Top > Search of International Patents > TECHNIQUE FOR ISOLATING/CULTURING COLORECTAL EPITHELIAL STEM CELL, AND TECHNIQUE FOR TRANSPLANTING COLORECTAL EPITHELIUM EMPLOYING SAID TECHNIQUE

TECHNIQUE FOR ISOLATING/CULTURING COLORECTAL EPITHELIAL STEM CELL, AND TECHNIQUE FOR TRANSPLANTING COLORECTAL EPITHELIUM EMPLOYING SAID TECHNIQUE

Foreign code F130007364
File No. S2012-0054-N0
Posted date May 15, 2013
Country WIPO
International application number 2012JP006897
International publication number WO 2013061608
Date of international filing Oct 26, 2012
Date of international publication May 2, 2013
Priority data
  • P2011-236469 (Oct 27, 2011) JP
Title TECHNIQUE FOR ISOLATING/CULTURING COLORECTAL EPITHELIAL STEM CELL, AND TECHNIQUE FOR TRANSPLANTING COLORECTAL EPITHELIUM EMPLOYING SAID TECHNIQUE
Abstract The purpose of the present invention is to provide: a culture medium for the in vitro culture of a colorectal epithelial stem cell or the like; a method for culturing a colorectal epithelial stem cell or the like in vitro using the culture medium; a prophylactic/therapeutic agent for bowel diseases, which comprises a colorectal epithelial stem cell or the like produced by culturing by the aforementioned method; a method for administering, i.e., transplanting, a colorectal epithelial stem cell or the like produced by culturing by the aforementioned method to a patient suffering from a bowel disease; and a method for isolating a colorectal epithelial stem cell or the like. The culture medium, the prophylactic/therapeutic agent and the methods are characterized by using a culture medium for the in vitro culture of a colorectal epithelial stem cell and/or a colorectal epithelial cell, which comprises serum albumin, Wnt3a and r-spondin-1.
Outline of related art and contending technology BACKGROUND ART
In the treatment of diseases in the digestive tract of the adult stem cells for possible therapeutic applications which are of interest. Stomach, small intestine and colon epithelial cells have the ability to generate the ability to self-replicate and gastrointestinal epithelial stem cells, which are parts of the digestive tract play an important role in the maintenance of tissue homeostasis (non-patent document 1-3) play. A recent study, stem cells of the gastrointestinal tract is largely increased biological findings are. Wnt, some BMP Notch and a cascade of signal transduction pathways and are simultaneously applied, adjusting the maintenance of stem cells, epithelial homeostasis control (non-patent document 4) are shown. Studies using the tracking system, the target gene is Wnt Lgr5, small intestine and colon crypts (non-patent document 5) as well as the bottom of the stomach (non-patent document 6) unit at the bottom of which is identified as a marker of stem cells.
Current, reliable markers can be recognized by stem cells from the gastrointestinal tract, the gastrointestinal tract many cells with stem cell therapy for the treatment of diseases refractory to fundamentally change being expected. However to this day, gastrointestinal epithelial cells isolated and cultured in vitro after incrementing the, cell therapy to repair the injured tissue used as the material of there is no report. In addition, culture and propagate gastrointestinal epithelial stem cell injury can be restored to a tissue at the site as yet not been found. Mesenchymal and epithelial component a small fragment of an intestinal component over a long period of culture can be a recently (non-patent document 7).
Non-patent document 8 is, colon crypt was isolated and embedded in the matrigel, the destination completely negative fossa culture added, the method comprising culturing a colon epithelial stem cells is described. A completely negative fossa culture point that is, Advanced DMEM/F12 to a basal medium, conditioned medium Wnt3a or Wnt3a, EGF, Noggin, r-spondin-1, (registered trademark) supplement(Invitrogen Corporation) B-27, gastrin, nicotinamide, A83-01 (TGF-β receptor kinase inhibitor 1), and, such as SB202190 (p38 inhibitor) which is a medium was added. (Registered trademark) supplement B-27 precise composition is not disclosed, biotin, carnitine L -, corticosterone, ethanolamine, D (+) galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, -l- chiromin toriodo, vitamin E, vitamin E acetate, bovine albumin, catalase, insulin, superoxide, dismutase, and the like known transferrin (non-patent document 9). Colon epithelial stem cells cultured in vitro in a serum-free medium, gastrin, nicotinamide, A83-01 (TGF-β receptor kinase inhibitor 1), SB202190 (p38 inhibitor), and, an essential component B-27 supplement are believed to, among, and is particularly essential B-27 supplement has been considered.
However, bovine serum albumin (bovine serum albumin; BSA) is, contained a lot in the bovine serum albumin, fatty acids and trace elements function as carriers has been known. Is Wnt3a, and the protein encoded by the gene WNT3A, β - catenin pathways known to activate. - Catenin pathway is Wnt / β -, or proliferation of the stem cell, is related to a maintenance anaplasia is known. R-spondin-1(Roof-plate-specific Spondin-1; Rspo1) is, and the protein encoded by the gene RSPO1, contribute to the differentiation of dorsal neural tube, of promoting proliferation of endothelial cells crypt intestinal anion is known. Epidermal growth factor (Epidermal Growth Factor; EGF) is, and the protein encoded by the gene EGF, promote proliferation of epithelial cells has been known. Hepatocyte growth factor (hepatocyte growth factor; HGF) is, HGF protein encoded by the gene, as well as a hepatocytes, promoting the growth of various cells are known. Noggin is, and the protein encoded by the NOG gene, transforming growth factor β (Transforming growth factor beta; TGF-β) signaling is known to inhibit.
However, the basic medium components (sugars of a carbon source, nitrogen source such as amino acids, inorganic salts) in addition to, serum albumin, Wnt3a, and, using r-spondin-1, culture in serum-free medium, a mammal such as a mouse or a human colon isolated from tissue such as colon epithelial stem cells in vitro for long periods of time, may amplify has not been known at all.
Scope of claims (In Japanese)請求の範囲 [請求項1]
血清アルブミン、Wnt3a、及び、r-spondin-1を含有する、大腸上皮幹細胞及び/又は大腸上皮細胞のインビトロ培養用培地。

[請求項2]
上皮細胞増殖因子(EGF)、肝細胞増殖因子(HGF)、Noggin、インスリン、トランスフェリン、亜セレン酸塩、ピルビン酸ナトリウム、抗酸化剤、コレラ毒素、核酸、セルロプラスミン、ビタミン、及び、血清からなる群から選択される1種又は2種以上をさらに含有する、請求項1に記載の培地。

[請求項3]
血清を含有しない、請求項1又は2に記載の培地。

[請求項4]
大腸上皮幹細胞及び/又は大腸上皮細胞が、哺乳動物の大腸へ移植するためのものである、請求項1~3のいずれか1項に記載の培地。

[請求項5]
請求項1~4のいずれか1項に記載の培地と、細胞外基質とを用いて、大腸上皮幹細胞及び/又は大腸上皮細胞を培養する工程Zを含む、大腸上皮幹細胞及び/又は大腸上皮細胞をインビトロで培養する方法。

[請求項6]
工程Zにおける大腸上皮幹細胞及び/又は大腸上皮細胞として、以下の工程A~Dの工程をその順序で含む方法で単離される大腸上皮幹細胞及び/又は大腸上皮細胞を用いる、請求項5に記載の方法;
工程A:大腸上皮幹細胞及び/又は大腸上皮細胞を含む大腸組織を、哺乳動物の大腸から採取する工程;
工程B:コラゲナーゼ、ディスパーゼ及び還元剤を用いて、大腸組織を消化処理する工程;
工程C:消化処理した大腸組織の組織片を破砕処理する工程;
工程D:破砕処理した大腸組織について濃度勾配遠心法を適用して、大腸上皮幹細胞及び/又は大腸上皮細胞を含む画分を得る工程;

[請求項7]
哺乳動物の大腸へ移植するための大腸上皮幹細胞及び/又は大腸上皮細胞をインビトロで培養する方法である、請求項5又は6に記載の方法。

[請求項8]
請求項5~7のいずれか1項に記載の方法で培養して得られる大腸上皮幹細胞及び/又は大腸上皮細胞を含有する、腸疾患の予防・治療剤。

[請求項9]
以下の工程A~Dの工程をその順序で含む、大腸上皮幹細胞及び/又は大腸上皮細胞の単離方法;
工程A:大腸上皮幹細胞及び/又は大腸上皮細胞を含む大腸組織を、哺乳動物の大腸から採取する工程;
工程B:コラゲナーゼ、ディスパーゼ及び還元剤を用いて、大腸組織を消化処理する工程;
工程C:消化処理した大腸組織の組織片を破砕処理する工程;
工程D:破砕処理した大腸組織について濃度勾配遠心法を適用して、大腸上皮幹細胞及び/又は大腸上皮細胞を含む画分を得る工程;

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPOPRATION TOKYO MEDICAL AND DENTAL UNIVERSITY
  • Inventor
  • WATANABE, Mamoru
  • NAKAMURA, Tetsuya
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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