WO2005049865A2 - Marqueurs pour cellules souches et compatibilite de transplantation - Google Patents

Marqueurs pour cellules souches et compatibilite de transplantation Download PDF

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Publication number
WO2005049865A2
WO2005049865A2 PCT/JP2004/017655 JP2004017655W WO2005049865A2 WO 2005049865 A2 WO2005049865 A2 WO 2005049865A2 JP 2004017655 W JP2004017655 W JP 2004017655W WO 2005049865 A2 WO2005049865 A2 WO 2005049865A2
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Prior art keywords
endomucin
polypeptide
acid sequence
seq
cell
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PCT/JP2004/017655
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English (en)
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WO2005049865A3 (fr
Inventor
Hiromitsu Nakauchi
Atsushi Iwama
Azusa Maeda
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Reprocell Inc.
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Publication of WO2005049865A2 publication Critical patent/WO2005049865A2/fr
Publication of WO2005049865A3 publication Critical patent/WO2005049865A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel gene relating to the undifferentiated state of a cell. More specifically, the present invention relates to a method for determining the undifferentiated state of a cell using such a gene, methods for isolation and preparation of stem cells, a composition and a system relating thereto, as well as a method and system for determining transplantation compatibility, and therapy and prevention using the same.
  • regeneration therapy regeneration therapy
  • regeneration therapy has not yet reached a point where it is usually applied to a number of patients suffering from organ or tissue dysfunctions.
  • organ transplantation or use of an auxiliary medical system or apparatus These therapies have problems in terms of shortage of donors, rejection, infection, durability, and the like.
  • donor shortage raises serious problems.
  • bone marrow transplantation bone marrow and umbilical cord blood banks have gradually become more widely used at home and abroad, though it is still difficult to provide a limited amount of samples to the number ofpatients inneed. Therefore, there is an increasingdemand for therapies using stem cells and regeneration medicine using the same in order to overcome the above-described problems .
  • Organs of organisms may be partially lost or seriously injured due to wounds or diseases during the life of the organism. Whether or not damaged organs can be regenerated depends on the organ (or the animal species) .
  • organs ortissues
  • Regeneration of tissues can be determined by confirming an improvement in the function of the tissue. Mammals have the ability to regenerate tissue and organs to some extent (e.g., regeneration of skin, liver, andblood) .
  • organs such as heart, lungs, brain, and the like, have poor regenerative ability. Therefore, it has been believed that if the organs are once damaged, their functions cannot be recovered. Conventionally, for example, when an organ is damaged, organ transplantation is substantially the only effective treatment.
  • stem cells are present in organs having a high level of regenerative ability. This presumption was proved by experimental bone marrow transplantation using animal models. Subsequent studies have demonstrated that stem cells in bone marrow are resources for regeneration of all kinds of blood cells. It has also been demonstrated that stem cells are present in organs having a high level of regenerative ability, such as bone marrow, skin, and the like. In addition, although it was long believed that the brain cannot be regenerated, it has been demonstrated that stem cells are present in the brain. It has been clarified that stem cells are present in any organ in the body and play a role in regeneration of the organs to some extent. Stem cells present in each tissue have plasticity to an extent beyond expectation, so- that stem cells in one organ may be used in regeneration of another organ.
  • stem cells can be used for regeneration of organs which have not been possible to date.
  • Regenerative medicine particularly stem cell therapy
  • stem cell therapy has increasingly attracted attention.
  • attention has been focused onto establishment of totipotent embryonic stem cells (ES cells) and preparation of cloned individuals from adult somatic cells. This is because techniques related to development and regeneration can be applied to stem cell therapies.
  • ES cells totipotent embryonic stem cells
  • stem cell therapies have proceeded into preliminary stages for the purpose of clinical applications .
  • stem cell therapies have been in actual use.
  • organs are reconstructed ex vivo; and organs are reconstructed in vivo.
  • stem cells are required for reconstruction of organs.
  • pluripotency and self-replication ability are required for stem cells for use in the above-described application.
  • Stem cells are roughly divided into two categories: embryonic stem cells and somatic (tissue) stem cells.
  • somatic stem cells hematopoietic stem cells have attracted attention for some time.
  • the self-replication ability and pluripotency of hematopoietic stem cells are required for maintaining mature blood cells having a short life time during the life-span of a human. This concept had already been proposed by 1961 (Till, J.E. , et al., Radiat. Res. 14: 21.3-222) .
  • a bone marrow transplantation method using a mouse model was established (Micklem, H.S., et al. : J.
  • hematopoietic stem cells are present at a low rate of several in about 100,000 bone marrow cells even in the case of mice. Therefore, in actual research or clinical application, hematopoietic stem cells need to be concentrated or purified.
  • hematopoietic stem cell transplantation therapies have been carried out using naturally-occurring cells, leading to various side effects.
  • a side effect is produced by pretreatment before transplantation using a large dose of an anticancer drug or radiation.
  • bacterial or fungal infectious diseases and hemorrhage due to suppression of bone marrow in the case of allotransplantation, when donor' s leucocytes survive and the number of the cells is increased, they recognize recipient organs as foreign and attack the organs (graft versus host diseases (GVHD) ) ; various pulmonary complications, mainly including cytomegalovirus (CMV) pneumonia; various visceral disorders due to disorders of vascular endothelial cells (cells lining the inner wall of blood vessels) ; various infectious diseases during immune suppression prolonged after survival of transplanted cells (at least 1-2 years) ; prolonged chronic GVHD exhibiting various symptoms; late-onset disorders (e.g., secondary cancers, gonad dysfunction, infertility, etc.); and the like.
  • CMV cytomegalovirus
  • transplantation often temporarily worsens systemic conditions.
  • the rate of death of patients due to complications is about 10 to 20% in autotransplantation and about 20 to 40% in allotransplantation. Even if patients overcome complications, some patients may relapse. Thus, the current transplantation therapies are inadequate.
  • stem cells are separated and purified and precursor cells are produced in large quantities from the stem cells, and the precursor cells as well as the stem cell are transplanted.
  • the method has already entered clinical trials.
  • Fluorescence activated cell sorting was developed in the 1980' s. Since then, techniques utilizing FACS have been employed for enrichment and purification of hematopoietic stem cells. It- has been revealed that high-purity hematopoietic stem cells are obtained by separating CD34 ⁇ KSL cells from multiply stained bone marrow cells (Osawa, M. et al . , Science, 273: 242-245, 1996). As described above, pluripotency and self-replication ability are the essential features of stem cells. To exploit these abilities, it is important to enrich and purify ste cells and expand the cells by in vitro culture.
  • FACS Fluorescence activated cell sorting
  • SCF stemcell factor
  • SCF is produced by bone marrow stromal cells and acts on pluripotent stem cells, bone marrow cells (e.g., CFU-M, CFU-GM, CFU-Meg, etc. ) , andlymphocyteprecursor cells to support their expansion and differentiation. That is, it is believed that SCF acts on cells from hematopoietic stem cells to precursor cells so as to aid other cytokines which induce differentiation toward the • final stage (S. Kitamura, Saitokain-no-Saizensen [Frontline of Cytokine] , Yodb-sha, editedbyT. Hirano, pp. 174-187, 2000) . However, such a factor cannot be a marker.
  • Cell surface markers such as CD34, Lin (lineage marker) , c-kit and Sca-1 and the like have been generally used for identifying a stem cell.
  • CD34 Lin (lineage marker)
  • c-kit c-kit and Sca-1 and the like have been generally used for identifying a stem cell.
  • a number of drawbacks are suggested where these markers are not particularlyspecific, andare not sufficient fordetermining transplantation compatibility, the stem cell undifferentiated state and the like by the use of each marker alone. Evenifthese arkers are combined, it is questionable whether stem cells can be separated to nearly 100 % purity.
  • transplantation compatibility evenifall themarkers areused, onlyamixture of cells comprising cells having transplantation compatibility and those having no such compatibility can be obtained. This is a reason why there are some patients who have successfully undergone bone marrow transplantation and those who have not successfully undergone the same.
  • stem cell characteristics and transplantation compatibility of hematopoietic stem cells it can be said that there is currently no
  • HSCs Hematopoietic stem cells
  • CD34 KSL Sca-1 lineage marker-negative (Lin ) (CD34 KSL) cells, which constitute 0.004 % of total nucleated BM cells, represent hematopoietic stem cells with long-term marrow repopulating ability, while CD34 + KSL cells are progenitors with short-term repopulating capacity (Osawa, M., Hanada,
  • HSC purification still include selection by negative markers for HSC, such as lineage-markers, CD34 and Flk2/Flt3 (Osawa, et al., 1996; Adolfsson, J., Borge, O.J., Bryder, D., Theilgaard-M ⁇ nch, K. , Astrand-Grundstr ⁇ , I., Sitnicka, E., Sasaki, Y., and Jacobsen, S.E.W. (2001). Immunity 15, 659-669; Christensen, J.L., andWeissman, I.L. (2001) . Proc. Natle. Acad. Sci. U.S.A.
  • HSCs develop from hemangioblasts, the common progenitor cells for HSCs andendothelial cells, or fromthe hemogenicendothelium (Choi, K. , Kennedy, M. , Kazarov, A. , Papadimitriou, J.C. , andKeller, G. (1998). Development 125, 725-732; de Bruijn, M.F., Ma, X., Robin, C, Ottersbach, K. , Sanchez, M.J., and Dzierzak, E. (2002) . Immunity 16, 673-83) . Therefore, HSCs and endothelial cells share a number of molecules crucial for their ontogeny.
  • endothelial cell surface molecules havebeen appliedto identifyHSC development; Flk-1 andVE-cadherin or ES cell-derivedhemangioblasts andTie-2, PCLP-1, and Sca-1 for he ogenic endothelium localized to the endothelial layer lining the wall of the dorsal aorta in intraembryonic aorta-gonads-mesonephros (AGM) region, from which the first definitive HSCs arise (Hamaguchi, I. , Huang, X.L., Takakura, N., Tada, J. , Yamaguchi, Y., Kodama, H., and Suda, T. (1999). .
  • c-kit and CD41 are absent on hemangioblasts or hemogenic endothelium, but mark the developing hamatopoietic stem and progenitor cells of both primitive and definitive types in theyolksacandAGM (Mikkola, H.K.A. , Fijiwara, Y. , Schlaeger, T.M., Traver, D. , and Orkin, S.H. (2003). Blood 101 , 508-516; Ferkowicz, M.J., Starr, M., Xie, X., Li, W. , Johnson, S.A., Shelley, W.C., Morrison, P.R., and Yoder, M.C. (2003) .Development 130, 4393-4403) . An increasing number of these marker antigens are facilitating progress in the precise identification of HSC development. SUMMARY OF THE INVENTION
  • the present invention achieved the above-identified object in part by the fact that the present inventors have found that Endomucin per se unexpectedly has a marker function to identify a stem cell such as a hematopoietic cell, and is an efficient marker for determining transplantation compatibility.
  • the inventors combined long-distance PCRamplification of full-length cDNA with the signal sequence trap by retrovirus-mediated expression screening (SST-REX) (Kojima, T., and Kitamura, T. (1999) . Nat. Biotech.17, 487-490) .
  • SST-REX retrovirus-mediated expression screening
  • Endomucin has been characterized to encode an endothelial sialomucin which is highly related to the CD34 molecule (Morgan, S.M., Samulowitz, U.
  • Endomucin is a novel cell surface antigen that marks both BM LTR-HSCs and developing definitive HSCs in embryos .
  • Endomucin expression in embryo was confined to definitive hematopoietic stem and progenitor cellsbutnottoprimitivehematopoieticprogenitors, leading to a successful tracking of definitive hematopoiesis in embryos.
  • Endomucin could be a useful marker to understand HSC ontogeny. Further, analysis of embryonic hematopoiesis demonstratedthat Endomucin expressionmarks the development of definitive HSCs .
  • a modified signal sequence trap was applied to mouse CD34 " c-kit + Sca-1 + lineage- (CD34 ⁇ KSL) hematopoietic stem cells (HSC) and identified Endomucin, an endothelial sialo ucin gene to be HSC-specific. Endomucin expression was confined to the KSL fraction and the majority of CD34 " cells within this- fraction exhibited the highest Endomucin expression. Of note, long-term repopulating (LTR) -HSCs were exclusively detected in the Endomucin "1" CD34 ⁇ KSL fraction, suggesting that all HSCs express Endomucin.
  • LTR long-term repopulating
  • Endomucin traces the development of definitive HSCs in both yolk sac and intraembryonic aorta-gonads-mesonephros (AGM) region.
  • AGM intraembryonic aorta-gonads-mesonephros
  • the present invention provides the following:
  • a arker composition for a stem cell comprising an agent specific to a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin polypeptide, or a portion thereof.
  • composition according to Item 1 wherein the stem cell is a hematopoietic cell.
  • the Endomucin polypeptide comprises: (a) a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO. 2, 4, 6, 8, or 10, or a fragment thereof; (b) a polypeptide having an amino acid sequence set forth in SEQ ID NO. 2, 4, 6, 8 or 10, or a fragment thereof, wherein at least one amino acid in the sequence has a mutation selected fromthe group consisting of substitution, addition, and deletion, and wherein the polypeptide has biological activity; (c) a polypeptide encoded by a spliced mutant or allelic mutant of a base sequence set forth in SEQ ID NO.
  • composition according to Item 1 wherein the Endomucin polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10.
  • compositionaccordingto Item1 wherein the nucleic acid molecule comprises a sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9.
  • composition according to Iteml further comprising at least one agent specific to a nucleic acid encoding a polypeptide selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2.
  • composition according to Itern 1 wherein said agent is selected from the group consisting of a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, an organic low molecular weight molecule, and a complex molecule thereof.
  • composition according to Item 8 wherein said agent is a nucleic acid molecule or a derivative thereof.
  • composition according to Item 9 wherein said agent is used as a .primer.
  • composition according to Item 8 wherein said agent is labeled or is capable of being labeled.
  • composition according to Item 12 wherein the label is selected from the group consisting of a radioactive material, chemilunescence material, fluorescence material, phosphorescence material, chromophore, ligand, hapten and raw material thereof.
  • a marker composition for a stem cell comprising an agent specifically binding to Endomucin polypeptide or a portion thereof.
  • composition according to Item 14 wherein said stem cell is a hematopoietic stem cell.
  • composition according to Item 14, wherein said Endomucin comprises: (a) a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; (b) a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 having at least one mutation selected from at least one amino acid substitution, addition and deletion, and having biological activity; (c) a polypeptide encoded by an allelic variant of a base sequence set forth in SEQ ID NO: 1 , 3 , 5, 7 or 9; (d) a polypeptide which is a species homolog of an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10; or (e) a polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides (a) to (d) , and having biological activity.
  • composition according to Item 14 wherein the Endomucin comprises a sequence set forth in SEQ ID NO: 2,
  • composition according to Item 14 further comprising at least one agent specific to a polypeptide selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2.
  • composition accordingto Item14 wherein saidagent is selected from the group consisting of a nucleic acid molecule, a polypeptide, a lipid / a sugar chain, an organic low molecular weight molecule,- and a complex molecule thereof.
  • compositionaccordingto Item14 wherein saidagent is an antibody or a derivative thereof.
  • composition accordingto Item14 wherein saidagent is labeled or is capable of being labeled.
  • composition according to Item 23 wherein the label is selected from the group consisting of a radioactive material, chemilunescence material, fluorescence material, phosphorescence material, chromophore, ligand, hapten and raw material thereof.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • APC allophycocyanin
  • a composition for determining transplantation compatibility of a transplant comprising an agent specific to a nucleic acidmolecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof.
  • the composition according to Item 26 wherein the transplant comprises a stem cell.
  • composition according to Item 26 further comprising at least one agent specific to a nucleic acid molecule encoding a polypeptide selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2.
  • the composition for determining transplantation compatibility of a transplant comprising an agent specific to Endomucin polypeptide or a fragment thereof.
  • composition according to Item 29 further comprising at least one additional agent specific to a marker selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2.
  • composition for identifying a stem cell comprising a nucleic acid encoding Endomucin or a portion thereof.
  • the composition according to Item 32, wherein the Endomucin comprises: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; (b) a variant polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, having at least one amino acid mutation selected from the group consisting of substitutions, additions, and deletions, the variant polypeptide having a biological activity; (c) a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or an allelic or splicing variant thereof; (d) a species homolog of a polypeptide having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10; or (e) a polypeptide having at least 70% amino acid sequence homology to any one of polypeptides (a) to (d) and having biological activity.
  • composition for identifying a stem cell comprising Endomucin polypeptide or a portion thereof.
  • the composition according to Item 36, wherein the Endomucin comprises: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; (b) a variant polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, having at least one amino acid mutation selected from the group consisting of substitutions, additions, and deletions, the variant polypeptide having a biological activity; (c) a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or an allelic or splicing variant thereof; (d) a species homolog of a polypeptide having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10; ' or (e) a polypeptide having at least 70% amino acid sequence homology to any one of polypeptides (a) to (d) and having biological activity.
  • composition according to Item 36 wherein the Endomucin is set forth in SEQ ID NO: 2 , 4, 6, 8 or 10.
  • a composition for identifying transplantation compatibility of a transplant comprising a nucleic acid molecule encoding Endomucin or a portion thereof.
  • the composition according to Item 40, wherein the Endomucin comprises: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; (b) a variant polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, having at least one amino acid mutation selected from the group consisting of substitutions, additions, and deletions, the variant polypeptide having a biological activity; (c) a polypeptide encoded by a nucleic acid sequence as set forth.in SEQ ID NO: 1, 3, 5, 7 or 9, or an allelic or splicing variant thereof; (d) a species homolog of a polypeptide having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10; or (e) a polypeptide having at least 70% amino acid sequence homology to any one of polypeptides (a) to (d) and having biological activity.
  • composition for identifying transplantation compatibility of a transplant comprising Endomucin polypeptide or a portion thereof.
  • composition according to Item 44 wherein the transplant comprises a stem cell.
  • the composition according to Item 44, wherein the Endomucin comprises: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; (b) a variant polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, having at least one amino acid mutation selected from the group consisting of substitutions, additions, and deletions, the variant polypeptide having a biological activity; (c) a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or an allelic or splicing variant thereof; (d) a species homolog of a polypeptide having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10; or (e) a polypeptide having at least 70% amino acid sequence homology to any one of polypeptides (a) to (d) and having biological activity.
  • a method for determining whether or not a candidate cell is a stem cell comprising the steps of: (I) providing a candidate cell to be determined; (II) contacting the cell with an agent specific to a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; and (III) detecting a specific reaction between the agent and the nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucinpolypeptide or a portion thereof to confirmwhether or not the Endomucin gene is expressed in the candidate cell; wherein the gene expression of the Endomucin in the candidate cell indicates that the candidate cell is a stem cell.
  • stem cell comprises a hematopoietic cell.
  • stem cell comprises a stem cell from bone marrow.
  • the method according to Item 48 further comprising the step of contacting the candidate cell with at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2, wherein the gene expression of the CD34, the Lin (Lineage marker) , the c-kit, and the Sca-1 in the candidate cell, indicates that the candidate cell is a stem cell, the negative gene expression of the Flt3/Flk2 gene indicates that the candidate is a stem cell.
  • a marker selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2, wherein the gene expression of the CD34, the Lin (Lineage marker) , the c-kit, and the Sca-1 in the candidate cell, indicates that the candidate cell is a stem cell, the negative gene expression of the Flt3/Flk2 gene indicates that the candidate is a stem cell.
  • the method according to Item 53, wherein the state of more undifferentiated comprises maintaining a capability selected from the group consisting of a capability of forming n EM colonies consisting of multisystem cells, and a capability of reconstructing and maintaining hematopoiesis of a living organism having had a lethal dose of radiation for a long period of time.
  • the state of less undifferentiation comprises capability selected from the group consisting of reduction of a capability of forming nmEM colonies consisting of multisystem cells, and a capability of reconstructing and maintaining hematopoiesis of a living organism having had a lethal dose of radiation for no time or a short period of time.
  • Amethod forpreparing a stem cell comprising the steps of: (I) providing a sample containing or suspected to contain a stem cell; (II) contacting the sample with an agent specific to a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; (III) determining whether or not a cell in the sample expresses a gene of the Endomucin; and (IV) separating or concentrating the cell expressing the gene of the Endomucin.
  • a method for determining transplantation compatibility of a transplant comprising the step of: (I) determining whether or not the transplant expresses a gene of Endomucin, wherein the gene expression of the Endomucin in the transplant indicates that the transplant is compatible for transplantation.
  • the method according to Item 70 further comprising the step of: (II) determining whether the transplant expresses at least one marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2, wherein the negative or weakly positive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2 indicates that the transplant has transplantation compatibility.
  • a kit for determining differentiation state of a cell comprising the steps of: (a) an agent specifically reacting with Endomucin gene or Endomucin gene product; and (b) means for determining whether or not the Endomucin gene is expressed in the cell. (74) The kit according to Item 73, further comprising means for determining expression of another stem cell marker.
  • the kit according to Item 73 further comprising means for determining whether or not at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2; and means for determining whether or not the agent is expressed in a cell of the sample.
  • the kit according to Item 73 further comprising at least one agent specific to Lin marker; and means for determining whether or not the agent is expressed in a cell of the sample.
  • said Lin marker comprises at least one marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220.
  • kits for preparing a cell of undifferentiated state comprising: (I) an agent specifically reacting with a nucleic acid molecule comprising a nucleic acid sequence encoding
  • a kit for determining transplantation compatibility of a transplant comprising: (I) an agent specifically reacting with a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; and . (II) means for determining whether or not the Endomucin gene is expressed in a cell of interest.
  • kit according to Item 79 further comprising means for determining whether or not at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2; and means for determining whether or nqt the agent is expressed in a cell of the sample.
  • a marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2; and means for determining whether or nqt the agent is expressed in a cell of the sample.
  • kit according to Item 79 further comprising at least one agent specific to Lin marker; and means for determining whether or not the agent is expressed in a cell of the sample.
  • Lin marker comprises at least one marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220.
  • a method for treating, preventing or providing a prognosis for a subject in need of stem cell transplantation comprising the steps of: (I) determining whether or not a transplant containing a stem cell expresses a gene encoding Endomucin; (II) separating or concentrating a transplant containing a stem cell expressing the gene encoding Endomucin; and (III) transplanting the separated or concentrated transplant into the subject.
  • the method according to Item 83 further comprising the steps of: (la) determining whether or not at least one marker selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2, in the transplant; and (Ha) separating or concentrating a transplant containing a stem cell with negative or weakly positive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2.
  • step (Ila) further comprising the step of separating or concentrating a transplant containing a stem cell with strongly positive expression of at least one gene selected from the group consisting of the c-kit and Sca-1 genes.
  • transplant containing stem cell is derived from the subject.
  • transplant containing the stem cell is derived from bone marrow, umbilical cord cell or peripheral blood.
  • a medicament for treating, preventing or providing a prognosis for a subject in need of stem cell transplantation comprising: A) cell populationwith stemcells expressingEndomucin concentrated.
  • the medicament according to Item 88, wherein the stem cells have negative or weakly positive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2.
  • stem cell comprises a hematopoietic stem cell.
  • the present invention allows identification of a stem cell in a simpler manner than before, and thereby allows simple separation and/or concentration of stem cells such as hematopoietic cells . Efficient transplantation was also achieved by the present invention, such as stem cell transplantation.
  • Diseases which can be targeted transplantation of stem cells of the present invention include 1) diseases for which stem cells with qualitative and/or quantitative abnormality should be replaced with normal stem cells; 2) diseases for which irreversible disorders of lymphoid tissues or hematopoietic tissues have resulted from extra large, doses of chemical therapy or radiotherapy, such as leukemia, malignant lymphoma, multiple myeloma, solid cancers such as breast cancer, neuroblastoma, lung small cell cancer, ovarian cancer and the like; 3) diseases in need of eternal supplementation of enzymes such as congenital abnormal metabolism.
  • the present invention allows efficient selection, separation and concentration of cells suitable for transplantation, and thus allows efficient treatment using stem cell transplantation as mentioned above and alleviation of adverse effects conventionally associated therewith. Further, as the invention achieves highly efficient selection of cells with transplantation compatibility, the present invention allows transplantation therapy using only sources from an autologous origin. It is also possible to prepare a necessary amount for therapy after a small amount of cells with transplantation compatibility are obtained by using expansion agents and the like. After preparation of a necessary amount of the cells, the method of the present invention for determining transplantation compatibility may be further used to concentrate more appropriate cells. In such a case, rejection reactions such as graft versus host disease (GVHD) and the like are reduced or never occur.
  • GVHD graft versus host disease
  • Figure 1 shows the result of Endomucin mRNA expression in a human andmouse, in a semi-quantitative RT-PCR assay. Indication of markers refers to cells positive for the marker, unless otherwise stated.
  • Figure 2 shows preparation of SP cell (left panel), middle panel refers to the case where only anti-rat PE was used, right panel refers to the case whereEndomucin + anti-rat PE was used.
  • Figure 3 shows the expression pattern in an exemplary case where Gr-1, a lineage marker, and Endomucin are used for assessing development.
  • Figure 4 shows the expression pattern in an exemplary case where Mac-1, a lineage marker, and Endomucin are used for assessing development.
  • Figure 5 shows the expression pattern in an exemplary case where TER119, a lineage marker, and Endomucin are used for assessing development.
  • Figure 6 shows the expression pattern in an exemplary case where CD4, a lineage marker, and Endomucin are used for assessing development.
  • Figure 7 shows the expression pattern in an exemplary case where CD8, a lineage marker, and Endomucin are used for assessing development.
  • Figure 8 shows the expression pattern in an exemplary case where B220, a lineage marker, and Endomucin are used for assessing development.
  • Figure 9 shows results where KSL cells were divided into four fractions by expression pattern of CD34 and Endomucin, using similar FACS sorting.
  • Upper left panel shows an outline of Lineage depletion.
  • cells corresponding to stem cells were sorted as shown in lower left panel, andconductedclassification into four categories by Endomucin and CD34 expressions.
  • Figure 10 shows results of a colony serum assay.
  • Left panel of Figure 10 shows a procedure for sorting. As in upper left panel, selection has made by c-kit-APC and Sca-1-PE-Cy5.5 antibodies for stem cell selection, and the stem cells were fractioned by observing the expression of CD34 and Endomucin.
  • immature refers to immature cells
  • m refers to macrophage
  • me refers to macrophage/erythroblast
  • nm refers to neutrophil/macrophage
  • mM refers to macrophage/megakaryocyte
  • eM refers to acrophage/erythroblast/megakaryocyte
  • nme refers to neutrophil/macrophage/erythroblast
  • nmM refers to neutrophil/macrophage/megakaryocyte
  • nmeM refers to neutrophil/macrophage/erythroblast/megakaryocyte.
  • Figure 11 shows results of a bone marrow reconstruction assay. Cells were sorted using FACS in accordancewith the cell number as shown ineach cell fraction.
  • FIG. 12 shows the distribution pattern of KSL cells of cell fractions gated by Endomucin.
  • Left panel shows classification according to five expression levels to 1-e (Endomucin PEexpression levels of 1-5, 5-35, 35-150, 150-400 and 400-1500, respectively). Each existence ratio is 18.6%, 56%, 20.1%, 1.58% and 0.26%, respectively.
  • Right panel shows results of these panels developed by Sca-1 and c-kit.
  • Figure 13 shows results of the expression interrelationship in lineage negative cells for Endomucin, c-kit (a), Sca-1 (b) and CD34 (c) . It has been found that CD34 " Endomucin high cells have high correlation with c-kit high , Sca-l h ⁇ gh cells, and thus Endomucin is an effective and efficient indicator for stem cells and transplantation compatibility.
  • Figure 14 shows vector construct for knocking-in of Endomucin.
  • Figure 15 shows a map of restriction sites of promoters.
  • Figure 16 shows SST screening of CD34 ⁇ KSL cells.
  • Cells analyzed are bone marrow CD34TSL HSCs, CD34 + KSL progenitors, Lineagemarker " cells (Lin-) , Gr-l + neutrophils, Mac-1 + monocytes/macrophages, TER119 + erythroblasts, B220 + B cells, spleen Thy-1.2 + T cells, NKl .1 + NK cells, B220 + B cells, and thymic CD4-CD8 ⁇ T cells (DN) , CD4 + CD8 + T cells (DP), CD4 + CD8 ⁇ T cells (CD4SP) , andCD4-CD8 + (CD8SP) .
  • DN CD4 + CD8 + T cells
  • DP CD4 + CD8 ⁇ T cells
  • CD8SP CD4-CD8 +
  • FIG. 17 shows Endomucin expression in BM hematopoietic cells.
  • Endomucin "1" cells were compared between Lin-c-kit + Sca-l + and
  • Percent chimerism of donor cells 6 months after transplantation is presented as mean ⁇ S.D.
  • Figure 19 shows that Endomucin can substitute for c-kit and Sca-1 in HSC purification.
  • Expression of Endomucin in CD34 ⁇ Lin ⁇ cells was also investigated (FACS profile) .
  • Lin " cells were divided into four populations by their expression of CD34 and Endomucin (a) .
  • Table (b) shows competitive lymphohematopoietic repopulation capacity of the Endomucin "1" CD34 ⁇ Lin- cells .
  • the indicatednumber of cells from each subpopulation (B6-Ly5.1) andB6-Ly5.2 competitor cells were mixed and injected ⁇ into lethally irradiated B6-Ly5.2 recipient mice.
  • Percent chimerism of donor cells 6 months after transplantation is presented as mean ⁇ S.D.
  • Figure 20 shows Expression of Endomucin on E14. 5 fetal liver hematopoietic stem cells. The vast majority of fetal liver hematopoietic stem cells (CD34 + KSL) were demonstrated to be Endomucin "1" . (left panel: Lin-negative-gated) .
  • Figure 21 shows Endomucin expression in YS (yolk sac) hematopoiesis.
  • A Expression of Endomucin in the CD45- yolk sac cells from embryonic day 8.5 (E8.5) embryo.
  • B Expression of Endomucin in the CD45- yolk sac cells from E10.5 embryo.
  • D Number of endothelial colonies, hematopoietic colonies and total hematopoietic cells generated from each population on OP-9 (bar graph with SD) .
  • Figure 22 shows Endomucin expression marks definitive HSC development in AGM region.
  • A Expression of Endomucin in the CD45- AGM cells from E10.5 embryo.
  • B Colony formation capacity of indicated cell fractions on OP-9 stromal cells in the presence of SCF, TPO, IL-3, and EPO.
  • C Proliferation activity of indicated cell fractions detected on OP-9 in the presence of SCF, TPO, IL-3, and EPO.
  • D Competitive bone marrow repopulating capacity of indicated cell fractions. Cells (750) from each fraction were transplanted into lethallyirradiatedmicebyintra-bone marrow injection.
  • E Chimerismof donor cells inperipheral blood reconstituted with CD45-CD41+Endomucin+ cells.
  • E Chimerism of each lineage in positive mouse reconstituted with CD45-CD41+Endomucin+ cells.
  • Figure 23 shows localization of Endomucin on HSC in contact with stromal cells by capping of Endomucin protein on hematopoietic cells on OP-9 stromal cells.
  • Endomucin + CD41- cells from day 10 AGM were cultured on OP-9 stromal cells, then cells were immunostained with anti-Endomucin monoclonal antibody.
  • Capping of Endomucin protein on some of the hematopoietic cells growing underneath stromal cells (B: light field) were detected (A, C) .
  • SEQ ID NO: 1 refers to a full length nucleic acid sequence of Endomucin (la; murine) .
  • SEQ ID NO: 2 refers to a full length amino acid sequence of Endomucin (la; murine) .
  • SEQ ID NO: 3 refers to a nucleic acid sequence of Endomucin (lb; murine) .
  • SEQ ID NO: 4 refers to an amino acid sequence of Endomucin (lb; murine) .
  • SEQ ID NO: 5 refers to a nucleic acid sequence of Endomucin (lc; murine) .
  • SEQ ID NO: 6 refers to an amino acid sequence of Endomucin (lc; murine) .
  • SEQ ID NO: 7 refers to a nucleic acid sequence of Endomucin (Id; murine) .
  • SEQ ID NO: 8 refers to an amino acid sequence of Endomucin (Id; murine) .
  • SEQ ID NO: 9 refers to a full-length nucleic acid sequence of Endomucin (human) .
  • SEQ ID NO: 10 refers to a full-length amino acid sequence of Endomucin (human) .
  • SEQ ID NO: 11 refers to a nucleic acid sequence of Flt3/Flk2 (human) .
  • SEQ ID NO: 12 refers to an amino acid sequence of Flt3/Flk2 (human) .
  • SEQ ID NO: 13 refers to a nucleic acid sequence of CD34 (murine) .
  • SEQ ID NO: 14 refers to an amino acid sequence of CD34 (murine) .
  • SEQ ID NO: 15 refers to a nucleic acid sequence of CD34 (human) .
  • SEQ ID NO: 16 refers to a amino acid sequence of CD34 (human) .
  • SEQ ID NO: 17 refers to a nucleic acid sequence of Mac-1 (murine) .
  • SEQ ID NO: 18 refers to an amino acid sequence of Mac-1 (murine) .
  • SEQ ID NO: 19 refers to a nucleic acid sequence of CD4 (murine) .
  • SEQ ID NO: 20 refers to an amino acid sequence of CD4 (murine) .
  • SEQ ID NO: 21 refers to a nucleic acid sequence of CD8 (murine) .
  • SEQ ID NO: 22 refers to an amino acid sequence of CD8 (murine) .
  • SEQ ID NO: 23 refers to a nucleic acid sequence of B220 (murine) .
  • SEQ ID NO: 24 refers to an amino acid sequence of B220 (murine) .
  • SEQ ID NO: 25 refers to a nucleic acid sequence of c-kit (murine) .
  • SEQ ID NO: 26 refers to an amino acid sequence of c-kit (murine) .
  • SEQ ID NO: 27 refers to a nucleic acid sequence of Sca-1 (murine) .
  • SEQ ID NO: 28 refers to an amino acid sequence of Sca-1 (murine) .
  • SEQ ID NO: 29 refers to a primer for identification of murine Endomucin (sense) .
  • SEQ ID NO: 30 refers to a primer for identification of murine Endomucin (antisense) .
  • SEQ ID NO: 31 ref ers to a primer for identification of human Endomucin (sense) .
  • SEQ ID NO: 32 refers to a primer for identification of human Endomucin (antisense) .
  • SEQ ID NO: 33 refers to an upsream sequence of coding region of Endomucin (-10269-+3) .
  • SEQ ID NO: 34 refers to a sense primer used in
  • SEQ ID NO: 35 refers to an antisense primer used in Example 16 for identification of Endomucin.
  • Endomucin refers to an agent having a sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9 (nucleic acid sequences) , and SEQ ID NO: 2, 4, 6, 8 or 10 (amino acid sequences) , and a corresponding agent (ortholog) in another species of animal.
  • Endomucin is a sialomucin protein, which was identified as a membrane surface factor specifically expressed by endothelial cells (Morgan S.M., et al . , Blood 93, pl65-175 (1999) ; Liu C.,et al., BBRC 288,129-136(2001)).
  • Encomudin is known to be a type I protein having threonine and serine residues in the extracellular portions (35 %) and being 2 . 48 amino acids in length (mice) . It is known to be susceptible to O-sialo glycoprotein endopeptidase (Morgan etal. supra ) . Inhuman, it is known as a molecule having 261 amino acids (Liu et al. supra) . This molecule is known to have an apparent molecular weight of 45 kDabythe actions of sialidase andO-glycosidase. Further, it has been reported that there are Endomucins 1 and 2 in human (Kinoshita M. et al.,FEBS Lett.
  • sequences are designated as set forth in SEQ ID NO: 1-2, SEQ ID NO: 3-4, SEQID O:5-6, SEQ ID NO: 7-8, respectively, where the odd number refers to a nucleic acid sequence, and the even number refers to amino acid number.
  • la is the full-length sequence, and has the sequence set forth in SEQ ID NO: 1 and 2 (where the odd number refers to a nucleic acid sequence) .
  • all these variants may be used as amarker or the purpose of the present inveniton.
  • a human also has Endomucin, and it has been shown that the human Endomucin has the sequences set forth in SEQ ID NO: 9 and 10 (where the odd number refers to a nucleic acid sequence) . Therefore, it should be. understood by those skilled in the art thatthe above-described variants may also correspondingly exist for human Endomucin.
  • Endomucin has recentlybeen reported to possibly be the as L-selectin ligand (Samulowitz U.et al.,Am.J. of Pathol., 160, 1669-1681, 2002) .
  • Endomucin is merely suggested to be specifically expressed in vessel endothelia (particularly, vein endothelia, capillary blood vessel; and no expression has been found in artenal endothelia) , and the functions thereof are not known to date .
  • the use for cancer diagnosis or relationship with extracellular matrices has been speculated. However, no indication or reports for Endomucin have been published with respect to stem cells.
  • Endomucin and the homologs thereof are known in mammals in general including rat and mouse, in addition to in a human. Accordingly, as used herein, Endomucin refers to any Endomucin and the homologs thereofpresent that inthemammals in general. Endomucin is known to have a variety of splicing variants or isoforms, and it is to be understood that all such splicing variants or isoforms may be used as a marker in the present invention. As used herein Endomucin may be prepared in an artificial manner or in a synthetic manner. Such artificially prepared Endomucins include but are not limited to Endomucins. with mutation introduced therein. In order to determine whehter a molecule is
  • Endomucin or not it should be sufficient herein to determine at least one of the activities of Endomucin.
  • activities include, for example, but are not limitedto having capability of being recognized by a monoclonal antibody specific to Endomucin, capable of abundant addition of sugar chains of O-glycoside type linkage, property of inhibiting adhesion by high expression in a specific cell line in vitro, and the like.
  • Methods to determining such activities include for example but are not limited to-, assay for identification of sugar chains, and assay for observing adhesion inhibition by transforming a cell with the molecule of interest, and the like.
  • Moieties to which sugar chains can be added to Endomucin used in the present invention include moieties to which a N-glucoside linkage can be made (asparagine-X-serine/threonine) such as
  • sugar chain refers to a compound in which at least one unit sugar/saccharide (a monosaccharide and/or derivative thereof) is associated in series. When two or more unit sugars are associated between each of such unit sugars, such association is mediated by binding dehydration condensation by glycoside linkage.
  • Such sugar chains include, but are not limitedto, forexample, a wide variety of polysaccharides such as those found in living ' organisms suchas glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid and the complexes and derivatives thereof, degraded polysaccharides, glycoproteins, proteoglyeans, glycosaminoglycans, glycolipids, and complexed biomolecules thereof, degraded or derivatized sugar chains derivedtherefrom, andthe like.
  • polysaccharides such as those found in living ' organisms suchas glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid and the complexes and derivatives thereof, degraded polysaccharides, glycoproteins, proteoglye
  • sucgar chain may be interchangeably used with “polysaccharide”, “carbohydrate”, and “sugar”. Further, unless otherwise- specified, as used herein, the term “sugar chain” may include sugar chains and sugar chain containing substances.
  • monosaccharide refers to a polyhydroxyaldehyde or polyhydroxy ketone containing at least one hydroxy group and at least one aldehyde or ketone group which is not hydrolysed into similar molecules, and derivatives thereof.
  • monosaccharides may be represented by the formula: C n H2nO n , however, the formula is not limited to this, and includes fucose (deoxy hexose) , N-acetylglucosamine andthe like.
  • monosaccharides are aldehydes or ketones of chain type polyalcohols, and the aldehyde-type is also called “aldose”, and the ketone-type is also called “ketose”.
  • derivatives of monosaccharides refer to substances resulting from at least one hydroxy group on a non-derivatized monosaccharide being substituted with another substituent.
  • Derivatives of such a monosaccharide include but are not limited to a sugar having a carboxyl group (for example, aldonic acid, a carboxylic acid resulting from oxidization of C-l position of aldose such as D-gluconic acidwhich D-glucose is oxidized, an uronic acid, a carboxylic acid resulting from oxidization of C-atom at the terminus of an aldose such as D-glucuronic acid) resulting from oxidization of D-glucose, sugar having an amino group or a derivative thereof (for example, an acetylated amino group) , such as N-ac ⁇ tylD-glucosamine, N-acetylD-galactosamine and the like, a sugar having both amino group and a carboxy group (for example, N-acetylneuraminic acid (sialicacid) , N-acetylmuramic acid and the like, a deoxy sugar such as 2-deoxy-D
  • N-acetyllactosamine (complex type) sugar chain is represented by (A) indicating the number of branches in the first three digits; (B) indicating 1 when a fucose is bound to N-acetylglucosamine of the reduced terminus by ⁇ l,6 linkage, and 0 when no linkage is found at the center of the three digits; (C) indicating 1 when N-acetylglucosamine is linked to a ⁇ -mannose, which is a core pentose thereof, by ⁇ l,4, and 0 when no linkage is found, at the end of the three digits.
  • High mannose type sugar chain is represented by indicating M first, and the number of mannose residues in the following digits. Thereafter, “.” is inserted and on the right hand side thereof is added an inherent number.
  • F is inserted after "M”.
  • Hybrid type sugar chain is represented by indicating H first, and the number of mannose residues in the following digits. Thereafter, ".” is insertedandontherighthandside thereof is addedaninherent number.
  • N-acetylgalactosamine When having galactose insted of N-acetylgalactosamine, after giving symbol for galactose, a lower case letter corresponding to the number is added to the bottom, such as if the number of N-acetylgalactosamine is one, then "a” is inserted, if two then "b” is inserted, if three then He” is inserted, and the like. 6) When it can be classified into any of N-acetyllactosamine type, and high mannose type or hybrid type such as core pentose, then the nomenclature of N-acetyl lactoseamine type is precedent.
  • Such designation methods include the following: galactose ⁇ l, 4-N-acetylglucosamine ⁇ l,2-mannose ⁇ l, 3- (mannose ⁇ l, 3- (mannose ⁇ l, 6-)mannose ⁇ l, 6-)mannose ⁇ l, 4-N-acetylglucosamine ⁇ l, 4-N-acetylglucosamine refers toH5.12 ; galactose ⁇ l, 4-N-acetylglucosamine ⁇ l,2-mannose ⁇ l, 3- (mannose ⁇ l, 6-) annose ⁇ l, 4-N-acetyl glucosamine ⁇ l, 4-N-acetylglucosamine refers to 110.4 and galactose ⁇ l, 4-N-acetylglucosamine ⁇ l,2- (galactose ⁇ l, 3-N-acetylglucosamine ⁇ l, 4-) annose ⁇ l, 3- (galactose ⁇ l, 4-N-acet
  • protein protein
  • polypeptide oligopeptide
  • peptide as used herein have the same meaning and refer to an amino acid polymer having any length.
  • This polymer may be a straight, branched or cyclic chain.
  • An amino acid may be a naturally-occurring or nonnaturally-occurring amino acid, or a variant amino acid.
  • the term may include those assembled into a complex of a plurality of polypeptide chains.
  • the term also includes a naturally-occurring or artificially modified amino acid polymer. Such modification includes, ⁇ for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety).
  • apolypeptide containingat least one amino acid analog (e.g., nonnaturally-occurring amino acid, etc.), a peptide-like compound (e.g., peptoid) , and other variants known in the art, " for example.
  • polypeptide as used herein may refer to Endomucin or Endomucin analogs.
  • polynucleotide refers to a nucleotide polymer having any length. This term also includes an "oligonucleotide derivative” or a "polynucleotide derivative”.
  • An "oligonucleotide derivative” or a “polynucleotide derivative” includes a nucleotide derivative, or refers to an oligonucleotide or a polynucleotide having linkages between nucleotides different from typical linkages, which are interchangeably used.
  • Examples of such an oligonucleotide specifically include 2 ' -O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a N3'-P5' phosphoroamidate bond, an oligonucleotide derivative in which a ribose and a phosphodiesterbond in an oligonucleotide are converted to a peptide-nucleic acid bond, an oligonucleotide derivative in which uracil in an oligonucleotide is substituted with C-5 propynyl uracil, an oligonucleotide derivative in which uracil in an oligonucleotide is substituted with C-5 thiazole
  • nucleic acid sequence also implicitly encompasses conservatively-modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be produced by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (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) ) .
  • nucleic acid is also used interchangeably with the terms “gene”, “cDNA”, “mRNA”, “oligonucleotide”, and “polynucleotide”.
  • a particular nucleic acid sequence includes a “splice variant”.
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants” are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternative) nucleic acid splice products encode different polypeptides.
  • splice variants vary, but include alternative splicing of exons.
  • Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Therefore, for example, the Endomucin gene may herein employ the form of a nucleic acid. Particularly, Endomucin is known to be subjected to a variety of splicing variation, and it is assumed that any of those variants will have a function as a marker. Therefore it shouldbe understood that all those variants will be used as a marker for undifferentiated state and/or transplantation compatibility.
  • gene refers to an element defining a genetic trait.
  • a gene is typically arranged in a given sequence on a chromosome.
  • a gene which defines the primary structure of a protein is called a structural gene.
  • a gene which regulates the expression of a structural gene is called a regulatory gene.
  • gene may refer to "polynucleotide”, “oligonucleotide”, “nucleic acid”, and “nucleic acid molecule” and/or “protein”, “polypeptide”, “oligopeptide” and “peptide”.
  • homoology of a gene refers to the proportion of identity between two or more gene sequences.
  • the greater the homology between two given genes the greater the identity or similaritybetweentheir sequences. Whether or not two genes have homology is determined by comparing their sequences directly or by a hybridization method under stringent conditions. When two gene sequences are directly compared with each other, these genes havehomologyif the DNAsequences of the genes have representatively at least 50% identity, preferably at least 70% identity, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with each other.
  • ligand refers to a substance capable of specifically binding to a certain protein.
  • examples of a ligand include lectin, antigens, antibodies, hormones, neurotransmitters, and the like, which are capable of specifically binding to various receptor proteinmolecules on cell membranes . Accordingly, it should be understood that ligands to the Endomucin of the present invention can be used as an agent for detecting a marker of the present invention.
  • polynucleotides hybridizing under stringent conditions refers to conditions commonly used and well known in the art.
  • a polynucleotide can be obtained by conducting colony hybridization, plaque hybridization, southern blot hybridization, or the like using a polynucleotide selected from the polynucleotides of the present invention.
  • a filter on which DNA derived from a colony or plaque is immobilized is used to conduct hybridization at 65°C in the presence of 0.7 to 1.0 M NaCl.
  • a 0.1 to 2-fold concentration SSC (saline-sodium citrate) solution (1-fold concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate) is used to wash the filter at 65°C.
  • SSC saline-sodium citrate
  • Polynucleotides identified by this method are referred to as "polynucleotides hybridizing under stringent- conditions".
  • Hybridization can be conducted in accordance with a method described in, for example, Molecular Cloning 2nded., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, SecondEdition, OxfordUniversityPress (1995) , andthe like.
  • sequences hybridizing under stringent conditions exclude, preferably, sequences containing only A (adenine) or T (thymine) . Therefore, the polypeptide of the present invention (e.g., Endomucin 1, splicing variants thereof, or the like) includes a polypeptide encoded by a nucleic acid molecule hybridizable to a nucleic acid molecule encoding a polypeptide particularly described herein under stringent conditions.
  • hybridizable polynucleotide refers to a polynucleotide which can hybridize other polynucleotides under the above-described hybridization conditions.
  • the hybridizable polynucleotide includes at least a polynucleotide having a homology of at least 60% to the base sequence of DNA encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, preferably a polynucleotide having a homology of at least 80%, and more preferably a polynucleotide having a homology of at least 95%.
  • the homology of a nucleic acid sequence may be represented by similarity evaluated with a score using, for example, a searchprogramBLAST usingan algorithmdevelopedbyAltschul et al., J. Mol. Biol., 215, 403-410(1990)).
  • highly stringent conditions refers to those conditions that are designed to permit hybridization of DNA strands whose sequences are highly complementary, and to exclude hybridization of significantly mismatched DNAs. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of “highly stringent conditions” for hybridization and washing are
  • Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodiumdodecyl sulfate (NaDodS0 4 or SDS) , Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) , and dextran sulfate, although other suitable agents can also be used.
  • concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions.
  • Hybridization experiments are ordinarily carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al .
  • N is the length of the duplex formed
  • [Na + ] is the molar concentration of the sodium ion in the hybridization or washing solution
  • % G+C is the percentage of (guanine+cytosine) bases in the hybrid.
  • the melting temperature is reduced by approximately 1°C for each 1% mismatch.
  • moderately stringent conditions refers to conditions under which a DNA duplex with a greater degree ofbasepairmismatching than could occur under “highly stringent conditions” is able to form.
  • typical “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50-65°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at
  • Tm (2°C per A-T base pair) + (4°C per G-C base pair) .
  • the term "isolated" biological agent refers to a biological agent that is substantially separated or purified from other biological agents in cells of a naturally-occurring organism (e.g., in the case of nucleic acids, agents other than nucleic acids and a nucleic acid having nucleic acid sequences other than an intended nucleic acid; and in the case of proteins, agents other than proteins and proteins having an amino acid sequence other than an intended protein) .
  • the "isolated" nucleic acids and proteins include nucleic acids and proteins purified by a standard purification method.
  • the isolated nucleic acids and proteins also include chemically synthesized nucleic acids and proteins.
  • purified biological agent e.g., nucleic acids, proteins, and the like
  • purified biological agent refers to one from which at least a part of naturally accompanying agents are removed. Therefore, ordinarily, the purity of a purified biological agent is higher than that of the biological agent in a normal state (i.e., concentrated).
  • purified and
  • isolated mean that the same type of biological agent is present preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight.
  • the term "expression" of a gene, a polynucleotide, apolypeptide, or the like indicates that the gene or the like is affected by a predetermined action in vivo to be changed into another form.
  • the term "expression” indicates that genes, polynucleotides, or the like are transcribed and translated into polypeptides .
  • genes may be transcribed into mRNA. More preferably, these polypeptides may have post-translational processing modifications.
  • such an expressed polypeptide may be glycosylated Endomucin.
  • expression is understood to mean both transcription and translation. Accordingly, when confirming expression of the Endomucin gene of the present invention, it should be understood that a confirmation assay should be conducted for either or both the presence of the transcript and the translated matter.
  • FI fluorescence intensity
  • Endomucin for example, it is called “Endomucin positive” and the like.
  • negative, “weakly positive”, “positive”, or “strongly positive” of "expression” can be alternatively referred to as “-”; “ ⁇ ” or “low”; “+”; or “++” or “high”, respectively.
  • Expression intensity in mRNA level can be measured using expression analysis using RT-PCR or microarrays and the like.
  • the expression level can be quantified using densitometer in a relative manner. More specifically, such expression levels can be digitizedinterms ofHRPT, ahouse keepinggene in amicroarray, and if less expression than that of HRPT is found, then such a gene can be regarded as "negative", if approximately the
  • interaction means that one substance influences the other substance via forces (e.g., intermolecular forces (van derWaals force) , hydrogenbonding, hydrophobic interactions, or the like) .
  • forces e.g., intermolecular forces (van derWaals force) , hydrogenbonding, hydrophobic interactions, or the like.
  • two substances interactingwith each other interact in themanner of association or binding. Accordingly, when using an agent specific to a marker in order to confirm the presence of the marker, the presence of the marker can be confirmed by confirming the interaction of the agent and the marker.
  • binding means the physical or chemical interaction between two proteins or compounds or associatedproteins or compounds or combinations thereof. Binding includes ionic, non-ionic, hydrogen, van der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another protein or compound, but instead are without other substantial chemical intermediates. Accordingly, it should be understood that when confirming an expression of the Endomucin of the present invention, such an expression of Endomucin can be confirmed by confirming the binding of an agent specific therefor.
  • the term “modulate” or “modify” refers to an increase, decrease or maintenance in a specific activity, or the amount, quality or effect of a protein.
  • a naturally-occurring nucleic acid encoding a protein e.g., Endomucin, or variants or fragments thereof, or the like
  • a preferable nucleic acid encoding Endomucin, or variants or fragments thereof, or the like is hybridizable to the whole or part of a sequence as set forth in SEQ ID N0:1, 3, 5, 7 or 9 under low stringency conditions defined by hybridization buffer essentially containing 1% bovine serum albumin (BSA) ; 500 mM sodium phosphate (NaP0 4 ) ; lmM EDTA; and 7% SDS at 4.2°C, and wash buffer essentially containing 2xSSC ( 600 mM NaCl; 60 mM sodium citrate) ; and 0.1% SDS at 50°C, more preferably under low stringent conditions defined by hybridization buffer essentially containing 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaP0 4 ) ; 15% formamide; 1 mM EDTA; and 7%
  • BSA bovine serum albumin
  • NaP0 4 200 mM sodium phosphate
  • wash buffer essentially containing 0.5xSSC (150 mM NaCl; 15 mM sodium citrate); and 0.1% SDS at 65°C.
  • the term "probe” refers to a substance for use in searching, which is used in a biological experiment, such as in vitro and/or in vivo screening or the like, including, but not being limited to, for example, a nucleic acid molecule having a specific base sequence or a peptide containing a specific amino acid sequence.
  • a nucleic acid molecule as a common probe include one having a nucleic acid sequence having a length of at least 8 contiguous nucleotides, which is homologous or complementary to the nucleic acid sequence of a gene of interest.
  • Such a nucleic acid sequence may be preferably a nucleic acid sequence having a length of at least 9 contiguous nucleotides, more preferably a length of at least 10 contiguous nucleotides, and even more preferably a length of at least 11 contiguous nucleotides, a length of at least 12 contiguous nucleotides, a length of at least 13 contiguous nucleotides, a length of at least 14 contiguous nucleotides, a length of at least 15 contiguous nucleotides, a length of at least 20 contiguous nucleotides, a length of at least 25 contiguous nucleotides, a length of at least 30 contiguous nucleotides, a length of at least 40 contiguous nucleotides, or a length of at least 50 contiguous nucleotides.
  • a nucleic acid sequence used as a probe includes a nucleic acid sequence having at least 70% homology to the above-described
  • Protein typically used as a probe includes, for example, but not limited to an antibodyor derivative thereof.
  • the protein When using a protein as a probe, the protein can be directly or indirectly labeled with a labeling agent such as fluorophore or radioisotope.
  • a labeling agent such as fluorophore or radioisotope.
  • search indicates that a givennucleic acid sequence is utilizedto findother nucleic acid base sequences having a specific function and/or property either electronically or biologically, or using other methods.
  • Examples of an electronic search include, but are not limited to, BLAST (Altschul et al. , J. Mol. Biol. 215:403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444-24.48 (1988)), Smith and Waterman method (Smith andWaterman, J. Mol. Biol.147:195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol.
  • Examples of a biological search include, but are not limited to, a macroarrayinwhich genomic DNAis attachedto a nylonmembrane or the like or a microarray (microassay) in which genomic DNA is attachedto a glass plate under stringent hybridization conditions, PCR and in situ hybridization, and the like. It is herein intended that Endomucin or another marker (CD34) and the like used in the present invention include corresponding genes identified by such an electronic or biological search.
  • the "percentage of sequence identity, homology or similarity (amino acid, nucleotide, or the like) is determinedby comparing two optimally aligned sequences over a window of comparison, wherein the portion of a polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps), as compared to the reference sequences (which does not comprise additions or deletions (if the other sequence includes an addition, a gap may occur) ) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • homology is evaluated by an appropriate technique selected from various sequence comparison algorithms and programs well known in the art. Examples of such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson andLipman, 1988, Proc. Natl. Acad. Sci. USA85 (8) -.2444-2448, Altannet al., 1990, J.
  • BLAST Basic Local Alignment Search Tool
  • 5 specialized-BLAST programs may be used to perform the following tasks to achieve comparison or search: (1) comparison of an amino acid query sequence with a protein sequence database using BLASTP and BLAST3; (2) comparison of a nucleotide query sequence with a nucleotide sequence database using BLASTN; (3 ) comparison of a conceptually translated product in which a nucleotide query sequence (both strands) is converted over
  • the BLAST program identifies homologous sequences byspecif ing analogous segments called "high score segment pairs" between amino acid query sequences or nucleic acid query sequences and test sequences obtained from preferably a protein sequence database or a nucleic acid sequence database.
  • a large number of the high score segment pairs are preferably identified (aligned) using a scoring matrix well known in the art .
  • the scoringmatrix is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445, Henikoff and Henikoff, 1993, Proteins 17:49-61) .
  • the PAM or PAM250 matrix may be used, although they are not .as preferable as the BLOSUM62 matrix (e.g., see Schwartz and Dayhoff, eds. , 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation) .
  • the BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments which satisfy a threshold level of significance independently defined by a user, such as a user set homology.
  • the statistical significance of high score segment pairs is evaluated using Karlin' s formula (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).
  • the term "primer” refers to a substance required for initiation of a reaction of a macromolecule compound to be synthesized, in a macromolecule synthesis enzymatic reaction.
  • a nucleic acidmolecule e.g., DNA, RNA, or the like
  • a nucleic acidmolecule which is complementary to part of a macromolecule compound to be synthesized may be used.
  • a nucleic acid molecule which is ordinarily used as a primer includes one that has a nucleic acid sequence having a length of at least 8 contiguous nucleotides, which is complementary to the nucleic acid sequence of a gene of interest.
  • Such a nucleic acid sequence preferably has a length of at least 9 contiguous nucleotides, more preferably a length of at least 10 contiguous nucleotides, even more preferably a length of at least 11 contiguous nucleotides, a length of at least 12 contiguous nucleotides, a length of at least 13 contiguous nucleotides, a length of at least 14 contiguous nucleotides, a length of at least 15 contiguous nucleotides, a length of at least 16 contiguous nucleotides, a length of at least 17 contiguous nucleotides, a length of at least 18 contiguous nucleotides, a length of at least 19 contiguous nucle
  • a nucleic acid sequence used as a primer includes a nucleic acid sequence having at least 70% homology to the above-described sequence, more preferably at least 80%, even more preferably at least 90% , and most preferably at least 95%.
  • An appropriate sequence as a primer may vary depending on the property of the sequence to be synthesized (amplified) . Those skilled in the art can design an appropriate primer depending on ' the sequence of interest. Such primer design is well known in the art and may be performed manually or using a computer program (e.g. , LASERGENE, Primer Select, DNAStar) .
  • amino acid may refer to a naturally-occurring or nonnaturally-occurring amino acid.
  • amino acidderivative or “amino acidanalog” refers to an amino acid which is different from a naturally-occurring amino acid and has a function similar to that of the original amino acid.
  • amino acid derivatives and amino acid analogs are well known in the art.
  • naturally-occurring amino acid refers to an L-isomer of a naturally-occurring amino acid.
  • the naturally-occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -carboxyglutamic acid, arginine, ornithine, and lysine. Unless otherwise indicated, all amino acids as used herein are L-isomers.
  • nonnaturally-occurring amino acid refers to an amino acid which is ordinarily not found in nature.
  • nonnaturally-occurring amino acids include D-f ⁇ rm of amino acids as described above, norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzyl propionicacid, D- or L-homoarginine, and D-phenylalanine.
  • amino acid analog refers to a molecule having a physical property and/or function similar to that of amino acids, but is not an amino acid. Examples of amino acid analogs include, for example, ethionine, canavanine, 2-methylglutamine, and the like.
  • An amino acid mimic refers to a compound which has a structure different from that of the general chemical structure of amino acids but which functions in a manner similar to that of naturally-occurring amino acids.
  • Amino acids may be referred to herein by either their commonly knownthree letter symbols or bythe one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Bases Symbol Meaning a adenine g guanine c cytosine t thyi ⁇ ine 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 or 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 bases.
  • corresponding amino acid or nucleic acid refers to an amino acid or nucleotide in a given polypeptide or polynucleotide molecule, which has, or is anticipated to have, a function similar to that of a predetermined amino acid or nucleotide in a polypeptide or polynucleotide as a reference for comparison.
  • enzymemolecules the term refers to an amino acid which is present at a similar position in an active site and similarly contributes to catalytic activity.
  • antisense molecules refers to a similar portion in an ortholog corresponding to a particular portion of the antisense molecule.
  • a corresponding amino acid may be a phosphorylated site, for example.
  • a corresponding amino acid may be an amino acid playing a role in dimerization.
  • Such a "corresponding" amino acid or nucleic acid may extend over a region or domain having a certain range. Therefore, in this case, such a region or domain is herein referred to as a "corresponding" region or domain.
  • corresponding gene refers to a gene (e.g., a polypeptide or polynucleotide molecule) in a given species, which has, or is anticipated to have, a function similar to that of a predetermined gene in a species as a reference for comparison.
  • the term refers to a gene having ' the same evolutionary origin. Therefore, a gene corresponding to a given gene may be an ortholog of the given gene. Therefore, genes corresponding to the mouse Endomucin gene and the like can be found in other animals (human, rat, pig, cattle, and the like) .
  • a corresponding gene in a given animal can be found by searching a sequence database of the animal (e.g. , human, rat) using the sequence of a reference gene (e.g., mouse Endomucin gene, and the like) as a query sequence.
  • a reference gene e.g., mouse Endomucin gene, and the like
  • nucleotide may be either naturally-occurring.or nonnaturally-occurring.
  • nucleotide derivative or “nucleotide analog” refers to a nucleotide which is different from naturally-occurring nucleotides and has a function similar to that of the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art.
  • nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, and peptide-nucleic acid (PNA) .
  • fragment refers to a polypeptide or polynucleotide having a sequence length ranging from 1 to n-1 with respect to the full length of the reference polypeptide or polynucleotide (of length n) .
  • the length of the fragment can be appropriately changed depending on the purpose.
  • the lower limit of the length of the fragment includes 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more nucleotides .
  • Lengths represented by integers which are not herein specified may be appropriate as a lower limit.
  • the lower limit of the length of the fragment includes 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 or more nucleotides. Lengths represented by integers which are not herein specified (e.g., 11 and the like) may be appropriate as a lower limit.
  • the term "specifically interact with” indicates that a first substance or agent interacts with a second substance or agent with higher affinity than that to substances.or agents other than the second substance or agent (particularly, other substances or agents in a sample containing the second substance or agent) .
  • Examples of a specific interaction with reference to a substance or agent include, but are not limited to, hybridization of nucleic acids, antigen-antibody reaction, ligand-receptor reaction, enzyme-substrate reaction, a reaction between a transcriptional agent and a binding site of the transcriptional agent when both a nucleic acid and a protein are involved, a protein-lipid interaction, a nucleic acid-lipid interaction, and the like. Therefore, when the first and second substances or agents are nucleic acids, "specifically interact with” means that the first substance or agent is at least partially complementary to the second substance or agent.
  • both the first and second substances or agents are proteins
  • “specifically interact with” includes, but is not limitedto, an interaction due to an antigen-antibody reaction, an interaction due to a receptor-ligand reaction, an enzyme-substrate interaction, and the like .
  • "specifically interact with” includes an interaction between a transcriptional agent and a binding region of a nucleic acid molecule targeted by the transcriptional agent.
  • the term "agent capable of specifically interacting with" a biological agent refers to an agent which has an affinity to the biological agent, such as a polynucleotide, a polypeptide or the like, which is representatively higher than or equal to an affinity to other non-related biological agents, such as polynucleotides, polypeptides or the like (particularly, those with identity of less than 30%), and preferably significantly (e.g., statistically significantly) higher.
  • an affinity can be measured with, for example, a hybridization assay, a binding assay, or the like.
  • the "agent” may be any substance or other agent (e.g., energy, such as light, radiation, heat, electricity, or the like) as long as the intended purpose can be achieved.
  • a substance include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA , genomic DNA , or the like, and RNA such as mRNA) , polysaccharides, oligosaccharides, lipids, low molecular weight organic molecules (e.g., hormones, ligands, information transfer substances, molecules synthesized by combinatorial chemistry, low molecular weight molecules (e.g., pharmaceutically acceptable low molecular weight ligands and the like) , and the like) , and combinations of these molecules .
  • an agent specific to a polynucleotide include
  • a polypeptide such as a transcriptional agent binding to a promoter region, and the like.
  • agent specific to a polypeptide include, but are not limited to, representatively, an antibody specifically directed to the polypeptide or derivatives or analogsthereof (e.g., single chainantibody) , a specific ligand or receptor when the polypeptide is a receptor or ligand, a substrate when the polypeptide is an enzyme, and the like.
  • the term "compound” refers to any identifiable chemical substance or molecule, including, but not limitedto, a lowmolecularweightmolecule, apeptide, a protein, a sugar, a nucleotide, or a nucleic acid. Such a co poundmaybe a naturally-occurringproduct or a synthetic product.
  • the term "low molecular weight organic molecule” refers to an organic molecule having a relatively small molecular weight. Usually, the low molecular weight organic molecule refers to a molecular weight of about 1,000 or less, or may refer to a molecular weight of more than 1,000. Low molecular weight organic molecules can be ordinarily synthesized by methods known in the art or combinations thereof. These low molecular weight organic molecules may be produced by organisms. Examples of the low molecular weight organic molecules include, but are not limited to, hormones, ligands, information transfer substances, synthesized by combinatorial chemistry, pharmaceutically acceptable low molecular weight molecules (e.g., low molecular weight ligands and the like), and the like.
  • the term "contact” refers to direct or indirect placement of a compound physically close to the polypeptide or polynucleotide of the present invention. Polypeptides or polynucleotides may be present in a number of buffers, salts, solutions, and the like.
  • the term “contact” includes placement of a compound in a beaker, a microtiter plate, a cell culture flask, a microarray (e.g., a gene chip) or the like containing a polypeptide encoded by a nucleic acid or a fragment thereof.
  • biological activity refers to activity possessed by an agent (e.g., a polynucleotide, a protein, etc.) within an organism, including activities exhibiting various functions.
  • an agent e.g., a polynucleotide, a protein, etc.
  • the biological activity thereof includes the activity of the ligand to bind to a corresponding receptor.
  • the Endomucin of the present invention the biological activity thereof includes at least one of the activities possessed by at least Endomucin (nuclear transfer ability, ability to bind to the Endomucin consensus sequence, and the like) .
  • an example of biological activity is an activity of Endomucin functioning as a transcription factor (e.g., ability to bind to a Endomucin consensus sequence) .
  • biological activity may be assayed by a method using assays for detecting or identifying Endomucin.
  • Polypeptides used in the present invention may be produced by, for example, cultivating primary culture cells producing the peptides or cell lines thereof, ollowed by separation or purification of the peptides from culture supernatant.
  • genetic manipulation techniques can be used to incorporate a gene encoding a polypeptide of interest into anappropriate expressionvector, transform an expression host with the vector, and collect recombinant polypeptides from the culture supernatant of the transformed cells.
  • the above-described host cell may be any host cell conventionally used in genetic manipulation techniques as long as they can express a polypeptide of interest while maintaining the physiological activity of the peptide (e.g., E. coli, yeast, an animal cell, etc.).
  • Polypeptides derived from the thus-obtained cells may have at least one amino acid substitution, addition, and/or deletion or at least one sugar chain substitution, addition, and/or deletion as long as they have substantially the same function as that of naturally-occurring polypeptides.
  • a given biological function ofaprotein is definedbythe interactive ability or other property of the protein. Therefore, a particular amino acid substitution may be performed in an amino acid sequence, or at the DNA code sequence level, to produce a proteinwhichmaintains the original property after the substitution. Therefore, various modifications of peptides as disclosed herein and DNA encoding such peptides may be performed without clear losses of biological usefulness.
  • hydrophobicity indices of amino acids may be taken into consideration.
  • the hydrophobic amino acid indices play an important role in providing a protein with an interactive biological function, which is generally recognized in the art (Kyte, J. and Doolittle, R.F., J. Mol. Biol. 157 (1) -.105-132, 1982).
  • the hydrophobic property of an amino acid contributes to the secondary structure of a protein and thus regulates interactions between the protein and other molecules (e.g., enzymes, substrates, receptors,
  • Each amino acid is given a hydrophobicityindex based on the hydrophobicity and charge properties thereof as follows: 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.
  • tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamicacid (-3.5) ; glutamine (-3.5) ; asparticacid (-3.5) ; asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the resultant protein may still have a biological function similar to that of the original protein (e.g., a protein having an equivalent enzymatic activity) .
  • 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 an amino acid substitution based on hydrophobicity is- efficient.
  • a hydrophilicity index is also useful for modification of an amino acid sequence of the present invention.
  • amino acidresidues are giventhe followinghydrophilicityindices : arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ⁇ 1); gluta ic acid (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.
  • an amino acid may be substituted with another amino acid which has a similar hydrophilicity index and can still provide a biological equivalent.
  • the hydrophilicity index is preferablywithin ⁇ 2, morepreferably ⁇ 1, and even more preferably +0.5.
  • conservative substitution refers to amino acid substitution in which a substituted amino acid and a substituting amino acid have similar hydrophilicity indices or/and hydrophobicity indices.
  • conservative substitution is carried out between amino acids having a hydrophilicity or hydrophobicity index of within +2, preferably within ⁇ 1, and more preferably within ⁇ 0.5.
  • conservative substitutions include, but are not limited to, substitutions within each of the following residue pairs': arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine, which are well known to those skilled in the art.
  • the term "variant" refers to a substance, such as a polypeptide, polynucleotide, or the like, which differs partially from the original substance.
  • examples of such a variant include a substitution variant, an addition variant, a deletion variant, a truncated variant, an allelic variant, and the like.
  • examples of such a variant include, but are not limited to, a nucleotide or polypeptide having one or several substitutions, additions and/or deletions or a nucleotide or polypeptide having at least one substitution, addition and/or deletion.
  • allele refers to a genetic variant located at a locus identical to a corresponding gene, where the two genes are distinguished fromeach other .
  • allelic variant refers to a variant which has an allelic relationship with a given gene.
  • allelic variant ordinarily has a sequence the same as or highly similar to that of the corresponding allele, and ordinarily has almost the same biological activity, though it rarely has different biological activity.
  • spectrum homolog or “homolog” as used herein refer to one that has amino acid or nucleotide homology with a given gene in a given species (preferably at least 60% homology, more preferably at least 80%, at least 85%, at least 90%, and at least 95% homology) . Amethod for obtaining such a species homolog is clearly understood from the description of the present specification.
  • ortholog also called orthologous genes refers to genes in different species derived from a common ancestry (due to speciation) .
  • human and mouse ⁇ -hemoglobin genes are orthologs, while the human ⁇ -hemoglobin gene and the human ⁇ -hemoglobin gene are paralogs (genes arising from gene duplication) .
  • Orthologs are useful for estimation of molecular phylogenetic trees. Usually, orthologs in different species may have a function similar to that of the original species. Therefore, orthologs of Endomucin of the present inventionmay be useful in the present invention.
  • conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences. Due to 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 everyposition 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 variations" which represent one species of conservatively modified variation.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • such modification may be performed while avoiding substitution of cysteine which is an amino acid capable of largely affecting the higher-order structure of a polypeptide.
  • Examples of a method for such modification of a base sequence include cleavage using a restriction enzyme or the like; ligation or the like by treatment using DNA polymerase, Klenow fragments, DNA ligase, or the like; and a site specific base substitution method using synthesized oligonucleotides (specific-site directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500(1983)). Modification can be performed using methods ordinarily used in the field of molecular biology.
  • amino acid additions, deletions, or modifications can be performed in addition to amino acid substitutions.
  • Amino acid substitution (s) refers to the replacement of at least one amino acid of an original peptide chain with different amino acids, such as the replacement of 1 to 10 amino acids, preferably 1 to 5 amino acids, and more preferably 1 to 3 amino acids with different amino acids .
  • Amino acid addition (s) refers to the addition of at least one amino acid to an original peptide chain, such as the addition of 1 to 10 amino acids, preferably 1 to 5 amino acids, and more preferably 1 to 3 amino acids to an original peptide chain.
  • Amino aciddeletion refers to the deletion of at least one amino acid, such as the deletion of 1 to 10 amino acids, preferably 1 to 5 amino acids, and more preferably 1 to 3 amino acids.
  • Amino acid modification includes, but is not limited to, amidation, carboxylation, sulfation, halogenation, truncation, lipidation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (e.g., acetylation) , and the like.
  • Amino acids to be substituted or added may be naturally-occurring or nonnaturally-occurring amino acids, or amino acid analogs. Naturally-occurring amino acids are preferable.
  • peptide analog refers to a compound which is different from a peptide but has at least one chemical or biological function equivalent to the peptide. Therefore, a peptide analog includes one that has at least one amino acid analog or amino acid derivative addition or substitution with respect to the original peptide .
  • a peptide analog has the above-described addition or substitution so that the function thereof is substantially the same as the function of the original peptide (e.g., a similar pKa value, a similar functional group, a similar bindingmannerto othermolecules, a similarwater-solubility, and the like) .
  • Such a peptide analog can be prepared using a technique well known in the art. Therefore, a peptide analog may be a polymer containing an amino acid analog.
  • a chemically-modified polypeptide composition in which a polypeptide of the present invention is attached to a polymer is included within the scope of the present invention.
  • This polymer may be water soluble so that the protein does not precipitate in an aqueous environment (e.g. , a physiological environment) .
  • An appropriate water soluble polymer may be selected from the group consisting of: polyethylene glycol (PEG) , monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly- (N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol.
  • the selected polymer is typically modified to have a single reactive group (e.g., active ester for acylation or aldehyde for alkylation) . As a result, the degree of polymerization may be controlled.
  • the polymer may be of anymolecular weight, andmay be branched or unbranched. Included within the scope of suitable polymers is a mixture of polymers. When the chemically modified polymer of the • present invention is used in therapeutic applications, a pharmaceutically acceptable polymer is selected.
  • the polymer When the polymer is modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, when the polymer is modified by reducing alkylation, the polymer should have a single reactive aldehyde group.
  • Apreferable reactive aldehyde is, for example, polyethylene glycol, propionaldehyde (which is water stable) , o onoCl-CIO alkoxy or aryloxy derivatives thereof (see U.S. Patent No. 5,252,714, which is herein incorporated by reference in its entity) .
  • Pegylation of the polypeptide of the present invention maybe carried outbyany of thepegylation reactions known in the art, as described for example in the following references: Focus on Growth Factors, 3, 4-10 (1992); EP 0 154 316; EP 0 401 384, which are herein incorporated by reference in their entity) .
  • pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-solublepolymer) .
  • Polyethylene glycol is a water-soluble polymer suitable for use in pegylation of the polypeptide of the present invention (e.g., Endomucin, an active form thereof or an activating agent therefor, and the like) .
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize proteins (e.g., mono(Cl-ClO) .alkoxy-polyethylene glycol or mono(Cl-ClO) aryloxy-polyethylene glycol (PEG) ) .
  • Chemical derivatization of the polypeptide of the present invention may be performed under any suitable conditions that can be used to react a biologically active substance with an activated polymer molecule.
  • Methods for preparing pegylated polypeptides of the present invention will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby Endomucin or an activating agent therefor becomes attached to one or more PEG groups, and (b) obtaining the reaction product (s) .
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • the optimal reaction conditions or the acylation reactions are easily selected by those skilled in the art based on known parameters and the desired result.
  • conditions may be alleviated or modulated by administration of the pegylated polypeptide of the present invention.
  • the polypeptide derivative of the polypeptide molecule of the present invention disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics (e.g., increased or decreased half-life) , as compared to the nonderivatized molecules.
  • the polypeptideofthepresent invention, and fragments, variants and derivatives thereof may be used singly or in combination, or in combination with other pharmaceutical compositions.
  • a substance of the present invention may be in the form of a nucleic acid (nucleic acid molecule) if the expression product of the substance is consistently or transiently Endomucin.
  • a nucleic acid molecule includes one in which a part of the sequence of the nucleic acid is deleted or is substituted with other base(s), or an additional nucleic acid sequence is inserted, as long as a polypeptide expressed by the nucleic acid has substantially the same activity as that of naturally-occurring Endomucin, as described above.
  • an additional nucleic acid may be linked to the 5' terminus and/or 3' terminus of the nucleic acid.
  • the nucleic acid molecule may include one that is hybridizable to a gene encoding a polypeptide under stringent conditions and encodes a polypeptide having substantially the same function. Such a gene is known in the art and can be used in the present invention.
  • the above-describednucleic acid can be obtained by a well-known PCR method, i.e. , chemical synthesis. This method may be combined with, for example, site-specific mutagenesis, hybridization, or the like.
  • substitution, addition or deletion for a polypeptide or a polynucleotide refers to the substitution, addition or deletion of an amino acid or its substitute, or a nucleotide or its substitute, with respect to the original polypeptide or polynucleotide, respectively. This is achieved by techniques well known in the art, including a site-specific mutagenesis technique and the like.
  • a polypeptide or a polynucleotide may have any number (>0) of substitutions, additions, or deletions. The number can be as large as a variant having such a number of substitutions, additions or deletions which maintains an intended function (e.g., the information transfer function of hormones and cytokines, etc.). For example, such a number may be one or several, and preferably within 20% or 10% of the full length, or no more than 100, no more than 50, no more than 25, or the like.
  • Polypeptides used in the present invention may be derived from any organism.
  • the organism is a vertebrate (e.g., the class Mammalia, the class Reptilia, the class Amphibia, the class Pisces, the class Aves, and the like, and more preferably a mammal (e.g., the order Rodentia (e.g., mice, rats, etc.), the order Primates (e.g., humans, etc.), etc.).
  • Polypeptides used in the present invention may be synthesized as long as they have the desired effect. Such polypeptides may be synthesized by synthesis techniques well known in the art.
  • Polypeptides used in the present invention may be expressed as fusion proteins with only a hinge region of an antibody, so that the resultant polypeptides form a dimer via a disulfide bond.
  • polypeptides used in the present invention may be expressed as fusion proteins which have disulfide bonds at the C-terminus, the N-terminus, or other positions while keeping the activity, so that the resultant polypeptides form a multimer which is a dimer or higher order oligomers.
  • sequences of the present invention such as a sequence set forth in SEQ ID NO:2, 4, 6, 8, 10 or the like, may be linked with one another in series to produce ayogric structure. Therefore, the polypeptides of the present invention in the form of any dimer or higher order oligomers, which are producedbygenetic engineering techniques, are within the scope of the present invention. Alternatively, the polypeptides of the present invention in the form of a dimer or higher order oligomers may contain monomers of the same or different species. Therefore, the dimer or higher order oligomers of the present invention may be a homodimer/homooligomer or a heterodimer/heterooligo er.
  • antibody is used in its ordinary sense, which is well known to those skilled in the art.
  • antibody encompasses the whole molecule and a fragment thereof, a derivative thereof, a conjugate thereof, and the like.
  • antibodies which are preferably used in the present invention, recognize a polypeptide of the present invention, more preferably in a specific manner.
  • Such antibodies may be polyclonal antibodies or monoclonal antibodies. In one embodiment of the present invention, such antibodies are also within the scope of the present invention.
  • the term "antigen" refers to a substance (e.g., but not limited to, a protein, a lipid, a sugar, or the like) which binds to an antibody or a specific receptor of a B lymphocyte, a T lymphocyte, or the like to elicit an immune reaction (e.g., antibody production and/or cytopathy, or the like) .
  • an immune reaction e.g., antibody production and/or cytopathy, or the like
  • the ability to bind to an antibody or a lymphocyte receptor is called "antigenicity”.
  • the ability to induce an immune response, such as antibody production or the like, is called “immunogenicity”.
  • a substance used as an antigen contains at least one material of interest (e.g., a protein, etc.).
  • a substance use as antigen with preferably has its full length, or alternatively, may have a partial sequence as long as the sequence contains at least one epitope capable of eliciting an immune response .
  • epitope or "antigenic determinant” refers to a site of an antigen molecule to which an antibody or a lymphocyte receptor binds. Techniques for determining epitopes are well known in the art. Epitopes can be determined by those skilled in the art using the well-known routine techniques if the primary sequence of a nucleic acid or amino acid is provided. Epitopes can be used even if their exact sites and detailed structures are not elucidated.
  • epitope includes a set of amino acid residues which are involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by a T cell receptor proteins and/or Major Histocompatibility Complex. (MHC) molecules. This term is also used interchangeably with "antigenic determinant” or "antigenic determinant site”.
  • an epitope is the feature of a molecule (e.g., primary, secondary and tertiary peptide structure, and charge) that forms a site recognizedbyan immunoglobulin,
  • An epitope including a peptide comprises 3 or more amino acids in a spatial conformation which is unique to the epitope.
  • an epitope consists of at least 5 such amino acids, and more ordinarily, consists of at least 6, 7, 8, 9 or 10 such amino acids.
  • the greater the length of an epitope the more the similarity of the epitope to the original peptide, i.e., longer epitopes are generally preferable. This is not necessarily the case when the conformation is taken into account.
  • Methods of 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.
  • epitopes in a given protein is readily accomplished using techniques well known in the art. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984) 81: 3998 (general method for rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); U. S. Patent No. 4,708,871 (procedures for identifying and chemically synthesizing epitopes of antigens); and Geysen et al., Molecular Immunology (1986) 23: 709 (technique for identifyingpeptides with high affinity for a given antibody) . Antibodies that recognize the same epitope can be identified in a simple immunoassay.
  • an epitope including a peptide is well known in the art.
  • Such an epitope can be, determined using a well-known, common technique by those skilled in the art once the primary nucleic acid or amino acid sequence of the epitope is provided.
  • an epitope including a peptide requires a sequence having a length of at least 3 amino acids, preferably at least 4 amino acids, more preferably at least 5 amino acids, at least 6 amino acids, at least 7 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, and at least 25 amino acids.
  • Epitopes may be linear or conformational.
  • polymers e.g., polypeptide structure
  • polypeptide structure The structure of polymers (e.g., polypeptide structure) may be described at various levels. This structure is generally described in, for example, Alberts et al., Molecular Biology of the Cell (3rd Ed., 1994), and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • the term "primary structure” refers to an amino acid sequence of a particular peptide.
  • secondary structure refers to a three-dimensional structure locally provided in a polypeptide. Sucha structure is generallyknownas a domain. A domain forms a small unit of polypeptide, and representatively, are portions having a length of about 50 to 350 amino acids.
  • tertiary structure refers to the complete structure of a polypeptide monomor.
  • quaternary structure refers to a three-dimensional structure formed by non-covalent association of separate three-dimensional units. Terms relating to anisotropy are used in the same sense as in the field of energy. Therefore, polypeptides of the present invention may encompass polypeptides having anyamino acid sequence as long as theirhigher order structure has substantially the same, ability as that of Endomucin.
  • the term “specifically expressed” in the case of genes indicates that a gene is expressed in a specific site or for a specific period of time at a level different from (preferably higher than) that in other sites or periods of time.
  • the term “specifically expressed” indicates that a gene may be expressed only in a given site (specific site) or may be expressed in other sites.
  • the term “specifically expressed” indicates that a gene is expressed only in a given site.
  • a gene encoding Endomucin of the present invention can be manipulated so as to be expressed at a specific site or at a specific period of time. Such a technique is well known in the art and is described in literature referenced herein.
  • Endomucin and the like, and fragments and variants thereofaswell as anagent thereto suchas an antibody as used herein can be produced by genetic engineering techniques.
  • vector refers to a vector capable of transferring a polynucleotide sequence of interest to a target cell.
  • a vector is capable of self-replication or incorporation into a chromosome in a host cell (e.g., a prokaryotic cell, yeast, an animal cell, a plant cell, an insect cell, an individual animal, and an individual plant, etc.), and contains a promoter at a site suitable for transcription of a polynucleotide of the present invention.
  • a vector may be a plasmid.
  • viral vector refers to a vector derived from a virus.
  • virus refers to an infectious small construct having DNA or RNA as a genome and being capable of proliferating only within infected cells. Viruses are selected from the group consisting of Retroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, andHepadnaviridae.
  • retrovirus refers to a virus which has genetic information in the form of RNA and- synthesizes DNA using reverse transcriptase based on the RNA information.
  • retrovirus vector refers to a retrovirus which is used as a carrier (vector) carrying a gene.
  • a retrovirus 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) , or the like.
  • MMLV Moloney Murine Leukemia Virus
  • MSCV Murine Stem Cell Virus
  • a retrovirus vector includes pGen-, pMSCV, and the like.
  • the term "expression vector” refers to a nucleic acid sequence comprising a structural gene and a promoter for regulating expression thereof, and in addition, various regulatory elements in a state that allows them to operate within host cells.
  • the regulatory element may include, preferably, terminators, selectable markers such as drug-resistance genes, and enhancers.
  • an expression vector used in the present invention may further include pCAGGS (Niwa H. , et al, Gene; 108: 193-9(1991) ) .
  • the term "recombinant vector” refers to a vector capable of transferring a polynucleotide sequence of interest to a target cell. Such a vector is capable of self-replication or incorporation into a chromosome in a host cell, and contains a promoter at a site suitable for transcription of a polynucleotide of the present invention.
  • a "recombinant vector" for animal cells includes pcDNA I/Amp, pcDNA I, pCDM8 (all commercially available from Funakoshi) , pAGE107 (Japanese Laid-Open Publication No. 3-22979; Cytotechnology, 3, 133(1990)), pREP4 (Invitrogen), pAGE103 (J. Biochem., 101, 1307(1987)), pAMo, pAMoA (J. Biol. Chem. , 268, 22782-22787 (1993) ) , pCAGGS (Niwa H., et al, Gene; 108: 193-9(1991)), and the like.
  • terminator refers to a sequence which is located downstream of a protein-encoding region of a gene and which is involved in the termination of transcription when DNA is transcribed into mRNA, and the addition of a poly A sequence. It is known that a terminator contributes to the stability of mRNA, and has an influence on the amount of gene expression. Examples of a terminator include, but are not limited to, terminators derived from mammals, the CaMV35S terminator, the terminator of the nopaline synthase gene (Tnos) , the terminator of the tobacco PRla gene, and the like.
  • promoter refers to a base sequence which determines the initiation site of transcription of a gene and is a DNA region which directly regulates the,frequency of transcription. Transcription is started by RNA polymerase binding to a promoter.
  • a promoter region is usually located within about 2 kbp upstream of the first exon of aputativeproteincoding region. Therefore, it is possible to estimate a promoter region by predicting a protein coding region in a genomic base sequence using DNA analysis software .
  • Aputative promoter region is usually located upstream of a structural gene, but depending on the structural gene, i.e., a putative promoter region may be located downstream of a structural gene. Preferably, a putative promoter region is located within about 2 kbp upstream of the translation initiation site of the first exon.
  • the term "site-specific" in relation to gene expression generally refers to specificity of the gene expression at a site (e.g. , the heart, myocardial cells, or the like in animals; etc.) of an organism (e.g., an animal) .
  • the term “time-specific” refers to specificity of gene expression with respect to a particular stage (e.g., upon stroke, or the like) of an organism (e.g., an animal) . Such specificity can be introduced into a desired organism using an appropriately selected promoter.
  • the term "constitutive" in relation to expression of a promoter of the present invention indicates that the promoter is expressed at substantially a constant level no matter whether the organisms are in the juvenile phase ormature phase of growth. Specifically, when northern blotting is performed under the same conditions as those described in the examples below, expression is defined as being constitutive if the expression is substantially observed at any time point (e. g. , at the same two sites or more, or corresponding sites) . Constitutive promoters are considered to play a role in homeostasis of organisms in ordinary growth environments .
  • stress responsive in relation to expression of a promoter of the present invention indicates that the level of expression of the promoter is changed when at least one stress (e.g., differentiating stimuli, or the like) is given to an organism.
  • stress inductive indicates that the expression level is increased
  • stress reductive indicates that the expression level is decreased.
  • Stress reductive expression is based on the premise that expression is normally observed, overlapping the concept of "constitutive” expression. These properties can be determined by extracting RNA from any portion of an organism and analyzing the level of expression by northern blotting or RT-PCR, or quantitating expressed proteins by western blotting.
  • a vector having an incorporated nucleic acid encoding a polypeptide used in the present invention with a stress inductive promoter is used in order to transform an animal or a part thereof (a specific cell, tissue, or thelike)
  • the peptide canbe expressedunder given conditions (e.g., stimulation for differentiation) using a stimulating agent having activity to induce the promoter.
  • the term “enhancer” refers to a sequence which is used so as to enhance the expression efficiency of a gene of interest. When used in plants, an enhancer region containing a sequence upstream of a human cytomegalo irus immediate-early enhancer is preferable. One or more enhancers may be used, or no enhancer may be used.
  • operatively linked indicates that a desired sequence is located such that expression (operation) thereof is under control of a transcription and translation regulatory sequence (e.g., a promoter, an enhancer, and the like) or a translation regulatory sequence.
  • a transcription and translation regulatory sequence e.g., a promoter, an enhancer, and the like
  • a promoter In order for a promoter to be operatively linked to a gene, typically, the promoter is located immediately upstream of the gene .
  • a promoter is not necessarily adjacent to a structural gene.
  • the present invention may be used in any animal. Techniques for use of the present invention in animals are well known in the art and are commonly used, as described in, for example, Ausubel F.A. etal., editors, (1988), Current Protocols inMolecular Biology, Wiley, New York, NY; Sambrook J. et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. and its 3rd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Special issue, Jikken Igaku [Experimental Medicine] "Experimental Method for Gene Introduction & Expression Analysis", Yodo-sha, 1997; and the like.
  • transformant refers to the whole or a part of an. organism, such as a cell, which is produced by transformation. Examples of a transformant include animal cells and the like. Transformants may be referred to as transformed cells, transformed tissue, transformed hosts, or the like, depending on the subject. As used herein, all of the forms are encompassed, however, a particular form may be specified in a particular context.
  • animal as used herein in its broadest sense refers to vertebrates and invertebrates (e.g., arthropods) .
  • animals include, but are not limited to, any of the class Mammalia, the class Aves, the class Reptilia, the class Amphibia, the class Pisces, the class Insecta, the class Vermes, and the like.
  • the animal may be a mammal.
  • polypeptides, nucleic acids, kits, systems, compositions, and methods for use in the present invention can function in all animals including mammals and other species. This is because ligands corresponding to Endomucin are known to be present in animals in addition to mammals .
  • the term "cell” is herein used in its broadest sense in the art, referring to a structural unit of tissue of a multicellular organism, which is capable of self replicating, has genetic information and a mechanism for expressing it, and is surrounded by a membrane structure which isolates the living body from the outside.
  • tissue in relation to organisms refers to an aggregate of cells having substantially the same function. Therefore, a tissue may be part of an organ. Organs usually have cells having the same function, an may have coexisting cells having slightly different functions. Therefore, as used herein, tissues may have various kinds of cells as long as a certain property is shared by the cells.
  • organ refers to a structure which has a single independent form and in which one or more tissues are associated together to perform a specific function.
  • organs include, but are not limited to, stomach, liver, intestine, pancreas, lung, airway, nose, heart, artery, vein, lymph node (lymphatic system) , thymus, ovary, eye, ear, tongue, skin, and the like.
  • the term "treat by Endomucin and an agent capable of activating Endomucin” indicates that cells are exposed to the Endomucin of the present invention, or an agent capable of activating the Endomucin, so as to expand the cells. Therefore, such a treatment includes techniques, such as: directly contacting cells with Endomucin; transforming cells so that the agent is transiently expressed therein; or the like.
  • transgenic refers to incorporation of a specific gene into an organism (e.g., animals (mice, etc.) or plants) or such an organism having an incorporated gene.
  • the transgenic organisms can be produced by a icroinjection method (a trace amount injection method), a viral vector method, an embryonic stem (ES) cell method, a sperm vector method, a chromosome fragment introducing method, an episome method, or the like.
  • a icroinjection method a trace amount injection method
  • viral vector method a viral vector method
  • ES cell method an embryonic stem (ES) cell method
  • sperm vector method a chromosome fragment introducing method
  • episome method or the like.
  • screening refers to selection of a target, such as an organism, a substance, or the like, a given specific property of interest from a population containing a number of elements using a specific operation/evaluation method.
  • an agent e.g., an antibody
  • Screening maybeperformed using libraries obtained in vi tro, in vivo, or the like (with a system using a real substance) or alternatively in silico (with a system using a computer) .
  • the present invention encompasses compounds having desired activity obtained by screening.
  • the present invention is also intended to provide drugs which are produced by computer modeling based on the disclosures- of the present invention.
  • the present invention provides an assay for screening candidate compounds or test compounds for a protein or polypeptide of the present invention, or a compound capable of binding to a biologically active portion thereof or modulating the activity thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries, spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable topeptide, nonpeptideoligomer, or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145) .
  • a compound is also provided, which is used as a tool for screening for an agent effective as an active component (e.g., a polypeptide or a nucleic acid) of the present invention and which is obtained by a quantitative structure activity relationship (QSAR) modeling technique using a computer.
  • an agent effective as an active component e.g., a polypeptide or a nucleic acid
  • QSAR quantitative structure activity relationship
  • the computer technique includes several substrate templates prepared by a computer, pharmacophores, homology models of an active portion of the present invention, and the like.
  • a method for modeling a typical characteristic group of a substance, which interacts with another substance, based on data obtained in vitro includes a recent CATALYSTTM pharmacophore method (Ekins et al., Pharmacogenetics, 9:477 to 489, 1999; Ekins et al., J. Pharmacol. & Exp. Ther. , 288 : 21 to 29, 1999; Ekins et al . , J. Pharmacol., & Exp. Ther., 290:429 to 438, 1999; Ekins et al., J. Pharmacol. & Exp.
  • molecule modeling software e.g., CATALYSTTM Version 4 (Molecular Simulations, Inc., San Diego, CA) , etc. .
  • the fitting of a compound with respect to an active site can be performed using any of various computer modeling techniques known in the art.
  • Visual inspection and manual operation of a compound with respect to an active site can be performed using a program, such as QUANTA (Molecular Simulations, Burlington, MA, 1992), SYBYL (Molecular Modeling Software, Tripos Associates, Inc., St. Louis, MO, 1992), AMBER (Weiner et al., J. Am. Chem. Soc, 106:765-784, 1984), CHARMM (Brooks et al., J. Co p. Chem., 4:187 to 217, 1983), or the like.
  • energy minimization can be performed using a standard force field, such as CHARMM, AMBER, or the like.
  • Examples of other specialized computermodelingmethods include GRID (Goodford et al., J. Med. Chem., 28:849 to 857, 1985), MCSS (Miranker and Karplus, Function and Genetics, 11:29 to 34, 1991), AUTODOCK (Goodsell and Olsen, Proteins: Structure, Function and Genetics, 8:195 to 202, 1990), DOCK (Kuntz et al., J. Mol. Biol., 161:269 to 288, 1982), and the like.
  • structural compounds can be newly constructed using an empty active site, an active site of a known small molecule compound with a computer program, such as LUDI (Bohm, J. Comp. Aid. Molec.
  • a cell is the target for which undifferentiated state and/or transplantation compatibility is to be determined.
  • stem cells may be used.
  • any cells can be employed as long as they can be changed to a desired cell by treatment using an agent of the present invention.
  • stem cell refers to a cell having self-replication ability and pluripotency, and in actual situations, a cell capable of regenerating a tissue, which has been injured, to some extent.
  • a stem cell for use in the present invention may be an embryonic stem (ES) cell or tissue stemcell (also calledtissue specific stem cell or somatic stem cell) .
  • ES embryonic stem
  • tissue stemcell also called tissue specific stem cell or somatic stem cell
  • Embryonic stem cells are pluripotent stem cells derived from early embryos . An embryonic stem cell was first established in 1981, which has been applied to the production of knockout mice since 1989. In 1998, a human embryonic stem cell was established, which is currently becoming available for regenerative medicine.
  • embryonic stemcells embryonic stemcells and embryonic germ cells
  • tissue stem cells e.g., bone marrow cells (e.g., hematopoietic stem cells)
  • bone marrow cells e.g., hematopoietic stem cells
  • colony assay methods proliferation capability and proliferation values can be evaluated by expressing colony number obtained by culturing stem cells a medium.
  • fluorescence histogram display determination is made as follows: after stained with anti-CD34 antibody with a fluorescent label, calculation is made using FCM, and specifically cells expressing a FL value equal to ormore than that of a control will be calculated as "positive cells”.
  • This display method is calculated including a portion of monocytes, thus the values may be extremely high.
  • FL-SSC method after staining with fluorescently labeled anti-CD34 antibody, calculation is made using FCM, and only the stem cell region which can be clearly distinguished from the other cells when drawing a two dimensional graph with FL values and SSC values, is calculated for positive cells. This can also be said to be a numerical evaluationmethod. Further, FL-SSCmethods have extremely good correlation with colony assay methods as substantially no effects by monocytes are found.
  • Markers or indicators for stem cells include, for example, but are not limited to CD34 (SEQ ID NOs: 13-16) ,
  • Lin Lineage marker
  • c-kit SEQ ID NOs: 25-26
  • Sca-1 SEQ ID NOs: 27-28
  • Flt3/Flk2 SEQ ID NOs: 11-12
  • Lineage (marker) and “differentiation antigen “marker)” are interchangeably used to refer to a marker for differentiation state, if the marker is positive, then cells of interest are differentiated, and if themarker is negative, then cells of interest are not differentiated.
  • Lin markers include, for example, but are not limited to 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) and the like.
  • Tissue stemcells have a relatively limited level of differentiation unlike embryonic stem cells .
  • Tissue stem cells are present in tissues and have an undifferentiated intracellular structure.
  • Tissue stem cells have a higher nucleus/cytoplasm ratio and have few intracellular organelles.
  • Most tissue stemcells have pluripotency, along cell cycle, and proliferative ability beyond the life of the individual. Therefore, in a preferred embodiment of the present invention, a tissue stem cell committed to a blood cell phenotype may be employed.
  • Tissue stem cells may be classified into three categories in accordance with their origins: ectoderm, mesoderm, andendoderm.
  • Ectoderm-derived tissue stem cells include neural stem cells existing in the brain, epidermal stem cells existing in the skin, follicular stem cells and pigment stem cells.
  • Mesoderm-derived tissue stem cells include vascular stem cells, hematopoietic stem cells and mesenchymal stem cells observed in bone marrow and blood. Endoderm-derived tissue stem cells are mainly present in organs, including liver stem cells, pancreatic stem cells, and intestinal epithelial stemcells.
  • germ line stem cells are present in the testes and the ovaries.
  • mesoderm-derived stem cells may be employed.
  • bone marrow cells e.g., hematopoietic stem cells
  • Tissue stem cells are separated into categories of sites from which the cells are derived, such as the dermal system, the digestive system, the bone marrow system, the nervous system, and the like .
  • Tissue stem cells in the dermal system include epidermal stem cells, hair follicle stem cells, and the like.
  • Tissue stem cells in the digestive system include pancreatic (common) stem cells, liver stem cells, and the like.
  • Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, and the like.
  • Tissue stem cells in the nervous system include neural stem cells, retinal stem cells, and the like.
  • it was unexpectedly revealed that bone marrow cells are preferable.
  • bone marrow cells may be used, as it is, as a cell source, or alternatively, a particular enriched or purified cell group (e.g., tissue stem cells) may be employed as a cell source.
  • the term "regeneration” refers to the recovery of injured tissue or organs to the original condition, and is also called pathological regeneration.
  • the body of an organism may lose a part of its organs or may be heavily injured by traumas or diseases in its life time. In this case, whether or not the injured organ can be regenerat d varies among organs (or among animal species) .
  • the branch of medicine that permits organs (or tissue) which cannot be naturally regenerated, to undergo regeneration so as to recover function, is regenerativemedicine. Whether or not tissue, has regenerated, can be determined based on whether or not the function is improved. Mammals have the capability of regenerating tissue and organs to some degree (e.g., regeneration of skin, liver, and blood).
  • heart transplant is substantially the only measure for the treatment of the organ.
  • stem cells are present in organs having a high level of regenerative ability. This presumption was proved by experimental bone marrow transplantation using animal models. Subsequent studies have demonstrated that stem cells in bone marrow are resources for regeneration of all kinds of blood cells. It has also been demonstrated that stem cells are present in organs having a high level of regenerative ability, such as bone marrow, skin, and the like. In addition, although it was long believed that the brain cannot be regenerated, it has been demonstrated that stem cells are present in the brain, the heart, and the like. It has been clarified that stem cells are present in all organs in the body and play ' a role in regeneration of the organs to some extent. Stem cells present in each tissue have plasticity to an extent beyond expectation, so that stem cells in one organ may be used in regeneration of another organ. Therefore, the expansion function found in the present invention is effective for various tissue stem cells and the number of applications of the present invention is infinite.
  • differentiation refers to a phenomenon that two or more types of cells having qualitative differences in form and/or function occur in a daughter cell population derived from the division of a single cell. Therefore, “differentiation” includes a process during which a population (family tree) of cells, which do not originally have a specific detectable feature, acquire a feature, such as production of a specific protein, or the like. Atpresent, cell differentiation is generally considered to be a state of a cell in which a specific group of genes in the genome are expressed. Cell differentiation can be identified by searching for intracellular or- extracellular agents or conditions which elicit the above-described state of gene expression. Differentiated cells are stable in principle.
  • undifferentiated refers to a state of a cell which does not have a difference in morphology and functionality.
  • the term “maintain” in relation to an undifferentiated state refers to keeping pluripotency. Therefore, maintenance of an undifferentiated state can be assessed by determing whether or not pluripotency is kept.
  • the term “expansion” in relation to stem cells indicates that the number of stem cells is increased while maintaining the autoexpansion ability and pluripotency of the stem cell. Although the expansion phenomenon of stem cells has been known, few molecules which specifically cause such an expansion has been found.
  • pluripotency may be at any level as long as expansion can be maintained.
  • totipotency, pluripotency in a narrow sense (as possessed by tissue stem cells) and the like are illustrated herein.
  • the present invention is not limited to this.
  • differentiated cell refers to a cell having a specialized function and form (e.g., muscle cells, neurons, etc.). Unlike stem cells, differentiated cells have little or no pluripotency. Examples of differentiated cells include epidermic cells, pancreaticparenchymal cells, pancreatic duct cells, hepatic cells, blood cells, cardiac muscle cells, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells, cartilage cells, andthe like. Therefore, in one embodiment of the present invention, if a certain material cell can be treated by an agent of the present invention (e. g. , Endomucin polypeptide or nucleic acid) to obtain differentiated cells, such as a leucocyte or the like, the differentiated cell may also be within the scope of the present invention.
  • an agent of the present invention e. g. , Endomucin polypeptide or nucleic acid
  • Cells used as a resource of the present invention can be differentiated into hematopoietic cells by treatment with a polypeptide, nucleic acid, composition, kit and/or medicament of the present invention.
  • pluripotency refers to a nature of a cell, i.e., an ability to differentiate into one or more, preferably two or more, tissues or organs.
  • the pluripotency of a cell is limited as the cell is developed, and in an adult , cells constituting a tissue or organ rarely alter to different cells, where the pluripotency is lost. Such alteration typically occurs in pathological conditions, and is called metaplasia.
  • mesenchymal cells tend to easily undergo metaplasia, i.e., alter to other mesenchymal cells, with relatively simple stimuli. Therefore, mesenchymal cells have a high level of pluripotency. Therefore, cells having pluripotency may be preferable as source cells in the present invention, though this is not required.
  • pluripotency As used herein, one type of pluripotency is "totipotency", which refers to an ability to differentiate into all kinds of cells which constitute an organism.
  • the idea of pluripotency encompasses totipotency.
  • An example of a totipotent cell is a fertilized ovum.
  • totipotency may be clearly separated frompluripotency. The former indicates an ability to be differentiated into all kinds of cells while the latter indicates an ability to be committed to a plurality of types of cells but not all types.
  • An ability to differentiate into only one type of cell is called “unipotency”.
  • totipotency andpluripotency can be determined based on the number of days which have passed after fertilization. For example, for mouse, totipotency is distinguished from pluripotency at about Day 8 after fertilization as a borderline.
  • cells develop over time after fertilization as follows. On .Day 6.5 after fertilization (also represented by E6.5), a primitive streak appears on one side of an epiblast, clarifying the future anteroposterior axis of the embryo .
  • the primitive streak indicates the. future posterior end of the embryo, extending across the ectoderm to reach the distal end of the epiblast.
  • the primitive streak is an area in which cell movement takes place.
  • the future endoderm and mesoderm are formed.
  • a head process appears ahead of the node, in which a notochord, and a future endoderm (lower layer) and a neural plate (upper layer) around the notochord, are formed.
  • the node appears around E6.5 and moves backward, so that the axial structure is formed from the head to the tail .
  • the embryo is elongated and a large head lamella mostly consisting of the anterior neural plate is formed at the anterior end of the embryo. Segments are formed at a rate of one per 1.5 hours from E8 from the head to the tail.
  • Cells prepared using an agent- of the present invention e.g., stem cells, cells differentiated therefrom (e.g., myocardinal cells)
  • cell compositions may be in any dosage form as long as the dosage form is suited for transfer into organisms .
  • Examples of the dosage formin include, but are not limited to, liquid drug, injection drug, sustained-release drug, and the like.
  • administration route examples include, but are not limited to, oral adir ⁇ inistration, parenteral administration (including for example, intravenous, administration, intramuscular administraion, percutaneous administration, mucous administration, intrarectal administration, intravaginal administration) , direct administration to affected portions, skin administration and the like.
  • Administration methods may be herein oral, parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, to mucosa, intrarectal, vaginal, topical to an affected site, to the skin, etc. ) .
  • a prescription for such administration may be provided in any formulation form.
  • Such a formulation form includes liquid formulations, injections, sustained preparations, and the like.
  • compositions and medicament of the present invention are systemically administered, an aqueous formulation orally acceptable having no pyrogen may be suitable.
  • aqueous formulation orally acceptable having no pyrogen may be suitable.
  • Preparation of such pharmaceutically acceptable solutions are within the skill of those in the art provided that significant attention is paid to pH, isotonicity, stability and the like.
  • Solvents used in formulations of the medicament of the present invention may have the property of either aqueous or non-aqueous.
  • vehicles therefor can include any other material for formulation such as altering or maintaining pH, volume osmolality concentration, viscosity, clearness, color, sterilization property, stability, isotonicity, disintegration speed, or smell.
  • the composition of the present invention may contain other formulation material for altering or maintaining release speed of an active ingredient, or accelerating absorption or permeation of active ingredient.
  • the medicament of the present invention may be prepared for storage by mixing a composition having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Japanese Pharmacopeia ver. 14, or a supplement thereto or the latest version; Remington' s Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990; and the like) , in the form of lyophilized cake or aqueous solutions.
  • a medicament of the present invention is administered in the formof a composition comprising an active component, of the present invention (e.g., a polypeptide, a nucleic acid, or the like) with at least one physiologically acceptable carrier, excipient or diluent.
  • an appropriate vehicle maybe injection solution, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other substances which are commonly used for compositions for parenteral delivery.
  • Acceptable carriers, excipients or stabilizers used herein preferably are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and preferably include phosphate, citrate, or other organic acids; antioxidants (e.g., ascorbic acid); low molecular weight polypeptides; proteins (e.g., serumalbu in, gelatin, or immunoglobulins) ; hydrophilic polymers (e.g., polyvinylpyrrolidone) ; amino acids (e.g., glycine, glutamine, asparagine, arginine or lysine) ; monosaccharides , disaccharides , and other carbohydrates (glucose, mannose, or dextrins) ; chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and/or nonionic surfactants (e.g.,
  • composition or kit of the present invention may further comprise at least one biocompatibility material such as silicone, collagen, gelatin, copolymer of glycolic acid and lactic acid, ethylene vinyl acetate copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyacrylate, polymethacrylate and the like. Silicone is preferable since it is amenable for shaping.
  • Exemplary biodegradable polymers include, collagen, gelatin, polymers, copolymers, and mixtures thereof synthesized by condensing at least one of ⁇ -hydroxy carboxylic acids (for example, glycolic acid, lactic acid, hydroxy butyric acid and the like), hydroxydicarboxylic acids (for example, malic acid and the like) , and hydroxy tricarboxylic acids (for example, citric acid and thelike) , and thelike; poly- ⁇ -cyano acrylate ester, poly amino acids (for example, poly- ⁇ -benzyl-L-glutamic acid and the like) , poly acid anhydride such as aleic acid copolymer anhydrides (for example, styrene-maleic acid copolymer and the like) , and thelike.
  • ⁇ -hydroxy carboxylic acids for example, glycolic acid, lactic acid, hydroxy butyric acid and the like
  • hydroxydicarboxylic acids for example, malic acid and the like
  • Forms ofpolymerization maybe random, block, graft and the like, and ⁇ -hydroxy carboxylic acids, hydroxy dicarboxylic acids, hydroxytricarboxylic acids have optical chirality in the molecule, any of D-, L-, and DL-species may be used.
  • copolymers of glycolic acid and lactic acid may be used.
  • Injection drugs can be prepared using techniques well known in the art. For example, an agent of the present invention is dissolved in an appropriate solvent (physiological saline, buffer (e.g., PBS, etc.), sterilized water, etc.), followedby filter sterilization using a filter, or the like. Thereafter, the solution is placed into a sterile container (e.g., an ampoule, or the like) . Thus, an injection drug can be prepared.
  • the injection drug may contain a commonly used pharmaceutical carrier if required.
  • An administration method using a non-irivasive catheter may be employed. Examples of .appropriate carriers include neutral buffered saline or saline mixed with serum albumin.
  • the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose).
  • excipients e.g., sucrose
  • Other standard carriers, diluents, and excipients maybe included as desired.
  • Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
  • the pH of the solution or various other pH should be selected based on the relative solubility of an active component of the present invention (e.g., a polypeptide, a nucleic acid, etc. ) .
  • a protocol for preparing a formulation of the present invention is known, in the art as described in, for example, the Japanese Pharmacopeia, the U.S. Pharmacopeia, Pharmacopeias of other countries, and the like. Therefore, those skilled in the art can determine the amount of a polypeptide or a cell tobe administered in view of the present specification without undue experimentation.
  • a composition and medicament of the present invention may be provided in a sustained-release form.
  • an active component e.g., a nucleic acid or a polypeptide
  • Any sustained-released dosage form may be used in the present invention.
  • sustained-release dosage forms include, but are not limited to, rod-like formulations (e.g., pellet-like, cylinder-like, needle-like formulations, etc.), tablet formulations, disk-like formulations, sphere-like formulations, sheet-like formulations, and the like.
  • Methods ' for preparing sustained-release dosage forms are well known in the art, as described in, for example, the Japanese Pharmacopeia, the U.S. Pharmacopeia, Pharmacopeias of other countries, and the like.
  • Examples of a method for producing sustained-release drugs include, but are not limited to, a method using disaggregation of a drug from a complex, a method for preparing an aqueous suspended liquiddrug, a ethod forpreparing an oil injection liquid or oil suspended injection liquid, a method for preparing an emulsified injection liquid (o/w or w/o type emulsified injection liquid, or the like), and the like.
  • Such pharmaceutical agents maybe anymedicament known in the art, such as, for example, any pharmaceutical agent known in pharmacology (e.g., antibiotics, etc.). Two or more of such pharmaceutical agents may be simultaneously used. These pharmaceutical agents are described in, for example, the Japanese Pharmacopeia (latest edition) , the U.S. Pharmacopeia (latest edition), Pharmacopeias (latest edition) of other countries, and the like.
  • a medicament or composition of the present invention may contain two or more kinds of cells prepared by a method of the present invention.
  • the cells may have similarproperties ormaybe derived fromsimilar origins, or may have different properties or may be derived from different origins.
  • the amount of a polypeptide, nucleic acid, compound, medicament, or cell used in the treatment method of the present invention can be easily determined by those skilled in the art with reference to the purpose of use, the subject's age, size, sex, and case history, the form or type of the polypeptide, nucleic acid, compound, medicament, or cell, and the like.
  • the frequency of the compound of the present invention which is applied to a subject is also determined by those skilled in the art with respect to the purpose of use, the subject's age, size, sex, case history, the progression of the therapy, and the like. Examples of the frequency include once per day to once per several months (e.g., once per week to once per month). Preferably, administration is performed once per week to once per month with reference to progression.
  • gene therapy using a nucleic acid encoding Endomucin or a variant or fragment thereof may be useful.
  • the nucleic acid molecule When a composition of the present invention comprising a nucleic acid molecule is administered, the nucleic acid molecule may be administered in the form of a non-viral vector or a viral vector, or naked DNA (direct administration) .
  • the administration forms are well known in the art as described in, for example, Special issue, Jikken Igaku [Experimental Medicine] "Idenshi Chiryo no
  • the nucleic acid sequence of a normal gene or a nucleic acid comprising a sequence encoding an antibody or a functional derivative thereof is administered for the purpose of gene therapy for treatment, inhibition, or prophylaxis of a disease or a disorder associated with abnormal expression and/or activity of a polypeptide of the present invention.
  • Gene therapy means that subjects are treated by administering an expressed or expressible nucleic acid thereto.
  • a protein encodedby a nucleic acid is produced and the protein mediates a therapeutic effect.
  • a nucleic acid may be introduced into organisms as follows : a method of introducing a nucleic acid molecule using a liposome (e.g., a liposome method, an HVJ-liposome method, a cationic liposome method, a lipofectin method, a lipofectamine method, etc.); a microinjection method; a gene gun method of transferring a nucleic aci molecule together with a carrier (e.g., gold particle, etc.); and the like.
  • a liposome e.g., a liposome method, an HVJ-liposome method, a cationic liposome method, a lipofectin method, a lipofectamine method, etc.
  • a microinjection method e.g., a gene gun method of transferring a nucleic aci molecule together with a carrier (e.g., gold particle, etc.); and the like.
  • an expression vector examples include, but are not limited to, pCAGGS (Gene 108: 193-9, Niwa H., Yamamura K., Miyazaki J. (1991)), pBJ-CMV, pcDNA 3.1, pZeoSV (available from Invitrogen, Stratagene, etc.), and the like.
  • the HVJ-liposome method comprises including a nucleic acid molecule in a liposome made of lipid double membrane and fusing the liposome with inactivated Sendai virus (Hemagglutinating virus of Japan, HVJ) .
  • the HVJ-liposome method has a higher level of activity to fuse with cell membrane than conventional liposome methods.
  • any strain thereof can be used (e.g., ATCC VR-907, ATCC VR-105, ' etc.), preferably Z strain.
  • a composition of the present invention is provided in the form of a nucleic acid of a viral vector
  • viral vectors such as recombinant adenoviruses, retroviruses, and the like
  • a gene can be introduced into cells or tissues as follows: a nucleic acid encoding Endomucin is introduced into a DNA or RNA virus (e.g., detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliomyelitis virus, Sindbis virus, Sendai virus, SV40, human immunodeficiency virus (HIV) , or the like; and cells or tissues are infectedwiththe recombinant virus .
  • adenovirus has a dramatically higher level of infection efficiency than that of ' other viral vectors.
  • Adenovirus vectors are preferable.
  • an expression plasmid (a non-viral vector as described) is dissolved in physiological saline or the like and the resultant solution is administered as it is.
  • a solution can be directly injected into a tissue of an.organ of an organism by a method described in, for example, Tsurumi Y., Kearney M. , Chen D., Silver M., Takeshita S., Yang J. , Symes J.F., Isner J.M., Circulation 98 (Suppl. II), 382-388(1997).
  • the amount of a polypeptide (e.g., Endomucin etc.) contained as an active component in a composition and kit of the present invention may be, for example, about 1 ⁇ g to about 1000 mg for an adult (weight: about 60 kg) , preferably about 5 ⁇ g to about 100 mg.
  • the lower limit of the amount range of the polypeptide is any value between about 1 ⁇ g and about 1 mg, such as, for example, about 1 ⁇ g, about 2 ⁇ g, about 3 ⁇ g, about 4 ⁇ g, about 5 ⁇ g, about 6 ⁇ g, about 7 ⁇ g, about 8 ⁇ g, about 9 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, etc.
  • the upper limit of the amount range of the polypeptide is any value between about 1000 mg and about 1 mg, such as, 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 25 mg, about 10 mg, about 5 mg, etc.
  • an active component of the present invention is in the form of a nucleic acid (e.g., a nucleic acid encoding Endomucin or a nucleic acid encoding Endomucin, etc.)
  • the amount of the nucleic acid may be about 1 ⁇ g to about 10 mg for an adult (weight: about 60 kg) , preferably about 1 ⁇ g to about 1000 ⁇ g, and more preferably about 5 ⁇ g to about 400 ⁇ g.
  • the lower limit of the amount range of the nucleic acid may be any value between about 1 ⁇ g and about 20 ⁇ g, such as, for example, about 1 ⁇ g, about 2 ⁇ g, about 3 ⁇ g, about 4 ⁇ g, about 5 ⁇ g, about 6 ⁇ g, about 7 ⁇ g, about 8 ⁇ g, about 9 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, etc.
  • the upper limit of the amount range of the nucleic acid may be any value between about 10 mg and about 10 ⁇ g, such as, 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 ⁇ g, about 500 ⁇ g, about 250 ⁇ g, about 100 ⁇ g, etc.
  • the above-described amounts are also applicable.
  • the amount is usually 0.0001 to 100 mg, preferably 0.001 to lO g, and more preferably 0.01 to 1 mg.
  • the frequency of administration includes, for example, daily to once per several months (e.g., once per week to once per month).
  • the composition of the present invention may contain cells prepared in the present invention in amounts, for example, about lxlO 3 cells to about IxlO 11 cells, preferably about IxlO 4 cells to about IxlO 10 cells, more preferably about 1x10 s cells to about IxlO 9 cells, and the like. These cells may be present as a solution, such as about 0.1 ml, 0.2 ml, 0.5 ml, or 1 ml of physiological saline, or the like.
  • the upper limit of the amount range of cells may be, for example, about IxlO 11 cells, about 5xl0 10 cells, about 2xl0 10 cells, about IxlO 10 cells, about 5xl0 9 cells, about 2xl0 9 cells, about IxlO 9 cells, about 5xl0 8 cells, about 2xl0 8 cells, about IxlO 8 cells, about 5xl0 7 cells, about2xl0 7 cells, about IxlO 7 cells, andthe like.
  • the lower limit of the amount of cells may be, for example, about IxlO 3 cells, about 2xl0 3 cells, about 5xl0 3 cells, about IxlO 4 cells, about 2xl0 4 cells, about 5xl0 4 cells, about IxlO 5 cells, about 2xl0 5 cells, about 5xl0 5 cells, about IxlO 6 cells, and the like.
  • compositions comprising concentrated or separated cells of the present invention such as explant, medicament and the like, may comprise stem cells at the ratio of usually for example, at least about 10 %, preferably at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 %, at least about 95 %, and the like.
  • the composition of such cells may be assayed for expression level or quantification of the Endomucin of the present invention.
  • polypeptide expression may be "detected” or "quantified” by an appropriate method, including mRNA measurement and immunological measurement methods.
  • the molecular biological measurement methods include a Northern blotting method, a dot blotting method, a PCR method, and the like.
  • the immunological measurement method include an ELISA method, an RIA method, a fluorescent antibody method, a Western blotting method, an immunohistological staining method, and the like, where a microtiter plate may be used.
  • a quantification method include an ELISA method, an RIA method, and the like.
  • the term “amount of expression” refers to the amount of a polypeptide or mRNA expressed in a cell of interest.
  • the amount of expression includes the amount of expression at the protein level of a polypeptide of the present invention evaluated by any appropriate method using an antibody of the present invention, including immunological measurement methods (e.g., an ELISA method, a RIA method, a fluorescent antibody method, a Western blotting method, an immunohistological staining method, and the like, or the amount of expression at the mRNA level of a polypeptide of the present invention evaluated by any appropriate method, including molecular biological measurement methods (e.g., a Northern blotting method, a dot blotting method, a PCR method, and the like) .
  • the term "change in the amount of expression” indicates that an increase ordecrease in the amount ofexpression at theprotein or mRNA level of a polypeptide of the present invention evaluated by an appropriate method including the above-described immunological measurement method or molecular biological measurement method.
  • instructions describe a method of administering a medicament of the present invention, or the like for persons who administer, or are administered, the medicament or the like (e.g., physicians, patients, and the like) .
  • the instructions may state that a medicament of the present invention is administered immediately ' after myocardial .infarction (e.g., within 48 hours, within 36 hours, within 24 hours, within 12 hours, within 6 hours, etc. ) .
  • the instructions may also state that the administration site is skeletal muscle (e.g., via injection, etc.).
  • the instructions are prepared in accordance with a format defined by an authority of a country in which the present invention is practiced (e.g., Health, Labor and Welfare Ministry in Japan, Food and Drug Administration (FDA) in U.S., and the like), explicitly describing that the instructions are approved by the authority.
  • the instructions are so-called package insert and are typically provided in paper media.
  • the instructions are not so limited andmaybe provided in the formof electronic media (e.g., web sites and electronic mail provided on the Internet) .
  • the present invention targets any "diseases" requiring an amount of stem cells, or cells, tissues, and organs differentiated therefrom.
  • the present invention may be intended to treat diseases or disorders related to differentiated cells, tissues, or organs which can be developed as a result of the differentiation of a stem cell of the present invention.
  • bone marrow cells orpurifiedhematopoietic stem cells orprecursor cells may be used for treatment of a disease including arteriosclerosis obliterans, Buerger's disease, angina cordis, myocardial infarction, ischemic heart disease such as a variety of arteriosclerosis.
  • a collateral vessel is formed. While not wishing to be bound to any theory, the newly formed vessel is related to either or both mechanisms where hematopoietic cells become blood vessels, and/or an agent from hematopoietic cells may stimulate vascularization.
  • the above-described differentiated cells, tissues, or organs may be of the circulatory system (blood cells, etc.).
  • diseases or disorders included, but not limitedto are anemia (e.g., aplastic anemia (particularly, severe aplastic anemia) , renal anemia, cancerous anemia, secondary anemia, refractory anemia, etc.), cancer or tumors (e.g., leukemia); and after chemotherapy therefor, hematopoietic failure, thrombocytopenia, acute myelocytic leukemia (particularly, a first remission (high-risk group) , a second remission and thereafter) , acute lymphocytic leukemia (particularly, a first remission, a second remission and thereafter) , chronic myelocytic leukemia (particularly, chronic period, transmigration period) , malignant lymphoma (particularly, a first remission (high-risk group) , ' a second remission and thereafter) ,
  • anemia
  • the above-described differentiated cells, tissues, or organs maybe of the nervous system.
  • diseases or disorders include, but are not limited to, dementia, cerebral stroke and sequela thereof, cerebral tumor, spinal injury, and the like.
  • the above-described differentiated cells, tissues, or organs may be of the immune system.
  • diseases or disorders include, but are not limited to, T-cell deficiency syndrome, leukemia, and the like.
  • the above-described differentiated cells, tissues, or organs may be of the motor organ and the skeletal system. Examples of such diseases or disorders include, but are not limited to, fracture, osteoporosis, luxation of joints, subluxation, sprain, ligament injury, osteoarthritis, osteosarcoma, Ewing's sarcoma, osteogenesis imperfecta, osteochondrodysplasia, and the like.
  • the above-described differentiated cells, tissues, or organs may be of the skin system.
  • diseases or disorders include, but are not limited to, atrichia, melanoma, cutis malignant lymphoma, hemangiosarcoma, histiocytosis, hydroa, pustulosis, dermatitis, eczema, and the like.
  • the above-described differentiated cells, tissues, or organs may be of the endocrine system.
  • diseases or disorders include, but are not limited to, hypothalamus/hypophysis diseases, thyroid gland diseases, accessory thyroid gland (parathyroid) diseases, adrenal cortex/medulla diseases, saccharometabolism abnormality, lipid metabolism abnormality, protein metabolism abnormality, nucleic acid metabolism abnormality, inborn error of metabolism
  • the above-described differentiated cells, tissues, or organs may be of the respiratory system.
  • diseases or disorders include, but are not limited to, pulmonary diseases (e.g., pneumonia, lung cancer, etc.), bronchial diseases, and the like.
  • the above-described differentiated cells, tissues, or organs may be of the digestive system.
  • diseases or disorders include, but are not limited to, esophagial diseases (e.g., esophagial cancer, etc.), stomach/duodenum diseases (e.g., stomach cancer, duodenum cancer, etc . ) , small intestine diseases/large intestine diseases (e. g. , polyps ofthe colon, colon cancer, rectal cancer, etc.
  • liver diseases e.g., liver cirrhosis, hepatitis (A, B, C, D, E, etc.), fulminant hepatitis, chronic hepatitis, primary liver cancer, alcoholic liver disorders, drug induced liver disorders, etc.
  • pancreatic diseases acute pancreatitis, chronic pancreatitis, pancreas cancer, cystic pancreas diseases, etc.
  • peritoneum/abdominal wall/diaphragm diseases hereinia, etc.
  • Hirschsprung' s disease and the like.
  • the above-described differentiated cells, tissues, or organs maybe of the urinary system.
  • diseases or disorders include, but are not limited to, kidney diseases (e.g., renal failure, primary glomerulus diseases, renovascular disorders, tubular function abnormality, interstitial kidney diseases, kidney disorders due to systemic diseases, kidney cancer, etc.), bladder diseases (e.g., cystitis, bladder cancer, etc.), and the like.
  • the above-described differentiated cells, tissues, or organs maybe of the genital system.
  • diseases or disorders include, but are not limited to, male genital organ diseases (e.g., male sterility, prostatomegaly, prostate cancer, testicular cancer, etc.), female genital organ diseases (e.g., female sterility, ovary function disorders, hysteromyoma, adenomyosis uteri, uterine cancer, endor ⁇ etriosis, ovarian cancer, villosity diseases, etc.), and the like.
  • male genital organ diseases e.g., male sterility, prostatomegaly, prostate cancer, testicular cancer, etc.
  • female genital organ diseases e.g., female sterility, ovary function disorders, hysteromyoma, adenomyosis uteri, uterine cancer, endor ⁇ etriosis, ovarian cancer, villosity diseases, etc.
  • the above-described differentiated cells, tissues, or organs may be of the circulatory system.
  • diseases or disorders include, but are not limited to, heart failure, angina pectoris, myocardial infarction, arrhythmia, valvulitis, cardiac muscle/pericardium diseases, congenital heart diseases (e.g., atrial septal defect, arterial canalpatency, tetralogy of Fallot, etc.), artery diseases (e.g., arteriosclerosis, aneurysm) , vein diseases (e.g., phlebeurysm, etc.), lymphoduct diseases (e.g., lymphedema, etc.), and the like.
  • the above-described diseases could be treated while avoiding conventional side effects of transplantation therapy of naturally-occurring stem cells or differentiation cells (particularly, causedbyforeignmatterorheterogenous cells, (e.g. , infection, graft-versus-host diseases, etc. ) ) .
  • This effect is efficientlyachieved onlyafter amethod is provided which can maintain the pluripotency and self-replication of a stem cell .
  • the effect cannot be conventionally achieved or is difficult to achieve.
  • Cells used inthe present invention maybe derived from any organism (e.g., vertebrates and invertebrates).
  • the organism is a vertebrate, andmore preferably a mammal (e.g., the order Primates, Rodentia, etc.). Even more preferably, the organism is the order Primates. Most preferably, the organism is human.
  • the term "in an organism” or in vivo” refers to in an intact organism.
  • the term “in an organism” or “in vivo” refers to a position, at which a tissue or organ of interest is to be placed, in a particular context.
  • in vi tro indicates that a part of an organism is excised or isolated “outside the organism” (e.g., in a test tube) for the purposes of various research. This term is in contrast to the term “in vivo” .
  • ex vivo means that a target cell for gene introduction has been isolated from a subject, and a therapeutic gene or agent is introduced in vi tro into the cell, and the cell is returned to the subject.
  • An expansion agent of the present invention is also useful for ex vivo therapy.
  • the term "subject” refers to an organismwhich is treated according to the present invention, also called “patient”.
  • a patient or a subject may be preferably a human.
  • the term "recipient” refers to an individual which receives a graft, also called a "host”. In contrast, an individual which,provides a graft is called "donor”.
  • autograft or “auto” interchangeably refers to a graft which is implanted into the same individual from which the graft is derived.
  • autograft may encompass a graft from a genetically identical individual (e.g. an identical twin) in a broad sense.
  • the term “allograft” or “allo” interchangeably refers to a graft which is implanted into an individual which is of the same species but is genetically different from that from which the graft is derived. Since an allograft is genetically different from an individual (recipient) to which the graft is implanted, the graft may elicit an immune reaction.
  • a graft includes, but is not limited to, for example, a graft derived from a parent.
  • xenograft As used herein, the term "xenograft" or
  • heterograft interchangeably refers to a graft which is implanted from a different species . Therefore, for example, when a human is a recipient, a porcine-derived graft is called a xenograft or heterograft.
  • cellular physiologically active substance refers to a substance capable of acting on a cell, which may be employed with an expansion agent.
  • Cellularly physiologically active substances include cytokines and growth factors .
  • a cellular physiologically active substance may be naturally-occurring or synthesized.
  • a cellularly physiologically active substance is one that is produced by a cell or one that has a function similar thereto.
  • a cellularly physiologically active substance may be in the form of a protein or a nucleic acid or in other forms. In actual practice, cytokines are typically proteins.
  • Cytokines are generally proteins or polypeptides having a function of controlling an immune response, regulating the endocrine system, regulating the nervous system, acting against a tumor, acting against a virus, regulating cell growth, regulating cell differentiation, or the like. Cytokines are herein in the form of a protein or a nucleic acid or in other forms. In actual practice, cytokines are typically proteins.
  • growth factor or “cell growth factor” are usedherein interchangeablyand each refers to a substance which promotes or controls cell growth.
  • Growth factors are also called “proliferation factors” or “development factors” . Growth factors may be added to cells or tissue culturemedium, substituting for serummacromolecules. It has been revealed that a number of growth factors have a function of controlling differentiation in addition to a function of promoting cell growth.
  • cytokines representatively include, but are not limited to, interleukins, chemokines, hematopoietic factors such as colony stimulating factors, a tumor necrosis factor, interferons, a platelet-derived growth factor (PDGF) , an epidermal growth factor (EGF) , a fibroblast growth factor (FGF) , a hepatocyte growth factor (HGF) , an endothelial cell growth factor (VEGF) , cardiotrophin, and the like, which have proliferative activity.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF endothelial cell growth factor
  • cytokines typically have redundancy in function
  • reference herein to a particular cytokine or growth factor by one name or function also includes any other names or functions by which the factor is known to those of skill in the art, as long as the factor has the activity of a cellularlyphysiologically active substance for use in thepresent invention.
  • Cytokines or growth factors can be used in a preferred embodiment of thepresent invention as long as theyhavepreferable activity as described herein.
  • any cellularly physiologicallyactive substance maybe used.
  • a cellularly physiologicallyactive substance a cytokine or growth factor having hematopoietic activity, colony stimulating activity, or cell proliferative activity.
  • cytokine havinghematopoietic activityor colony stimulating activity examples include a leukemia inhibitory factor (LIF) , a granulocyte macrophage colony stimulating factor (GM-CSF) , a macrophage colony stimulating factor (M-CSF) , a granulocyte colony stimulating factor (G-CSF) , a multi-CSF (IL-3), erythropoietin (EPO), c-kit ligand (SCF), and the like.
  • LIF leukemia inhibitory factor
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL-3 multi-CSF
  • EPO erythropoietin
  • SCF c-kit ligand
  • a growth factor having cell proliferative activity examples include a platelet-derived growth factor (PDGF) , an epidermal growth factor (EGF) , a fibroblast growth factor (FGF) , a hepatocyte growth factor (HGF) , a vascular endothelial cell growth factor (VEGF) , an insulin-like growth factor (IGF), and the like.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial cell growth factor
  • IGF insulin-like growth factor
  • a cellularly physiologically active substance e.g., a cytokine or a growth factor having cell proliferative activity may be used.
  • such a cellularly physiologically active substance includes SCF, TPO, and Flt-3L.
  • Cellularly physiologically active substances such as cytokines and growth factors, can also be divided into categories in accordance with their receptors (e.g., cytokine receptors, etc.) .
  • Cytokine receptors are divided into non-kinase type and kinase type. Examples of the non-kinase type include G-protein binding type receptors, anNGF/TNFreceptor family, an IFNreceptor family, a cytokine receptor superfamily, and the like.
  • kinase type examples include a growth factor type receptor (tyrosine kinase type, such as c-met (for .HGF)), a TGF ⁇ receptor family (serine/threonine kinase type) , andthe like.
  • cellularly physiologically active substances share a receptor subunit. Therefore, a cytokine or growth factor, which shares a receptor subunit with the above-described preferable cytokines or growth factors, may be a preferable cytokine or growth factor.
  • Cellularly physiologically active substances, such as cytokines and growth factors may also be divided into categories in accordance with homology comparison when they are provided in the form of a protein or a nucleic acid.
  • a cellularly physiologically active substance having homology to a preferable cellularly physiologically active substance of the present invention may be used.
  • Such a cellularly physiologically active substance has at least about 30% homology to a control cellularly physiologically active substance, when BLAST is employed to perform comparison with default parameters, preferably about 35%, about 40% , about 45%, about 50%, about 55%, about 60%, about 65%, about 70% , about 75%, about 80%, about 85%, about 90%, about 95%, and about 99% homology.
  • the present invention provides technology where states of stem cells such as hematopoietic stem cells can be efficiently and/or simply determined.
  • markers which allow determination that stem cells such as hematopoietic stem cells can be simply separated and concentrated, and further transplantation compatibility can also be determined, is considered unexpectedly superior by those skilled in the art and thus provides significant effects.
  • the present invention provides a marker composition for stem cells comprising an agent capable of specifically interacting with a nucleic- acid molecule encoding the Endomucin polypeptide.
  • Endomucin has been elucidated in the present invention for the first time to be specifically expressed in hematopoietic stem cells. There have been no such findings in the art, and thus the present invention takes such findings into account for providing a marker for stem cells. Accordingly, in the present invention any agent may be used as long as Endomucin expression (such as mRNA) can be confirmed.
  • an effective amount of the composition for regeneration, diagnosis, prophylaxis, treatment, or prognosis can be determined by those skilled in the art based on the disclosure of the present specification using techniques well known in the art with reference to various parameters. For example, such an amount can be determined by those skilled in the art with reference to the purpose of use, a target disease (type, severity, andthe like) , thepatient' s age, weight, sexandcase history, the form or type of the cells, and the like.
  • a target disease type, severity, andthe like
  • the patient' s age, weight, sexandcase history the form or type of the cells, and the like.
  • regeneration of nerves occurs due to inhibition of neurite outgrowth being disruptedbyblocking of the Endomucin signal transduction pathway (by an agent capable of specifically interacting with Endomucin) .
  • the present invention provides an effect more excellent than the prior art.
  • effective amounts in diagnosis, prevention, treatment and prognosis are readily determined by those skilled in the art in view of a variety of parameters including for example, purpose of use, disease of interest (variety, severity and the like) , age, bodily weight, sex, and disease history of a patient, forms and species of the cells to be used.
  • the expression of Endomucin corresponds to undifferentiated state (in particular, pluripotency, more particularly, totipotency) of a stem cell (in particular, hematopoietic stem cell) . Therefore, the present invention can be used to identify the state and/or property of such a cell in an efficient manner.
  • the agent of the present invention may be an agent selected from the group consisting of a nucleic acid molecule, polypeptide, lipid, sugar chain, organic low molecular weight molecule, and a complex molecule thereof. It should be understood that such an agent may be any agent , as long as the agent can bind to the nucleic acid molecule of the present invention in a specific manner.
  • the agent of the present invention may be advantageously an agent which is labeled or is capable of binding to a label.
  • a variety of states which can be measured by the present agent may be directly and/or simply measured.
  • Any lables may be used as such a label, as long as a target of the present invention can be distinguishably labeld, and include, but are not limited to, for example, a fluorophore, phosphorophore, chemilunescence agent, radioactive agent, color producing agent, ligand, hapten, enzyme substrate, and antibody-antigen reaction and the like .
  • systems widely used in the art of immunological reactions such as biotin-streptavidin may be used.
  • the agent of the present invention is a nucleic acidmolecule.
  • the agent of the present invention is a nucleic acid molecule
  • such a nucleic acid molecule may have a length of at least 8 contiguous nucleotides, and preferably specifically reacts with a nucleic acid sequence of Endomucin, such as a sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or the like.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the purpose of use of thepresent invention.
  • thenucleic acid molecule of the present invention may have a length of at least 10 contiguous nucleotides, even more preferably at least 15 contiguous nucleotides, and still even more preferably at least 20 contiguous nucleotides. These lower limits of the nucleotide length may be present between the above-specified numbers (e.g., 9, 11, 12, 13, 14, 16, and the like) or above the above-specified numbers (e.g., 21, 22, ...30, and the like) .
  • the upper limit of the length of the polynucleotide of the present invention may be greater than or equal to the full length of the sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or the like, as long as the polynucleotide can be used for the intended purpose (e.g. antisense, RNAi, marker, primer, probe, capable of interacting with a given agent) .
  • the nucleic acid molecule of the present invention when used as a primer, the nucleic acid molecule typically may have a nucleotide length of at least about 8, preferablya nucleotide length of about 10.
  • the nucleic acid molecule typically may have a nucleotide length of at least about 15, and preferably a nucleotide length of about 17.
  • the agent of the present invention may be a nucleic acid molecule sequence having a sequence complementary to a nucleic acid sequnce of a polynucleotide encoding Endomucin or a sequence having at least 70% identity thereto.
  • the agent of the present invention may be a nucleic acid molecule hybridizable to any of the nucleic acid sequences of a polynucleotide encoding Endomucin.
  • the agents of the present invention may be a nucleic acid molecule comrpising: (a) a polynucleotide having the base sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9 or a complementary sequence or fragment thereof; (b) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof; (c) a polynucleotide encoding a variant polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10 having at least one mutation selected from the group consisting of one ormore amino acid substitutions, additions and deletions, and having a biological activity; (d) a polynucleotide which is a splice variant or allelic variant of the base sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9; (e) a polynucleotide encoding a species homo
  • the agent ofthepresent invention is specificto anucleic acidmolecule comprising a sequence set forth -in SEQ ID NO: 1, 3, 5, 7, or 9 or a complementary sequence thereof.
  • a sequence can be used for identifying the undifferentiated state of hematopoietic stem cells, and therefore is useful in determining the level of differentiation of a hematopoietic tissue or individual, but thepresent invention is not limited to this.
  • a stem cell found by the marker of the present invention is a tissue stem cell, preferably hematopoietic stem cell, however, the present invention is not limited to this, and . may typically include vessel endothelial cells, hemangioblast, a common precursor cell for vessel endothelial cells and blood cells as a target of the present invention.
  • Endomucin of the present invention includes: (a) a polypeptide consisting of an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment thereof; (b) a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10 having at least one mutation selected from the group consisting of.one ormore amino acid substitutions, additions and deletions, and having biological activity; (c) a polypeptide encoded by a splice variant or allelic variant of a base sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9; (d) a polypeptide which is a species homolog of the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10; or (e) a polypeptide having an amino acid sequence having at least 70% identity to any one of the polypeptides described in (a) to (d) , and having biological activity.
  • Endomucin of the present invention comprises a sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10.
  • the nucleic acidmolecule of the present invention encoding Endomucin may comprise a sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9.
  • the number of substitutions, additions and deletions in the above (c) may be limited to 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, 2 or less.
  • biological activities to be possessed by the above variants of the polypeptide of the present invention include: specific interaction to a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 2, 4 , 6, 8 or 10, or a fragment thereof; maintenance of undifferentiated state; interaction with extracellular matrices and the like.
  • such activity includes maintenance of undifferentiated state of a cell.
  • Endomucin is believed to play an important role in maintaining the undifferentiated state of a cell.
  • gene introduction experiments, gene deletion experiments, RNAi experiments, protein function inhibition experiments using antibodies and the like may be conducted.
  • an allelic variant of the present invention has preferably at least 90 % homology with a nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9.
  • the species homologs may be identified by conducting a search using as query, the entire amino acid sequence of the present invention set forth in SEQ ID NO: 2, 4, 6, 8 or 10, or aportionthereof, orthe entirenucleicacidsequence of the present invention set forth in SEQ ID NO: 1, 3, 5, 7 or 9 encoding Endomucin polypeptide or " a portion thereof, when a gene sequence database for the species of interest exists.
  • nucleic acid sequence of Endomucin of the present invention or a portion thereof may be used as a probe or primer to screen a gene library for the species of interest to identify such homologs.
  • identification methods are well known in the art and are also described herein.
  • Species homologs have preferably for example, about 30 % homology with a nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9.
  • Species homology maybe preferablyat least about 50 % homology with the nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9.
  • the identity against one polynucleotide of the above mentioned (a) to (e) , or the complementary sequence thereof may be at least about 80 %, more preferably at least about 90 %, yet more ' preferably at least about 98 %, and most preferably at least about 99 %.
  • the nucleic acid molecule of the present invention encoding p21 or fragments and variants thereof may have a length of at least 8 contiguous nucleotides.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the purpose of use of the present invention. More preferably, the nucleic acid molecule of the present invention may have a length of at least 10 contiguous nucleotides, even more preferably at least 15 contiguous nucleotides, and still even more preferably at least 20 contiguous nucleotides.
  • the upper limit of the length of the polynucleotide of the present invention may be greater than or equal to the full length of the sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9, as long as the polynucleotide can be used for the intended purpose (e.g. antisense, RNAi, marker, primer, probe, capable of interacting with a given agent) .
  • the nucleic acid molecule of the present invention when used as a primer, the nucleic acid molecule typically may have a nucleotide length of at least about 8, preferably a nucleotide length of about 10.
  • the nucleic acid molecule When used as a probe, the nucleic acid molecule typically may have a nucleotide .length of at least about 15, and preferably a nucleotide length of about 17.
  • the present invention may be (a) a polynucleotide having a base sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or a fragment sequence thereof; or (b) a polunucleotide encoding a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a fragment.
  • the nucleic acid of the present invention may have in its structure of the polypeptide encoded thereby a number of serine and threonin residues and about 25 sites of potential O-glycosyl sites in addition to two N-glycosylation sites (Asn-X-Ser/Thr) in the extracellular domains.
  • a number of serine and threonin residues and about 25 sites of potential O-glycosyl sites in addition to two N-glycosylation sites (Asn-X-Ser/Thr) in the extracellular domains.
  • Ser/Thr-X-Lys protein kinase C
  • the present invention preferably conserves sites corresponding to these specific domains in an embodiment, but, the present invention is not limited to this.
  • the identity to one polynucleotide of any of the above (a) -(d) or a complementary sequence thereof may be at least about 80 %, more preferably at least 90 %, more preferably at least about
  • the nucleic acid molecule encoding Endomucin of the present invention or a fragment or variant thereof may have a length of at least 8 contiguous nucleotides.
  • the appropriate nucleotide length of the nucleic acid molecule of the present invention may vary depending on the purpose of use of the present invention. More preferably, the nucleic acid molecule of the present invention may have a length of at least 10 contiguous nucleotides, even more preferably at least 15 contiguous nucleotides, and still even more preferably at least 20 contiguous nucleotides.
  • nucleotide length may be present between the above-specified numbers (e.g., 9, 11, 12, 13, 14, 16, and the like) or above the above-specified numbers (e.g., 21, 22, ... 30, and the like) .
  • the upper limit of the length of the polynucleotide of the present invention may be greater than or equal to the full length of the sequence as set forth in SEQ ID NO: 1, 3, 5, 7 or 9 as long as the polynucleotide can be used for the intended purpose (e.g. antisense, RNAi, marker, primer, probe, capable of interacting with a given agent) .
  • the nucleic acid molecule when used as a primer, typically may have a nucleotide length of at least about 8, preferably a nucleotide length of about 10.
  • the nucleic acid molecule When used as a probe, typically may have a nucleotide length of at least about 15, and preferably a nucleotide length of about 17.
  • the composition of the invention may further comprise at least one additional specific agent to a nucleic acid molecule encoding a polypeptide selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2.
  • the addition of such an additional agent may preferably include the addition of at least two kinds, more preferably the addition of at least three kinds, more preferably the addition of at least four kinds, yet more preferably the addition of at least five kinds.
  • Lin refers to a combination of a plurality of markers, and thus it should be understood that the markers actually used may vary. Lin markers include for example, but are not limited to Gr-1, Mac-1, TER119, CD4, CD8 and B220 and the like.
  • the CD34 molecule used in the present invention comprises a nucleic acid sequence set forth in SEQ ID NO: 13 or 15, or a portion or variant thereof.
  • the Lin used in the present invention preferably includes the nucleic acid sequence set forth in SEQ ID NO: 17, 19, 21 and/or 23, or a portion or variant thereof.
  • the c-kit used in the present invention may include the nucleic acid sequence set forth in SEQ ID NO: 25, or a portion or variant thereof.
  • the Sca-1 used in the present invention may include a nucleic acid sequence set forth in SEQ ID NO: 27, or a portion orvariant thereof.
  • the Flt3/Flk2 usedinthepresent invention may include a nucleic acid sequence set forth in SEQ ID NO: 11, or a portion or variant thereof.
  • Gr-1 granulocyte-differentiation antigen used herein recognizes Ly-6G (which is a glyco-phospho-inositol (GPI) anchored membrane proteinsof about 21-25 kDa) (see TJ Fleming, ML Fleming and TR Malek, The Journal of Immunology, Vol 151, Issue 52399-2408, 1993) . Accordingly, Gr-1 is used as a marker for differentiation.
  • Ly-6G which is a glyco-phospho-inositol (GPI) anchored membrane proteinsof about 21-25 kDa
  • GPI glyco-phospho-inositol
  • B220 is recognized by an antibody TER119 (Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL,
  • B220 is also used as a marker for differentiation.
  • these additional agents may also be labeled.
  • labels to be used are preferably different to each other depending on each of the agents to be labeled.
  • labels such as Texas-Red, PE, FITC, APC and biotin and the like.
  • Hoechst Blue, Hoechst 33342, Hoechst Red and the like may be used for staining DNA.
  • the present invention provides a marker composition for identifying a stem cell comprising an agent specifically .interacting or binding to Endomucin polypeptide or a portion thereof.
  • Such marker composition may be, for example, but are not limited to: polypeptides such as an antibody or single chain antibody, polynucleotides, a lipid, a sugar chain, an organic low molecular weight molecule and a composite molecule thereof.
  • an agent may be any molecule as long as the agent specifically binds to the polypeptide of the present invention. More preferably, the agent of the present invention is an antibody or a derivative thereof such as a single-chain antibody, Accoridingly, the agent of the present invention can be used as a probe and/or an inhibitor.
  • the agent of the present invention is advantageously an agent labeled or capable of binding to a label. If such an agent is labeld or capable of binding to a label, those skilled in the art can readily determine a variety of states in a direct and/or simple manner.
  • a label may be any label as long as the label is distinguishably labeled, and include for example, but are not limited to: technologies relating to fluorophore, phosphorophore, che iluminescence, radioactivity, enzyme-substrate interaction, and antibody-antigen interaction and the like.
  • a sysmet often used in immunological reactions such as biotin-streptavidin may be used.
  • Labels used in the present invention include fluorescein isothiocyanate (FITC) ,• phycoerythrin (PE) , allophycocyanin (APC) , Texas Red and biotin and the like. Hoechst Blue, Hoechst 33342, Hoechst Red can be used for staining DNA in this regard.
  • the agents of the present invention may be an antibody.
  • Antibodies may be for example, monoclonal or polyclonal antibodies, and further may be humanized, chimeric, and anti-idiotypic antibodies, and fragments thereof such as F(ab' ) 2 a d Fab fragments, as well as any other conjugate produced by recombinant technologies. Such antibodies may be used as a tool for determining an expression of a gene of the present invention, and thus can be used for screening of the present invention.
  • Endomucin polypeptide of the present invention may comprise: (a) a polypeptide having an amino acid sequence as set forth in
  • a species homolog of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a polypeptide having an amino acid sequence having at least 70% homology to any one of the polypeptides described in (a) to (d) , and having biological activity.
  • Endomucin of the present invention may comprise a sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10.
  • the number of substitutions, additions anddeletions describedin (b) above may be limited to, for example, preferably 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.
  • the number of substitutions, additions and deletions is preferably small, but may be large as long as the biological activity is maintained (preferably, the activity is similar to or substantially the same as that of Endomucin having amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10) .
  • the above-described homology to any one of the polypeptides described in (c) above may be at least about 80%, more preferably at least about 90%, even more preferably at least about 98%, and most preferably at least about 99% to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10.
  • the species homolog preferably can be readily identified as described herein using known technology, has at least about 30% homology to the amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10, more preferably at least about 40% homology, at least about 50% homology, at least about 60% homology, at least about 70% homology, at least about 80% homology, at least about 90% homology, at least about 95% homology, or at least about 98% homology thereto.
  • Species- homologs may have at least about 50% homology, at least about 60% homology, at least about 70% homology, at least about 80% homology, at least about 90% homology, at least about 95% homology, or at least about 98% homology with the nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9.
  • the biological activity as possessed by the varient polypeptide of (e) above include but are not limited to: specific interaction to a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, or a fragment thereof; maintenance of undifferentiated state; interaction with extracellular matrices and the like.
  • a biological activity includes maintenance of undifferentiated state.
  • Endomucin is believed to play an important role in maintaining the undifferentiated state of a cell.
  • identity to any one polypeptide of the above (a) -(d) maybe at least about 80%, more preferably, at least about 90%, yet more preferably at least about 98%, most preferably at least about 99%.
  • the polypeptide of the present invention typically has a sequence of at least 3 contiguous amino acids .
  • the amino acid length of the polypeptide of the present invention may be short as long as the peptide is suitable for an intended application, but preferably a longer sequence may be used.
  • the amino acid length may be preferably at least 4, more preferably at least 5, at least 6, at least 7, at least 8, at least 9 and at least 10, even more preferably at least 15, and still even more preferably at least 20.
  • These lower limits of the amino acid length may be present between the above-specified numbers (e.g., 11, 12, 13, 14, 16, and the like) or above the above-specified numbers (e.g., 21, 22, ..., 30, and the like).
  • the upper limit of the length of the polypeptide of the present invention may be greater than or equal to the full length of the sequence as set forth in SEQ ID NO: 2, 4, 6, 8 or 10 as long as the polypeptide can be used for the application of interest such as an im unogen, a marker and the like.
  • the polypeptide of the present invention may have in its structure a number of serine andthreoninresidues and about 25 sites ofpotential O-glycosyl sites in addition to two N-glycosylation sites (Asn-X-Ser/Thr) in the extracellylar domains.
  • Asn-X-Ser/Thr N-glycosylation sites
  • the present invention preferably conserves sites corresponding to these specific domains in an embodiment, but, the present invention is not limitedto this. Therefore, in thepolypeptide of thepresent invention, it is usual that at least one out of the corresponding sites of the characteristic domains and preferable all the domains are conserved, but the present invention is not limited to this.
  • the above-mentioned domains may be one correponding to sequence in the amino acid sequence set orth in SEQ ID NO: 2, 4, 6, 8 or 10; or a polypeptide corresponding thereto (for example, Endomucin polypeptide of different species than those disclosed in SEQ ID NO: 2, 4, 6, 8 or 10) , however, the present invention is not limited to this.
  • Such a sequence may be identified using well known technology in the art.
  • identity to any one of the polypeptides described in (a) to (c) above may be at least about 80%, more preferably at least about 90%, even more preferably at least about 98%, and most preferably at least about 99% .
  • a composition of the present invention comprising the polypeptide of the present invention may further comprise at least one agent specific to a polypeptide selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2. More preferably, it should be understood that the present inventionmay comprise at least two, more preferably at least three, yet more preferably at least four of these agents.
  • Lin marker is a combination of a plurality of markers, and thus such markers contained therein may vary dependeing on the situation, and those skilled in the art can readily select an appropriate combination based on the present description of the present application.
  • CD34 as used in the present invention may comprise an amino acid sequence set forth in SEQ ID NO: 14 or 16 or a fragment or variant thereof.
  • Lin may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO: 18, 20, 22 and 24, or a fragment or variant thereof.
  • c-kit as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 26, or a fragment or variant thereof.
  • Sca-1 as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 28, or a fragment or variant thereof.
  • Flt3/Flk2 as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 12, or a fragment or variant thereof.
  • the present invention provides a composition for determining transplantation compatibility of a transplant comprising comprising an agent specific to a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof. It has been demonstrated in the present invention that a cell determined to be positive with respect to the expression of Endomucin of the present invention has transplantation compatibility. On the other hand, it has also been demonstrated that a cell determined not to be positive with respect to the expression of Endomucin of the present invention has less or no transplantation compatibility.
  • a specific agent of the present invention specific to a nucleic acid molecule comprising a nucleic acid sequence of Endomucin polypeptide or a fragment or variant thereof, has utility in that the agent can be used for determining transplantation compatibility of a transplant such as a composition comprising a stem cell such as a hematopoietic stem cell.
  • the present invention allows one to determine transplantation compatiblity of a transplant comprising a stem cell.
  • the stem cell comprises a hematopoietic stem cell.
  • An agent specific to a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, as used in the composition for determining transplantation compatibility of a transplant may be used in any form as described herein such as in the section above (Agent capable of interacting with stem cell marker in the nucleic acid form) .
  • the composition of the present invention may further comprise at least one agent specific to a nucleic acid molecule encoding a polypeptide selected from the group consisting of CD34, Lin (Lineage marker), c-kit, Sca-1 and Flt3/Flk2, or the like.
  • the addition of such an additional agent may preferably include the addition of at least two kinds, morepreferablythe addtion of at least three kinds, more preferably the .addtion of at least four kinds, yet more preferably the addtion of at least five kinds.
  • Lin refers to a combination of a plurality of markers, and thus it should be understood that the markers actually usedmay vary.
  • Lin markers include for example, but are not limited to Gr-1, Mac-1, TER119, CD4, CD8 and B220 and the like.
  • the present composition comprises an agent capable of determining negative or weakly positive expression of CD34.
  • the composition may comprise an . agent capable of determining positive, particularly strongly positive expression of c-kit and/or Sca-1.
  • the composition of the present invention may further comprise an agent capable of determining negative expression of Lin.
  • Such negative, weakly positive, positive, and strongly positive may preferably be based on the expression level employed in quantitative PCR. In such a case, such an expression level is determined by a level relatively determined using PCR technology.
  • such an expression level may be determined by using a microarray based on the expression level of a housekeeping gene such as hypoxanthine-guanine phosphoribosyltransferase (HPRT) .
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • an agent is preferably an agent capable of determing the level of positive expression in a sensitive manner, and include but are not limited to for example, a nucleic acid molecule comprising a sequence having perfect matching complementarity, a nucleic acid comprising complementary sequence with one base mismatch, a nucleic acid molecule having completelyrandomsequence, anda combination thereof.
  • the CD34 used in the present invention comprises a nucleic acid sequence set forth in SEQ ID NO: 13 or 15, or a portion or variant thereof.
  • the Lin used in the present invention preferably include the nucleic acid sequence set forth in SEQ ID NO: 17, 19, 21 and/or 23, or a portion or variant thereof.
  • the c-kit used in the present invention mayinclude the nucleic acid sequence set forth in SEQ ID NO: 25, or a portion or variant thereof.
  • the Sca-1 used in the present invention may include a nucleic acid sequence set forth in SEQ ID NO: 27, or a portion or variant thereof.
  • the Flt3/Flk2 used in thepresent invention may include a nucleic acid sequence set forth in SEQ ID NO: 11, or a portion or variant thereof.
  • labels to be used are preferably different to each other dependeing on each of the agents to be labeled.
  • labels such as Texas-Red, PE, FITC, APC and biotin and the like.
  • Hoechst Blue, Hoechst 33342, Hoechst Red and the like may be used for staining DNA.
  • the present invention provides a composition for determining transplantation compatibility of a transplant, comprising an agent specific to Endomucin polypeptide or a fragment or variant thereof. It has been demonstrated in the present invention that a cell determined to be positive with respect to the expression of Endomucin of the present invention has transplantation compatibility. On the other hand, it has also been demonstrated that a cell determined not to be positive with respect to the expression of Endomucin of the present invention has less or no transplantation compatibility. Accordingly, it has been demonstrated in the present invention for the first time that the determination of positive/negative expression of Endomucin can be used for directly determining transplantation compatibility.
  • a specific agent of the present invention specific to Endomucin polypeptide or a fragment or variant thereof has utility in that the agent can be used for determining transplantation compatibility of a transplant such as a composition comprising a stem cell such as a hematopoietic stem cell.
  • the agent specific to a Endomucin polypeptide or a fragment or variant thereof allows one to determine transplantation compatibility of a transplant comprising a stem cell. More preferably, the stem cell comprise a hematopoietic cell.
  • the agent specific to Endomucin polypeptide or a fragment or variant thereof as used in the composition for determining transplantation compatibility of a transplant of the present invention may be any agent as described herein above in the section (Agents against a stem cell marker in a polypeptide form) .
  • the composition of the present invention may further comprise at least one agent specific to a polypeptide selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2, or the like.
  • the addition of such an additional agent may preferably include the addition of at least two kinds, more preferably the addition of at least three kinds, more preferably the addition of at least four kinds, yet more preferably the addition of at least five kinds.
  • Lin refers to a combination of a plurality of markers, and thus it should be understood that the markers actually used may vary. Lin markers include for example, but are not limited to Gr-1, Mac-1, TER119, CD4, CD8 and B220 and the like.
  • the present composition comprises an agent capable of determining negative or weakly positive expression of CD34.
  • the composition may comprise an agent capable of determining positive, particularly strongly positive expression of c-kit and/or Sca-1.
  • the composition of the present invention may further comprise an agent capable of determining negative expression of Lin.
  • Such negative, weakly positive, positive, and strongly positive may preferably be based on the expression level employed in technology using FACS or magnetic beads. In such a case, such an expression level is determined by a level relatively determined using technology using FACS or magnetic beads.
  • such an expression level may be determined by using a microarray (protein/antibody array) based on the expression level of a housekeeping gene such as hypoxanthine-guanine phosphoribosyl transferase (HPRT) .
  • a microarray protein/antibody array
  • HPRT hypoxanthine-guanine phosphoribosyl transferase
  • an agent is preferably an agent capable of determining the level ofpositive expression in a sensitive manner, and includes but is not limited to for example, a monoclonal antibody having high specificity, a specific ligand or a variant or fragment thereof, and the like.
  • CD34 as used in the present invention may comprise an amino acid sequence set forth in SEQ ID NO: 14 or 16 or a fragment or variant thereof.
  • Lin may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NO: 18, 20, 22 and 24, or a fragment or variant thereof.
  • c-kit as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 26, or a fragment or variant thereof.
  • Sca-1 as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 28, or a fragment or variant thereof.
  • Flt3/Flk2 as used herein may comprise an amino acid sequence set forth in SEQ ID NO: 12, or a fragment or variant thereof.
  • labels to be used are preferably different to each other .depending on each of the agents to be labeled.
  • labels such as Texas-Red, PE, FITC, APC andbiotin andthe like maybe employed.
  • Hoechst Blue, Hoechst 33342, Hoechst Red and the like may be used for staining DNA.
  • An exemplary specific procedure for analysis using FACS with these specific agents includes but is not limited to the following:
  • a sample such as a cell from bone marrow in femur, tibia or the like and suspend in a buffer such as phosphate buffered saline (PBS) or the like, pass the sample though a filter such as a nylon filter (for example, having 70 ⁇ m pore size) to remove tissue contaminated with cell masses or muscles or the like.
  • a filter such as a nylon filter (for example, having 70 ⁇ m pore size) to remove tissue contaminated with cell masses or muscles or the like.
  • Count cell number Sample cells with a number of about IxlO 7 to IxlO 8 are usually obtained, but it may vary depending to the conditions actually used.
  • biotinylated anti-lineage antibody for example, a mixture of at least one of Gr-1 antibody such as anti RB6-8C5, anti Mac-1 antibody such as Ml/70,, anti-Terll9 antibody, anti B220 antibody such asRA3-6B2, anti CD4 antibody such as RM4-5, anti-CD8 antibody such as 53.6.7, and the like, these antibodies are available from for example eBioscience, San Diego, CA, USA) , to the resulting sample and incubate for about 30 minutes on ice.
  • biotinylated anti-lineage antibody for example, a mixture of at least one of Gr-1 antibody such as anti RB6-8C5, anti Mac-1 antibody such as Ml/70,, anti-Terll9 antibody, anti B220 antibody such asRA3-6B2, anti CD4 antibody such as RM4-5, anti-CD8 antibody such as 53.6.7, and the like, these antibodies are available from for example eBioscience, San Diego, CA, USA
  • SM twice and resuspend the sample in 100
  • Whether or not a sample is compatible with transplantation may also be determined by determining the compatibility after transplantation of the sample.
  • An exemplary specific procedure is described as follows:
  • IM EDTA-Na 2 /H 2 0 is obtained in a capillary tubing such as 100 ⁇ l by the use of capillary phenomenon.
  • biotinylated anti-Ly5.1 antibody A-20
  • FITC-anti-Ly5.2 antibody 104
  • PE-anti-Mac-1 antibody PE-anti-Gr-1 antibody
  • APC-anti-B220 antibody APC-anti-B220 antibody.
  • the staining is used for determining reconstruction of myeloid lineage and B lymph ⁇ id lineage.
  • biotinylated anti-Ly5.1 antibody A20
  • FITC-anti-Ly5.2 antibody 104
  • PE-anti-CD4 antibody PE-anti-CD4 antibody
  • P ⁇ -anti-CD8 antibody P ⁇ -anti-CD8 antibody
  • Total chimerism (%) (%Ly5.1 cellsxlOO) / (%Ly5.1 cells + % FI cells)
  • B lymphoid cell lineage chimerism (%) (%B220 + Ly5.1 cells 100)/(%B220 + Ly5.1 cells + %B220 + Fl cells)
  • RU (%Ly5.1 cells (the number of competitive cells /10 5 ) )/%Fl cells.
  • CRU (%Ly5.1 cells x 2)/%Fl cells
  • Stem cells can be represented by CRU.
  • Frequency of CRU may be calculated by a formula according to the maximum likelihood method using a ratio of the number of test cells to the number of negative mice.
  • positive mice refer to those having 1 % more chimerism in all three lineages, whereas “negative mice” refer to those having less than the same. Calculation formula is described in St.Groth S . F. , J. Immunol .Methods 49, R11-R23, 1982.
  • compatibility to transplantation refers to the state where chimerism value is 1 % or more, preferably, 10 % or more.
  • Transplantation methods used in the present invention typically include stem cell transplantation.
  • diseases of interest for stem cell transplantation include, for example: 1) diseases in which stem cells with qualitative and/or quantitative abnormality should be replaced with normal stem cells such as severe aplastic anemia, myelo dysplasia syndrome, thalassemia, congenital immunodeficiency syndrome and the like; 2) diseases in which irreversible lymphoid tissues or hematopoietic tissues disorders are inevitable due to for example a large amount of chemotherapy or radiation therapy such as leukemia (chronic lymphoid leukemia (CLL) is excluded) , malignant lymphoma, multiple myeloma, solid cancers (for example, breast cancer, neuroblastoma, lung small cell cancer, ovarian cancer and the like) and the like; 3) diseases in which permanent enzyme supplement is necessary such as nborn error of metabolism and the like.
  • CLL chronic lymphoid leukemia
  • the present method allows one to separate, concentrate and enrich for cells suitable fortransplantation in an efficient and effective manner, and thus allows efficient therapy and alleviation of adverse effects in stem cell transplantation as mentioned above. Further, as the method of the present invention provides highly efficient selection of cells with transplantation compatibility, it is now possible by the present invention to conduct transplantationtherapyusingonlysources derivedfromself. Further, by using expansion agents, it is also possible to prepare a necessary amount for therapy after obtaining a small amount of cells with transplantation compatibility in an efficient manner. After preparation of a necessary amount of such cells ' , it is also possible to concentrate or enrich for appropriate cells by the method for determining transplantation compatibility of the present invention.
  • graft-versus-host disease GVHD
  • the present invention attains an effect of achieving therapeutic effects much faster than those not using the method for determining transplantation compatibility.
  • the present invention provides a composition for identifying a stem cell, or a composition for identiftying transplant compatibility of a transplant, comprising a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof.
  • Endomucin per se may be a marker for undifferentiated state of a stem cell and/or a marker for transplantation compatibility of a transplant in a cell, the molecule per se can be used as a source for preparing a marker composition or as a marker composition per se.
  • the present invention is useful as ' a composition for identification of a stem cell such as hematopoietic stem cell, and/or determination of compatibility of a transplant .
  • a screening method includes, but is not limited to, for example, providing a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a portion thereof, subjecting a candidate chemical substance to the nucleic acid molecule, and selecting a candidate chemical substance bound to the nucleic acid molecule.
  • the above mentioned stemeelIs arepreferablyhematopoietic stemcells .
  • the transplant as mentioned above preferably comprises a stem cell.
  • a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin as used in the present invention may be any form and/or embodiment as described herein above such as those described in the section (Agent capable of interacting with stem cell marker in the nucleic acid form) . More preferably, the nucleic acid molecule used in the present invention comprises a nucleic acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, or a portion thereof, or a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10.
  • the present invention provides a composition for identifying a stem cell or for identifying transplantation compatibility of a transplant, comprising Endomucin polypeptide or a portion thereof.
  • Endomucin per se is a marker for the undifferentiated state of a stem cell and/or a marker for transplantation compatibility of a transplant in a cell
  • the polypeptide or a portion thereof per se can also be used for preparing a marker composition or a composition for preparing the same.
  • Endomucin is used for screening an agent specific thereto, it is understood that the present invention is useful as a composition for identifying a stem cell and/or determining transplantation compatibility of a transplant.
  • Screening methods may comprise the step of providing Endomucin polypeptide or a portion thereof, the step of contacting a chemical substance or an antibody of interest to the polypeptide or the portion thereof, the step of selecting a candidate specifically binding to the polypeptide or the portion thereof.
  • the stem cell may be hematopoietic stem cells.
  • the transplant preferably comprises a stem cell.
  • the Endomucin may be any form or embodiment as described herein above in the sections (Agents against a stem cell marker in a polypeptide form) and (Transplantation compatibilitymarker as apolypeptide form) . More preferably, the Endomucin of the present invention comprises an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 or a portion thereof.
  • the present invention provides a method for determining whether or not a candidate cell is a stem cell, comprising the steps of: (I) providing a candidate cell to be determined; (II) contacting the cell with an agent specific to nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; and (III) detecting a specific reaction between the agent and the nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof to confirm whether or not the Endomucin gene is expressed in the candidate cell; wherein the gene expression of the Endomucin in the candidate cell is a stem cell.
  • the step of providing a candidate cell tobe determined maybe carried out according to any method well known in the art, based on the description of the present application.
  • Such cells may be for transplantation purposes, differentiation purposes, and thus it is to be understood that the cells used are not limited to those for a specific purpose.
  • Such cells may be obtained from for example, bone marrow, peripheral blood, umbilical cord blood, embryo and the like of an animal of interest, and the like.
  • Anucleic acidmolecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or
  • Endomucin polypeptide or a portion thereof, as used in the present invention may be any form or embodiment such as those described above in the section (Agents against a stem cell marker in a nucleic acid form) and (Agents against a stem cell marker in a polypeptide form) .
  • agents may be prepared in accordance with any method well known in the art such as chemical measures such as chemical synthesis and the like, molecular biological measures such as polymerase chain reaction (PCR) and the like, and genetic engineering methods, and the like.
  • an agent when an agent is an antibody, as well known in the art, it may be obtained by immunizing an animal of interest such as a rabbit with a target of interest such as Endomucinpolypeptide or a fragment thereof, and boosting if necessary, obtaining serum from the immunized animal, and purifying if necessary the serum.
  • an agent may be prepared by preparing a hybdridoma, and preparing the cells producing the monoclonal antibody.
  • an agent of interest is a low molecular weight molecule, based on the identified chemical structure, it is possible to synthesize the molecule using well known technologies in the art and/or if the structure is the same as that known as a natural substance, such an agent may be purified or separated from a raw material containing such a natural substance using means for purification such as chromatography.
  • Contacting such an agent and a cell or a sample containing a cell may be carried out by simply mixing or retaining such an agent on a carrier such as a chromatography resin and passing the cells through a column carrying the chromatography resin. Detection of a specific reaction may be carried out by determining whether or not the cell of interest is bound to the agent of interest.
  • a carrier such as chromatography resins
  • stem cells capable of being determined by the present invention include hematopoietic stem cells but the present invention is not limited to this.
  • a stem cell preferably comprises a stem cell derived from bone marrow, embryo, and the like, but the present invention is not limited to this.
  • a sample from bone marrow, peripheral blood, umbilical cord blood, embryo and the like, which are believed to be rich in stem cells particularly hematopoietic stem cells, may be used as such a cell source.
  • the stem cell determination method of the present invention further comprises the step of contacting the candidate cell with at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineagemarker), c-kit, Sca-1 and Flt3/Flk2, wherein the gene expression of the CD34, the Lin (Lineagemarker) , thec-kit, andthe Sca-1 inthe candidate cell, indicates that the candidate cell is a stem cell, the negative gene expression of the Flt3/Flk2 gene indicates that the candidate is a stem cell, and more specifically, negative or weakly positive expression of CD34, negative expression of Lin, positive (preferably strongly positive) expression of c-kit, and/or an positive (preferably strongly positive) expression of Sca-1, indicate that the candidate cell is a stem cell.
  • a marker selected from the group consisting of CD34, Lin (Lineagemarker), c-kit, Sca-1 and Flt3/Flk2
  • such cells when the expression of CD34 is negative or weakly positive, such cells are enriched for cells determined to be in a high undifferentiated state.
  • Such high undifferentiated state may be characterized by a capability of forming nmEM colonies consisting of a variety of lineage of cells, and capability of reconstructing and maintaining hematopoiesis of a living organism irradiated with a fatal dose of radiation for a long period of time.
  • the cells of interest are enriched for cells with low undifferentiated state.
  • Reduction of undifferentiated state may be characterized by reduction of capability of forming nmEM colonies, and capability of reconstructing and maintaining hematopoiesis of a living organism irradiated with a fatal dose of radiation for only a short period of time, or lack of such capability.
  • the determination of the above-mentioned expression may be carried out using magnetic bead or FACS. Magnetic beads and FACS are well known in the art, and can also be combined.
  • the method for determining a stem cell of the present invention further comprises the step of depleting differentiation antigen positive (lineage positive) cells.
  • the step of depletion may be conducted by MACS.
  • MACS technology is a system for separating a cell of interestbysubjectingcells labeledwithmagneticmicrobeads through a column set to a strong magnetic field. MACS technology is exemplified as follows: First, cells are labeled with magnetic icrobeads. Labels may be direct labels or indirect labels, which can be used in an arbitrary manner depending on the situation used. Direct labeling refers to a method for immunologically reacting a cell with these microbeads in a direct manner.
  • Indirect labeling refers to labeling in which when there is no antibody magnetic microbeads against cells of interest, after labeling a specific antibody with a primary label, an antibody against this specific antibody is "secondary" labele with the antibody labeled microbeads.
  • Direct labeling has a number of advantages including but not limited to for example, that separation rate is fast, that a cell fraction of a high degree ofpurity canbe obtained.
  • Indirect labeling also has a number advantages including but not limited to for example, that a variety of combinations are available, effects of enhancing antibody labeling and the like. Labeled cells may be separated using either positive selection where a labeled cell is labeled to isolate the same, and depletion method.
  • Positive selection refers to a method for isolating a cell using a label as an indicator.
  • Depletion method re ers to a method for separating a negative cell passed through a magnetic field which is labeled with microbeads, in which cells to be separated are not activated with an antibody, and thus is advantageous in that cells can be separated even in the case where no specific antibody against that cell is not available. Both positive selection and depletion can be combined.
  • the depletion step may be carried out using at least one Lin marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220.
  • the Lin marker may be conjugated with an IgG MACS bead.
  • a candidate cell or a cell of interest may comprise a mononuclear cell separated by density gradient centrifugation.
  • density gradient centrifugation may use a separation medium such as Ficoll, Percoll, sucrose and other sugar or sugar derivatives.
  • the method for determining a stem cell of the present invention further comprises the step of preparing a candidate cell by subjecting the specimen comprising or being suspected to comprise a stem cell, to density gradient centrifugation.
  • the present invention provides a method for preparing a stem cell, comprising the steps of: (I) providing a sample containing or suspected to contain a stem cell; (II) contacting the sample with an agent specific to nucleic acidmolecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucinpolypeptide oraportionthereof; (III) determining whether or not a cell ' in the sample expresses a gene of the Endomucin; and (IV) separating or concentrating the cell expressing the gene of the Endomucin.
  • Sample containing or suspected to contain a stem cell may be provided in accordance with well known technology in the art, and includes for example, bone marrow collection, peripheral blood collection, umbilical cordblood collection, and the like.
  • Endomucin may be any form or embodiment as described herein in the above in the section (Stem cell determination method) . Separation, concentration/enrichment of cells expression Endomucin may be carried out using FACS and/or magnetic beads. In particular, in such a case, an antibody is preferably used. Such an antibody is preferably labeled, and such a label may be any label described herein elsewhere in the specification.
  • preferable separation or concentration as used in the present invention includes, but is not limited to panning using antibody inactivated dishes, FACS, or magnetic beads, and a combination thereof.
  • the present invention provides a stem cell prepared by the method for preparing a stem cell according to the present invention.
  • a stem cell or a cell population or composition/preparation containing such a stem cell, are enriched for Endomucin expressing cells, and thus is different from cells of the prior art.
  • Endomucin expressing cells are preferable to be isolated. Further, as enrichment or concentration of the Endomucin expressing cells rendered unexpectedly significant improvement or enhancement in transplantation compatibility, the stem cell or cell population, composition/preparation containing the stem cell, attained unexpectedly superior effects to conventional stem cells.
  • the present invention thus provides a cell population in which cells expressing
  • Endomucin was not used as a marker for a cell, and thus cells selected/concentrated using Endomucin as a marker or indicator, didnot simply exist previously. Therefore, such a cell population is significantly different from the prior art technology in that the stem cell ratio is significantly improved or enhanced, and transplantation compatibility is significantly improved.
  • the present invention provides a method for determining transplantation compatibility of a transplant comprising the step of: (I) determining whether or not the transplant expresses a gene of Endomucin, wherein the gene expression of the Endomucin in the transplant indicates that the transplant is compatible to transplantation.
  • the present invention attained significant effects in that the expression of Endomucin is correlatedto transplantation compatibilityto an unexpected extent, andthus attainedsimple detection oftransplantation compatibility.
  • the method for determining transplantation compatibility may further comprise the step of: (II) determining whether the transplant expresses at least one marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2, whereinthe negative orweaklypositive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2 indicates that the transplant has transplantation compatibility.
  • strongly positive expression of at least one of c-kit and Sca-1 genes used in the transplantation compatibility determination method of the present invention indicates that the transplantation in tissue has transplantation compatibility.
  • the strongly positive expression in thepresent invention has demonstrated a strong correlation with transplantation compatibility.
  • the present invention provides a kit for determining differentiation state of a cell comprising the steps of: (a) an agent specifically reacting Endomucin gene or Endomucin gene product; and (b) means for determining whether or not the Endomucin gene is expressed in the cell.
  • an agent specifically reacting Endomucin gene or Endomucin gene product
  • means for determining whether or not the Endomucin gene is expressed in the cell may be any form or embodiment described herein above in the sections (Agent capable of interacting with stem cell marker in the nucleic acid form) and (Agent capable of interacting with stem cell marker in the polypeptide form) .
  • Means for determining whether or not Endomucin is expressed includes but is not limited to: a label and a moiety binding to the label .
  • Such a label may be any label as long as it can label the target of interest in a distinguishable manner, including a fluorophore, phosphorophore, chemilunescence agent, radioactive agent, color producing agent, ligand, hapten, enzyme substrate, and antibody-antigen reaction and the like.
  • a fluorophore, phosphorophore, chemilunescence agent, radioactive agent, color producing agent, ligand, hapten, enzyme substrate, and antibody-antigen reaction and the like when such an agent interacts with its partner such as an antibody using immunological reaction, systems widely used in the art of immunological reactions such as biotin-streptavidin may be used.
  • Labels used in the present invention may be Texas-Red, PE, FITC, APC and biotin and the like. Hoechst Blue, Hoechst 33342, Hoechst Red and the like may be used for staining DNA.
  • the present kit includes means for determining the expression of another stem cell marker.
  • a means includes but is not limited to: means for determining whether or not at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2 ; and means for determining whether or not the agent is expressed in a cell of the sample.
  • a marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2 ; and means for determining whether or not the agent is expressed in a cell of the sample.
  • the kit of the present invention further comprises at least one agent specific to Lin marker; and means for determining whether or not the agent is expressed in a cell of the sample.
  • Such a means may include at ' least one marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8andB220.
  • markers are described herein above in the section (Stem cell determination method) and determination standard described herein may be employed. In brief, these Lineage (Lin) markers are preferably negative.
  • the present invention provides a kit forpreparing a cell of undifferentiated state, comprising: (I) an agent specifically reacting with a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; (II) means for determining whether or not the Endomucin gene is expressed in a cell of interest; and (III) means for separating or concentrating a cell expressing the Endomucin gene.
  • the above agent means for determination may be any form or embodiment as described in the above mentioned (Cell differentiation state determination kit) .
  • the present invention provides a kit fordeterminingtransplantation compatibility of a transplant comprising: (I) an agent specifically reacting with a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a fragment thereof, or Endomucin polypeptide or a portion thereof; and (II) means for determining whether or not the Endomucin gene is expressed in a cell of interest.
  • Such an agent may be any form or embodiment as described herein above in the sections (Transplantationcompatibilitymarker as anucleicacidform) and (Transplantation compatibility marker as a polypeptide form) . Determination of whether or not a transplantation containing a stem cell expresses gene encoding Endomucin may also be carried out in any form or embodiment as described herein above in the section (Stem cell preparation kit) .
  • the transplantation compatibility determination kit of the present invention may further comprise means for determining whether or not at least one agent specific to a marker selected from the group consisting of CD34, Lin (Lineagemarker), c-kit, Sca-1 and Flt3/Flk2; and means for determining whether or not the agent is expression in a cell of the sample. Determination of that CD34 is negative or weakly positive, Lin is negative, c-kit and Sca-1 are positive preferably strongly positive, allows one to sort or select a high ratio of stem cells, and improves transplantation compatibility of a transplant selected according to the present invention.
  • the kit for determining transplantation compatibility of the present invention further comprises at least one agent specific to Lin marker; and means for determining whether or not the agent is expressed in a cell of the sample.
  • said Lin marker comprises at least one marker selected from the group consisting of Gr-1, Mac-1, TER119, CD4, CD8 and B220.
  • the present invention provides a method for treating, preventing or providing a prognosis a subject in need of stem cell transplantation, comprising the steps of: (I) determining whether or not a transplantation containing a stem cell expresses a gene encoding Endomucin; (II) separating or concentrating a transplant containing a stem cell expressing the gene encoding Endomucin; and (III) transplanting the separated or concentrated transplant into the subject.
  • determination of gene expression of Endomucin may be carried out by using a specific agent thereto and determining and/or detecting such a specific binding.
  • a means for separating or concentrating a cell that was identified ' to express Endomucin includes, as described herein, for example, but is not limited to, FACS using specific antibodies, and/or separation using magnetic beads and the like.
  • Such a concentrated or separated cell preparation or cell population may advantageously include Endomucin positive cells only, as this increases transplantation compatibility to a significant extent. It has also been demonstrated that the correlation of Endomucin expression and transplantation compatibility is very high, being as high as about 100%.
  • the present invention further comprises the steps of: (la) determining whether or not at least one marker selected from the group consisting of CD34, Lin (Lineage marker) , c-kit, Sca-1 and Flt3/Flk2, in the transplant; and (Ha) separating or concentrating a transplant containing a stem cell with negative or weakly positive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2. It should be noted that such a further selection or sorting attains additional effects such as improvement in reliablity of transplantation compatibility, and that these markers have been used in the art, and that thus the knowledge already known in the art can alsobe applied for analysis, diagnosis and/or determination.
  • the above step (Ila) of the present invention further comprises a step of separating or enriching/concentrating a transplant comprising a stem cell expressing strongly at least one gene selected from the group consisting of c-kit and Sca-1 genes.
  • the strong expression of these markers were found to have strong correlation with transplantation compatibility, in order to more clearly determine transplantation compatibility, it is preferable to observe the strong expression of these markers.
  • a transplant comprising a stemcellmaybe prepared ex vivo.
  • the transplant is preferably derived from a subject of interest per se, but the present invention is not limited to this.
  • an immunosuppressive method may be conducted.
  • Amethod for avoiding rejection reactions is known in the art and is described in, for example, "Shin-Geka-Taikei, Shinzo-Ishoku • Hai-Ishoku Gijyutsuteki, Rinriteki-Seibi-kara-Jissi-nimukete [Heart Transplant • Lung Transplant - From Technical and Ethical Development to Practice]" (3rd Version).
  • Examples of such a method include use of an immunosuppressant or a steroid drug, and the like.
  • Immunosuppressants for preventing rejection reactions currently include “cyclosporine” (SANDIMMUNE/NEORAL) , “tacrolimus” (PROGRAF) , “azathioprine” (IMURAN), "steroid hormone” (prednine, methylprednine) , and “T-cell antibodies” (OKT3, ATG, etc.) .
  • a method which is used for preventative immunosuppressive therapy in a number of facilities in the world is three-drug therapy using "cyclosporine, azathioprine, and steroid hormone".
  • An immunosuppressant is preferably, but not necessarily, administered conco itantly with a medicament of the present invention. Therefore, an immunosuppressant may be administered before or after a prevention/regeneration/therapeutic/prognosis method of the present invention as long as an immunosuppression effect can be achieved.
  • the transplant comprising a stem cell is advantageously derived from bone marrow, umbilical cord blood, peripheral blood, embryo, and fetus. These sources are known to contain a hematopoietic stem cell.
  • the present invention provides a medicament for treating, preventing or providing aprognosis for a subject inneedofa stemcell transplantation, comprising cell population concentrated with stem cells expressing Endomucin.
  • a stem cell has negative or weakly positive expression of CD34, negative expression of Lin (Lineage marker) , positive expression of c-kit, positive expression of Sca-1 and negative expression of Flt3/Flk2.
  • Lin Lineage marker
  • c-kit positive expression of c-kit
  • Sca-1 positive expression of Sca-1
  • Flt3/Flk2 negative expression of Flt3/Flk2.
  • the present invention provides use of Endomucin for detecting or identifying a stem cell.
  • the ste cell comprises a hematopoietic stem cell.
  • Endomucin may be any form or embodiment as described herein above.
  • the present invention provides use of Endomucin for determining transplantation compatibility of a transplant.
  • Transplant to be investigated for transplantation compatibility preferably comprise a stem cell.
  • Endomucin may be any form or embodiment as described herein above.
  • the present invention provides use of an agent specific to Endomucin polypeptide or a portion thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a portion thereof, for detecting or identifying a stem cell.
  • the stem cell preferably comprises a hematopoietic stem cell .
  • Endomucin and the agent may be any form or embodiment as described herein above.
  • the present invention provides use of an agent specific to Endomucin polypeptide or a portion thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a portion thereof, for determining transplantation compatibility of a transplant.
  • a transplant to be investigated for transplantation compatibility preferably comprises a hematopoietic stem cell.
  • Endomucin and the agent may be any form or embodiment as described herein above.
  • the present invention provides use of an agent specific to Endomucin polypeptide or a portion thereof, or a nucleic acid molecule comprising a nucleic acid sequence encoding Endomucin or a portion thereof, for manufacturing a medicament comprising a stem cell having transplantation compatibility.
  • the transplant to be investigated for transplantation compatibility preferably comprises a stem cell.
  • Endomucin can be in any form described herein above.
  • the present invention provides use of a stem cell expressing Endomucin in manufacture of a medicament for stem cell transplantation. Description of a medicament and Endomucin are also described herein above, and any forms and enbodiments thereof may be used for the present use.
  • the present invention provides an isolated cell expressing Endomucin.
  • the expression of CD34 of the cells is negative or weakly positive .
  • the expression of c-kit and Sca-1 of the cells is positive, and yet more preferably strongly positive.
  • the expression of Lin of the cells is negative.
  • the present invention provides relates to a cell obtained or obtainable by the method of the present invention, and a tissue and organ obtained by the cell
  • the present invention provides a pharmaceutical composition comprising a cell obtained by the method of the present invention.
  • Such a pharmaceutical composition may comprise a necessary pharmaceutical carrier, and/or an additional pharmaceutically effective ingredient.
  • the present invention provides a method for treating, preventing or prognosis of a disease or a disorder in need of a stem cell or a differentiated cell derived therefrom, comprising: A) administering to a subject in need of such a treatment, prevention or prognosis, a cell obtained by the present invention.
  • the present invention provides use of a cell obtained by the present invention, for treating, preventing or prognosis of a disease or disorder in need of a stem cell or a differentiated cell derived therefrom. Technologies for such use of the cells are well known in the art, and those skilled in the art can select an appropriate method therefor.
  • the present invention provides a stem cell prepared by a method of expanding the stem cells of the present invention, and/or maintaining the pluripotency or self-proliferative capability thereof. This method can be used in vivo or in vi tro.
  • a stem cell or a cell population prepared according to the present invention allows a certain quality and mass production of the cells to be attained, which are not available from the state of the art. Therefore, the present invention allows preferable therapeutic effect.
  • the present invention allows efficient provision of a stem cell such as a hematopoietic stem cell. Accordingly, such effects are not expected from the prior art and thus the utility thereof should be noted.
  • Example 1 Search for molecules expressed on a membrane of hematopoietic stem cells
  • search for molecules expressed in the cellular membrane of CD34-KSL cells CD34-, Kit + , Sca-1 + , lineage- myelocytes fraction
  • CD34-KSL cells CD34-, Kit + , Sca-1 + , lineage- myelocytes fraction
  • CD34 ⁇ KSL cells Preparation of CD34 ⁇ KSL cells was conducted as follows: Obtain bone marrow fluid from C57BL/6 Ly5.1 mice of age of 8-10 weeks and mononuclear cells were separated by subjecting the sample to density gradient centrifugation.
  • Lineage marker mixture (CD4, CD8, B220, Gr-1, Mac-1 and
  • FACS Vantage cell sorter (Becton Dickinson) is used to the select hematopoietic stem cell fraction of C D34 - l0W c-kit + Sca-l + Lineage-marker- ( CD34-K S L ) on to
  • Serum free medium (X-vivo-10 (BioWhittaker) ) was used as a culture solution at the volume of 200 ⁇ l/well, and SCF (Stem cell factor) and TPO (thrombopoietin) were used as cytokines at a concentrations of 100 ng/ml for each.
  • CD34 + KSL cells, KSL cells, TER119 cells, Gr-1 cells, Mac-1 cells, B220 cells, THy-1 cells, NK1.1 cells, DN cells, DP cells, CD4SP cells, CD8SP murine cells; CD34 + /38- cells, CD34 + cells, CD34 + /13 + cells, CD14 + cells, CD15 + cells, GPA + cells, GPA + cells, CD71 + cells, CD41 + cells, CD3 + cells, CD19 + cells, CD56 + human cells were also prepared.
  • cDNA libraries were prepared according to well known technologies in the art, and a search was conducted using a variant signal sequence trapping method to identify a molecule expressed on the membrane of a hematopoietic stem cell.
  • Endomucin (la) was identified as a candidate molecule.
  • Endomucin a candidate molecule, expression levels of Endomucin genes in a variety of cell fractions was determined by conducting RT-PCR. The protocols therefor are described as follows:
  • the primers used for RT-PCR are as follows :
  • Bone Marrow refers to a cell from the bone marrow
  • Spleen refers to a cell derived from the spleen
  • Thymus refers to a cell from the thymus .
  • Endomucin expression was observed only in the cell groups which are believed to be enriched for stem cells .
  • Endomucin was demonstrated to specifically expressed in a hematopoietic stem cell by the mRNA level.
  • Example 2 Analysis of Endomucin expression using antibodies
  • the present Example investigated how stem cells express Endomucin polypeptides using a variety of antibodies. Antibodies as used in the present Example, were obtained from Dr. Vestwebe . r D.
  • SP cell fraction which is not stained with Hoechst 33342, due to a good excretion property for the stain, is present about 0.1 % in the bone marrow cells, and thus hematopoietic stem cells are concentrated (Goodell MA etal, J. Exp.Med.183 : 1797, 1996).
  • An exemplary protocol for SP cells is described as follows. Instead of labeled antibodies used in the preparation procedures of CD34 ⁇ KSL cells described in Example 1, Hoechst Blue, Hoechst 33342 and Hoechst Red were used to conduct FACS assays in the present Example. Here, weakly positive cells for Hoechst Blue and Hoechst Red were extracted as an SP cell (see Figure 2, left panel) .
  • Bone marrow cells were obtained from femur and tibia ofmice (Ly5.1) using a 25 G syringe and a 2.5 ml syringe, and bone marrow was extracted with 5 ml of PBS .
  • the bone marrow cell suspension was overlaid onto 6ml of Ficoll-Paque (Amersham-Pharmacia Biotech), and subjected to centrifugation at 400 x g, for twenty minutes . Thereafter, the middle layer containing mononuclear cells was collected. The collectedcells were washedwith 10ml of staining solution (SM, 5% FCS + 0.05%NaN 3 + PBS) , andnon-labeleddifferentiation antigen (Lineage) antibody was added thereto. After reaction on ice for twenty minutes, the sample was washed with SM, and mixed with magnetic beads bound to anti-rat IgG antibody.
  • SM staining solution
  • Lineage antigen negative cells not bound to magnetic beads were passed through a depletion column fixed to a magnetic stand, and cells flowed from the column without being trapped therewith were collected (named as Lineage-negative cells) .
  • the middle panel refers to the case where only anti-rat PE was used, and the right panel refers to the case where Endomucin and anti-rat PE were used.
  • the SP cells were demonstrated to be enriched for Endomucin expressing cells. Further, 0.8 % of total bone marrow cells were Endomucin positive. Further, cell groups using other differentiation markers (prepared by Example 1) were observed with similar Endomucin expression. The results are shown in Figures 3-8.
  • bone marrow KSL cells were about 90 % Endomucin positive.
  • Figure 9 shows the results where KSL cells were divided into four fractions by expression pattern of CD34 and Endomucin, using similar FACS sorting.
  • Upper left panel shows an outline of Lineage depletion. These cells, corresponding to stem cells were sorted as shown in lower left panel, and classed into four categories by Endomucin and CD34 expression.
  • the ratio of CD34 " Endomucin- fraction was about 3 % in total, and the ratio of CD34-Endomucin + fraction was about 23 %, and the ratio of CD34 + Endomucin + fraction was abotu 65 %, and the ratio of CD34 + Endomucin- fraction was about 9%. From these, about 90 % or more of CD34-KSL cells are Endomucin positive, and thus Endomucin was demonstrated to be one of the markers for hematopoietic stem cells.
  • Example 3 colony forming assay
  • MACS magnetic cell sorting system
  • magnetic microbeads were labeled with an antibody specific to a Lin positive cell, and the labeled cell was subj ected to the depletion method-, in which negative cells passed through the magnetic field without label of microbead, were separated to reduce the number ofLinpositive cells to about 1/50 of the original
  • KSL cells were gated by expressing CD34 and Endomucin.
  • the protocols therefor are as follows:
  • Bone marrow cells were obtained from femur and tibia ofmice (Ly5.1) using a 25 G syringe and a 2.5 ml syringe, and bone marrow was extracted with 5 ml of PBS.
  • the bone marrow cell suspension was overlaid onto 6ml of Ficoll-Paque (Amersham-Pharmacia Biotech), and subjected to centrifugation at 400 x g, for twenty minutes. Thereafter, the middle layer containing mononuclear cells was collected.
  • the collectedcells werewashedwith 10ml of staining solution (SM, 5% FCS + 0.05% NaN 3 + PBS), and non-labeled differentiation antigen (Lineage) antibodywas added thereto.
  • SM staining solution
  • FCS + 0.05% NaN 3 + PBS non-labeled differentiation antigen
  • Left panel of Figure 10 shows a procedure for sorting. As in upper left panel, selection was ' ade by c-kit APC and Sca-1 PE-Cy5.5 for stem cell selection, and the stem cells were fractioned by observing the expression of CD34 and Endomucin.
  • Endomucin was demonstrated to be an efficient and good marker for stem cells.
  • Example 4 Bone reconstruction assay
  • hybrid colonies were also observed to comprise cell lineage comprising neutropb.il/macrophage/erythroblast/megakaryocyte from CD34-Endomucin " fractions, in order to directly observe hematopoietic stem cells, bone marrow reconstruction assay was conducted.
  • the protocols are described below:
  • Cells were sorted using FACS in accordance with the cell number as shown in each cell fraction in Figure 11, and mixed with 2 x 10 5 cells of C57BL/6 Ly5.2 murine bone marrow cells.
  • FIG. 11 shows results by sorting the sample with Mo-flo fifteen weeks after the bone marrow transplantation. As shown in the Table in
  • Endomucin has been demonstrated to be a good indicator for determining transplantation compatibility.
  • Example 5 Correlation of Endomucin expression amount and undifferentiated state
  • Example 5 distribution of KSL cells of the cellular fraction separated by Endomucin gating, was observed for confirming correlation between the Endomucin expression amount and undifferentiated state of the cell.
  • Endomucin PE was used as in Figure 12, left panel, and classified according to five expression levels to 1-e (Endomucin PE expression levels of 1-5, 5-35, 35-150, 150-400 and 400-1500, respectively) . Each existence ratio is 18.6%, 56%, 20.1%, 1.58% and 0.26%, respectively. Results of these panels developed by Sca-1 and c-kit are shown in Figure 12, right panel. As a result, it is understood that high expression of Endomucin is correlated with both high expression of Sca-1 and c-kit from d-e panel therein.
  • Example 6 Distribution of c-kit high , Sca-l high cells whichwere gatedbythe expression of CD34 andEndomucin
  • mononuclear cells were treated using MACS technology to deplete lineage positive cells to 1/21 of the original. These lineage-positive cell-depleted cells were used for demonstrating the relationship between c-kit, Sca-1 and CD34 expressions, and Endomucin expression.
  • the • depleted cells were subjected to the depletion process as described above in the Examples.
  • the cells were observedfor expression andthe inter-relationship between (1) c-kit, (b) Sca-1, and (c) CD34, in lineage negative cells, which is shown in Figure 13.
  • Figures 13a, 13b and 13c correspond to c-kit, Sca-1 and CD34, respectively.
  • the right lower panel refers to c-kit and Sca-1 expression distribution in a cell of CD34 ⁇ Endomucin h ⁇ gh cells which corresponds to 0.1 in c.
  • Endomucin is not co-expressed with lineage surface marker in a bone marrow cell and a spleen cell, and it has been demonstrated that 70-90 % of CD34-KSL cells are Endomucin positive, and about 100 % of CD34-Lin-c-kit ++ Sca-l ++ cells are Endomucin positive.
  • CD34 ⁇ KSLEndomucin + cells were shown to retain similar pluripotency as CD34-KSL cells.
  • CD34-KSLEndomucin + cells are preferable for reconstructing hematopoietic cells.
  • Example 7 Separation and Concentration of stem cells using Endomucin
  • Anti-Endomucin is conjugated with magnetic beads, and react it with bone marrow cells.
  • the cells of interest are passed through a column chromatography fixed onto a magnetic stand, and cells trapped to the column are recovered to purify Endomucin positive cells.
  • the cell fraction obtained have a cell fraction containing a purified and concentrated hematopoietic stem cell, and thus hematopoietic stem cells can be concentrated using only one purification step.
  • Example 8 Induced expression of Endomucin gene
  • Hematopoietic stem cells (CD34-KSL; 100 cells) were placed in a well of a 96-well plate and sorted by FACS.
  • Pre-culture is conducted in a S clone medium (Sanko Pure Chemical) in the presence of 100 ng/ml murine SCF and 100 ng/ml human TPO for 24 hours. Thereafter, concentrated Endomucin retrovirus is added to the cells at MOI 600 and the cells are infected with the virus for 24 hours. CD34 " KSL cells with the gene introduced therein by the retrovirus are further liquid cultured in the presence of 20 ng/ml murine SCF, 100 ng/ml human TPO. On Days 7 and 14 of the culture, a colony assay is conducted using methyl cellulose in the presence of 20.
  • a portion of the cultured cells are transplanted via the tail vein of a mouse (Ly5.2) which was radiated at a fatal dose of 9.5 Gy, together with 2 * 10 5 cells of competitive cells (Ly5.2).
  • chimerism of the peripheral blood of the cells with Endomucin gene introduced therein is assayed to determine the effect of the induced expression of the Endomucin gene in hematopoiteic stem cells. This is called a competitive bone marrow transplantation experiment.
  • Endomucin was directly used to investigate whether or not capabilities of a stem cell can be maintained.
  • End-Fc protein A chimeric protein with Endomucin extracellular domain and human IgG Fc portion is prepared (hereinafter called End-Fc protein) .
  • Endomucin positive cells are sorted using FACS and change in the number of CFU-Mix/CFU-nmeM are evaluated by neutralizing Endomucin molecule functions on hematopoietic stem cells by adding End-Fc proteins, when conducting colony assays using methyl cellulose in the presence of 20ng/ml murine SCF, 50ng/ml human TPO, 20ng/ml murine IL-3 and unit/ml EPO.
  • Example 10 effects by Endomucin variants
  • splice variants such as Endomucin la, lb, lc and Id are investigated with respect to the stem cell undifferentiation state, and transplantation compatibility.
  • FACS analysis is conducted on hematopoietic stem cells CD34 " KSL (100 cells) in a 96-well plate for sorting.
  • Pre-culture is conducted in a S clone medium (Sanko Pure Chemical) in the presence of 100 ng/ml murine SCF and 100 ng/ml human TPO for 24 hours.
  • concentrated Endomucin retrovirus is added to the cells at MOI 600 and the cells are infected with the virus for 24 hours.
  • CD34 ' KSL cells with the gene introduced therein by the retrovirus are further liquid cultured in the presence of 20 ng/ml murine SCF, 100 ng/ml human TPO.
  • a colony assay is conducted using methyl cellulose in the presence of 20 ng/ml murine SCF, 50 ng/ml human TPO, 20 ng/ml murine Interleukin-3, and 5 unit/ml of Erythropoietin.
  • the mixed colony (CFU-Mix/CFU-nmeM) is counted.
  • a portion of the cultured cells are transplanted via the tail vein of a mouse (Ly5.2) which was radiated at a fatal dose of 9.5 Gy, together with 2 * 10 5 cells of competitive cells (Ly5.2).
  • chimerism of the peripheral blood of the cells with the Endomucin gene introduced therein is assayed to determine the effect of the induced expression of the Endomucin gene in hematopoiteic stem cells (competitive bone marrow transplantation experiment) .
  • the effects of induced expression of these splice variants of Endomucin genes in hematopoietic stem cells can be determined.
  • Example 11 screening for substances interacting with Endomucin
  • agents interactingwith Endomucin are found to be useful. Therefore, the present Example demonstrates screening for an agent interacting with Endomucin. Endomucin with no bound substances is prepared. By determining whether the Endomucin is activated by providing a candidate agent, it is possible to screen for an interacting agent of Endomucin to determine and identify such agents.
  • Endomucin can specifically interact with Endomucin, such compounds can be used as a marker or agent relating to the undifferentiated state of a stem cell or transplant compatibility. Such compounds can bind to Endomucin, and/or di erize with Endomucin, or a low molecular weight molecule activating Endomucin, or the like.
  • a substance of target canbedeterminedtobeEndomucinornotbydetermining whether ornot the agent is present onthemembrane and/orbyconfirming whether or not such is capable of binding to an Endomucin specific consensus sequence.
  • a cell is transfected with the Endomucin gene, and anti-Endomucin antibody is used to immunostain the cell, and thereafter it is possible to determine whether or not a candidate is Endomucin or not by confirming the localization to the me brane is present when the candidate compound is administered. If such an agent is a candidate compound such as a protein, such a compound may be directly introduced into a cell, and subsequently, whether or not the agent is translocated to the membrane . can be determined by immmunostain with a specific antibody to the agent in order to confirm that the agent is Endomucin. Such technologies are well known in the art .
  • a gel shift assay is conducted using double-stranded oligonucleotide comprising a base sequence used for the Endomucin binding base sequence .
  • a candidate compound is administered, after a nucleic acid molecule comprising the consensus sequence and an Endomucin molecule are reacted, electrophoresed and separated on a polyacrylamide gel, the ability of such a candidate compound can be determined by identifying the formation of a complex with the molecule containing the consensus sequence and the Endomucin molecule. Usually, if the formation of the complex is significantly confirmed, then the agent has a similar function as Endomucin.
  • Example 12 Use of Endomucin interacting agent
  • an agent interacting with Endomucin as prepared in Example 11 it is possible to determine the undifferentiated state of a stem cell, determine transplantation compatibility, enhance the undifferentiated state of a stem cell, and enhance transplantation compatibility of a transplant and the like.
  • a compound activates Endomucin expression, it should be understood that administration of a stem cell and culture for a certain period of time, allow the stem cell to be undifferentiated, and/or transplantation compatibility of a stem cell or a composition/transplant containing the same to be obtained and/or enhanced.
  • Example 13 Knock-in of Endomucin fused to a fluorescence stain
  • a sequence encoding Endomucin of the present invention was fused to a sequence encoding a fluorescence stain (Venus stain) to construct a knock-in vector containing a fused nucleic acid sequence.
  • Anucleotide sequenceATG within Endomucin first exon was inserted with a sequence encoding Venus to prepare a knock-in vector which is then used to transfect ES cells for preparing a knock-in cell by using technologies well known in the art (see knock-in construct in Figure 14).
  • This ES cell is differentiated in vi tro, and it is determined that Endomucin is expressed in the stem cell by observing the expression of Venus in vitro.
  • the resultant ES cells are used for preparing a mouse individual using technologies well known in the art, and confirms that Endomucin is expressed in the stem cell for observingthe expressionofVenus .
  • the Endomucin gene is deleted, and thus Endomucin function is specified by analyzing function abnormalities in the stem cells, such a hematopoietic stem cell in a mouse in which Endomucin gene is deleted.
  • Example 14 Promoter sequence of Endomucin
  • a promoter sequence of Endomucin is analyzed.
  • the upstream sequence of Endomucin was determined and the determined sequence was used for preparing a construct in which the 5' terminus of lOkbp upstream of the transcriptional starting point was truncated (see Figure 15 for the used restriction endonuclease sites) for analyzing promoter activity of Endomucin sequences.
  • the luciferase assay was evaluated by determining the luminescence level of luciferase. As a result, it has been determined that an element positively controlling transcription is located between the transcriptional starting point and lOkbp upstream therefrom (see SEQ ID NO: 33).
  • Example 15 Endomucin as a marker for stem cells in embryos
  • Endomucin is a novel cell surface antigen that marks both BM LTR-HSCs and developing definitive HSCs in embryos.
  • Endomucin expression in an embryo was confined to definitive hematopoietic stem and progenitor cells but not to primitive hematopoietic progenitors, leading to a successful tracking of definitive hematopoiesis in the embryo.
  • Endomucin could be a useful marker to understand HSC ontogeny.
  • C57BL/6 -Ly5.2 mice Eight to 10 week old C57BL/6 strain mice were used throughout this experiment.
  • C57BL/6 -Ly5.2 mice were purchased from SLC (Shizuoka, Japan) .
  • C57BL/6 -Ly5.1 mice were bred andprovidedby Sankyo Laboratories (Tokyo, Japan) .
  • Ly5.1 x Ly5.2 FI mice were bred and maintained in the Animal Research Facility of the Institute of Medical Science, University of Tokyo.
  • RT-PCR Semi-quantitative RT-PCR was carried out using normalized cDNA by the quantitative PCR using TaqMan rodent GAPDH control reagent (Perkin-Elmer Applied Biosystem, Foster City, CA) as described before (Osawa, M. , Hanada, K. -I., Hamada, H. and Nakauchi, H. (1996). Science 273, 242-245) .
  • the primer sequences are as follows: Endomucin sense 5'-ACA ACT GAA GGT CCC CTA AGG-3' (SEQ ID NO: 34), antisense 5'-TTG GTT TTC CCC TGT GCA GAC-3' (SEQ ID NO: 35) .
  • PCR parameters were denaturation at 94 °C for 20 sec, annealing at 58 °C for 20 sec, and extension at 72 °C for 30 sec. The amplification proceeded for 35 cycles. PCR products were separated on an agarose gel and visualized by ethidium bromide staining.
  • Mouse hematopoietic stem cells (CD34 ⁇ KSL cells) were purified from bone marrow cells of 2-mo-old mice. In brief, low-density cells were isolated on Lymphoprep (1.086 g/ml; Nycomed, Oslo, Norway) . The cells were stained with an antibody cocktail consisting of biotinylated anti-Gr-1, Mac-1, B220, CD4, CD8, and Ter-119 mAbs (PharMingen, San Diego, CA) . Lineage-positive cells were depleted with streptavidin-magnetic beads (M-280; Dynal Biotech, Oslo, Norway) .
  • the cells were further stained with fluorescein isothiocyanate (FITC) -conjugated anti-CD34, phycoerythrin (PE) -conjugated anti-Sca-1, and allophycocyanin (APC) -conjugated anti-c-kit antibodies (PharMingen).
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • APC allophycocyanin
  • Biotinylated antibodies were detected with streptavidin-Texas Red (Molecular Probes, Eugene, OR) .
  • Four-color analysis and sorting was performed on a .
  • FACS Vantage Becton Dickinson, San Jose, CA
  • BM mononuclear cells were first bound with a purified anti-lineage antibody mixture (Gr-1, Mac-1, CD4, CD8, B220, and TER119) (eBioscience, San Diego, CA) , followed by anti-Rat IgG-Microbeads (Miltenyi Biotech, Bergisch Gladback, Germany) . Magnetic labeled cells were then passed through a LD column (Miltenyi Biotech) and the flow-through cells were recovered as lineage-depleted cells.
  • a purified anti-lineage antibody mixture Gr-1, Mac-1, CD4, CD8, B220, and TER119
  • eBioscience San Diego, CA
  • anti-Rat IgG-Microbeads Magnetic labeled cells were then passed through a LD column (Miltenyi Biotech) and the flow-through cells were recovered as lineage-depleted cells.
  • FACS analysis and cell sorting was performed on a FACS Vantage using CELLQuestTM software (Becton Dickinson) , a FACS AriaTM using FACS Diva TM software (Becton Dickinson) , or a Mo-floTM using SummitTM software (DakoCytomation, Grostrup, Denmark) .
  • Data Analysis was performed using Flow-JoTM software (Tree Star, Inc., Ashland, OR).
  • Colony formation assay Single cells were directly sorted into 96 well round-bottomplates containing150 ⁇ l S-clone (Sanko Junyaku, Tokyo, Japan), supplemented with 10 % FBS (Sigma) , 2 mM L-glutamine (Sigma), 5 X 10 -4 M 2- ⁇ -merca ⁇ toethanol (Sigma), 100 U/ml penicillin (Sigma) , 100 ⁇ g/ml streptomycin (Sigma) , 10 ng/ml mouse IL-3, 10 ng/ml mouse stem cell factor (mSCF) , 2 U/ml Erythropoietin (EPO) , and 50 ng/ml Thrombopoietin (TPO) (Peprotech, Rocky Hill, NJ) .
  • 10 % FBS Sigma
  • 2 mM L-glutamine Sigma
  • 5 X 10 -4 M 2- ⁇ -merca ⁇ toethanol Sigma
  • 100 U/ml penicillin Sigma
  • peripheral blood cells of recipient mice were collected and were stained with biotinylated anti-Ly5.1 (eBioscience) , FITC-conjugated anti-Ly5.2 antibody, APC-conjugated anti-Gr-1 antibody, APC-conjugated anti-Mac-1 antibody, PE-conjugated anti-CD4, PE-conjugated anti-CD8 antibody (PharMingen), PE-Cy7-conjugated anti-B220 antibody (Caltag), and streptavidin-Texas Red (Molecular Probes) .
  • chimerism total (%) donor-derived cells / (donor-derived cells + competitor-derived cells) lOO
  • chimerism lineage (%) donor-derived lineage positive cells / (donor-derived lineage positive cells + competitor-derived lineage positive cells) lOO. Mice that had > 1 % chimerism in each lineage were considered to be multilineage reconstituted. Median and standard deviation was calculated among the reconstituted recipients.
  • EB formation and staining ES cells were maintained on irradiated mouse embryonic fibroblasts in DME/15% inactivated fetal serum (IFS), 0.1 mM non-essential amino acids (GIBCO-BRL) , 2 mM glutamine, 50 U/ml penicillin, 50 g/ml streptomycin, 0.1 mM 2-mercaptomethanol, 1000 U/ml leukemia inhibitory factor (Peprotech) .
  • ES cells were trypsinized, collected in EBD [Iscove' s modified Dulbecco' s medium (IMDM) , 15 % IFS, 200 ⁇ g/ml iron-saturated transferrin (Sigma), 4 , 5 mM monothioglycerol (Sigma), 50 ⁇ g/ml ascorbic acid (Sigma) , 2 mM glutamine] .
  • EBD Iscove' s modified Dulbecco' s medium
  • IFS IFS
  • 200 ⁇ g/ml iron-saturated transferrin Sigma
  • 4 5 mM monothioglycerol
  • 50 ⁇ g/ml ascorbic acid Sigma
  • 2 mM glutamine 2 mM glutamine
  • EBs were then collected from the hanging drops and further cultured in 10 ml of EBD in 10 cm bacterial Petri dishes. At day 3, EBs were fed by exchanging half of their spent medium for fresh EBD. EB-forming days correspond to the time point of EB collection counting from the day when they were plated in hanging drops (EB 0) . Collected EBs were mildly trypsinized and washed twice with EBD. Single cells (5xl0 5 ) were stained with PE-conjugated anti-Flkl, FITC-conjugated anti-CD41 antibody, or PE-conjugated anti-CD45 antibody (PharMingen) , and biotinylated anti-Endomucin antibody (V7C7) .
  • Streptavidin-APC was used for detecting Endomucin .
  • Cells were analyzed on a FACS AriaTM. Each subpopulation was sorted on a Mo-floTM and plated onto OP-9 stromal cells in the presence of 20 ng/ml VEGF (Peprotech) to allow endothelial differentiation and in the presence of 10 ng/ml mouse IL-3, 10 ng/ml murine SCF, 2 U/ml human EPO, and 50 ng/ml human TPO to allow hematopoietic differentiation.
  • RESULTS Identification of Endomucin as an HSC-specific gene In order to identify cell surface molecules specific to HSCs, we tookadvantage of SST-REX cloningmethod.
  • This method detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a c-Mpl to the cell surface, thereby permitting interleukin-3 (IL-3) -independent growth of Ba/F3 cells (Kojima, T., and Kita ura, T. (1999). Nat. Biotech. 17, 487-490).
  • IL-3 interleukin-3
  • Endomucin is a sialomucinproteinhighlyglycosylatedwith 0-linkedglycans ( Figure 16c) . Structurally, it belongs to a sialomucin family that includes CD34, a well-characterized molecule specific to hematopoietic and endothelial systems. Endomucin was originally identified as an endothelial-specificmarkerbut has not yetbeen investigated in hematopoietic lineages (Morgan, S.M., Samulowitz, U., Darley, L., Simmons, D.L., and Vestweber, D. (1999). Blood 93, 165-75) . We therefore focused on Endomucin for its specificity to HSCs.
  • Endomucin was extensively analyzed on adult hematopoietic cells from BM, spleen, and thymus. Endomucin was not expressed on a vast majority of lineage marker-positive cells including Gr-1-, Mac-1-, B220-, CD4-, CD8- and TER119-positive cells, although only a small population of B220 + cells fromBM (0.66%) and spleen (1.8 %) , which appeared to be B220 + CD43 " IgM + mature B cells, were Endomucin positive (data not shown) .
  • Mac-1 is an antigen that is highly expressed on myeloid cells, but is also moderately expressed on a portion of short-term repopulating hematopoietic progenitor cells (Morrison, S.J., and eissman, I.L. (1994). Immunity 1 , 661-673) .
  • Endomucin-positive cells Within lineage-depleted BM cells, 85 %, 50 %, and 86 % of Endomucin-positive cells were demonstrated to be c-kit-positive, Sca-1-positive, and CD34-positive, respectively. This indicates that Endomucin protein stays at a high level even on CD34 + KSL cells, although Endomucin is markedly down-regulated in CD34 + KSL cells at the mRNA level. Importantly, 14 % of Endomucin-positive cells were CD34-negative and 30 % were c-kit + Sca-l high , suggesting that CD34-KSL HSCs are enriched in this CD34-Endomucin + L- fraction.
  • Hoechst33342 dye staining provides an alternative way for HSC enrichment, in which side population (SP) cells that efficiently efflux this dye are enriched for primitive hematopoietic cells including HSC (Goodell, et al. , 1996) . Sixty-three percent of SP cells were revealed to be Endomucin "1" (data not shown) . All these FACS profiles indicate that positive selection by Endomucin expression would provide a good yield of HSCs and multipotential progenitor cells.
  • SP side population
  • CD34-KSL cells are highly enriched for long-term repopulating (LTR) -HSCs, while CD34 + KSL cells are progenitors with short-term repopulating capacity (Osawa, etal., 1996).
  • LTR long-term repopulating
  • CD34 + KSL cells progenitors with short-term repopulating capacity
  • Endomucin expressionmarks all HSCs and correlates well with those of c-kit and Sca-1 we checked if Endomucin could substitute for Sca-1.
  • Sca-1 is a well-known positive marker for HSC purification, because of its variable expression among species, it cannot be used as a universal marker.
  • CD34-KSL Endomucin + Lin- cells (0.18% of Lin- cells) represented comparable number of cells with CD34-KSL cells (0.20 % of Lin ' cells), indicating that Endomucin can be universally used as a substitute for Sca-1 in HSC purification.
  • Endomucin could substitute for both c-kit and Sca-1 we further asked if Endomucin could substitute for both c-kit and Sca-1.
  • Lineage marker-positive cells were strictly gated out by direct staining, then Lin- cells were fractionated by their expression of CD34 and Endomucin (Figure 19a, FACS profile) .
  • Figure 20 shows expression of Endomucin on E14.5 fetal liver hematopoietic stem cells.
  • the vast majority of fetal liver hematopoietic stem cells (CD34 + KSL) were demonstrated to be Endomucin "1" . (left panel: Lin-negative-gated) .
  • the first adult repopulating HSCs arise from the hemogenic endothelium localized to the endothelial layer lining the wall of the dorsal aorta in the AGM region (Muller et al . , 1994: de Bruijn, etal., 2002).
  • yolk sac cells can reconstitute long-termhematopoiesis only in conditioned newborn mice, indicating their inability to home and engraft in the BM (Yoder and Hiatt, 1997) .
  • HSCs develop from hemangioblasts, the common progenitor cells for HSCs and endothelial cells, or from the hemogenic endothelium (Choi, K, , Kennedy, M.
  • HSCs and endothelial cells share a number of molecules crucial for their ontogeny.
  • AGM intraembryonic aorta-gonads-mesonephros
  • c-kit and CD41 are absent on hemangioblasts or hemogenic endothelium, but mark the developing hematopoietic stem and progenitor cells of both primitive and definitive types in the yolk sac and AGM (12, 13). An increasing number of these marker antigens are facilitating progress in the precise identification of HSC development.
  • Endomucin is a novel cell surface antigen that marks both BM LTR-HSCs and developing definitive HSCs in embryos. Importantly, Endomucin expression in embryos was confined to definitive hematopoietic stem and progenitor cells but not to primitive hematopoietic progenitors, leading to a successful tracing of definitive hematopoiesis in the embryo. Endomucin could be a useful marker to understand HSC ontogeny.
  • sorted cells were suspended in ⁇ -MEM based 1.2% methylcellulose (Aldrich Chemical), 30% FBS (Sigma), 1% BSA (Sigma), 10 -4 2-mercaptomethanol , 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (Sigma) , 10 ng/ml mouse recombinant IL-3, 10 ng/ml mSCF, 2 U/ml EPO, and 50 ng/ml TPO. Numbers of primitive erythroid colonies (Ery-P) were determined at day 2-3 of culture. Numbers of CFU-Cs were determined at dayl2 of culture.
  • Ery-P primitive erythroid colonies
  • OP9 co-culture OP9 cells at the density of 40, 000 cells per well were plated in 4 chamber culture slides (BD Falcon, MA) or on 24-well culture plates (Corning, NY) a day before co-culture.
  • Yolk sac orAGM-derived sorted cells were placed onto OP9 the layer in triplicate and were co-cultured in ⁇ -MEM supplemented with 10 % FBS, 2 mM L-glutamine, 5 X 10 -4 M 2-mercaptoethanol, 100 U/ml penicillin (Sigma) , 100 ⁇ g/ml streptomycin, 10 ng/ml mouse recombinant IL-3, lOng/mlmSCF, 2 U/ml EPO, and 50 ng/ml TPO (PeproTech, Rocky Hill, NJ) .
  • the first adult repopulating HSCs arise from the hemogenic endothelium localized to the endothelial layer lining the wall of the dorsal aorta in AGM region (Muller et al., 1994: de Bruijn, M.F., Ma, X. , Robin, C, Ottersbach, K. , Sanchez, M.J., and Dzierzak, E. (2002) . Immunity 16, 673-83) .
  • yolk sac cells can reconstitute long-term hematopoiesis only in conditioned newborn mice, indicating their inability to home and engraft in the BM (Yoder, M.C. andHiatt, K.
  • c-kit and CD41 are absent on hemangioblasts or hemogenic endothelium, but mark the developing hematopoietic stem and progenitor cells of both primitive and definitive types in the yolk sac and AGM (12, 13) . An increasing number of these marker antigens are facilitating progress in the precise identification of HSC development.
  • CD45 ⁇ CD41 + Endomucin 4" cells were detected in yolk sac from E10.5 embryo ( Figure 21b), but again these cells rearly contained colony-forming unit-erythroid (CFU-E) ( Figure 21c) .
  • CD45- AGM cells were divided into 4 subgroups from their expression of Endomucin and CD41 ( Figure 22a) .
  • CD45-CD41 + Endomucin + cells showed highest plating efficiency on OP9 stromal cells ( Figure 22b) , and they presented high-level potential of proliferation and multilineage differentiation ( Figure 22c) .
  • Figure 22b shows highest plating efficiency on OP9 stromal cells
  • Figure 22c high-level potential of proliferation and multilineage differentiation
  • Figure 23 shows localization of Endomucin on HSC in contact with stromal cells by capping of Endomucin protein on hematopoietic cells on OP-9 stromal cells.
  • Endomucin + CD41- cells from day 10 AGM were cultured on OP-9 stromal cells, then cells were immunostained with anti-Endomucin monoclonal antibody.
  • Capping of Endomucin protein on some of the hematopoietic cells growing underneath stromal cells (B: light field) were detected (A, C) .
  • A, C light field
  • the present invention provides for the first time a composition for determining undifferentiated state of a stem cell and/or determining transplantation compatibility in a simple and efficient manner.
  • a composition may be used for a variety of treatment, prevention, diagnosis, detection, prognosis and the like, in that the hematopoietic stem cells and differentiation prepared according to the present invention may be used therefor.
  • the present composition can be used as a medicament, pharmaceutical composition and diagnostic agent, and thus, in particular are useful in the pharmaceutical field.

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Abstract

La présente invention concerne l'identification d'un facteur permettant la détermination de la compatibilité de transplantation et/ou la discrimination efficace de cellules souches. Cette invention a aussi trait à l'objet identifié susmentionné reposant sur le fait que l'endomucine présente per se de manière inattendue une fonction de marqueur destinée à identifier une cellule souche, telle qu'une cellule hématopoïétique, et l'endomucine constitue un marqueur efficace dans la détermination de la compatibilité de transplantation. De ce fait, cette invention a pour objet une composition de marqueur pour cellule souche qui contient un agent spécifique d'une molécule d'acides nucléiques renfermant une séquence d'acides nucléiques codant un polypeptide d'endomucine ou une partie correspondante et similaire.
PCT/JP2004/017655 2003-11-20 2004-11-19 Marqueurs pour cellules souches et compatibilite de transplantation WO2005049865A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012660A2 (fr) * 1999-08-17 2001-02-22 Sagami Chemical Research Center Proteines humaines a domaines hydrophobes et adn codant pour ces proteines
WO2001034628A1 (fr) * 1999-11-12 2001-05-17 Human Genome Sciences, Inc. 35 proteines humaines secretees

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012660A2 (fr) * 1999-08-17 2001-02-22 Sagami Chemical Research Center Proteines humaines a domaines hydrophobes et adn codant pour ces proteines
WO2001034628A1 (fr) * 1999-11-12 2001-05-17 Human Genome Sciences, Inc. 35 proteines humaines secretees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; November 2001 (2001-11), BRACHTENDORF GERTRUD ET AL: "Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta" XP002341890 Database accession no. PREV200100558461 & DEVELOPMENTAL DYNAMICS, vol. 222, no. 3, November 2001 (2001-11), pages 410-419, ISSN: 1058-8388 *

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