JP5560393B2 - 心筋細胞系列細胞への霊長類多能性幹細胞の分化 - Google Patents
心筋細胞系列細胞への霊長類多能性幹細胞の分化 Download PDFInfo
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Description
本出願は、2005年6月22日出願の米国特許出願第60/693,141号の優先権を主張する。
本発明は、インビトロにおいて霊長類多能性幹細胞を心筋細胞系列細胞へと分化させる分野に関する。
再生医学研究の主な課題は、心機能の再構築を助けることができる細胞組成物を開発することである。男女約5人に1人が何らかの形態の心血管疾患を有すると推定される(非特許文献1)。広範に及ぶ病態には、冠動脈性心疾患(人口の5%)、先天性心血管障害(0.5%)および先天性心不全(3%)が含まれる。これまで医薬品の技術分野では、心疾患により生じる損傷を抑える助けとなる低分子薬および生物学的化合物が製造されてきたが、損傷した組織の再生を助ける薬剤は市販されていない。
National Health and Nutrition Examination Survey III, 1988−94, Center of Desease Control and the American Heart Association Perin, et al., Circulation 107:2294, 2003 Strauer, et al., Circulation 106:1913, 2002 Zeiher, et al., Circulation 106:3009, 2002 Tse, et al., Lancet 361:47, 2003 Stamm, et al., Lancet 3661:45, 2003 Menasche, et al., J.Am. Coll. Cardiol. 41:1078, 2003 Pagani, et al., J.Am. Coll. Cardiol. 41:879, 2003 Hagege, et al., Lancet 361:491, 2003 Reffelmann, et al., J. Mol. Cell Cardiol. 35:607, 2003 Yao, et al., J. Molec. Cell. Cardiol. 35:607, 2003
本発明は、霊長類多能性幹細胞から心筋細胞系列細胞を得る方法を提供する。心筋細胞系列細胞は、多くの可能性ある利用法を有しており、これには、潜在的な製剤のスクリーニング、細胞毒性化学物質のスクリーニング、および損傷または疾患のある心臓のin vivo修復等の治療的用途が含まれるが、これらに限定されない。
「心筋細胞系列細胞」とは一般的に、心筋細胞の前駆体細胞および成熟心筋細胞双方を指す。特に指示がない限り、本開示内容における心筋細胞系列細胞、前駆体または心筋細胞の言及は、上に定義した通り、心筋細胞の個体発生の制限のない何れかの段階の細胞にも一様に適用することができる。以下に記載の通り、心筋細胞系列細胞は、以下の一覧の1つ以上のマーカー(時には少なくとも3個または5個のマーカー)を有する場合がある:心臓トロポニンI(cTnI)、心臓トロポニンT(cTnT)、サルコメアミオシン重鎖(MHC)、GATA−4、Nkx2.5、N−カドヘリン、β1−アドレナリン受容体(β1−AR)、ANF、転写因子のMEF−2ファミリー、クレアチンキナーゼMB(CK−MB)、ミオグロビン、または心房性ナトリウム利尿因子(ANF)。
一般的技法:本発明の実施に有用な一般的技法の詳細については、細胞生物学、組織培養、胎生学および心臓生理学の標準的な文献およびレビューを参照されたい。
本発明は、霊長類多機能性幹細胞を心筋細胞系列細胞に分化させる方法を提供する。本発明の方法に使用される場合がある霊長類多機能性幹細胞には、胚幹細胞が含まれるが、これに限定されない。胚幹細胞は、霊長類種の胚芽細胞から単離することができる(米国特許第5843780号;Thomson, et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995)。ヒト胚性幹(hES)細胞は、例えばThomson, et al. (米国特許第6200806号;Science282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff.,1998)およびReubinoff, et al., Nature Biotech. 18:399, 2000に記載の技法を使用して、ヒト胚芽細胞から調製することができる。その他の霊長類多機能性幹細胞型には、国際公開第WO 01/51610号(Bresagen)に概説される原始外胚葉様(EPL)細胞、およびヒト胚性生殖(hEG)細胞(Shamblott, et al., Proc. Natl. Acad. Sci. USA95:13726, 1998)が含まれるが、これらに限定されない。
霊長類多機能性幹細胞は、種々の基質、培地および他の補給剤並びに当技術分野で既知の因子を使用して培養される場合がある。霊長類多機能性幹細胞は、増殖を促進するものの、分化を抑制する培養条件を使用して、継続的に培養物中で繁殖させることができる。例示的な培地を80% DMEM(例えば、Knock−Out DMEM、Gibco)、既に明らかなウシ胎仔血清(FBS、Hyclone)若しくは血清代替品(米国特許公開第2002/0076747 Al号,Life Technologies Inc.)の何れか20%、非必須アミノ酸1%、Lグルタミン1mM並びにβメルカプトエタノール0.1mMから精製する。
本発明は、とりわけ、初めにアクチビンの存在下で、霊長類多能性幹細胞を逐次培養し、BMPの存在下でその後に培養することにより、霊長類多能性幹細胞を心筋細胞系列細胞に分化させる方法を提供する。BMPは、アクチビンによる培養工程時に除外されるが、アクチビンは、場合によりその後のBMP培養工程の間も含む場合がある。
本発明は、富化工程なしに高純度心筋細胞系列細胞集団を得る方法を提供する。しかし、1回以上の富化工程を加えることで、さらに高純度心筋細胞系列細胞集団を生成することができる。それゆえ、本発明の方法には、本発明の分化工程により得られた心筋細胞系列細胞を富化および/または拡大する工程が含まれる場合がある。特有の細胞型を富化する種々の方法は、当業者に既知であり、これには、機械的分離法、密度分離法、細胞分別、磁気分別および遺伝選択法が含まれるがこれらに限定されない(細胞分離の概説については、Freshney, Culture of Animal Cells, Wiley−Liss, New york, 2000−Chapter 14を参照)。これらの方法論の一部の例を以下に説明する。
特定の実施形態において、心筋細胞系列細胞は、例えばパーコール(例えば、本明細書の実施例3およびXu, et al., Circ. Res. 91(6):501−08, 2002)、Ficoll(Pharmacia)、メトリザミド(Nygaard)、RediGrad(GE Healthcare)およびデキストランを含むがこれらに限定されない密度勾配培地を使用する密度勾配分離法により富化される。
多くの細胞分別法が、非心筋細胞系列細胞から心筋細胞系列細胞を分別するために利用可能である。これらの細胞分別法には、陰性免疫選択および陽性免疫選択が含まれるが、これらに限定されない。
本発明の技法に従って得られた心筋細胞系列細胞を、多くの表現型の基準に従って特徴付けすることができる。
・心臓トロポニンI(cTnI)、横紋筋収縮の制御の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
・心臓トロポニンT(cTnT)
・Nkx2.5、マウス初期胚発生中の心臓中胚葉に発現する心臓転写因子であり、これは発達中の心臓内で持続する。
・心房性ナトリウム利尿因子(ANF)、発達中の心臓および胎児心筋細胞に発現するが、成人では下方制御されるホルモン。心臓細胞中に高度な特異様式で発現するが、骨格筋細胞には発現しないため心筋細胞に適したマーカーであると考えられている。
・ミオシン重鎖(MHC)、特に心臓特異的であるβ鎖。
・チチン、トロポミオシン、αサルコメアアクチニンおよびデスミン
・GATA−4、心臓中胚葉に高度に発現し、発達中の心臓内に持続する転写因子。多くの心臓遺伝子を調節し、心臓発生の役割を果たす。
・MEF−2A、MEF−2B、MEF−2CおよびMEF−2D;心臓中胚葉に発現し、発達中の心臓内に持続する転写因子。
・N−カドヘリン、心臓細胞間の付着を媒介する。
・コネキシン43、心筋細胞間のギャップ結合を形成する。
・β1アドレナリン受容体(β1−AR)
・クレアチンキナーゼMB(CK−MB)およびミオグロビン、これは、心筋梗塞後の血清において上昇する。
・α−心臓アクチン、初期成長応答(遺伝子)−IおよびサイクリンD2。
本発明の細胞は、分化の前後の何れかに細胞を遺伝的に操作することにより1つ以上の遺伝的変化を含むように生成することができる(米国特許公開第2002/0168766 A1号)。例えば、細胞を、発達制限系列細胞または最終分化細胞になる前後の何れかに、細胞がテロメラーゼ逆転写酵素を発現するように遺伝的に変化させることにより複製能を増加するように加工することができる(米国特許公開第2003/0022367 A1号)。
本発明は、多数の心筋細胞系列の細胞を生成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、開発および商業化目的に使用することができる。
本発明の心筋細胞は、このような細胞およびこれらの種々の後代の特徴に影響する因子(例えば、溶剤、低分子薬、ペプチド類、オリゴヌクレオチド類)または環境条件(例えば、培養条件または操作)をスクリーニングするために商業的に使用することができる。
本発明はまた、心筋細胞およびその前駆体を利用して、代謝機能の先天異常、疾患状態の影響または有意な外傷性障害の結果等、必要と感じる心筋組織の維持または修復を高める方法も提供する。
十分に試験した後、本発明の分化細胞をヒト患者またはこのような治療が必要な他の試料の組織再構築または再生に使用することができる。細胞を目的の組織部位にグラフトまたは移動させ、機能的に不完全な領域を再生する様式で投与する。特別なデバイスを、直接心機能を再構築することが可能な細胞を所望の部位にて心臓腔、心膜または心筋内部に投与するために使用することが可能である。
ゼラチン/FBSにおける3因子分化
ゼラチン/FBS被覆表面の調製:ゼラチン0.5%溶液1mL/ウェルを6−ウェルプレートのウェルに添加し、37℃にて一晩インキュベートした。ゼラチン溶液を除去し、十分な20%FBS含有培地(例えば、20%FBS(Sigma)含有knockout DMEM)をウェルの表面を被覆するよう添加した。プレートを37℃にてさらに5〜6時間インキュベートした。ヒト胚性幹細胞を添加する前に培地をウェルから除去した。
FACS分析:培地を吸着により培養物から除去した。ウェルをカルシウム/マグネシウム非含有PBS5mLで1回洗浄した。1ウェル当たり0.25%トリプシン/500mM EDTA溶液0.5mLを添加し、細胞を37℃にて20分間インキュベートした。細胞を単一細胞懸濁液が得られるまでピペットで粉砕した。トリプシン消化は、20%FBS含有培地1mL(20%FBS含有knockout DMEM)を添加することにより停止した。細胞濃度を計数により評価し、約500,000個の細胞を染色毎に分類した(EMA、アイソタイプ、cTnI、cTnI+EMA;各々15mLコニカルチューブ)。
マトリゲル被覆表面における3因子直接分化
増殖因子減少型マトリゲルのアリコート(予め、冷Knockout DMEMで1:2に希釈し、−20℃で保存)。マトリゲル溶液をさらに冷Knockout DMEMで1:15に希釈した。6ウェルプレート中の空のウェルに希釈マトリゲル溶液1mL/ウェルを被覆し、プレートを室温で4〜5時間インキュベートした。マトリゲル溶液を除去した後、ヒトES細胞を、ウェルを前洗浄することなく以下のようにプレートに入れた。
4相遠心分離法の富化の実施例
心筋細胞は、4日間の胚様体形成により、H7株のhES細胞から発生し、次いで、17日間ゼラチン被覆プレートにおいて増幅させた(5−アザ−デオキシ−シチジンおよび増殖因子を使用しなかった)。次いで、細胞を、コラゲナーゼBを使用して分離し、分化培地に再懸濁した。次いで細胞懸濁液をパーコール不連続勾配に層状に重ね、1,500g、30分間遠心分離した。以下の4つの画分を収集した:I、上部の界面;II、40.5%層;III、下部の界面;IV、58.5%層。細胞を洗浄し、分化培地に再懸濁した。免疫染色のための細胞を1ウェル当たり細胞104個にてチャンバースライドに播種し、2または7日間培養し、その後固定し染色した。
心臓体生成による収縮細胞の富化の実施例
この実施例は、心臓体である心筋細胞群のその後の培養により治療による使用および他の目的に望ましい特徴を有する細胞に富化することについて説明する。
H7hES細胞の分化を実施例1のように行った。但し、分化は6ウェルプレートの代わりに24ウェルプレートにて行い、因子量はウェル当たり1mLであった。さらに、BMP−2およびBMP−4を25ng/mL、50ng/mLおよび100ng/mLの濃度で使用した。各濃度で3回行った。図4は、対照の相対的倍率として表し、これはアクチビン、BMPまたはIGF−Iを添加しないプロトコルを実施した。また、BMP−2は分化プロトコルにおいて効果があったことが認められた。
H7hES細胞が密集する6ウェルプレートをPBS2mLで洗浄した。次いで、PBS中0.5mM EDTA溶液2mLを各ウェルに添加し、プレートを10分間、37℃でインキュベートした。EDTA溶液をマウス胚繊維芽細胞馴化培地(MEF−CM)1mL+bFGF8ng/mL(「培地A」)に入れ換えた。未分化ES細胞を2〜3回ピペットで分離した後、培地A中の細胞400,000個/ウェルにて24ウェルプレートに播種した。細胞を2日間37℃でインキュベートした。
Claims (15)
- ヒト胚性幹(hES)細胞から心筋細胞系列細胞を得るための方法であって、
a)アクチビンAの存在下であるがBMPの非存在下で該hES細胞を培養する工程;および
b)その後、該細胞をBMPの存在下であるがアクチビンAの非存在下で培養する工程
を包含する、方法。 - 前記BMPがBMP−2またはBMP−4である、請求項1に記載の方法。
- 前記BMPがBMP−2である、請求項2に記載の方法。
- 前記BMPがBMP−4である、請求項2に記載の方法。
- 前記hES細胞が、アクチビンAの存在下で約1日間培養された後、前記BMPの存在下で約4日間培養される、請求項1に記載の方法。
- 前記BMP培養工程に続いて、培地がアクチビンAもBMPも含まない追加の培養工程をさらに包含する、請求項1に記載の方法。
- 前記追加の培養工程が少なくとも1週間にわたり行われる、請求項6に記載の方法。
- 前記追加の培養工程が少なくとも2週間にわたり行われる、請求項6に記載の方法。
- 前記追加の培養工程がIGF−IまたはIGF−IIを含有する培地中で行われる、請求項6に記載の方法。
- 前記追加の培養工程がIGF−Iを含有する培地中で行われる、請求項9に記載の方法。
- 胚様体を形成させる工程を含まない、請求項1に記載の方法。
- 前記BMP培養工程に続いて、前記培養物から細胞を回収する工程、および該回収した細胞集団を心筋細胞系列細胞について富化させる工程をさらに包含する、請求項1に記載の方法。
- 前記回収した細胞集団がパーコール勾配によって富化される、請求項12に記載の方法。
- 前記富化が心臓体の形成を伴う、請求項12に記載の方法。
- hES細胞から心筋細胞系列細胞の富化集団を得る方法であって、
a)アクチビンAの存在下であるがBMPの非存在下で、無血清培地中で約1日間該hES細胞を培養する工程;
b)その後、BMPの存在下であるがアクチビンの非存在下で、無血清培地中で約4日間培養する工程;
c)該培養物から細胞を回収する工程;および
d)該回収した細胞集団を心筋細胞系列細胞について富化させる工程
をこの順序で包含する、方法。
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KR20080030026A (ko) | 2008-04-03 |
CA2611809A1 (en) | 2007-01-04 |
US20150284684A1 (en) | 2015-10-08 |
CN101228264B (zh) | 2017-12-26 |
GB0800264D0 (en) | 2008-02-13 |
JP2013215206A (ja) | 2013-10-24 |
EP1910518A2 (en) | 2008-04-16 |
IL187611A0 (en) | 2008-03-20 |
AU2006262329B2 (en) | 2011-04-07 |
HK1109913A1 (en) | 2008-06-27 |
JP2008543338A (ja) | 2008-12-04 |
KR20130099253A (ko) | 2013-09-05 |
WO2007002136A3 (en) | 2007-04-19 |
GB2441718B (en) | 2010-10-06 |
US9062289B2 (en) | 2015-06-23 |
IL187611A (en) | 2012-12-31 |
AU2006262329A1 (en) | 2007-01-04 |
CN101228264A (zh) | 2008-07-23 |
CA2611809C (en) | 2018-06-19 |
WO2007002136A9 (en) | 2008-03-06 |
EP1910518A4 (en) | 2015-03-11 |
US20070010012A1 (en) | 2007-01-11 |
EP1910518B1 (en) | 2019-05-08 |
WO2007002136A2 (en) | 2007-01-04 |
GB2441718A (en) | 2008-03-12 |
KR101529317B1 (ko) | 2015-06-16 |
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