(19) TZZ Z _T (11) EP 2 270 226 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) G01N 33/569 (2006.01) 18.05.2016 Bulletin 2016/20 G01N 33/68 (2006.01) (21) Application number: 10013575.5 (22) Date of filing: 30.03.2006 (54) Method for distinguishing mesenchymal stem cell using molecular marker and use thereof Verfahren zur Unterscheidung mesenchymaler Stammzellen mittels molekularem Marker und Nutzung dieses Marker Procédé de distinction d’une cellule souche mésenchymateuse en utilisant un marqueur moléculaire et son usage (84) Designated Contracting States: (74) Representative: Müller Hoffmann & Partner AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Patentanwälte mbB HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI St.-Martin-Strasse 58 SK TR 81541 München (DE) (30) Priority: 31.03.2005 JP 2005104563 (56) References cited: EP-A1- 1 475 438 WO-A1-02/46373 (43) Date of publication of application: WO-A1-03/016916 WO-A1-03/106492 05.01.2011 Bulletin 2011/01 WO-A2-2004/025293 WO-A2-2004/044142 JP-A- 2004 290 189 US-A1- 2003 161 817 (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: • DESCHASEAUX FREDERIC ET AL: "Direct 06730606.8 / 1 870 455 selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals (73) Proprietor: Two Cells Co., Ltd their CD45med,low phenotype", BRITISH Minami-ku JOURNAL OF HAEMATOLOGY, Hiroshima City WILEY-BLACKWELL PUBLISHING LTD, GB, vol. Hiroshima 732-0816 (JP) 122, no. 3, 1 August 2003 (2003-08-01), pages 506-517, XP002499042, ISSN: 0007-1048 (72) Inventors: • SEGUCHI T ET AL: "DECREASED EXPRESSION •Kato,Yukio OF FILAGGRIN IN ATOPIC SKIN", ARCHIVES OF Hiroshima-shi DERMATOLOGICAL RESEARCH, SPRINGER, Hiroshima 734-8551 (JP) INTERNATIONAL, BERLIN, DE, vol. 288, 1 • Kawamoto, Takeshi January 1996 (1996-01-01), pages 442-446, Hiroshima-shi XP002950708, ISSN: 0340-3696, DOI: Hiroshima 734-8551 (JP) DOI:10.1007/S004030050080 • Tsuji, Koichiro • "AFFYMETRIX CATALOG; PRODUKT: HUMAN Hiroshima-shi GENOME U133A ARRAY; ARRAY FINDER", Hiroshima 732-0811 (JP) AFFYMETRIX PRODUCT CATALOG, XX, XX, 1 • Igarashi, Akira July 2002 (2002-07-01), page 1, XP002267612, & Saitama 350-0209 (JP) "genecards", , 13 October 2009 (2009-10-13), • Shimizu, Masakazu Retrieved from the Internet: Kyoto-shi URL:http://www.genecards.org/cgi-bin/cardd Kyoto 606-8304 (JP) isp.pl?gene=FLG&search=filaggrin [retrieved on 2011-02-02] Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 270 226 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 270 226 B1 • HIROSHI KUBO ET AL: "Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunohistochemistry", GENES TO CELLS, vol. 14, no. 3, 1 March 2009 (2009-03-01), pages 407-424, XP055104674, ISSN: 1356-9597, DOI: 10.1111/j.1365-2443.2009.01281.x 2 EP 2 270 226 B1 Description TECHNICAL FIELD 5 [0001] The present invention relates to a method for detecting, distinguishing, and separating mesenchymal stem cells, especially, to a method for distinguishing mesenchymal stem cells from connective tissue cells such as fibroblasts, osteoblasts, chondrocytes, adipose cells, etc. by using a gene marker, a protein marker, and/or the like marker for detecting mesenchymal stem cells, the markers being expressed in a different way in mesenchymal stem cells and in the other connective tissue cells. 10 BACKGROUND ART [0002] Mesenchymal stem cells are present in mammalian marrows etc. and known as pluripotential stem cells, which can differentiate into adipose cells, cartilage cells, and bone cells. Due to its pluripotency, mesenchymal stem cells are 15 highly expected as transplantation material for use in regenerative medicine for many kinds of tissues. That is, the use of mesenchymal stem cell enables "regenerative medicine by cell transplantation" for regenerating lost tissues lost due to diseases or impairment and have not been able to be regenerated by a conventional remedy method. More specifically, therapeutic treatments have been started or planed, which are for example, transplantation of marrow mesenchymal stem cells to a patient of lower limb ischemia (Buerger’s disease), transplantation of marrow mesenchymal stem cells 20 to a patient of a periodontal disease, transplantation of marrow mesenchymal stem cells to a patient of osteoarthritis, transportation of amniotic epithelium sheet to burn injured portion, transportation of amniotic stem cells to a patient of diabetes mellitus, and the other transplantation. [0003] In order to use mesenchymal stem cells for regenerative medicine, the stem cells should be collected from a living tissue and then multiplied without differentiation, and the multiplied and undifferentiated stem cells should be 25 induced to differentiate to desired cells in order to prepare tissue for the regenerative medicine. [0004] The inventors of the present invention have reported a method of easily collecting mesenchymal stem cells by separating mesenchymal stem cells from an oral cavity tissue, which method is safe for an individual from which the mesenchymal stem cells are collected (see Patent Citation 1). Moreover, the inventors of the present invention have reported a culturing method, which can give a significantly larger amount of mesenchymal stem cells than can a con- 30 ventional culturing method. The culturing method having been reported by the inventors of the present invention is based on a fact found by the inventors that mesenchymal stem cells can be multiplied at a dramatically fast rate by culturing the mesenchymal stem cells in the presence of an extracellular matrix of a basement membrane or in a medium containing fibroblast growth factor (FGF) etc. and this culturing method can multiply mesenchymal stem cells without the differen- tiating ability thereof (see Patent Citation 2). 35 [0005] These arts are not enough to make the regenerative medicine using the mesenchymal stem cells practically applicable. To speak specifically, for the preparation of the tissue for regenerative medicine by inducing the differentiation of the cultured and multiplied mesenchymal stem cells to desired cells, the cultured cells should be confirmed beforehand that they are mesenchymal stem cells. That is, it is necessary to develop a method of detecting and distinguishing the mesenchymal stem cells after the culturing and multiplication. 40 [0006] To solve this technical problem, the inventors of the present invention have developed a method of effectively identifying and separating mesenchymal stem cells and fibroblast, which are morphologically similar and thus difficult to be distinguish, the method using a gene maker and/or a protein marker for detecting mesenchymal stem cells (see Patent Citation 3). 45 [Patent Citation 1] Japanese Patent Application Publication, Tokukai, No. 2003-52365 (published on February 25, 2003). [Patent Citation 2] Japanese Patent Application Publication, Tokukai, No. 2003-52360 (published on February 25, 2003). [Patent Citation 3] Japanese Patent Application Publication, Tokukai, No. 2005-27579 (published on February 3, 50 2005). WO 03/106492 A1 discloses a marker for mesenchymal stem cells (MSC) comprising an integrin alpha 10 chain and/or an integrin alpha 11 chain expressed on the cell surface of or intracellular in a MSC. The marker is used in methods for identification of mammalian MSC and in methods for isolation of MSC. Also included are isolated cellular populations 55 of mammalian MSC and a cellular composition comprising the latter. Moreover, uses of said marker for isolation, mod- ulation and identification of mammalian MSC are disclosed. 3 EP 2 270 226 B1 DISCLOSURE OF INVENTION [Technical Problems] 5 [0007] As described above, mesenchymal stem cells, which differentiate to bones, cartilages, fats, muscles, ten- dons/ligaments, nerves, etc., have been highly expected to be applicable to the regenerative medicine as cells for transplantation to remedy impairment of these tissues. Conventionally, the confirmation of the mesenchymal stem cells can be carried out in vitro or by providing the differentiation ability thereof in vivo. The practical use of the tissue regen- erative medicine of the mesenchymal stem cells cannot be 10 DISCLOSURE OF INVENTION [Technical Problems] 15 [0008] As described above, mesenchymal stem cells, which differentiate to bones, cartilages, fats, muscles, ten- dons/ligaments, nerves, etc., have been highly expected to be applicable to the regenerative medicine as cells for transplantation to remedy impairment of these tissues. Conventionally, the confirmation of the mesenchymal stem cells can be carried out in vitro or by providing the differentiation ability thereof in vivo. The practical use of the tissue regen- erative medicine of the mesenchymal stem cells cannot be attained without exact, accurate, and easy method to confirm 20 that the cells are mesenchymal stem cells and the mesenchymal stem cells keep its pluripotentcy. [0009] It is true that the method disclosed in Patent Citation 3 is sufficient to identify and distinguish the mesenchymal stem cells and fibroblast. However, bone marrows etc. contain many other connective tissue cells other than fibroblasts, such as osteoblasts, chondrocytes, adipose cells, etc. [0010] Therefore, the art to distinguish the mesenchymal stem cells from fibroblast is not enough to realize practical 25 regenerative medicine using the mesenchymal stem cells. Accordingly, there have been a high demand to develop an art to distinguish and separate the undifferentiated mesenchymal stem cells from the other connective tissue cells such as fibroblasts, osteoblasts, chondrocytes, adipose cells, etc.