JP4891884B2 - 分化中の胚幹細胞、成体幹細胞および胚生殖系細胞を細胞特異的および成長特異的に選択するためのシステム - Google Patents
分化中の胚幹細胞、成体幹細胞および胚生殖系細胞を細胞特異的および成長特異的に選択するためのシステム Download PDFInfo
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Description
1. 心臓α-MHCプロモーターにより制御されるピューロマイシン耐性遺伝子(α-MHC-pur);および
2. 心臓α-MHCプロモーターにより制御される強化緑色蛍光タンパク質の遺伝子(強化緑色蛍光タンパク質=EGFP)(α-MHC-EGFP)。
1. 第一に、ES細胞を簡単に二重トランスフェクトできることは、驚くべきものであった。本発明者らの実験により、トランスフェクトされたクローンのほとんどでは、両方の構築物を用いた効率的なトランスフェクションが行なわれたことが実証された。
2. さらに、抗生物質耐性の効率には、分化の初期、特に心臓細胞の分化の初期に選択剤を添加するのが重要であることが判明した。これにより、例えばインビトロにおける心筋発生効率が増加すると考えられるが、これは周辺細胞が、陰性シグナルを放出するからである可能性が最も高い。初期とは播種の2日後〜4日後を指すが、これは、特に播種を伴う懸滴法では、増殖(細胞が依然として増殖している)ならびにイオンチャネル発現(チャネルが依然として全心筋細胞で発現されている場合、心室細胞を含む全細胞型がこのイオンチャネルを発現し、自発的に拍動する)およびその調節(一酸化窒素系のムスカリン作動性アゴニストによる、L型Ca2+流入の基底阻害)について、初期パターンを依然として示している段階である。
3. 12時間〜24時間以内に全非心筋細胞の99%の排除をもたらしたピューロマイシンの高度に効率的な作用も、驚くべきものであった。
4. 本発明の重要な利点とは、非播種EBおよび攪拌培養物中のEBそれぞれの選択もまた可能であることである。なぜなら、本明細書においては、死滅細胞を問題なく洗い流すことができ、それにより、ES細胞由来の純粋な細胞型特異的培養物を初めて得ることができたためである。非生存細胞の排除は、酵素的消化(例えばトリプシン、コラーゲン)によりある程度改善される。非播種EBにおける心筋細胞により本方法の効率をさらに実証でき、これは、細胞網内に置かれた場合には再び収縮し始める。
材料および方法
ベクター
マウスα-MHC遺伝子の調節性断片5.5kbを含むベクターは、J.Robbins博士により提供された(Children Hospital Medical Center、Cincinnati、USA)(Gulickら、1991)。
ES細胞クローンの増殖および選択の全段階を、以下からなるES細胞増殖培地で実施した:
非必須アミノ酸(0.1mM)、L-グルタミン(2mM)、ペニシリンおよびストレプトマイシン(5μg/ml)、β-メルカプトエタノール(0.1mM)、LIF(ESGRO(商標))(500u/ml)、ウシ胎仔血清(FCS)(15%V/V)を添加した、グルコース強化DMEM培地。
細胞:0.8mlPBS中、4×106個〜5×106個(Ca2+、Mg2+非含有);
ベクターDNA:20μg〜40μg;
エレクトロポレーションキュベット:0.4cm(Bio-Rad Laboratories、Hercules、CA、USA);
エレクトロポレーター:Gene Pulser(商標)(Bio-Rad Laboratories);
電気インパルス条件:240V、500μF。
電気インパルス後、細胞懸濁液を氷上で20分間冷却し、その後、10cm組織品質ぺトリ皿に、10mlのES細胞増殖培地中のG418耐性線維芽細胞支持層と共に移した。2日後、ゲネチシンG418(GibcoBRL)300μg/mlをG418耐性細胞の選択のために加えた。G418(300μg/ml)を含む培地を、1日おきに交換した。8日間〜10日間の選抜後、薬物耐性クローンが出現した。クローンを選び、別々に0.1%トリプシン/EDTA溶液中でトリプシン処理し、ES細胞増殖培地中のG418耐性線維芽細胞支持層およびG418(300μg/ml)を含む48穴プレートに播種した。2日間〜4日間増殖させた後、続いてES細胞クローンをトリプシン処理し、24穴プレート中で、およびその後5cm組織ペトリ皿上で増殖させた。G418(300μg/ml)およびG418耐性線維芽細胞支持層は、ES細胞クローン増殖の全段階で存在していた。
15%FCSが20%FCSへと置き換えられた、LIFを除く前述の「ES細胞増殖培地」の全成分からなる「分化培地」中で、分化プロトコルの全段階を実施した。増殖後、選択したG418耐性ESクローンをトリプシン処理し、「分化培地」に再懸濁して最終濃度を0.020×106細胞〜0.025×106細胞/mlとした。続いて、この懸濁液20μl(400個〜500個の細胞)を細菌用ペトリ皿(Greiner Labortechnik、ドイツ)の蓋の上に配置することにより、懸滴を形成した。37℃、5%CO2における2日間のインキュベートの後、ES細胞は凝集物すなわち「胚様体」を形成した。これを細菌用ペトリ皿中で分化培地を用いて洗浄し、さらに5日間インキュベートした。その後、胚様体を、ゼラチンを含む分化培地で予め調整しておいた24穴組織品質プレート上に別々に播種した。平行実験において、いくつかの胚様体が懸濁液中に残ったが、これらを播種したものと同様に処理した。
FACS分析のために、成長および選択の段階の異なる10個〜20個の胚様体をPBSで洗浄し、その後、2分間〜3分間のトリプシン処理により単一細胞懸濁液に解離した(120μlトリプシン/EDTA溶液)。続いて、1mlのDMEM+単一細胞懸濁液の20%FCSを加えた。5分間遠心分離(1000upm)した後、細胞を、Ca2+(1mM)およびMg2+(0.5mM)を含む0.5ml〜1.0mlのPBSに再懸濁した。
pαMHC-EGFPおよびpαMHC-purベクターに関してトランスジェニックであるES細胞を培養し、心臓分化プロトコルにおいて使用した。ES細胞状態において、およびEB形成の後から播種の日(「懸滴」の形成から7日後)まで、全被験クローンにおけるEGFP蛍光は顕微鏡的に確認されなかった。播種後第一日目から第二日目に(8日齢〜9日齢のEB)、第一のEGFP蛍光領域が出現し、これは通常、1日後に自発的に拍動し始める。驚くべきことに、拍動クラスター外にあるEB細胞の圧倒的大多数が、顕微鏡的に測定可能な蛍光レベルを示さず、このことは、ES細胞心筋発生の際のEGFP発現が高度に組織特異的であることを示す。
最初のベクターとして、pIRES2-EGFP(CLONETECH Laboratories、Palo Alto、CA)を使用した。このベクターは、マルチクローニング部位(MCS)とEGFP遺伝子との間に、脳心筋炎ウイルスの内部リボソーム侵入部位(IRES)を含む。これにより、ピューロマイシン耐性およびEGFP遺伝子が、1つの単一のバイシストロン性mRNAから別々に翻訳されることが可能になる。pIRES2-EGFPベクターを、制限酵素AseIおよびECO47IIIで平滑末端化し、再ライゲーションして、サイトメガロウイルス最初期(CMV-IV)プロモーターを欠失させた。得られたベクターをSmaIで消化し、SacIおよびClaIにより上述のα-MHC-purベクターから切り出しておいたα-MCH-pur-カセットとライゲーションした。得られたpα-MHC-IRES-EGFP(pα-PIG)ベクターが正しい方向であるかを、SacI/SmaIによる消化で確認した。
ピューロマイシン選択法は、その後、心臓の損傷が模倣された自己由来マウスモデルで試験され、これにより確認された。ES細胞のインビトロ分化(10,000個〜100,000個の細胞)により得られた胚幹細胞または心臓細胞を、心臓が低温処理により部分的に損傷を受けたレシピエントに注射した、マウス移植モデルを、この目的のために使用した。腫瘍の成長を、マウス全体、単離心臓、および組織スライドを用いて形態学的に調べ、これらの検査を、操作後2日間から2ヶ月までの様々な時点で実施した。このアプローチにより、異なる細胞調製物の腫瘍成長可能性についての正確な評価が可能になる。非分化ES細胞を寒冷損傷(100,000個の細胞)に注射した際、大きな腫瘍がマウス内で成長した。操作から10日後に、動物はこれらの腫瘍のために死亡した。しかし、ES細胞がインビトロで心臓細胞へと分化した場合にも腫瘍が成長し、かつES細胞由来の心筋細胞に典型的な拍動領域を分離および単離し、その10,000個〜50,000個の細胞をマウスに注射した。これにより、心臓の胚幹細胞が腫瘍になる可能性が高いこと、および、高度に特異的な選択法で行なわなければならない必要性が高いことが実証される。
1. 発現ベクターpαMHC-EGFPおよびpαMHC-purで同時トランスフェクトされた安定なトランスジェニック胚幹細胞クローンを調製した。
2. インビトロにおける分化中のトランスジェニック胚幹細胞のピューロマイシン処理により、以前に作製したpαMHC-HygES細胞系のハイグロマイシン処理と比較して高効率の心臓特異的選択が示された(データは示していない)。
3. 選択された分化細胞は、その非処理対照物よりも高度な形態学的および機能的な生存度および長寿を示し、このことにより、遺伝的選択アプローチは、周辺細胞の負の影響から、分化中の胚幹細胞を効率的に解放することを示唆する。
4. 共通の細胞型特異的プロモーター下の生蛍光レポーターおよび薬物耐性遺伝子の併用により、分化および細胞特異的選択を含む、全手順の緊密なモニタリングおよび定量が可能となった。得られた細胞は、さらなる移植実験に適用可能であり、これにより、導入細胞のモニタリングが可能となった。
5. それぞれの細胞型における高度に特異的なプロモーターまたは特定の成長段階が同定されクローニングされれば、提示されたアプローチを、ES分化系における任意の細胞型特異的選択にも適用できる。原則的に、本システムにより、それぞれ2色のインビトロ蛍光タンパク質(例えば黄色型(EYEP)およびシアン(青色)型(ECFP)のEGFP)を有する2つの異なるプロモーター、および2つの薬物耐性遺伝子の併用が可能となる。このようなアプローチにより、全手順の選択性および効率性は増加し得る。本発明により提供される胚幹細胞、好ましくは胚様体を、例えば重金属および薬学的物質などの物質の毒性試験に使用できる(上記のリストも参照されたい)。この目的のために、二重ベクター構築物を用いて胚幹細胞培養物を利用し、細胞の典型的な分化開始後に選択剤を添加する(蛍光の検出)。細胞精製後またはすでにES細胞培養中に、様々な被験物質を細胞培養物に加え、様々な時点で、蛍光単一細胞および全蛍光それぞれを、様々な読み取り法(例えばフローサイトメトリー、蛍光読み取り器)で対照と比較して測定する。
Claims (21)
- 以下の(a)および(b)の工程を含む、分化中の幹細胞または分化した細胞を製造する方法:
(a)細胞非損傷性の蛍光タンパク質をコードする少なくとも1つのレポーター遺伝子および少なくとも1つの耐性遺伝子の情報を有するDNA配列を含む少なくとも一つのベクターを、霊長類またはげっ歯類由来の幹細胞に導入する工程であって、該DNA配列が、どちらも同じプロモーターの制御下にあり、該プロモーターが、該遺伝子に機能的に連結された少なくとも1つの細胞特異的および/または成長特異的なプロモーターより選択される、前記工程;
(b)蛍光の検出後に、該少なくとも1つの耐性遺伝子に対して選択的な少なくとも1つの選択剤を添加し、該ベクターを含む細胞を選択する工程であって、該ベクター含有細胞の選択が、
(i)安定にトランスフェクトされた細胞を選択するための第一の選択剤を添加する段階;
(ii)レポーター遺伝子を発現する細胞を検出する段階;
(iii)レポーター遺伝子を発現する細胞を選択するための第二の選択剤を添加する段階;ならびに
(iv)細胞特異的および/または組織特異的なプロモーターの制御下で幹細胞から成長する分化中のまたは分化した細胞を単離する段階
を含む、前記工程。 - 前記細胞が、マウス、ラットもしくはウサギに由来するか、またはヒトを起源とする、請求項1記載の方法。
- 前記細胞非損傷性の蛍光タンパク質が、(強化)緑色蛍光タンパク質(EGFPおよびGFP)、赤色蛍光タンパク質(RFP)、青色蛍光タンパク質(BFP)、黄色蛍光タンパク質(YFP)およびシアン蛍光タンパク質(CFP)より選択される、請求項1または2記載の方法。
- 前記細胞非損傷性の蛍光タンパク質がGFPである、請求項1〜3のいずれか一項記載の方法。
- 前記耐性遺伝子が、ヌクレオシド系抗生物質またはアミノグリコシド系抗生物質に対する耐性を付与する、請求項1〜4のいずれか一項記載の方法。
- 前記耐性遺伝子が、メトトレキサート耐性、または、ピューロマイシン、ストレプトマイシン、ネオマイシン、ゲンタマイシンもしくはハイグロマイシンに対する耐性、または、ビンブラスチン、ドキソルビシン、およびアクチノマイシンDに対する耐性、または多剤耐性の原因となる、請求項1〜5のいずれか一項記載の方法。
- 前記プロモーターが、中胚葉細胞、外胚葉細胞または内胚葉細胞に特異的なプロモーターである、請求項1〜6のいずれか一項記載の方法。
- 前記中胚葉細胞が、心臓細胞、造血細胞、内皮細胞、平滑筋細胞または骨格筋細胞であり;前記外胚葉細胞が、ニューロンおよび/またはグリア細胞であり;ならびに前記内胚葉細胞が上皮細胞である、請求項7記載の方法。
- 前記プロモーターが、心臓細胞、ニューロンに特異的、または骨格筋細胞、軟骨細胞もしくは線維芽細胞に特異的である、請求項1から8のいずれか一項記載の方法。
- 前記心臓特異的プロモーターが、Nkx-2.5プロモーター、ヒトα-アクチンプロモーター、α-MHCプロモーター、β-MHCプロモーターおよびMLC-2Vプロモーターより選択される、請求項1〜9のいずれか一項記載の方法。
- 前記プロモーターが、さらなる機能的DNA配列に連結され、該機能的DNA配列が、エンハンサー配列、リプレッサー配列またはIRES配列である、請求項1〜10のいずれか一項記載の方法。
- 前記細胞特異的プロモーターが、ピューロマイシン耐性遺伝子に機能的に連結された心臓特異的プロモーターである、請求項1〜11のいずれか一項記載の方法。
- 前記IRES配列が、前記レポーター遺伝子と前記耐性遺伝子の間に配置されている、請求項1〜12のいずれか一項記載の方法。
- 前記細胞が、前記ベクター構築物により安定にトランスフェクトされた細胞を選択するための追加的な耐性遺伝子を含む、請求項1〜13のいずれか一項記載の方法。
- 前記細胞が胚様体の形状である、請求項1〜14のいずれか一項記載の方法。
- 前記1つまたは複数のベクターをトランスフェクション、エレクトロポレーション法、ウイルスベクターまたはリポフェクションにより導入する、請求項1〜15のいずれか一項記載の方法。
- 前記幹細胞を胚様体の形状で培養する、またはその他の細胞との共培養で培養する、請求項1〜16のいずれか一項記載の方法。
- 前記細胞を懸濁液中で培養する、および/または前記細胞を胚様体として培養する、請求項1〜17のいずれか一項記載の方法。
- 前記レポーター遺伝子を発現する細胞を選択するための第二の選択剤としてピューロマイシンを適用する、および/またはさらなる濃縮のために、選択した細胞を細胞選別機に通す、請求項1〜18のいずれか一項記載の方法。
- 前記中胚葉細胞、外胚葉細胞または内胚葉細胞が、中胚葉細胞、外胚葉細胞または内胚葉細胞に特異的なプロモーターの使用により得られる、請求項1〜19のいずれか一項記載の方法。
- 前記中胚葉細胞が分化中の心臓細胞であり、前記中胚葉細胞特異的プロモーターが心臓特異的プロモーターである、請求項20記載の方法。
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US5602301A (en) * | 1993-11-16 | 1997-02-11 | Indiana University Foundation | Non-human mammal having a graft and methods of delivering protein to myocardial tissue |
US6015671A (en) * | 1995-06-07 | 2000-01-18 | Indiana University Foundation | Myocardial grafts and cellular compositions |
DE4441327C1 (de) * | 1994-11-22 | 1995-11-09 | Inst Pflanzengenetik & Kultur | Embryonale Herzmuskelzellen, ihre Herstellung und ihre Verwendung |
DE19615232A1 (de) * | 1996-04-18 | 1997-10-23 | Merck Patent Gmbh | Neue Carbamoylderivate und deren Verwendung als 5-HT ¶1¶¶A¶-Antagonisten |
DE19727962A1 (de) * | 1997-07-02 | 1999-01-14 | Juergen Hescheler | Fluoreszierende Proteine als zelltypspezifische Reporter |
US6080576A (en) * | 1998-03-27 | 2000-06-27 | Lexicon Genetics Incorporated | Vectors for gene trapping and gene activation |
EP1641912B1 (en) * | 2003-06-20 | 2016-04-27 | Axiogenesis Ag | Tissue modelling in embryonic stem (es) cell system |
CA2558946C (en) * | 2003-07-08 | 2013-05-21 | Axiogenesis Ag | Novel method for the preparation of embryoid bodies (ebs) and uses thereof |
-
2001
- 2001-12-27 US US10/451,816 patent/US20040096432A1/en not_active Abandoned
- 2001-12-27 BR BR0116549-6A patent/BR0116549A/pt not_active IP Right Cessation
- 2001-12-27 JP JP2002553468A patent/JP4159358B2/ja not_active Expired - Fee Related
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- 2001-12-27 EP EP01272041A patent/EP1348019B9/de not_active Expired - Lifetime
- 2001-12-27 CA CA2431197A patent/CA2431197C/en not_active Expired - Lifetime
- 2001-12-27 CN CNB018213588A patent/CN100557016C/zh not_active Expired - Fee Related
- 2001-12-27 AU AU2002217164A patent/AU2002217164B9/en not_active Ceased
- 2001-12-27 WO PCT/EP2001/015337 patent/WO2002051987A1/de active IP Right Grant
- 2001-12-27 GB GB0313260A patent/GB2386609C2/en not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
---|---|
JP2004520029A (ja) | 2004-07-08 |
WO2002051987A1 (de) | 2002-07-04 |
EP1348019A1 (de) | 2003-10-01 |
GB2386609A (en) | 2003-09-24 |
GB0313260D0 (en) | 2003-07-16 |
AU2002217164B2 (en) | 2005-12-15 |
BR0116549A (pt) | 2003-12-23 |
US20110059456A1 (en) | 2011-03-10 |
JP2008118991A (ja) | 2008-05-29 |
IL156410A0 (en) | 2004-01-04 |
GB2386609C2 (en) | 2014-01-08 |
US20040096432A1 (en) | 2004-05-20 |
GB2386609B (en) | 2004-12-15 |
EP1348019B9 (de) | 2012-04-18 |
CN1483074A (zh) | 2004-03-17 |
EP1348019B1 (de) | 2010-07-28 |
JP4159358B2 (ja) | 2008-10-01 |
GB2386609C (en) | 2013-12-18 |
CN100557016C (zh) | 2009-11-04 |
AU2002217164B9 (en) | 2006-02-09 |
CA2431197C (en) | 2012-03-13 |
CA2431197A1 (en) | 2002-07-04 |
AU2002217164B8 (en) | 2006-01-05 |
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