 or a part thereof. Alternatively, the nucleic acid molecule encoding endmucin of the present invention comprises a sequence encoding the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 or a part thereof.

  In another aspect, the present invention provides a composition for identification of adult hematopoietic stem cells, comprising an endomucin polypeptide or a part thereof. Endomucin itself serves as a marker for adult hematopoietic stem cells, so that itself can also be used as a composition for detecting adult hematopoietic stem cells or a composition for preparing the same. In particular, it is understood that the present invention is useful as a composition for identification and / or determination of adult hematopoietic stem cells because endmucin can be used to screen for specific factors. . Such screening methods include, for example, a step of providing an endomucin polypeptide or a part thereof, a step of contacting a candidate chemical substance or antibody with the polypeptide or a part thereof, a candidate for specific binding. Including a step of selecting.

  Preferably, any form of the polypeptide shown in the above (Adult hematopoietic stem cell marker-factor for polypeptide form) can be used for this endomucin. More preferably, the endomucin of the present invention comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10 or a part thereof.

(Adult hematopoietic stem cell determination method)
In another aspect, the present invention provides a method for determining whether a candidate cell is an adult hematopoietic stem cell, the method comprising (I) providing a candidate cell to be determined; (II) a nucleic acid encoding endomucin Contacting the candidate cell with a molecule or part thereof, or an agent that specifically reacts with an endmucin polypeptide or part thereof; and (III) a nucleic acid molecule or part thereof encoding the factor and the endmucin. Or confirming whether the gene for the endmucin is expressed in the candidate cell by detecting a specific reaction with the endomucin polypeptide or a part thereof, wherein the candidate cell The gene expression of endomucin in provides a method indicating that the candidate cell is an adult hematopoietic stem cell.

  Here, the step of providing candidate cells can be performed using any method known in the art. It is understood that such cells may be for transplantation purposes or for differentiation purposes and are not limited to such purposes. Such cells can be obtained from, for example, bone marrow, peripheral blood, umbilical cord blood and the like of the subject animal.

  In addition, a nucleic acid molecule encoding endomucin or a part thereof, or an agent that specifically reacts with endomucin polypeptide or a part thereof is the above-mentioned (adult hematopoietic stem cell marker-nucleic acid form) and (adult hematopoietic stem cell marker). -Polypeptide forms) are used. For example, in the case of a nucleic acid molecule, such an agent can be prepared by a known technique such as a chemical technique such as synthesis, a molecular biological technique such as PCR, or a genetic engineering technique. When the factor is an antibody, as is well known in the art, the animal to be immunized, such as a rabbit, is immunized with a specific target, such as endomucin, and boosted as necessary, and serum is obtained from the immunized animal. Can be obtained by purifying the serum and purifying the serum as necessary, or antibodies can be prepared by preparing hybridomas and preparing cells that produce monoclonal antibodies. If the factor is a low molecular weight compound, it can be synthesized based on the identified structure using techniques well known in the art, or if it is the same as the natural product, it can be chromatographed from the raw material containing such natural product. For example, it can be obtained by purification or separation.

  Contact of such factors with a sample containing cells can be accomplished by simply mixing them or by passing the cells through the column with the factors retained on a carrier such as chromatography.

  The detection of a specific reaction can be performed simply by confirming whether the target cell is bound to a factor. In the case of using a carrier such as chromatography, it can be used as an index whether or not there are bound cells.

  In a preferred embodiment, stem cells that can be determined by the present invention include, but are not limited to, hematopoietic stem cells. Such stem cells preferably include those derived from bone marrow cells, but are not limited thereto. Alternatively, a sample predicted to be rich in adult hematopoietic stem cells such as ES cells or differentiated cells thereof can be used as such candidate cells.

  In a preferred form, adult hematopoietic stem cells can be determined with an indicator that CD41 is positive and / or CD45 is negative.

  In a preferred embodiment, the adult hematopoietic stem cell determination method of the present invention comprises at least one factor specific for a marker selected from the group consisting of CD41, CD34, Lin (Lineage marker), and c-Kit, The method further comprises a step of contacting the candidate cell, wherein the CD34 and CD41 positive expression, the Lin negative expression, and the c-Kit positive expression (preferably strong positive expression) in the candidate cell are the candidate cell. Is a stem cell.

  In one embodiment, when the negative expression of CD34 is negative or weakly positive, the candidate cell is characterized in that there are many cells that are determined to be more undifferentiated. A state in which the undifferentiated state is high is, for example, the ability to be selected from the group consisting of the ability to form nmEM colonies consisting of multilineage cells, and the ability to reconstruct and maintain the hematopoiesis of a lethal dose of irradiated organisms over time. It can be characterized by maintaining

  Alternatively, when the negative expression of CD34 is positive, the candidate cells are characterized in that there are many cells that are determined to have a lower undifferentiated state. A decrease in the undifferentiated state is, for example, a decrease in the ability to form nmEM colonies consisting of multi-lineage cells, and a lack of the ability to reconstruct and maintain the hematopoiesis of long-lived irradiated organisms over a long period or only for a short period of time. It can be characterized by ability selected from the group consisting of maintenance ability.

  The confirmation of the expression can be performed using magnetic beads or FACS. For magnetic beads and FACS, methods well known in the art can be used, and these can also be combined.

  In a preferred embodiment, the method for determining adult hematopoietic stem cells of the present invention further includes a step of depleting differentiation antigen-positive (lineage-positive) cells. This depletion process is performed by MACS. The MACS technology is a system for separating target cells by passing the cells labeled with magnetic microbeads through a column set in a strong magnetic field. The MACS technology is as follows.

  First, the cells are labeled with magnetic microbeads. As this label, there are a direct label and an indirect label, which can be properly used depending on the situation. The method of directly immunoreacting these microbeads with cells is called direct labeling. If there are no antibody magnetic microbeads for the target cells, the antibody against this antibody is first labeled with a specific antibody on hand. The method of secondary labeling of labeled microbeads is called indirect labeling. As advantages of direct labeling, there are advantages such as a high separation rate and a high-purity cell fraction. Advantages of indirect labeling include, but are not limited to, effects such as taking various combinations and sensitizing antibody labeling.

  Labeled cells can be separated by either positive selection (a method of labeling and isolating target cells) or depletion. Positive selection is a method of isolating a label using an indicator. The depletion method is a method of separating negative cells that are not labeled with microbeads and pass through a magnetic field, and the cells to be separated are not activated by antibodies, and can be separated even when there are no specific antibodies in the cells. It is unique in that it can combine positive selection with the depletion method.

  This depletion step can be performed using at least one Lin marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220.

  In preferred embodiments, the Lin marker may be conjugated to IgG MACS beads.

  In a preferred embodiment of the present invention, candidate cells can include cells separated by density gradient centrifugation. For such density gradient centrifugation, a separation medium such as Ficoll, Percoll, or sucrose (many are sugars or sugar derivatives) can be used. Therefore, in a preferred embodiment of the present invention, the method for determining adult hematopoietic stem cells of the present invention further comprises a step of preparing candidate cells by subjecting a specimen containing adult hematopoietic stem cells to density gradient centrifugation.

(Adult hematopoietic stem cell preparation method)
In another aspect, the present invention provides a method for producing a composition in which adult hematopoietic stem cells are isolated or enriched, comprising the step of A) providing a source comprising or expected to contain adult hematopoietic stem cells. Providing a method comprising: B) contacting the source with an identification means of endmucin; C) isolating or enriching endmucin positive cells.

  Here, a sample containing or predicted to contain adult hematopoietic stem cells can be provided using techniques well known in the art, such as ES cells or differentiation from ES cells, bone marrow collection, peripheral blood Examples include, but are not limited to, collection and cord blood collection.

  Any form as described in the above (Adult Hematopoietic Stem Cell Determination Method) can be adopted for contact with the factor and the sample and confirmation of the expression of endomucin.

  Preferably, the identification means used is an anti-endomucin antibody and the enrichment is performed by flow cytometry.

  FACS and / or magnetic beads can be used to separate or concentrate cells that express endomucin. In such a case, it is particularly preferable to use an antibody. Such antibodies are preferably labeled, and any such label can be used as described elsewhere herein.

  In one preferred embodiment, preferred separations or enrichments used in the methods of the invention include, but are not limited to, panning with antibody-immobilized dishes, FACS or magnetic beads or combinations thereof.

  The present invention is also a kit for producing a composition in which adult hematopoietic stem cells are isolated or concentrated, and includes or is expected to contain A) means for identifying endmucin; B) the means for identifying and adult hematopoietic stem cells. There is also provided a kit comprising: a means for contacting the source; and C) a means for isolating or concentrating endomucin positive cells.

  The identification means is as described above in the present specification, and can include, for example, an antibody against endomucin, a nucleic acid molecule containing a complementary sequence, and the like.

  Preferably, the present invention is a kit for preparing adult hematopoietic stem cells, comprising: (I) a factor specific to an endmucin gene or an endmucin gene product; (II) the endmucin gene is expressed in cells in the sample And (III) means for isolating or concentrating cells in which the endomucin gene is expressed. It will be understood that any form described herein above (Adult Hematopoietic Stem Cell Preparation Method) can also be used for the separation or concentration of cells.

  Here, the factor or the identification means can take any form described in (Adult hematopoietic stem cell marker-factor for nucleic acid form) and (Adult hematopoietic stem cell marker-factor for polypeptide form). Understood. As a means for confirming whether endmucin is expressed, for example, a label or a moiety that binds to the label can be used. Such a label may be any label as long as it is distinguishably labeled, and examples thereof include techniques such as fluorescence, phosphorescence, chemiluminescence, radioactivity, enzyme substrate reaction, and antigen-antibody reaction. It is not limited. Alternatively, when the factor interacts using an immune reaction such as an antibody, a system often used in an immune reaction such as biotin-streptavidin may be used. The label used in the present invention may be Texas-Red, PE, APC, FITC, biotin and the like. Hoechst Blue, Hoechst 33342, Hoechst Red and the like can be used as DNA staining dyes.

  In one embodiment, the kit of the present invention further comprises means for confirming whether other stem cell markers are expressed. The kit may preferably include factors specific for CD41 and / or CD45 markers. Such means include, for example, at least one factor specific for a marker selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit, and the factor is a cell in the sample. Means for confirming whether or not it is expressed in (1) is not limited thereto. For such another marker, the criteria described in the above (Adult Hematopoietic Stem Cell Determination Method) can be employed.

  In another embodiment, the kit of the invention further comprises at least one factor specific for the lineage marker, as well as means for ascertaining whether the factor is expressed in cells in the sample. Such means include, for example, at least one marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220 as the Lin marker. For such another marker, the criteria described in the above (Adult Hematopoietic Stem Cell Determination Method) can be employed. For simplicity, these lineage markers are preferably all negative.

(Adult hematopoietic stem cell preparation)
In another aspect, the present invention relates to a stem cell prepared by the adult hematopoietic stem cell preparation method of the present invention. Such adult hematopoietic stem cells can be said to be different from conventional cells because endmucin-expressing cells are concentrated. Such expression cells are preferably isolated. In addition, since the compatibility of transplantation has increased unexpectedly due to the concentration of endmucin-expressing cells, it can be said that the stem cells of the present invention have a remarkable effect that cannot be predicted from conventional stem cells.

  In an alternative aspect, the present invention provides a cell population that is enriched for cells expressing endomucin. Conventionally, endomucin has not been used as a cell marker, and therefore, there has been no cell enriched with such a marker. Such a cell population can be said to be significantly different from the prior art in that it contains abundant stem cells and has a markedly improved transplantation compatibility.

(Methods and medicines for treatment, prevention, and prognosis using adult hematopoietic stem cells)
In another aspect, the present invention relates to a method for treating, preventing or prognosing a subject in need of transplantation of adult hematopoietic stem cells, wherein (I) whether the gene for endomucin is expressed in the transplant (II) a step of isolating or concentrating a transplant containing stem cells expressing the endomucin gene; and (III) a step of transplanting the isolated or concentrated transplant into the subject. A method is provided. Here, confirmation of endomucin gene expression (which may include expression of transcripts and / or translations) can be performed by using a factor specific thereto and confirming / detecting its specific binding. . Thus, means for isolating or enriching cells that have been confirmed to express endomucin can also be separated by, for example, FACS and / or magnetic beads using specific antibodies, as described herein. However, it is not limited to them. The cells enriched or separated in this way preferably contain only endomucin positive cells. This is because transplantation compatibility is remarkably increased. Moreover, it was found that the correlation between transplantation compatibility and the expression of endomucin was extremely high (almost 100%).

  In a preferred embodiment, the present invention relates to (Ia) wherein at least one marker selected from the group consisting of CD41, CD45, CD34, Lin (Lineage marker), and c-Kit is expressed in the transplant. And (IIa) in the transplant, the CD41 positive is an indicator that it is a stem cell, and CD45 negative is an indicator that it is a stem cell, and the CD34, the Lin (Lineage marker) Further comprising isolating or concentrating a transplant containing stem cells in which the c-Kit and Sca-1 genes are negatively expressed or weakly positively expressed, negatively expressed, positively and positively expressed, respectively. . The further selection with these markers significantly increases the reliability of transplantation compatibility, and the fact that these markers have been used in the past has the added benefit of being able to utilize conventional knowledge. Is done.

  In a further preferred embodiment, the step (IIa) in the present invention isolates a transplant containing adult hematopoietic stem cells in which at least one of the c-Kit and Sca-1 genes is strongly positively expressed. Or further concentrating. Since the strong expression of these markers was found to have a stronger correlation with transplantation compatibility, it is preferable to look at these strong expression to more reliably determine the transplantation compatibility of adult hematopoietic stem cells. .

  In the transplantation method of the present invention, the transplant containing adult hematopoietic stem cells is performed ex vivo. Therefore, this transplant is preferably derived from the subject itself, but is not limited thereto. When an immune reaction is anxious (for example, derived from cord blood), an immunosuppressive method may be performed. Procedures for avoiding rejection are well known in the art. For example, the New Surgery System, Vol. 12, heart transplantation / lung transplantation, from technical and ethical development to revision (3rd revised edition), Nakayama Shoten It is described in. Examples of such methods include methods such as the use of immunosuppressants and steroids. Immunosuppressants that prevent rejection are currently "cyclosporine" (Sandimune / Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (Predonin, Methylpredonin), "T cells There are “antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs “cyclosporine, azathioprine, steroid hormone”. The immunosuppressive agent is desirably administered at the same time as the combination therapy of the present invention, but it is not necessary. Therefore, as long as the immunosuppressive effect is achieved, the immunosuppressive agent can be administered before or after the combination therapy of the present invention.

  Alternatively, in another preferred embodiment, the transplant comprising adult hematopoietic stem cells is advantageously derived from ES cells, cells differentiated from ES cells, bone marrow, umbilical cord blood or peripheral blood. This is because such raw materials contain or are expected to contain adult hematopoietic stem cells.

  In another aspect, the present invention relates to a medicament for treatment, prevention or prognosis of a subject requiring transplantation of adult hematopoietic stem cells, and A) cells enriched with adult hematopoietic stem cells expressing endomucin Providing a medicament, including a population. Such cells are preferably positive for CD41 and / or negative for CD45. More preferably, it is advantageous that the genes of CD34, CD41, Lin (Lineage marker) and c-Kit have positive expression, positive expression, negative expression and positive expression, respectively. This is because it has been revealed by the present invention that such cells have transplantation compatibility. Such pharmaceutical preparation methods are described herein above and any form thereof can be used.

(use)
In another aspect, the present invention provides the use of endomucin for the determination of adult hematopoietic stem cells. Here, as an end mucin, the arbitrary forms described above in this specification can be used.

  In another aspect, the present invention provides the use of endomucin for determining transplant suitability. The transplant to be examined for transplantation compatibility preferably contains adult hematopoietic stem cells. Here, as the endmucin and the factor or identification means, any form described above in this specification can be used.

  In another aspect, the present invention provides for the determination of an adult hematopoietic stem cell of an agent specific for an endomucin polypeptide or a part thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding endomucin or a part thereof. Provide the use of.

  In another aspect, the present invention relates to a medicament comprising an adult hematopoietic stem cell, which is an agent specific for an endmucin polypeptide or a part thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding endmucin or a part thereof. Provide use for manufacturing. Here, as the endmucin and the factor or identification means, any form described above in this specification can be used.

  In another aspect, the present invention provides the use of stem cells expressing endomucin for producing a medicament for transplantation of adult hematopoietic stem cells. Descriptions relating to pharmaceuticals and endomucin are described herein above, and any of the forms described above can be used.

(Cell composition)
In another aspect, the present invention provides a composition comprising cells that express endomucin. Preferably, the cell is CD41 positive or CD45 negative. More preferably, the cells are CD41 positive and CD45 negative. The cells are further negative or weakly positive for CD34 expression. More preferably, the cells are positive (more preferably strong positive) for c-Kit and Sca-1 expression. Preferably, the cell is negative for Lin expression.

  Accordingly, the present invention provides an adult hematopoietic stem cell composition that is enriched for cells expressing endomucin and expressing CD41. In addition, in the adult hematopoietic stem cell composition of the present invention, cells that do not express CD45 are enriched in the adult hematopoietic stem cell composition.

  In another aspect, the present invention relates to cells obtained by the method of the present invention, tissues and organs obtained from the cells.

  In a preferred aspect of the present invention, the present invention provides a pharmaceutical composition comprising an adult hematopoietic stem cell composition obtained by the method of the present invention. Such a pharmaceutical composition may optionally contain a pharmaceutical carrier and further medicinal ingredients.

  In another aspect of the present invention, the present invention provides a method for treating or preventing a disease or disorder requiring an adult hematopoietic stem cell composition or differentiated cells derived therefrom. This method comprises A) administering the cells obtained by the present invention to a subject in need of such treatment or prevention.

  In another aspect of the present invention, the present invention treats or prevents a disease or disorder requiring an adult hematopoietic stem cell composition or differentiated cells derived from the adult hematopoietic stem cell composition obtained by the present invention. For use. Techniques for using such cells are well known in the art, and those skilled in the art can appropriately select them.

  In another aspect, the present invention provides a stem cell prepared by a method for amplifying the above-mentioned adult hematopoietic stem cell composition and maintaining its pluripotency or self-renewal ability. This method can be used in vivo or in vitro. The adult hematopoietic stem cell composition prepared by the method of the present invention has characteristics that are unconventional so that a certain quality can be ensured and it can be prepared in large quantities. Can have.

  Thus, the present invention can provide an adult hematopoietic stem cell composition more efficiently. Therefore, it can be said that such an effect is an exceptional effect that is not found in the prior art, and its usefulness is hard to beat.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

  Reagents used in the following examples were obtained from Wako Pure Chemicals, Sigma, and cell-related reagents from Gibco unless otherwise stated. Animal breeding, National Society for Medical Researchg created "Principles of Laboratory Animal Care" and the Institute of Laboratory Animal Resource is created, National Institute of Health has published "Guide for the Care and Use of Laboratory Animals" (NIH Publication, No. 86-23, 1985, revised), in accordance with the standards stipulated by the University of Tokyo and the Japanese government, and in accordance with the spirit of animal welfare.

(Example 1: Search for molecules expressed on the membrane of hematopoietic stem cells)
In this example, a search was performed for molecules expressed on the membrane in CD34-KSL cells (CD34-, c-Kit +, Sca-1 +, lineage-bone marrow cell fraction) containing hematopoietic stem cells at a high frequency.

  CD34-KSL cells were prepared as follows.

Bone marrow fluid from 8-10 week old C57BL / 6 Ly5.1 mice was collected and mononuclear cells were separated by specific gravity (density gradient) centrifugation. After staining mononuclear cells with various monoclonal antibodies (anti-CD34, anti-c-Kit, anti-Sca-1, Lineage-marker mixture (CD4, CD8, B220, Gr-1, Mac-1, Ter119)), FACS Vantage cell sorter (Becton Dickinson) using ^{CD34 - / low c-Kit +} Sca-1 + Lineage-marker - (CD34 - KSL) hematopoietic stem cell fraction sorting by 100 cells / well in 96 multi-titer plates did. As a culture solution, X-vivo-10 (BioWhittaker), which is a serum-free medium, was used at 200 μl / well, and SCF (Stem cell factor) and TPO (thrombopoietin) were added at 100 ng / ml as cytokines.

As controls, CD34 ⁺ KSL cells, KSL, TER119, Gr-1, Mac-1, B220, THy-1, NK1.1, DN, DP, CD4SP, CD8SP (mouse); CD34 ⁺ / 38 ⁻ , CD34 ⁺ , CD34 ⁺ / 13 ⁺ , CD14 ⁺ , CD15 ⁺ , GPA ⁺ , GPA ⁺ , CD71 ⁺ , CD41 ⁺ , CD3 ⁺ , CD19 ⁺ , CD56 ⁺ (human) cell populations were also prepared.

  Next, a cDNA library was prepared from the 5000 cells by a conventional method, and molecules expressed on the membrane of hematopoietic stem cells were searched using a modified signal sequencing strap method.

  As a result, endomucin (1a) was identified as a candidate molecule.

Therefore, the endmutin gene expression level in various cell fractions was compared by RT-PCR for this candidate molecule, endmucin. The protocol is shown below. The following sequences were used as primers for RT-PCR (mouse)
Sense: 5'-ACAACTGAAGGTCCCCTAAGG-3 '(SEQ ID NO: 29);
Antisense: TTGGTTTTCCCCTGTGCAGAG-3 ′ (SEQ ID NO: 30).

(Human)
Sense 5'-ACCAGGTAGTGTTCTACAACC-3 '(SEQ ID NO: 31);
Antisense 5′-AGTGCTCACCAGACTCATGAG-3 ′ (SEQ ID NO: 32).

  As a result, expression of endomucin was observed only in the cell group considered to be enriched with stem cells. Endomucin was shown to be specifically expressed in hematopoietic stem cells at the mRNA level.

(Example 2: Endomucin expression analysis using antibody)
Next, in this example, it was examined how endomucin polypeptides are expressed in stem cells using various antibodies. The antibody used in this example is Dr. Vestweber D.W. Institute of Cell Biology, ZMBE, University of Munster, Germany. However, it will be appreciated by those skilled in the art that endomucin polypeptides can be prepared as appropriate to generate antibodies using techniques well known in the art.

  First, SP (side population) cells were prepared. As a common feature of stem cells, it has been reported that Hoechst 33342 is often discharged. It is known that about 0.1% of SP cell fractions that do not stain in order to excrete Hoechst 33342 well into the bone marrow are present in the bone marrow cells and that hematopoietic stem cells are concentrated. (Goodell MA et al, J. Exp. Med. 183: 1797, 1996). The procedure for preparing SP cells is shown below.

  FACS was performed using Hoechst Blue, Hoechst 33342, and Hoechst Red instead of the labeled antibody in the procedure for preparing CD34-KSL cells described in Example 1. Here, weakly positive cells in Hoechst Blue and HoechstRed were taken out as SP cells. Since it was reported that hematopoietic stem cells were concentrated, this cell was used in this Example as a simple method for concentrating hematopoietic stem cells.

  Next, cells were selected using anti-rat PE and endomucin. The protocol is shown below.

Bone marrow cells were collected from the femur and tibia of a mouse (Ly5.1) using a 25G needle and a 2.5 ml syringe, and the bone marrow was extracted using 5 ml of PBS. The bone marrow cell suspension was overlaid on 6 ml of Ficoll-Paque (Amersham Pharmacia Biotech) and centrifuged at 400 × g for 20 minutes at room temperature, and then mononuclear cells in the intermediate layer were collected. The collected cells were washed with 10 ml of staining solution (SM: 5% FCS + 0.05% NaN ₃ + PBS), and an unlabeled differentiation antigen (Lineage) antibody was added. After reacting on ice for 20 minutes, it was washed with SM and mixed with magnetic beads bound with anti-rat IgG antibody. The reaction was further allowed to proceed on ice for 15 minutes, and the cells were passed through a depletion column in which Lineage antigen-negative cells not bound to the magnetic beads were fixed to a magnet stand, and the cells that had flowed out without being trapped on the column were collected. (Lineage-negative cells). Biotin-labeled anti-endomucin antibody, PE-Cy5.5-labeled anti-Sca-1 antibody, APC-labeled anti-c-Kit antibody, and FITC-labeled anti-CD34 antibody were added to the collected cells, and after 30 minutes on ice, streptavidinated PE was further added. The reaction was performed for 60 minutes. The cells were washed with SM and made turbid in 4 ml of SM, and then the endomucin positive cell fraction was separated into a 96-well plate using FACS Vantage (Becton Dickinson).

As a result, it was revealed that SP cells were enriched with endmutin-expressing cells. In addition, approximately 0.8% of all bone marrow cells were positive for endmucin. Also,
Similar endomucin expression was observed using a group of cells selected using other differentiation markers (prepared in Example 1).

  From this result, end mucin cells were detected in less than 1% of Mac-1 weakly positive and B220 positive cells. From these results, although endmucin is hardly expressed in differentiation antigen positive cells, some of B220 positive cells (less than 1%) and some of Mac-1 weakly positive cells that contain undifferentiated cells are included. It was shown that

  Taken together, about 90% of bone marrow KSL cells were positive for endomucin.

  Next, by the same FACS selection, four KSL cells were fractionated by the expression pattern of CD34 and endomucin. As a result, the CD34-endomucin-fraction is present at about 3%, the CD34-endomucin + fraction is present at about 23%, the CD34 + endomucin + fraction is present at about 65%, and the CD34 + endomucin-fraction is about 9%. % Existed. As a result, about 90% or more of CD34-KSL are positive for end mucin, and it was demonstrated that end mucin is one of the markers for hematopoietic stem cells.

(Example 3: Colony formation assay)
Next, in this example, the four fractions separated in Example 2 were subjected to an in vitro culture assay to observe colony formation. This aims to confirm by culture that stem cells are contained. The cells in each fraction were cultured under differentiation induction conditions. The protocol is shown below.

  Mononuclear cells were depleted to about 1/50 or less of Lin positive cells using MACS (Magnetic Cell Separation System) technology. The MACS technology is a system for separating target cells by passing the cells labeled with magnetic microbeads through a column set in a strong magnetic field. The MACS technology is as follows.

  First, cells are labeled with magnetic microbeads having an antibody specific to Lin positive cells, and the labeled cells are separated by a depletion method to separate negative cells that are not labeled with microbeads and pass through a magnetic field. Was reduced to about 1/50 or less.

  KSL cells were gated by expression of CD34 and enmutin. The protocol is as follows.

  Bone marrow cells were collected from the femur and tibia of a mouse (Ly5.1) using a 25G needle and a 2.5 ml syringe, and the bone marrow was extracted using 5 ml of PBS. The bone marrow cell suspension was overlaid on 6 ml of Ficoll-Paque (Amersham Pharmacia Biotech), centrifuged at 400 × g for 20 minutes at room temperature, and the mononuclear cells in the intermediate layer were collected. The collected cells were washed with 10 ml of staining solution (SM, 5% FCS + 0.05% NaN3 + PBS), and an unlabeled differentiation antigen (Lineage) antibody was added. After reacting on ice for 20 minutes, it was washed with SM and mixed with magnetic beads bound with anti-rat IgG antibody. The reaction was further allowed to proceed for 15 minutes on ice, and the cells were passed through a depletion column in which Lineage antigen-negative cells not bound to the magnetic beads were fixed to a magnetic stand, and the cells that flowed out without being trapped on the column were collected (Lineage-negative cells). .

  Biotin-labeled anti-Endomucin antibody, PE-Cy5.5-labeled anti-Sca-1 antibody, APC-labeled anti-c-Kit antibody and FITC-labeled anti-CD34 antibody were added to the collected cells, and after 30 minutes on ice, streptavidinated PE was further added. The reaction was performed for 60 minutes. The cells were washed with SM and made turbid in 4 ml of SM. Then, using FACSVantage (Becton Dickinson), various cell fractions were separated into 96 well plates one by one in each well and cultured.

  48 cells were extracted from each fraction into S-clone medium (supplemented with 10 ng / ml stem cell factor (SCF), 10 ng / ml mIL3, 50 ng / ml hTPO, 2 U / ml EPO and 10% fetal bovine serum). Plated. The cells were cultured for 15 days and the phenotype was stained by May-Giemmsa staining. The protocol is as follows.

  After culturing for 15 days, a cytospin specimen was prepared by spraying the cells grown in each well onto a slide glass. After staining and fixing the cells with May staining solution for 5 minutes, the cells were further stained with Giemsa staining solution for 20 minutes and washed with water. Air-dried and observed cell morphology under light microscope.

  As a result, a large number of mixed colonies containing neutrophil / macrophage / erythroblast / megakaryocyte cell lineages, which are indicators of pluripotency, were found in CD34-endomucin + cells. Thus, endmucin is shown to be an efficient marker for stem cells.

(Example 4: Bone marrow reconstruction assay)
In Example 3, since a mixed colony containing the cell lineage of neutrophil / macrophage / erythroblast / megakaryocyte was also observed from the CD34-endomucin- fraction, in order to observe hematopoietic stem cells directly, A reconstitution assay was performed. The protocol is shown below.

Cells were sorted using FACS and mixed with 2 × 10 ⁵ C57BL / 6 Ly5.2 mouse bone marrow cells, respectively. Cells were injected through the tail vein into 57BL / 6 Ly5.2 mice irradiated with a lethal dose of radiation. Peripheral blood was collected periodically after transplanting the cells, and the chimerism of the transplanted cells in the peripheral blood was observed using a flow cytometer. At 15 weeks after bone marrow transplantation, bone marrow reconstitution was observed in the group transplanted with cells of the endmucin positive fraction, whereas bone marrow reconstitution was observed in the group transplanted with cells of the endmucin negative fraction. It was not recognized at all. Therefore, it became clear that endomucin is an index for judging transplant suitability.

(Example 5: Correlation between endmutin expression level and undifferentiation)
In this example, the distribution of KSL cells in the cell fraction separated by the end mucin gate was observed, and its correlation with undifferentiation was verified.

  First, classification of 1-e (the strength of endmucin PE is 1-5, 5-35, 35-150, 150-400, 400-1500, respectively) is performed using endomucin PE according to five types of expression intensity. It was. The abundance ratios were 18.6%, 56%, 20.1%, 1.58%, and 0.26%, respectively.

  As a result, it can be seen that high expression of endomucin correlates with high expression of both Sca-1 and c-Kit.

(Example 6: Distribution of c-Kit ^high and Sca-1 ^high cells gated by the expression of CD34 and endomucin)
Next, the relationship between c-Kit, Sca-1 and CD34 and endomucin expression was further verified using mononuclear cells that had been depleted of lineage positive cells to 1/21 by MACS technology.

  Depleted cells were depleted as described in the above examples. The cells were examined for expression correlation in lineage negative cells for endomucin, c-Kit (a), Sca-1 (b) and CD34 (c).

As a result, it was confirmed that CD34 ^- endomucin ^high cells are highly correlated with c-Kit ^high and Sca-1 ^high cells, that is, they are more efficient indicators of stem cells and transplantation compatibility.

The above results indicate that endmucin is not co-expressed with lineage surface markers in bone marrow and spleen cells, 70-90% of CD34 ^- KSL cells are endmucin positive, and CD34 ^- Lin ^- c. -Kit ⁺⁺ Sca-1 ⁺⁺ cells were found to be 100% positive for endomucin.

CD34 ⁻ KSL endomucin ⁺ cells retained the same pluripotency as CD34 ⁻ KSL cells.

It has been found that CD34 ⁻ KSL endomucin ⁺ cells are preferred for reconstitution of hematopoietic cells.

(Example 7: Endomucin expression in ES cells-embryoid body formation and cell staining)
(Formation of embryoid bodies and cell staining)
E14.1 ES cells were cultured on irradiated fetal fibroblasts. Culture is DMEM, 15% fetal bovine serum, 0.1 mM non-essential amino acid (GIBCO / Invitrogen), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol (Sigma), 1000 U / ml leukemia inhibitory factor (LIF) (Chemicon, Temecula, CA). When differentiating ES cells, the ES cells are first loosened with 0.25% trypsin / EDTA (GIBCO / Invitrogen), and then EBD culture solution [Iscove's modified Dulbecco's medium (IMDM), 15% fetal bovine serum, 200 μg / ml human transferrin (Sigma), 150 μM monothioglycerol (Sigma), 50 μg / ml ascorbic acid (Sigma), 2 mM L-glutamine]. In order to remove fetal fibroblasts, the cells were cultured in a T25 flask (Corning) for 45 minutes, and non-adherent cells were collected. The cells were suspended in 5 μl of culture solution at a concentration of 100 cells and suspended in a petri dish for bacteria inverted in 50 μl of culture drops by the hanging drop method. After 24 hours, cells were collected from each culture drop and further cultured in a 10 cm bacterial petri dish using EBD culture solution. The culture medium was changed by half every 3 days. The day when the hanging drop was started was designated as EB day 0. The collected embryoid bodies were loosened with 0.25% trypsin (Sigma) and then washed twice with PBS containing 2% serum. 5 × 10 ⁵ floating cells were stained with PE-labeled anti-Flk1 antibody and FITC-labeled anti-CD41 antibody, or PE-labeled anti-CD45 antibody (BD Pharmingen) and biotinylated anti-endomucin antibody (clone V7c7), APC-labeled streptavidin, and FACSAria ^™ (Becton Dickinson) or Mo-flo (Dako Cytomation) was used to analyze the expression of cell surface antigens and to sort the cells.

(Culture of OP9 cells)
OP9 cells were collected on the day before the start of co-culture with ES cells at a concentration of 40,000 cells per well in a 4-chamber culture slide (BD Falcon, MA) or 24-well culture plate (Corning, NY) treated with 0.1% gelatin. Sowing. A portion of embryoid somatic cells sorted by FACS was seeded on OP9 cells, α-MEM, 10% fetal calf serum, 2 mM L-glutamine, 5 × 10 ⁻⁴ M 2-mercaptoethanol, 100 U / ml penicillin ( Sigma), 100 μg / ml streptomycin, 10 ng / ml mouse recombinant IL-3, 10 ng / ml mouse SCF, 2 U / ml erythropoietin, and 50 ng / ml thrombopoietin (PeproTech, Rocky Hill, NJ) did. The number of formed colonies was observed on the second day of culture. Next, on the 6th day, cells by trypsin treatment were suspended, and the number of proliferated blood cells was assayed using a hemocytometer.

(result)
The results of the expression analysis are shown in FIG. 1 (endomucin expression in embryoid bodies formed from mouse ES cells (1)). The relationship between the expression of Flk-1, CD41, CD45 and endomucin in embryoid body constituent cells was examined using FACS. Flk-1 is a receptor tyrosine kinase that is expressed early in a group of cells that have been determined to differentiate into the mesoderm system, and CD41 is a specific marker for hematopoietic progenitor cells that develop in the embryoid body (non-reactive). Patent Documents 17 to 18). On the other hand, CD45 is used as a pan-blood cell marker. Endomucin begins to express around day 5 after forming embryoid bodies from ES cells. Most of the embryoid bodies 4 to 7 days after culture are CD45 negative cells, and are divided into four cell groups: CD41 ⁺ Endomucin ⁺ , CD41 ⁺ Endomucin ⁻ , CD41 ⁻ Endomucin ⁺ and CD41 ⁻ Endomucin ^−. Became clear. Further, more mature blood cells positive for CD45 appeared in the embryoid bodies after the seventh day, but most of them were Endomucin positive.

FIG. 2 shows another result of endomucin expression in embryoid bodies formed from mouse ES cells. (A) The changes in the expression of Flk-1, CD41, CD45 and the expression of endomucin after embryoid body formation are shown by a line graph. (B) Embryoid somatic cells 5 to 7 days after culture are fractionated into four cell groups: CD41 ⁺ Endomucin +, CD41 ⁺ Endomucin ⁻ , CD41 ⁻ Endomucin ⁺ and CD41 ⁻ Endomucin ⁻ . (C) Endometin positive cells of EB9,75 in b are divided into three fractions of CD41 negative, CD41 weak positive, and CD41 positive, and the majority of CD41 weak positive and CD41 positive cells are CD45 positive blood cells. On the other hand, the majority of CD41 negative cells are CD45 negative and are assumed to be vascular endothelial cells.

Day 4 and day 6 embryoid somatic CD45 ⁻ cells were divided into four cell groups ^: CD41 ⁺ Endomucin ⁺ , CD41 ⁺ Endomucin ⁻ , CD41 ⁻ Endomucin ⁺ and CD41 ⁻ Endomucin ⁻ . Cells were collected and co-cultured with OP9 cells. The result is shown in FIG. As a result, CD41-positive cells, which are markers of nascent hematopoietic progenitor cells, contain endomucin-positive cells at a rate of 2 to 3%, and this CD41 ⁺ Endomucin ⁺ cell fraction is highly pluripotent with high hematopoietic activity. It became clear that the progenitor cells were concentrated.

  Thus, in the present invention, it can be seen that endomucin expression is an indicator of adult hematopoietic stem cells.

(Example 8: Analysis of fetal hematopoiesis)
The yolk sac and fetus were separated from the fetus at 8.5 days of gestation, and the yolk sac and the para-aortic reproductive midrenal region were separated and collected from 10.5 days, respectively. Each tissue was treated with 0.05% collagenase to form floating cells, and the target cell fraction was collected using FACS after staining with an antibody as in the case of embryoid bodies, and co-cultured with OP9 cells. . Analysis of colony-forming ability was performed using α-MEM 1.2% methylcellulose (Aldrich Chemical), 30% fetal calf serum (Sigma), 1% BSA (Sigma), 10 ⁻⁴ 2-mercaptoethanol, 10 ng / ml mouse IL-3, It was performed using culture conditions of 10 ng / ml mouse SCF, 2 U / ml erythropoietin and 50 ng / ml thrombopoietin. Colonies formed from fetal erythroid progenitor cells were observed on day 2-3 of culture, and colonies formed from adult hematopoietic progenitor cells were observed on day 12 of culture.

(result)
The results are shown in FIG. 4 (endomucin expression in yolk sac). We examined the expression of endomucin in the yolk sac where fetal hematopoiesis is performed. (A) In the embryonic yolk sac, the expression of endomucin was hardly observed in the CD45-negative CD41-positive fetal hematopoietic progenitor cells. On the other hand, some of CD45-positive and CD41-positive were endomucin-positive. Methylcellulose colony assay to examine the distribution of early erythroid progenitor cells (EryP), colony-forming unit-macrophages (CFU-M), fetal hematopoietic progenitor cells As a result, it was revealed that fetal hematopoietic progenitor cells do not express Endomucin regardless of CD45 positive or negative.

  (B) In the embryonic yolk sac of 10.5, a part of CD41 positive was Endomucin positive regardless of CD45 positive or negative. When the methylcellulose colony assay was performed, endmutin was expressed in adult erythroblast progenitor cells (BFU-E, colony-forning unit-erythroid: CFU-E) regardless of whether CD45 was positive or negative. However, it was revealed that Endomucin selectively forms in mixed colony-forming units exhibiting pluripotency.

  Thus, it can be seen that endomucin is not expressed in fetal hematopoietic stem cells, and that adult hematopoietic stem cells can be identified.

(Example 9: Competitive bone marrow reconstruction ability test)
(Reconstruction ability test)
Test cells from C57BL / 6-Ly5.2 mice were sorted by FACS and lethal dose irradiated Ly5.1 mice tibial bone marrow with 1 × 10 ⁵ competitor cells (total bone marrow cells of C57BL / 6-Ly5.1 mice) Ingested directly. Further, 2 × 10 ⁵ competitor cells were injected from the tail vein (the number of competitor cells was 3 × 10 ^{5 in} total). After transplantation, peripheral blood was collected and biotin-labeled anti-Ly5.1 antibody (eBioscience), FITC-labeled anti-Ly5.2 antibody, APC-labeled anti-Gr-1 antibody, APC-labeled anti-Mac-1, PE-labeled anti-CD4 antibody, PE Staining was performed with a labeled anti-CD8 antibody (PharMingen), PE-Cy7-labeled anti-B220 (Caltag), Texas Red-labeled streptavidin (Molecular Probes), and chimerism was analyzed by FACS. Chimerism was calculated as follows. Total chimerism (%) = donor-derived cell / (donor-derived cell + competitor cell-derived cell) × 100, chimerism of each differentiation line cell (%) = donor-derived differentiation line cell / (donor-derived differentiation line cell + competitor cell) Derived each differentiation line cell) × 100.

(result)
FIG. 5 shows the expression results of endomucin in the para-aortic reproductive middle kidney region. (A) Endomucin expression in the para-aortic reproductive mid-renal region on embryonic day 10.5 when adult hematopoietic stem cells capable of engrafting in adult bone marrow appear for the first time was examined. When the expression of CD41 and endomucin in CD45 ⁻ cells was observed, it was divided into four fractions of CD41 ⁻ Endomucin ⁻ , CD41 ⁻ Endomucin ⁻ , CD41 ⁺ Endomucin ⁻ and CD41 ⁺ Endomucin ⁺ . (B) In the co-culture system with OP9, in the presence of SCF, TPO, IL-3, and EPO, the number of colonies formed per 1000 cells of each fraction was examined. As a result, CD41 ⁺ Endomucin ⁺ was significantly While showing high colony formation ability, (C) high proliferation activity and pluripotency were shown. (D) In examining competitive bone marrow reconstruction ability, the long-term bone marrow remodeling activity of each fraction was assayed. Each fraction of 750 cells was inoculated into a lethal dose irradiated recipient mouse together with the competitor cells, and the engraftment after transplantation was observed. As a result, only all mice transplanted with CD41 ⁺ Endomucin ⁺ cells survived engraftment. confirmed. (E) Although the peripheral blood donor cells have low chimerism in this recipient mouse, long-term contribution to hematopoiesis was confirmed. (F) Although the differentiation tendency of the engrafted cells was superior to B cells, myelocytes and T cells were also observed although they were low, and engraftment of pluripotent hematopoietic stem cells was confirmed.

  Thus, it can be seen that the adult hematopoietic stem cells of the present invention can be clinically applied.

(Example 10: Separation and concentration of stem cells using endomucin)
Anti-endomucin antibody is immobilized on magnetic beads and reacted with bone marrow cells. The cells are passed through a column fixed on a magnetic stand, and endmucin-positive cells are purified by collecting the cells trapped on the column. This cell fraction is a cell fraction in which hematopoietic stem cells are highly concentrated, and hematopoietic stem cells can be concentrated by a one-step operation.

(Example 11: Compulsory expression of endomucin gene)
100 hematopoietic stem cells CD34-KSL are selected into 96-well plates by FACS, and precultured with S clone medium (Sanko Junyaku) for 24 hours in the presence of 100 ng / ml mouse SCF and 100 ng / ml human TPO. The cells are then added with concentrated endomucin retrovirus at MOI 600 and allowed to infect the virus for 24 hours. CD34-KSL cells transfected with retrovirus were further liquid cultured with 20 ng / ml, mouse SCF, 100 ng / ml, human TPO, and then 20 ng / ml, mouse SCF, 50 ng / ml, human on day 7 and 14 of culture. A colony assay using methylcellulose medium in the presence of TPO, 20 ng / ml, mouse IL-3 and 5 unit / ml EPO is performed, and the number of mixed colonies (CFU-Mix / CFU-nmeM) is counted on the 10th day.

Simultaneously with the colony assay, a part of the cultured cells was transplanted together with 2 × 10 ⁵ competitor cells (Ly5.2) from the tail vein of a mouse (Ly5.2) irradiated with a 9.5 Gy lethal dose. The effect of forced expression of adult murine hematopoietic stem cells of endmutin gene is determined by measuring the chimerism of endomucin gene-introduced cells in peripheral blood at 3 months (competitive bone marrow transplantation experiment).

  In this way, the effect of forced expression of the endmucin gene is demonstrated.

Example 12 Direct Introduction of Endomucin Polypeptide
Instead of the above-described form using the gene, it was examined whether endomucin could be used directly to retain the ability of stem cells.

  A chimeric protein is produced with an endomucin extracellular domain and the Fc portion of human IgG (End-Fc protein). When selecting endomucin positive cells using FACS and conducting a colony assay using methylcellulose medium in the presence of 20 ng / ml mouse SCF, 50 ng / ml human TPO, 20 ng / ml mouse IL-3 and 5 unit / ml EPO, By adding End-Fc protein, the change in the number of CFU-Mix / CFU-nmeM due to neutralization of endomucin molecule function on hematopoietic stem cells is assayed. In addition, a liquid bone culture is performed for a certain period in a state where End-Fc protein is added to an endmucin-positive cell, and then a competitive bone marrow transplantation experiment is performed to measure the chimerism of the endmutin gene-introduced cell in the peripheral blood at 3 months.

  This can determine the effect of neutralizing the function of endomucin molecules on hematopoietic stem cells.

  It was shown that when colony assay and bone marrow transplantation assay were performed using this endmucin, the same effect was obtained although the effect was weak.

(Example 13: Effect of modified endmucin)
The correlation between the difference in the types of the above-mentioned end mucins 1a, 1b, 1c and 1d splice variants and stem cell undifferentiation and transplantation compatibility is examined.

  100 hematopoietic stem cells CD34-KSL are sorted into 96-well plates by FACS, and precultured with S clone medium (Sanko Junyaku) in the presence of 100 ng / ml mouse SCF and 100 ng / ml human TPO for 24 hours. The cells are then added with each splice variant endomucin retrovirus enriched at MOI 600 and allowed to infect the virus for 24 hours. CD34-KSL cells transfected with a retrovirus were further liquid cultured with 20 ng / ml mouse SCF, 100 ng / ml human TPO, and then cultured on days 14 and 20 with 20 ng / ml mouse SCF, 50 ng / ml human TPO, 20 ng / ml. A colony assay using methylcellulose medium in the presence of ml mouse IL-3 and 5 units / ml EPO is performed, and the number of mixed colonies (CFU-Mix / CFU-nmeM) is counted on the 10th day.

Simultaneously with the colony assay, a part of the cultured cells was transplanted from the tail vein of a mouse (Ly5.2) irradiated with a 9.5 Gy lethal dose together with 2 × 10 ⁵ competitor cells (Ly5.2). The effect of forced expression in adult hematopoietic stem cells of each splice variant of the endmucin gene is determined by measuring the chimerism of the endometin gene-introduced cells in the peripheral blood at 3 months (competitive bone marrow transplantation experiment).

(Example 14: Screening of a substance that interacts with endomucin)
Endmucin can also be used in the present invention, but factors that interact with endmucin are also useful. Therefore, in this example, screening for factors that interact with endomucin is performed. Endometin-interacting factors can be screened and identified by providing unbound endmucin and confirming that the endmucin is activated by providing a candidate factor.

  Since the compound thus selected specifically interacts with endomucin, it can be used as a factor relating to stem cell undifferentiation or a marker for transplantation compatibility. Such a substance can be a small molecule that activates endomucin by binding to and polymerizing endmucin.

  In this example, whether a substance is endomucin is determined by determining that the factor can be present on the membrane and / or has the ability to bind to an endmucin-specific consensus sequence. Can do. In this example, it can be determined by transfecting cells with the endmucin gene, immunostaining with an anti-endomucin antibody, and confirming the presence of membrane localization when the candidate compound is administered. it can. If such a factor is a candidate compound such as a protein, it is introduced directly into the cell, and then whether or not such a factor is transferred to the membrane is immunostained using an antibody specific for that factor. I can confirm. Such techniques are well known in the art.

In order to judge the ability to bind to an endmucin consensus sequence, a gel shift assay using a double-stranded oligonucleotide containing a base sequence used as an endmucin-binding base sequence is examined. When a candidate compound is administered, a nucleic acid molecule containing this base sequence reacts with a certain end mucin, and after electrophoresis and separation on a polyacrylamide gel, a complex of endomucin and a nucleic acid molecule containing a consensus sequence is formed. It can be determined by identifying. Usually, if formation of a complex can be significantly confirmed, it can be determined that the factor has the same function as endomucin.

  To determine whether it is endmucin, it is sufficient that a significant activity can be determined by at least one of the above.

(Example 15: Use of endomucin interacting factor)
Using the factor that interacts with endomucin prepared in Example 14, it is possible to determine the undifferentiated state of stem cells, determine the suitability for transplantation, and undifferentiate stem cells and enhance the adaptability for transplantation. When such compounds activate endomucin expression, it can be seen that when administered to stem cells and cultured for a period of time, the stem cells become adult hematopoietic stem cells, or transplant compatibility is acquired or enhanced.

(Example 16: Knock-in of endomucin fused with fluorescent dye)
A knock-in vector containing a fusion nucleic acid sequence obtained by fusing a nucleic acid sequence encoding endomucin of the present invention with a sequence encoding a fluorescent dye (Venus dye) was constructed.

  ES cells obtained by knocking-in Venus, which is a fluorescent dye, to ATG in end mucin 1st exon are prepared using techniques well known in the art. This ES cell is differentiated in vitro, and it is confirmed by using an in vitro system that endomucin is expressed in stem cells using Venus as an index.

  Using this ES cell, a mouse individual is prepared by a technique well known in the art, and it is confirmed in the mouse individual that endomucin is expressed in stem cells using Venus as an index. Since the endomucin gene is deficient in homo mice, the function of endmucin can be identified by analyzing functional abnormalities in the adult hematopoietic stem cells in the endmutin gene deficient mice.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a composition for easily and efficiently determining the undifferentiated state and / or transplantation compatibility of stem cells is provided. Such a composition is used and very useful for using hematopoietic stem cells and differentiated cells for various treatments. Since such a composition can be used as a medicine or a diagnostic agent, it is very useful particularly in the pharmaceutical industry.

As an effect of endomucin in the present invention, the expression of endomucin in embryoid bodies formed from mouse ES cells is shown. The result of having investigated the relationship between the expression of Flk-1, CD41, and CD45 of an embryoid body constituent cell and endomucin using FACS is shown. From the left, the results on day 1, day 3, day 5, day 7 and day 10 are shown. The vertical axis is endmucin, and the horizontal axes are Flk-1, CD41, and CD45, respectively. FIG. 2 shows another result of endomucin expression in embryoid bodies formed from mouse ES cells. (A) The change (relative value) of Flk-1, CD41, and CD45 expression and endomucin expression after embryoid body formation is shown by a line graph. The scale on the horizontal axis (number of days) indicates the second day, the fourth day, the sixth day, the eighth day, and the tenth day. (B) embryoid bodies Cells CD41 ⁺ Endomucin 5-7 days after culture ^{+, CD41 + Endomucin -, CD41} - Endomucin +, CD41 - Endomucin - were divided into four cell groups. The panel shows EB 5.75 (5th day), 7.75 (7th day), 9.75 (9th day) from the left. (C) According to the horizontal axis of b, distribution was shown based on CD45 positive / negative. EB9,75 endmucin positive cells in b are divided into three fractions of CD41 negative, CD41 weak positive, and CD41 positive, and the majority of CD41 weak positive and CD41 positive cells are CD45 positive blood cells. The majority of CD41 negative cells are CD45 negative and are assumed to be vascular endothelial cells. FIG. 3 shows that embryonic somatic CD45 ⁻ cells on day 4 (EB4) and day 6 (EB6) were divided into four cell groups: CD41 + Endomucin +, CD41 ⁺ Endomucin ⁻ , CD41 ⁻ Endomucin ⁺ , CD41 ⁻ Endomucin ^−. The results are shown in which cells of each fraction were collected and co-cultured with OP9 cells. The tables in FIGS. 3a and 3b show the number of cells in each fraction seeded on OP-9, the number of blood cells grown on OP-9 observed at 6 days after the middle, and the number of blood cells grown on the right. A value obtained by converting the number per 1000 fractional cells is shown. C is a graph of the result of conversion per 1000 pieces. E + indicates endomucin positive, DP indicates both positive, 41+ indicates CD41 positive, and DN indicates both negative. FIG. 4 shows endomucin expression in the yolk sac. In this experiment, we examined the expression of endomucin in the yolk sac where fetal hematopoiesis is performed. (A) In the embryonic yolk sac, the expression of endomucin was hardly observed in the CD45-negative CD41-positive fetal hematopoietic progenitor cells. On the other hand, some of CD45-positive and CD41-positive were endomucin positive. Methylcellulose colony assay to examine the distribution of early erythroid progenitor cells (EryP), colony-forming unit-macrophages (CFU-M), fetal hematopoietic progenitor cells As a result, it was revealed that fetal hematopoietic progenitor cells do not express Endomucin regardless of CD45 positive or negative. (B) In the embryonic yolk sac on day 10.5, a part of CD41 positive was positive for endmucin regardless of CD45 positive or negative. When the methylcellulose colony assay was performed, endmutin was expressed in adult erythroid progenitor cells (BFU-E, colony-forning unit-erythroid: CFU-E) regardless of whether CD45 was positive or negative. However, it was revealed that endomucin is selectively formed in mixed colony-forming units exhibiting pluripotency. E ⁺ indicates endomucin positive, E ⁻ indicates endomucin negative, 41 ⁺ indicates CD41 positive, and 41 ⁻ indicates CD41 negative. CD45 ⁺ indicates CD45 positive and CD45 ⁻ indicates CD45 negative. FIG. 5 shows the expression results of endomucin in the para-aortic reproductive middle kidney region. (A) is a FACS pattern of a CD45 negative fraction in which hematopoietic stem cell activity is observed in AGM cells of embryonic 10.5, and is divided into four fractions depending on the expression of CD41 and endomucin. The numbers indicate the abundance ratio. (B, C) A total of 5 fractions obtained by adding a CD45 positive fraction to these 4 fractions were tested for the colony formation rate in co-culture with OP-9 cells. (B) Colony formation rate per 1000 seeded cells (classified as CD45 positive and CD45 negative cells, and for CD45 negative, classified as CD41 positive / negative (+/-) and endmucin positive / negative (+/-)) Show). The colony formation rate per 1000 seeded cells was significantly higher in CD45 negative CD41 positive endmucin positive cells. (C) The number of blood cells grown on OP-9, observed at the time after 6 days, was converted to a value per 5000 seeded cells. Also in this case, it was confirmed that the number of CD45 negative CD41 positive endomucin positive cells was significantly large. (D) When 5 fractions of cells were transplanted directly into the bone marrow, 750 per lethal radiation-irradiated mouse, only in mice transplanted with CD45 negative CD41 positive endomucin positive cells were all hematopoietic by donor cells. Reconstruction was observed. The contribution rate of this reconstruction is low (E), and B cells are significantly reconstructed (F). Such a tendency is observed when E10.5AGM cells are transplanted into adult mice. This is a characteristic finding. This contribution to hematopoiesis has been observed over a long period of time, indicating that only the transplanted CD45 negative CD41 positive endomucin positive cell fraction has hematopoietic stem cell activity.

(Explanation of sequence listing)
SEQ ID NO: 1 is the nucleic acid sequence of endomucin (1a = full length) (mouse).
SEQ ID NO: 2 is the amino acid sequence of endomucin (1a = full length) (mouse).
SEQ ID NO: 3 is the nucleic acid sequence of Endomucin (1b) (mouse).
SEQ ID NO: 4 is the amino acid sequence of endomucin (1b) (mouse).
SEQ ID NO: 5 is the nucleic acid sequence of Endomucin (1c) (mouse).
SEQ ID NO: 6 is the amino acid sequence of endomucin (1c) (mouse).
SEQ ID NO: 7 is the nucleic acid sequence of endomucin (1d) (mouse).
SEQ ID NO: 8 is the amino acid sequence of endomucin (1d) (mouse).
SEQ ID NO: 9 is the nucleic acid sequence of full length human endmucin.
SEQ ID NO: 10 is the amino acid sequence of the full length human endmucin.
SEQ ID NO: 11 is the nucleic acid sequence of Flt3 / Flk2 (human).
SEQ ID NO: 12 is the amino acid sequence of Flt3 / Flk2 (human).
SEQ ID NO: 13 is the nucleic acid sequence of CD34 (mouse).
SEQ ID NO: 14 is the amino acid sequence of CD34 (mouse).
SEQ ID NO: 15 is the nucleic acid sequence of CD34 (human).
SEQ ID NO: 16 is the amino acid sequence of CD34 (human).
SEQ ID NO: 17 is the nucleic acid sequence of Mac-1 (mouse).
SEQ ID NO: 18 is the amino acid sequence of Mac-1 (mouse).
SEQ ID NO: 19 is the nucleic acid sequence of CD4 (mouse).
SEQ ID NO: 20 is the amino acid sequence of CD4 (mouse).
SEQ ID NO: 21 is the nucleic acid sequence of CD8 (mouse).
SEQ ID NO: 22 is the amino acid sequence of CD8 (mouse).
SEQ ID NO: 23 is the nucleic acid sequence of B220 (mouse).
SEQ ID NO: 24 is the amino acid sequence of B220 (mouse).
SEQ ID NO: 25 is the nucleic acid sequence of c-Kit (mouse).
SEQ ID NO: 26 is the amino acid sequence of c-Kit (mouse).
SEQ ID NO: 27 is the nucleic acid sequence of Sca-1 (mouse).
SEQ ID NO: 28 is the amino acid sequence of Sca-1 (mouse).
SEQ ID NO: 29 is a primer (sense) for identifying mouse endomucin.
SEQ ID NO: 30 is a primer (anti) for identifying mouse endmucin.
SEQ ID NO: 31 is a primer for human endmutin identification (sense).
SEQ ID NO: 32 is a primer for human endmucin identification (anti).
SEQ ID NO: 33 is the nucleic acid sequence of mouse CD41.
SEQ ID NO: 34 is the amino acid sequence of mouse CD41.
SEQ ID NO: 35 is the nucleic acid sequence of human CD41.
SEQ ID NO: 36 is the amino acid sequence of human CD41.
SEQ ID NO: 37 is the nucleic acid sequence of mouse CD45.
SEQ ID NO: 38 is the amino acid sequence of mouse CD45.
SEQ ID NO: 39 is the nucleic acid sequence of human CD45.
SEQ ID NO: 40 is the amino acid sequence of human CD45.

Claims (49)

A composition for detecting adult hematopoietic stem cells, comprising means for identifying endomucin. The composition according to claim 1, wherein the adult hematopoietic stem cell comprises a cell having a long-term bone marrow remodeling ability. The composition according to claim 1, wherein the identification means comprises a factor specific for a polypeptide comprising endomucin or a part thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding endmucin or part thereof. The end mucin is
(A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof;
(B) in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, at least one amino acid has at least one mutation selected from the group consisting of substitution, addition and deletion, and an organism A polypeptide having a biological activity;
(C) a polypeptide encoded by an allelic variant or splice variant of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9;
(D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10; or (e) identity to any one polypeptide of (a)-(d) A polypeptide having an amino acid sequence that is at least 70% and having biological activity;
The composition of claim 1 comprising: The composition of claim 1, wherein the endmucin comprises the sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10. 4. The composition of claim 3, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9. The composition according to claim 1, wherein the means for identifying endmucin is an anti-endomucin antibody. The composition according to claim 1, wherein the means for identifying endmucin is a nucleic acid molecule comprising a sequence complementary to a nucleic acid sequence encoding endmucin or a part thereof. The composition according to claim 1, wherein the identification means is labeled or labelable. The composition according to claim 9, wherein the label is selected from the group consisting of a radioactive substance, a chemiluminescent substance, a fluorescent substance, a phosphorescent substance, a color former, a ligand, a hapten and their raw materials. 2. The composition of claim 1 comprising further marker identifying means. 12. The composition of claim 11, wherein the additional marker comprises at least one marker selected from the group consisting of CD34, Lin (Lineage marker), c-Kit, Sca-1, Flt3 / Flk2, CD41 and CD45. . 12. The composition of claim 11, wherein the additional marker comprises at least one marker selected from the group consisting of CD41 and CD45. 12. The composition of claim 11, wherein the additional markers include CD41 and CD45. The composition according to claim 1, wherein the marker has the ability to distinguish between fetal hematopoietic progenitor cells and adult hematopoietic stem cells. The composition according to claim 1, wherein the adult hematopoietic stem cells are cells that retain pluripotency. The composition according to claim 1, wherein the adult hematopoietic stem cells are embryonic stem cell-derived cells. The composition according to claim 1, wherein the adult hematopoietic stem cells are cells used for bone marrow transplantation. The composition according to claim 1, wherein the adult hematopoietic stem cell is a cell produced by forming an embryoid body from an embryonic stem cell. 10. The composition of claim 9, wherein the label comprises at least one dye selected from the group consisting of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin. A method for detecting adult hematopoietic stem cells in a sample comprising:
A) A method comprising the step of contacting the sample with a means for identifying endomucin. further,
B) determining that adult hematopoietic stem cells are present when the endmucin-positive cells are found,
The method of claim 21, comprising: C) further comprising contacting the sample with a marker factor identification means selected from the group consisting of CD41 and CD45.
The method of claim 21.   The adult hematopoietic stem cell is determined to be present when the CD41-positive cell is found and at least one selected from the group consisting of when the CD45-positive cell is found. 24. The method according to 23. 24. The method of claim 21, wherein the sample is a sample comprising or expected to contain embryonic stem cells. A kit for detecting adult hematopoietic stem cells in a sample, the kit comprising:
A) A kit comprising means for identifying endomucin. A method for producing a composition in which adult hematopoietic stem cells are isolated or concentrated, comprising:
A) providing a source comprising or expected to contain adult hematopoietic stem cells;
B) contacting the source with endumin identification means;
C) separating or concentrating endomucin positive cells,
Including the method. 28. The method of claim 27, wherein the identification means is an anti-endomucin antibody and the enrichment is performed by flow cytometry. 28. The method of claim 27, further comprising the step of concentrating CD41 positive cells. 28. The method of claim 27, further comprising concentrating CD45 negative cells.   Determining the reactivity of the sample to a further marker selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit, wherein the gene expression of the CD34, CD41 and c-Kit is determined by the candidate cell 28. The method of claim 27, further comprising the step of further enriching adult hematopoietic stem cells by indicating that the candidate cell is a stem cell, wherein the negative expression of the Lin (Lineage marker) gene indicates that the candidate cell is a stem cell. The method described. 28. The method of claim 27, wherein the separation or concentration is performed using panning with antibody immobilized dishes, flow cytometry or magnetic beads. 28. The method of claim 27, wherein the separation or concentration is performed using a combination of FACS and magnetic beads. A kit for producing a composition in which adult hematopoietic stem cells are isolated or concentrated,
A) means for identifying endomucin;
B) means for contacting said identifying means with a source comprising or expected to contain adult hematopoietic stem cells; and C) means for isolating or concentrating endomucin positive cells;
A kit comprising: An adult hematopoietic stem cell composition prepared by the method according to claim 27. An adult hematopoietic stem cell composition in which cells expressing endomucin and CD41 are concentrated. The adult hematopoietic stem cell composition according to claim 36, wherein the adult hematopoietic stem cell composition is enriched with cells that do not express CD45. A method for determining whether a candidate cell is an adult hematopoietic stem cell, comprising:
(I) providing a candidate cell to be determined;
(II) contacting the candidate cell with a nucleic acid molecule encoding endomucin or a part thereof, or an agent that specifically reacts with endomucin polypeptide or a part thereof; and (III) the factor and the endmucin. Confirming whether or not the gene for the endmutin is expressed in the candidate cell by detecting a specific reaction with the encoding nucleic acid molecule or a portion thereof, or an endomtin polypeptide or a portion thereof,
Wherein the gene expression of the endomucin in the candidate cell indicates that the candidate cell is an adult hematopoietic stem cell. 39. The method of claim 38, wherein the confirmation of expression is performed by flow cytometry. Further comprising contacting said candidate cell with at least one factor specific for a marker selected from the group consisting of CD34, CD41, Lin (Lineage marker) and c-Kit,
The gene expression of the CD34, CD41 and c-Kit in the candidate cell indicates that the candidate cell is a stem cell, and the negative expression of the Lin (Lineage marker) gene indicates that the candidate cell is a stem cell 40. The method of claim 38, wherein: 40. The method of claim 38, further comprising depleting lineage positive cells. 40. The method of claim 38, wherein the depletion step is performed by MACS. 40. The method according to claim 38, wherein the depletion step is performed using at least one LIN (Lineage) marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220. A kit for determining whether a candidate cell is an adult hematopoietic stem cell,
(I) means for identifying endomucin;
(II) A kit comprising means for detecting the expression of endomucin in a candidate cell by the identification means, wherein the gene expression of the endmucin in the candidate cell indicates that the candidate cell is an adult hematopoietic stem cell. A method for the treatment, prevention or prognosis of a subject in need of transplantation of adult hematopoietic stem cells, comprising:
(I) a step of confirming whether or not the gene for endomucin is expressed in the cells to be transplanted;
(II) separating or concentrating cells expressing the endomucin gene; and (III) transplanting the separated or enriched cells to the subject.
Including the method. A system for the treatment, prevention or prognosis of a subject in need of transplantation of adult hematopoietic stem cells,
(I) means for identifying endomucin;
(II) means for isolating or concentrating cells expressing the gene for endomucin; and (III) means for transplanting the isolated or enriched cells into the subject,
A system comprising: A medicament for the treatment, prevention or prognosis of a subject requiring transplantation of adult hematopoietic stem cells,
A) a cell population enriched with stem cells expressing endomucin,
Containing a pharmaceutical. Use of endomucin for determination of adult hematopoietic stem cells. Use of stem cells expressing endomucin for the manufacture of a medicament for transplantation of adult hematopoietic stem cells.