JP2006506970A - 神経細胞の延長されたインビトロ培養のための方法および系 - Google Patents
神経細胞の延長されたインビトロ培養のための方法および系 Download PDFInfo
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Abstract
Description
本出願は、米国仮特許出願番号60/395,973(2002年7月12日出願)(その全体が、本明細書中で参考として援用される)の利益を主張する。
(発明の分野)
本発明は、一般に、神経細胞の延長されたインビトロ培養を提供する細胞培養系に関する。本発明は特に、網膜神経細胞の延長された培養に関する。この細胞培養系は、神経変性疾患(特に、網膜の疾患および障害)を処置するために使用され得る生物活性薬剤を同定するために有用である。本発明はまた、網膜変性疾患または網膜変性障害を処置するために有用であり得る細胞を同定するための細胞培養方法を使用することに関する。
一般には、神経細胞の、そして、特に、網膜神経細胞のインビトロ培養が、問題であった。何年にもわたって、完全に成熟なニューロンは、可塑性ならびに損傷後の修復能および再生能を欠くと考えられていた。成熟な中枢神経系(CNS)ニューロンが培養され得、かつ、再生するように刺激され得るなら、損傷または疾患状態のCNS組織の移植および機能の修復が実現可能になり得る。
簡単に述べると、本発明は、生物活性薬剤を同定するために使用され得る神経細胞、および、神経変性疾患(神経変性性網膜疾患および神経変性性網膜障害を含む)の処置に有用な細胞の延長された細胞培養のための組成物および方法を提供する。本発明の1つの局面は、成熟な神経細胞と毛様体から単離された細胞の混合物を含む細胞培養系を提供する。本発明の特定の実施形態において、成熟な神経細胞は、成熟な網膜神経細胞を含み、この成熟な網膜神経細胞は、双極細胞、水平細胞、アマクリン細胞、神経節細胞および/または光受容細胞である。
本発明を詳細に示す前に、以下の用語を定義することが、本発明の理解の助けとなり得る。
神経変性性疾患は、罹患率の主要な原因であり、インビトロ神経細胞培養モデルは、この領域における薬物の発見に有益であり得る。減数***後の神経細胞の培養は、非常に難しいので、神経学的疾患および眼科疾患に関連する薬物をスクリーニングする場合、良好なパラダイムを有することが重要である。以前に記載したように、標的分子の潜在的な薬物候補に対する応答は、標的分子の細胞内環境に依存する傾向がある。従って、スクリーニングアッセイにおいて、最終的に薬物で処置される細胞型に密接に関連する培養細胞を使用することが重要である。
他の群が、胚性網膜ニューロンのインビトロ培養において報告されているが、これらの細胞は、成熟な網膜細胞により発現される網膜特異的なタンパク質の全てを発現しないか、または、これらの細胞は、短い時間だけ培養され得る。X.Luoら(IOVS 42:1096−1106,2001)は、インビトロでの網膜細胞の培養を報告したが、彼らの系は、本発明のインビトロ細胞培養系が、毛様体細胞(または、幹細胞の供給源)との同時培養を含むという点で、本明細書中に開示されるものとは異なった。
本明細書中に記載されるインビトロ細胞培養系は、2ヶ月を超える、成熟な霊長類網膜ニューロンの培養における生存を可能にする。現在までに、成熟な網膜ニューロンを使用して薬物候補をスクリーニングする能力は、霊長類の培養におけるニューロンの寿命までに制限されていた。除核の遅延、および、組織の解離の遅延が、ニューロンの回収および生存に有害な影響を及ぼしている(例えば、Gaudinら、前出を参照のこと)。ニューロンは、神経組織から解離した直後に劣化し始める。結果として生じるニューロンの劣化は、製薬産業による適切な化合物スクリーニングを妨げる。また、現在では、突出ニューロンまたは光受容細胞を分析することは困難である。光受容体は、黄斑変性症において影響を受ける主な細胞型であり、盲目の主要な原因である。神経節細胞は、網膜における突出ニューロンであり、これらの細胞は、緑内障の患者において影響を受け、そしてまた、盲目の主要な原因である。
記載されるものは除いて、全ての化合物および試薬は、Sigma Chemical Corporation(St.Louis,MO)から入手した。
Macaca nemestrinaおよびMacaca fascicularisの眼を、the Tissue Distribution Programにより、the University of Washington(Seattle,WA)のthe Regional Primate Research Centerから入手した。これらの実験における動物の使用は、the National Institute of Healthおよびthe University of Washington Animal Care Committeeにより確立されたガイドラインに従った。4〜17歳のサルを、網膜組織の供給源として使用した(サルは、4歳で完全に成熟する)。ニワトリを、透明なNalgeneケージ中、約25℃にて飼育した。ニワトリに、水およびPurinaニワトリスターターを自由に与え、16時間明、8時間暗(6:00AMに点灯する)のサイクルを維持した。クロロホルム過麻酔を使用してニワトリを屠殺し、眼球摘出した。
摘出した眼を、その赤道に沿って半分に切断し、神経網膜(毛様体を含んでも含まなくてもよい)を、1mM Hepes緩衝液(pH7.4)および2%スクロースを含むHankの緩衝化生理食塩水溶液(HBSS;Gibco BRL)中で、眼の前側表面から解剖した。各網膜を、Ca2+、Mg2+を含まない、HBSS(0.125% トリプシン(Gibco BRL,Invitrogen Life Technologies,Carlsbad,CA)、100U/ml ヒアルロニダーゼ、10U/ml コラゲナーゼ、および0.1mg/ml DnaseIを含有)5ml中、37℃にて15分間インキュベートしながら解離し、その後、5%ウシ胎仔血清(FBS;Gibco BRL)と共に不活性化した。酵素的に解離された細胞を、5mlのプラスチックピペットを用いて10分間摩砕し、次いで、先端熱加工したガラスピペットを用いて20分間摩砕した。解離した細胞を、1500×gにて10分間の遠心分離により回収し、25μg/mlのインシュリン、100μg/mlのトランスフェリン、60μM プトレッシン、30μM セレニウム、20nM プロゲステロン、100U/mlのペニシリン、100μg/mlのストレプトマイシン、0.05M Hepesおよび1% FBSを含有するダルベッコの改変イーグル培地(DMEM)/F12培地(Gibco BRL)中に再懸濁した。細胞を、24ウェルプレートの1ウェルあたり200,000細胞で、ポリD−リジンおよびMatrigel(Becton Dickinson Biosciences,Franklin Lakes,NJ)でコーティングしたカバーガラス上に、プレーティングした。各ウェルの培地の半分を48時間毎に交換した。細胞を37℃および5% CO2にてインキュベートし、14日〜3ヶ月維持した。いくつかの場合において、E6ニワトリ胚性網膜細胞(24ウェルプレートの1ウェルあたり300,000細胞)を、成熟なサル毛様体細胞および網膜細胞をプレーティングする1日前に、コーティングされたカバーガラス上にプレーティングした。簡単にいうと、E6ニワトリ胚性網膜細胞を得るために、卵をH&N International WAから入手し、加湿したインキュベーター内に6日間維持した(胚日数6または「E6」に対応する)。6日目の胚を卵から取り出し、胚性網膜細胞を得て、上記のようにプレーティングした。
培養網膜細胞の免疫細胞化学分析を、当該分野で周知の方法に従って実施した。杆体光受容体を、ロドプシン特異的抗体4D2(1:1000希釈;Dr.R.Moday,University of British Columbia,Vancouver,British Columbia,Canadaから提供された)を用いて標識することにより同定した。Tuj 1抗体(これは、β3−チューブリンを認識し(そして、神経節細胞に特異的である))を使用して、神経節細胞を同定した。リカバリンに対する霊長類固有(すなわち、ニワトリの細胞または組織には結合しない)抗体(1:1000希釈、Dr.J.Hurley,University of Washington,Seattle,WAから提供された)を使用して、霊長類の光受容細胞を同定した。ビシニンに対する抗体を使用して、ニワトリの光受容細胞を同定した。ブロモデオキシウリジン(BrdU、1:80希釈;Developmental Studies Hybridoma Bank,University of Iowa,Iowa City,Iowa)に対する抗体を使用して、BrdU含有細胞を同定した。4’,6−ジアミジノ−2−フェニルインドール(DAPI)で核染色した。一次抗体の免疫反応性を検出するために、Alexa 488結合体化ヤギ抗体またはAlexa 568結合体化ヤギ抗体(Molecular Probes,Eugene,OR)を使用した。Axioplan2顕微鏡(Carl Zeiss,NY)に取り付けられたSpotスライダー−RTカメラ(Diagnostic Instruments,Inc.,MI)を使用して画像を取得した。
成熟な網膜ニューロンを、毛様体の上皮の不在下にて培養した場合、全ての網膜ニューロンが1〜2日で死滅した。毛様体細胞を、成熟な網膜ニューロンと共に同時培養した場合(毛様体の上皮の不在下における成熟なニューロンの培養に使用した条件と同一の条件下で)、種々のニューロンが、実施例2に記載されるような免疫細胞化学技術により同定された。これらの培養物は、延長された期間維持するに従って、網膜ニューロンの持続的な生存が観察された。図1A〜1Cは、インビトロ培養の3ヶ月後の、霊長類の成熟な網膜ニューロンの生存を示す。生存細胞の存在を、DAPIを用いる核染色により確認した(図1Aおよび1Bを参照のこと;例示的な細胞の薄い染色を、白矢印により示した)。標識された抗−β3−チューブリン抗体を用いた免疫染色は、長期の培養における神経節細胞の存在を示す(図1Aおよび1B;例示的な細胞を、黒矢印により示す)。アマクリン細胞および水平細胞を、カルレチニンに特異的な抗体を用いる免疫染色により同定した(図1A;例示的な細胞を円で囲む)。培養物中に存在する光受容細胞を、抗リカバリン抗体および抗ロドプシン抗体を使用する免疫組織化学により同定した(図1C;例示的な細胞を円で囲む)。
Claims (21)
- 成熟な神経細胞および毛様体から単離された細胞の混合物を含む、細胞培養系。
- 前記成熟な神経細胞が成熟な網膜神経細胞を含む、請求項1に記載の細胞培養系。
- 前記成熟な網膜神経細胞が、双極細胞、水平細胞、アマクリン細胞、神経節細胞および光受容細胞からなる群から選択される、請求項2に記載の細胞培養系。
- 成熟な網膜神経細胞および毛様体から単離された細胞の混合物を含む、網膜細胞培養系。
- 前記成熟な網膜神経細胞が、双極細胞、水平細胞、アマクリン細胞、神経節細胞および光受容体からなる群から選択される、請求項4に記載の網膜細胞培養系。
- 成熟な網膜神経細胞および毛様体から単離された細胞の混合物を含む網膜細胞培養系であって、該成熟な網膜神経細胞は、双極細胞、水平細胞、アマクリン細胞、神経節細胞および光受容細胞からなる群から選択される、網膜細胞培養系。
- (i)成熟な網膜神経細胞;(ii)毛様体から単離された細胞;および(iii)胚性網膜細胞の混合物を含む、網膜細胞培養系。
- 前記胚性網膜細胞が網膜幹細胞を含む、請求項7に記載の細胞培養系。
- 前記胚性網膜細胞が胚性網膜前駆細胞を含む、請求項7に記載の細胞培養系。
- 前記成熟な網膜神経細胞が、双極細胞、水平細胞、アマクリン細胞、神経節細胞および光受容細胞からなる群から選択される、請求項7に記載の細胞培養系。
- 成熟な網膜神経細胞と、毛様体から単離された細胞とを同時に培養する工程を包含する、網膜細胞培養系を生成するための方法。
- 成熟な網膜神経細胞と、毛様体から単離された細胞とを同時に培養する工程を包含する、インビトロにおいて成熟な網膜神経細胞の生存を増強するための方法。
- (i)成熟な網膜神経細胞;(ii)毛様体から単離された細胞;および(iii)胚性網膜細胞を同時に培養する工程を包含する、請求項11または請求項12のいずれかに記載の方法。
- 前記胚性網膜細胞が、網膜幹細胞および胚性網膜前駆細胞からなる群から選択される、請求項13に記載の方法。
- 神経細胞の生存を増強し得る生物活性薬剤を同定するための方法であって、(i)請求項1〜10のいずれか1項に記載の細胞培養系の神経細胞と、候補薬剤との間の相互作用を許容する条件下で、かつ、該相互作用を許容するのに十分な時間、該候補薬剤と該細胞培養系とを接触させる工程;および(ii)該候補薬剤の存在下での該細胞培養系の神経細胞の生存と、該候補薬剤の不在下での該細胞培養系の神経細胞の生存とを比較し、そこから、該神経細胞の生存を増強し得る生物活性薬剤を同定する工程、を包含する、方法。
- 神経細胞の神経変性を阻害し得る生物活性薬剤を同定するための方法であって、(i)請求項1〜10のいずれか1項に記載の細胞培養系の神経細胞と候補薬剤との間の相互作用を許容にする条件下で、かつ、該相互作用を許容するのに十分な時間、該生物活性薬剤と該細胞培養系とを接触させる工程;および(ii)該生物活性薬剤の存在下での該細胞培養系の神経細胞の構造と、該生物活性薬剤の不在下での該細胞培養系の神経細胞の構造とを比較し、そこから、該神経細胞の神経変性を阻害し得る生物活性薬剤を同定する工程、を包含する、方法。
- 網膜疾患を処置し得る生物活性薬剤を同定するための方法であって、請求項1〜10のいずれか1項に記載の細胞培養系の神経細胞と候補薬剤との間の相互作用を許容する条件下で、かつ、該相互作用を許容するのに十分な時間、該生物活性薬剤と該細胞培養系とを接触させる工程;および(ii)該生物活性薬剤の存在下での該細胞培養系の神経細胞の神経変性と、該生物活性薬剤の不在下での該細胞培養系の神経細胞の神経変性とを比較し、そこから、網膜疾患を処置し得る生物活性薬剤を同定する工程、を包含する、方法。
- 前記神経細胞が網膜神経細胞である、請求項15〜17のいずれか1項に記載の方法。
- 前記網膜疾患が、黄斑変性症、緑内障、糖尿病性網膜症、網膜剥離、網膜血管閉塞、網膜色素変性症およびアルツハイマー病に関連する網膜障害からなる群から選択される、請求項17に記載の方法。
- 単離された網膜幹細胞の導入を必要とする被験体の網膜組織に、該単離された網膜幹細胞を導入する工程を包含する、網膜疾患を処置するための方法。
- 前記網膜疾患が、黄斑変性症、緑内障、糖尿病性網膜症、網膜剥離、網膜血管閉塞、網膜色素変性症およびアルツハイマー病に関連する網膜障害からなる群から選択される、請求項20に記載の方法。
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WO2001058460A1 (en) * | 2000-02-11 | 2001-08-16 | The Schepens Eye Research Institute, Inc. | Isolation and transplantation of retinal stem cells |
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WO2004007749A2 (en) | 2004-01-22 |
US20110044957A1 (en) | 2011-02-24 |
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CA2493849A1 (en) | 2004-01-22 |
AU2003249227A8 (en) | 2004-02-02 |
EP1551956A4 (en) | 2006-12-27 |
US7312025B2 (en) | 2007-12-25 |
WO2004007749A3 (en) | 2005-05-06 |
US20040147019A1 (en) | 2004-07-29 |
US20080175826A1 (en) | 2008-07-24 |
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