JP4971131B2 - 再生医学に適した高純度心筋細胞調製物を作製するための方法 - Google Patents
再生医学に適した高純度心筋細胞調製物を作製するための方法 Download PDFInfo
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Description
本出願は、2004年3月19日に提出された米国特許出願第10/805,099号(Geron整理番号第099/004号);2004年3月26日に提出された米国特許出願第60/556,722号(第099/005x号);および2005年2月3日に提出された米国特許出願第60/650,194号(第099/030x号)の優先権の恩典を主張する。
再生医学における研究の中心となる課題は、心機能を再構成するために役立ちうる細胞組成物を開発することである。男女5人にほぼ1人が何らかの型の心血管疾患を有すると推定される(米国国立健康栄養研究所調査III、1988〜94、米国疾病管理センターおよび米国心臓協会)。広範な病態には、心血管心疾患(集団の5%)、先天性心血管欠損(0.5%)、およびうっ血性心不全(3%)が含まれる。薬学の技術分野によって、心疾患の結果として起こる損傷を制限するために役立ちうる低分子薬および生物学的化合物が生成されたが、損傷した組織を再生するために役立つ市販の化合物はない。
本発明は、インビトロで増殖することができ、そしてインビトロまたはインビボで治療的に有用な表現型へとさらに分化することができる最終段階の心筋細胞または心筋細胞前駆体のいずれかである心筋細胞系列細胞の比率が非常に高められた分化細胞集団へと、ヒト起源の多能性幹細胞を分化させるためのシステムを提供する。
本発明は、霊長類の多能性幹細胞から心筋細胞およびその前駆細胞を調製および特徴付けするための改善されたプロトコール、技術、および試薬を提供する。
本発明の技術および組成物は、pPS由来心筋細胞およびその前駆体に関する。心筋細胞の表現型特徴は、本開示の後の章に提供される。心筋細胞前駆体に関して決定的である特定の特徴はないが、通常の個体発生の過程において、非分化型pPSは最初に中胚葉細胞に分化した後、様々な前駆体段階を通して機能的な(最終段階)心筋細胞へと分化すると認識されている。
本発明の実践において有用な全般的技術にさらに手を加えるために、実践者は細胞生物学、組織培養、発生学、および心臓生理学における標準的なテキストおよび論評を参照することができる。
本発明は、様々なタイプの多能性幹細胞、特に、先に記述したように胚組織に由来して、三つの胚芽層の全ての子孫を産生することができる特徴を有する幹細胞に関して実践することができる。
胚幹細胞は、霊長類種の胚盤胞から単離することができる(米国特許第5,843,780号;Thomson, et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995)。ヒト胚幹(hES)細胞は、Thomsonら(米国特許第6,200,806号;Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998)およびReubinoffら(Nature Biotech. 18:399, 2000)によって記述された技術を用いてヒト胚盤胞細胞から調製することができる。hES細胞と同等の細胞タイプには、国際公開公報第01/51610号(Bresagen)に概要されるように、原始外胚葉様(EPL)細胞のような、その多能性誘導体が含まれる。
pPS細胞を、分化を阻害しながら増殖を促進する培養条件を用いて培養において持続的に増殖させることができる。例としての血清含有ES培地は、80%DMEM(ノックアウトDMEM、Gibcoのような)、20%のディファインドウシ胎児血清(FBS, Hyclone)または血清代用剤(US 2002/0076747 A1, Life Technologies Inc.)のいずれか、1%非必須アミノ酸、1 mM L-グルタミンおよび0.1 mMβ-メルカプトエタノールで構成される。
心筋細胞系列細胞は、所望の表現型を有する細胞の比率を高める特殊な増殖環境において培養または分化させることによって、非分化型の幹細胞から得ることができる(所望の細胞の増殖によって、または他の細胞タイプの阻害もしくは死滅によって)。
・トランスフォーミング増殖因子β関連リガンド(TGF-β1、TGF-β2、TGF-β3、および下記のTGF-βスーパーフェミリーの他のメンバーによって示される)。TGF-β受容体に結合するリガンドは、I型およびII型セリンキナーゼを活性化して、Smadエフェクターの燐酸化を引き起こす。
・モルフォゲン様アクチビンAおよびアクチビンB(TGF-βスーパーファミリーのメンバー)
・インスリン様増殖因子(IGFIおよびIGFIIのような)
・骨形態形成タンパク質(TGF-βスーパーファミリーメンバー、BMP-2およびBMP-4によって示される)
・線維芽細胞増殖因子(bFGF、FGF-4、およびFGF-8によって示される)、細胞質キナーゼraf-1、およびマイトゲン活性化タンパク質キナーゼ(MAPK)を活性化する他のリガンド、ならびに上皮細胞増殖因子(EGF)のような他のマイトゲン
・DNAメチル化および心筋細胞関連遺伝子の発現を変化させるヌクレオチド類似体(例えば、5-アザ-デオキシ-シチジン)
・下垂体ホルモンオキシトシン、または酸化窒素(NO)
・同じ受容体に関するアゴニスト活性を有する特異的抗体または合成化合物。
既に述べたように、pPS細胞の分化の模範例は従来、異なる細胞タイプ間の相互干渉を可能にする胚様体を形成する段階を含み、胚を暗示するように組織形成を促進すると考えられる。しかし、胚様体を形成する必要がないことがしばしば都合がよく、それによって分化プロセスはより制御され、得られた細胞集団はより均一になる傾向がある(国際公開公報第01/51616号;US 2002/0151053 A1)。本開示は、胚様体を形成することなく、および血清または血清補助物質を用いることなくhES細胞を心筋細胞へと直接分化させるための新規方法を提供する。
pPS由来心筋細胞の調製物は、カーディアックボディズ(商標)と呼ばれるクラスターにおいてそれらを増殖させることによってさらに増殖または比率を高めることができることも同様に発見されている。
本発明の技術に従って得られる細胞は、多くの表現型基準に従って特徴を調べることができる。pPS細胞株に由来する心筋細胞および前駆細胞は、しばしば他の起源からの心筋細胞の形態学的特徴を有する。それらは紡錘形、丸形、三角形、または多角形の形状となりえて、それらは免疫染色によって検出可能なサルコメリック構造の特徴を有する線条を示すことがある(図1)。それらは平坦なシート状の形をとることがあり、または基質に結合するもしくは懸濁液において浮遊するクラスターの形であってもよく、電子顕微鏡によって調べた場合に典型的なサルコメアおよび心房顆粒を示す。
・横紋筋収縮を制御するためのカルシウム感受性分子スイッチを提供するトロポニン複合体のサブユニットである心トロポニンI(cTnI)
・心トロポニンT(cTnT)
・発達中の心臓において持続する初期マウス胚発達の際の心中胚葉において発現される心転写因子であるNkx2.5
・発達中の心臓および胎児心筋細胞において発現されるが、成人においてダウンレギュレートされているホルモンである心房性ナトリウム利尿因子(ANF)。これは、心細胞において非常に特異的に発現されるが、骨格筋細胞には発現されないことから、心筋細胞の良好なマーカーであると考えられる
・ミオシン重鎖(MHC)、特に心特異的であるβ鎖
・タイチン、トロポミオシン、α-サルコメリックアクチニン、およびデスミン
・心中胚葉において高度に発現されて、発達中の心臓において持続する転写因子であるGATA-4。これは多くの心遺伝子を調節し、心臓発生において役割を有する
・MEF-2A、MEF-2B、MEF-2C、MEF-2D;心中胚葉において発現され、発達中の心臓において持続する転写因子。
・心細胞において接着を媒介するN-カドヘリン。
・心筋細胞間のギャップ接合を形成するコネキシン43。
・β1-アドレナリン受容体(β1-AR)。
・心筋梗塞後に血清中で上昇するクレアチニンキナーゼMB(CK-MB)およびミオグロビン。
・α心アクチン、初期増殖反応-1、およびサイクリンD2。
本発明の細胞は、分化の前後いずれかで細胞を遺伝子操作することによって、一つまたはそれ以上の遺伝子の変化を含むように調製することができる(US2002/0168766 A1)。細胞は、任意の適した人工的な操作手段によってポリヌクレオチドが細胞に移入されている場合、または細胞がポリヌクレオチドを遺伝している当初改変された細胞の子孫である場合、に「遺伝子改変」されていると言われる。例えば、細胞は、それらが発達的に限定された系列の細胞または最終分化細胞へと進行する前または後に、テロメラーゼ逆転写酵素を発現するように細胞を遺伝子改変することによって、その複製能を増加するように処理することができる(US 2003/0022367 A1)。
本発明は、心筋細胞系列細胞を大量に産生する方法を提供する。これらの細胞集団は、多くの重要な研究、開発、および商業的目的のために用いることができる。
本発明の心筋細胞は、そのような細胞およびその様々な子孫の特徴に影響を及ぼす因子(溶媒、低分子薬、ペプチド、オリゴヌクレオチドのような)または環境的条件(培養条件または操作のような)をスクリーニングするために商業的に用いることができる。
本発明はまた、代謝機能における生来の欠損、疾患状態の影響、または有意な外傷の結果のような、任意の認められた必要性に関して心筋の組織の維持または修復を増強するために、心筋細胞およびその前駆体を用いることを提供する。
適当な試験の後、本発明の分化細胞を、そのような処置を必要とするヒト患者または他の対象における組織再構成または再生のために用いることができる。意図される組織部位へそれらを移植または遊走させて、機能的に欠損領域を再構成または再生させるように、細胞を投与する。心室、心膜、または心筋内部の所望の位置に直接、心機能を再構成することができる細胞を投与するように適合させた特殊な装置が利用可能である。
実施例1:hES細胞の心筋細胞への分化
hES細胞株、H1、H7、H9、およびH9.2(H9に由来するクローン株)は、国際公開公報第01/51616号に記述されるように、当初フィーダー細胞上で確立され、その後無フィーダー条件で維持された。分化は、hES細胞懸濁液を培養して胚様体を形成させることによって開始した。懸濁培養において4日後、EBをゼラチンコーティングプレートまたはチャンバースライドガラスに移した。拍動する心筋細胞を、分化15〜29日目にEB増殖物から機械的に単離して、回収して洗浄した。調べたhES細胞株は全て、50代より多い継代回数(集団倍加〜260)維持した後でさえも拍動する心筋細胞の産生能を有したが、いくつかの株(例えば、H7)は他より多く産生した。
H1またはH9細胞株からの胚様体を、DNAメチル化に影響を及ぼすシトシン類似体である5-アザ-デオキシ-シチジンによって分化1〜4、4〜6、または6〜8日目に処置した。細胞を15日目に回収して、心臓MHCに関してリアルタイムRT-PCRによって分析した。1〜10 μM 5-アザ-デオキシ-シチジンを6〜8日目に処置すると、心臓α-MHCの発現が有意に増加して、培養における拍動領域の比率の増加と相関した。
本実施例は、ヒトES細胞の心筋細胞分化に影響を及ぼす増殖因子添加の併用効果を調べる。
胚様体を4日間形成させた後、ゼラチンコーティングプレートにおいて17日間増殖させることによって(5-アザ-デオキシ-シチジンおよび増殖因子を用いなかった)、心筋細胞をH7株のhES細胞から生成した。細胞をコラゲナーゼBを用いて解離させて、分化培地中で再懸濁した。細胞懸濁液をPercoll(商標)の不連続勾配にローディングして、1500 gで30分間遠心した。画分4個を得た:I、上部界面;II、40.5%層;III、下界面;IV、58.5%層。細胞を洗浄して、分化培地で再懸濁した。免疫染色のための細胞をチャンバースライドガラスに104個/ウェルでプレーティングして、2または7日間培養した後、固定および染色した。
本実施例において、H7細胞株のhES細胞を、基質上にプレーティングして、分化因子を含む無血清培地において培養することによって、心筋細胞系列細胞に分化させた。
本実施例は、治療的用途および他の目的にとって望ましい特徴を有する細胞の比率を高めるために、心筋細胞クラスターのカーディアックボディズ(商標)としてのその後の培養を説明する。
本実施例において、心筋細胞の分化培養は、Percoll(商標)分離およびカーディアックボディズ(商標)形成の前に異なる期間行った。
インビボでのカーディアックボディズの生存能を評価するために、H7-由来細胞を、成体ヌードラットの非損傷心臓に移植した。H7 hES細胞を用いて、20%FBS含有培地において胚様体を形成した。胚様体を懸濁培養において4日間培養した後、ゼラチンコーティングフラスコに接着させ、胚様体をそこでさらに2週間培養した。カーディアックボディズ(商標)を、上記のように調製して、2〜3日毎に培地交換しながら20%FBS含有培地において1週間懸濁培養において維持した。
Claims (24)
- 以下の段階を下記の順序で含む、霊長類の多能性幹(pPS)細胞から心筋細胞系列細胞を得る方法:
a)それに対して心筋細胞系列細胞が接着する基質を有する固体表面に、胚様体を形成せずにヒト胚盤胞由来のpPS細胞株からの非分化細胞を直接プレーティングする段階;
b)プレーティングしたpPS細胞を基質上で確立させる段階;
c)血清またはフィーダー細胞の非存在下であるがアクチビンおよび骨形態形成タンパク質の存在下で、確立された細胞を培養する段階。 - 段階a)におけるpPS細胞が、確立されたヒト胚幹細胞株である、請求項1記載の方法。
- 細胞が、段階b)においてゼラチンまたはフィブロネクチンによってコーティングされた基質上にプレーティングされる、請求項1または2記載の方法。
- 細胞が、段階c)において、基質上で非分化pPS細胞として4日間またはそれ以上培養することによって確立される、請求項1〜3のいずれか一項記載の方法。
- 細胞が、段階d)において、アクチビンAおよびBMP-4と共に4日間またはそれ以上培養される、請求項1〜4のいずれか一項記載の方法。
- 細胞が、段階e)において、アクチビンまたは骨形態形成タンパク質のいずれかの非存在下で1週間またはそれ以上培養される、請求項1〜5のいずれか一項記載の方法。
- 細胞が、段階d)および段階e)において、BMP-2の非存在下であるが、インスリン、トランスフェリン、およびセレンを含む培地補助物質の存在下で培養される、請求項1〜6のいずれか一項記載の方法。
- 段階g)における細胞比率を高める段階が以下の段階を含む、請求項1〜7のいずれか一項記載の方法:
回収された細胞をその密度に従って分画する段階;および、
次に内因性の遺伝子からMHCを発現する画分を採取する段階。 - 以下の段階をさらに含む、請求項1〜8のいずれか一項記載の方法:
a)細胞比率が高められた細胞集団において単細胞として存在する細胞を、クラスターとして存在する細胞から分離する段階;
b)クラスターとして存在する細胞を栄養培地において再懸濁する段階;
c)再懸濁した細胞を栄養培地において再培養する段階;および
d)再培養した細胞を採取および洗浄して、それによってクラスターの少なくとも50%が自発的収縮を行う細胞クラスターを作製する段階。 - クラスター形成細胞を懸濁液から沈降させ、懸濁液に残っている細胞を除去することによって、単細胞がクラスター形成細胞から分離される、請求項9記載の方法。
- 再懸濁した細胞を培養する栄養培地が20%の血清または血清代用剤を含む、請求項9または10記載の方法。
- 細胞を分離、再懸濁、および再培養する段階を3回またはそれ以上含む、請求項9〜11のいずれか一項記載の方法。
- クラスターの少なくとも80%が再培養後自発的収縮を行う、請求項9〜12のいずれか一項記載の方法。
- 細胞クラスターがゼラチンまたはMatrigel(登録商標)によってコーティングされた表面にプレーティングされる、請求項13記載の方法。
- クラスターの少なくとも50%が自発的収縮を行う細胞クラスターを、単細胞および/またはより小さい細胞クラスターの懸濁液に分散させる段階をさらに含む、請求項9記載の方法。
- 以下を含む、ヒト胚盤胞細胞由来の心筋細胞系列細胞の産生の際に培養される複数の細胞集団:
a)pPS細胞株からの非分化細胞;および
b)請求項1〜9のいずれか一項記載の方法に従ってpPS細胞株から分化した心筋細胞系列細胞の集団。 - クラスターの少なくとも50%が自発的収縮を行う細胞クラスターが自発的収縮を行う細胞のクラスターとして定義され、心トロポニンI(cTnI)、心トロポニンT(cTnT)、または心房性ナトリウム利尿因子(ANF)、または内因性遺伝子由来のα-心臓ミオシン重鎖(MHC)を発現する多数の細胞を含む、請求項9記載の方法に従って得られたクラスターの少なくとも50%が自発的収縮を行う細胞クラスターの組成物。
- 哺乳類対象に対する組成物の投与によって、対象においてクラスターの少なくとも50%が自発的収縮を行う細胞クラスター由来の細胞の生存および移植が可能となるように、賦形剤で製剤化された請求項16または17記載の心筋細胞系列細胞またはクラスターの少なくとも50%が自発的収縮を行う細胞クラスター。
- 心疾患の処置における組成物の使用に関する情報と共に包装される、請求項16〜18のいずれか一項記載の細胞。
- 心疾患を処置するための薬剤の調製における、請求項1〜15のいずれか一項記載の方法に従って得られた細胞の使用。
- 心臓の筋系またはその周囲に細胞集団を投与するように適合した装置において、請求項1〜15のいずれか一項に従って得られた細胞を含む、心疾患を処置するための産物。
- 請求項1〜15のいずれか一項に従って得られた細胞を、化合物を含む培地と混合する段階、および細胞に及ぼす化合物の影響を決定する段階を含む、心細胞に及ぼす化合物の作用に関して化合物をスクリーニングする方法。
- 化合物が細胞に対して有毒であるかどうかを決定する段階を含む、請求項22記載の方法。
- 化合物が細胞の拍動回数を増加または減少させるかどうかを決定する段階を含む、請求項22または23記載の方法。
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CA (1) | CA2559854C (ja) |
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WO (1) | WO2005090558A1 (ja) |
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US7410798B2 (en) | 2001-01-10 | 2008-08-12 | Geron Corporation | Culture system for rapid expansion of human embryonic stem cells |
WO2005118784A1 (en) * | 2004-06-01 | 2005-12-15 | Es Cell International Pte Ltd | Improved cardiomyocyte differentiation |
WO2006015127A2 (en) | 2004-07-30 | 2006-02-09 | Mayo Foundation For Medical Education And Research | Treating cardiovascular tissue |
GB2441488C (en) | 2005-06-22 | 2011-10-05 | Geron Corp | Suspension culture of human embryonic stem cells |
JP5560393B2 (ja) * | 2005-06-22 | 2014-07-23 | アステリアス バイオセラピューティクス インコーポレイテッド | 心筋細胞系列細胞への霊長類多能性幹細胞の分化 |
EP1962719A4 (en) | 2005-08-29 | 2011-05-04 | Technion Res And Dev Of Foundation Ltd | MEDIA FOR BREEDING STEM CELLS |
AU2012227337B2 (en) * | 2006-01-31 | 2014-10-09 | Daiichi Sankyo Company, Limited | A method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses |
JP5312804B2 (ja) * | 2006-01-31 | 2013-10-09 | 第一三共株式会社 | 幹細胞及び胎児由来の心筋細胞及び予定心筋細胞の精製方法 |
US9765298B2 (en) | 2006-07-24 | 2017-09-19 | Mayo Foundation For Medical Education And Research | Methods and materials for providing cardiac cells |
DK3441459T3 (da) | 2006-08-02 | 2021-06-07 | Technion Res & Dev Foundation | Fremgangsmåder til ekspansion af embryonale stamceller i en suspensionskultur |
WO2008034929A1 (es) * | 2006-09-19 | 2008-03-27 | Fundación Progreso Y Salud | Método para el cultivo y mantenimiento de células troncales pluripotenciales y de células progenitoras de mamífero en estado no diferenciado |
CA2665183A1 (en) * | 2006-10-02 | 2008-04-10 | Cellartis Ab | Novel toxicity assay based on human blastocyst-derived stem cells and progenitor cells |
EP2172542B1 (en) * | 2007-07-31 | 2017-03-22 | Daiichi Sankyo Company, Limited | Method for constructing mass of myocardial cells and use of the myocardial cell mass |
EP2028268A1 (en) * | 2007-08-20 | 2009-02-25 | Université Libre De Bruxelles | Generation of neuronal cells from pluripotent stem cells |
WO2009145761A1 (en) | 2008-05-27 | 2009-12-03 | Mayo Foundation For Medical Education And Research | Methods and materials for using cells to treat heart tissue |
EP2499236B1 (en) | 2009-11-12 | 2020-01-01 | Technion Research & Development Foundation Ltd. | Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state |
EP3858979A1 (en) | 2010-06-13 | 2021-08-04 | Institute of Biophysics Chinese Academy of Sciences | Methods and compositions for preparing cardiomyocytes from stem cells and uses thereof |
AU2011293052A1 (en) | 2010-08-27 | 2013-03-28 | University Health Network | Methods for enriching pluripotent stem cell-derived cardiomyocyte progenitor cells and cardiomyocyte cells based on SIRPA expression |
KR101364965B1 (ko) * | 2010-10-06 | 2014-02-18 | (주)차바이오앤디오스텍 | 인간 배아줄기세포 유래 심근세포의 배양 및 정제 방법 |
EP2691512B1 (en) * | 2011-03-29 | 2019-05-01 | Asterias Biotherapeutics, Inc. | Enriched populations of cardiomyocyte lineage cells from pluripotent stem cells |
EP2594635A1 (en) * | 2011-11-18 | 2013-05-22 | Univercell Biosolutions | Method for generating primate cardiovascular progenitor cells for clinical and drug cells testing use from primate embryonic stem cells or embryonic-like state cells, and their applications |
US20140336074A1 (en) * | 2011-12-06 | 2014-11-13 | Singapore Health Services Pte Ltd | Method for preparing improved cardiomycocyte-like cells |
US20150184129A1 (en) | 2012-07-17 | 2015-07-02 | Kyoto University | Novel cardiomyocyte marker |
EP2891712A4 (en) | 2012-07-23 | 2016-04-06 | Inst Biophysics Cn Acad Sci | METHOD OF INDUCING THE DIFFERENTIATION OF PLURIPOTENT STEM CELLS IN VITRICULAR MYOCYTES IN VITRO |
FR3024462A1 (fr) * | 2014-07-29 | 2016-02-05 | Univ Pierre Et Marie Curie Paris 6 | Procede de production in vitro de progeniteurs adipocytaires et d'adipocytes |
JP2017108705A (ja) * | 2015-12-18 | 2017-06-22 | 国立大学法人京都大学 | 心筋細胞の製造方法 |
RU2749986C1 (ru) * | 2020-11-27 | 2021-06-21 | Государственное бюджетное учреждение здравоохранения Московской области "Московский областной научно-исследовательский клинический институт им. М.Ф. Владимирского" (ГБУЗ МО МОНИКИ им. М.Ф. Владимирского) | Способ выделения кардиомиоцитов из ткани сердца человека |
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US6667176B1 (en) * | 2000-01-11 | 2003-12-23 | Geron Corporation | cDNA libraries reflecting gene expression during growth and differentiation of human pluripotent stem cells |
IL154159A0 (en) * | 2000-08-01 | 2003-07-31 | Yissum Res Dev Co | Directed differentiation of ebryonic cells |
KR20040022448A (ko) * | 2001-07-12 | 2004-03-12 | 제론 코포레이션 | 인간 다분화능 줄기 세포로부터 제조된 심근 세포 계통의세포 |
US20030232430A1 (en) * | 2001-11-26 | 2003-12-18 | Advanced Cell Technology | Methods for making and using reprogrammed human somatic cell nuclei and autologous and isogenic human stem cells |
US7247477B2 (en) * | 2002-04-16 | 2007-07-24 | Technion Research & Development Foundation Ltd. | Methods for the in-vitro identification, isolation and differentiation of vasculogenic progenitor cells |
CN1471971A (zh) * | 2003-06-20 | 2004-02-04 | 中山大学 | 重组人粒细胞集落刺激因子在制备治疗心肌梗死的药物中的应用 |
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EP1725654A1 (en) | 2006-11-29 |
EP1725654B1 (en) | 2019-05-01 |
AU2005224670B2 (en) | 2010-11-11 |
JP2007529227A (ja) | 2007-10-25 |
CN1969040B (zh) | 2014-12-10 |
CA2559854C (en) | 2014-12-02 |
CN1969040A (zh) | 2007-05-23 |
WO2005090558A1 (en) | 2005-09-29 |
GB2427873B (en) | 2008-09-10 |
GB0619719D0 (en) | 2006-11-15 |
AU2005224670A1 (en) | 2005-09-29 |
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CA2559854A1 (en) | 2005-09-29 |
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