JP6591434B2 - 内皮コロニー形成細胞様細胞の生成方法 - Google Patents
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Description
本出願は、2014年3月11日に出願された米国仮出願61/951,103号、(これは、すべての目的のために参照により組み込まれる)の優先権を主張する。
a)多能性幹細胞を提供するステップ;
b)多能性幹細胞を内皮分化へと誘導するステップであって、前記誘導は、
i)アクチビンA、BMP−4、VEGF、及びFGF−2を含む内皮分化培地で約24時間多能性幹細胞を培養すること;及び
ii)その後、1または2日ごとにBMP−4、VEGF、及びFGF−2を含む内皮分化培地でステップi)の培地を交換すること
を含み;及び
c)分化誘導された細胞から、ECFC様細胞を単離するステップであって、前記ECFC様細胞はCD31+NRP−1+でありコブルストーン形態を示す。
本願に記載の前記細胞集団の少なくとも1つを被験物質に露出するステップ、及び
細胞増殖及び細胞生存能のうち1つ以上において、前記被験物質の前記効果を観察するステップを含む、被験物質の細胞活動修正能力を調べる方法が提供される。
A:多能性幹細胞を提供するステップ;
B:前記多能性幹細胞の内皮系統の細胞への分化を誘導するステップ;及び、
C:分化した内皮系統の細胞からECFC様細胞を単離するステップ。
D.単離したECFC様細胞を拡大させるステップ。
前記選択された細胞集団からECFC様細胞を生成するために、ウェルあたり約2500の選択細胞が、コラーゲンでコーティングした12ウェルプレート上に播かれた。2日後、培養培地は内皮成長培地と内皮分化培地の比率が3:1である培養培地と交換された。ECFC様コロニーはしっかりとした接着性の細胞として出現し、拡大7日目にコブルストーン形態を示した。
少なくとも、以下の特徴を有する約35〜50%のECFC様細胞を含む。
A.特徴的ECFC様分子表現型、
B.インビトロにおいてマトリゲルTM上で毛細管状のネットワークを作る能力、
C.高い増殖能、
D.自己補充能、
E.インビボにおける共培養細胞非存在下での血管形成能、及び
F.増加した細胞生存能、及び/または減少した老化。
一の態様において、本明細書で提供されるECFC様細胞は、コラーゲン、フィブロネクチンまたは合成材料から成るマトリックスに懸濁することができる、そしてこのゼリー状の懸濁液は、十分な血流が不足している組織(医者により判断)に、直接注射することができる。組織に注射するECFC様細胞の濃度は適宜調節することができ、例えば、送達ビヒクルまたはマトリックス材料に対して約10,000から約100,000細胞/マイクロリットルとすることができる。いくつかの態様において、他の組織が、適切な血流を回復するために長時間の多回及び連続的な注射を必要とする一方で、細胞は適切な血流の回復と共に一回で送達される。
8〜16時間のインキュベーションの後、対物レンズ10×CP−ACHROMAT/0.12 NAを装着した倒立顕微鏡ツァイスAxiovert25CFLを用いて、各ウェルの10倍の顕微鏡写真を撮影した。イメージは、SPOT RTカラーカメラ(Diagnostic Instruments)を用い、メーカーのソフトウェアにより得た。位相差コントラストイメージは、乾燥系対物レンズで撮影した。
一旦、リードカウントを得て、グループ2で発現せず、グループ3または4で少なくとも発現した遺伝子を考察した。DESeqを用いて、これら候補から異なる発現遺伝子を決定した。DESeqモデルにおいて、従来のポアソンモデル(すなわち、バリエーションは過小評価されるかもしれない)における過分散の問題を解決するために、負の二項分配(NB)を通して、サンプルにおけるデータを数えた。DESeqはまた、反復数が高くなく(この場合3つの反復)てもデータあてはめを改善するために、他のグループからのいくつかのモデルも含み、モデル予測は、違いを決定するためには十分強固なものである。
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Claims (28)
- 以下のa)からc)のステップを含む、ヒト多能性幹細胞からヒト内皮コロニー形成細胞様細胞(ECFC様細胞)の単離集団を生成する方法。
a)多能性幹細胞を提供するステップ;
b)多能性幹細胞を内皮分化へと誘導するステップであって、前記誘導は、
i)アクチビンA、BMP−4、VEGF、及びFGF−2を含む内皮分化培地で約24時間多能性幹細胞を培養すること、及び
ii)その後、1または2日ごとにBMP−4、VEGF、及びFGF−2を含む内皮分化培地でステップi)の培地を交換すること
を含み;及び
c)分化誘導された細胞から、ECFC様細胞を単離するステップであって、前記ECFC様細胞はCD31+NRP−1+でありコブルストーン形態を示す。 - 前記単離したECFC様細胞が、さらにCD144+、KDR+、及びα−SMA−発現、のうち1つ以上であることを特徴とする、請求項1に記載の方法。
- アクチビンA、BMP−4、及びFGF−2のうち1つ以上が、約5〜25ng/mLの濃度で提供される、請求項1または2に記載の方法。
- VEGFが、約5〜50ng/mLの濃度で提供される、請求項1〜3のいずれか一項に記載の方法。
- 前記誘導ステップが、共培養細胞、胚葉体形成、及び外来性TGF−β阻害のうち、一以上の不在下で実行される、請求項1〜4のいずれか一項に記載の方法。
- 前記単離ステップが、分化10日目、11日目、または12日目に実行される、請求項1〜5のいずれか一項に記載の方法。
- 前記単離ステップが、分化12日目に実行される、請求項6に記載の方法。
- 前記細胞の単離が、フローサイトメトリー、または磁気活性化セルソーティング(Nagmetic activated cell sorting)により行われる、請求項1〜7のいずれかに記載の方法。
- 前記単離されたECFC様細胞が、哺乳動物に同時移植細胞なしに移植された場合、血管形成能力を有する、請求項1〜8のいずれか一項に記載の方法。
- 前記単離されたECFC様細胞集団の少なくとも約95%のECFC様細胞集団が増殖する、請求項1〜9のいずれか一項に記載の方法。
- 前記単離されたECFC様細胞集団の少なくとも約35〜50%のECFCsが、高増殖可能な(HPP)ECFC様細胞である、請求項1〜10のいずれか一項に記載の方法。
- 前記HPP−ECFC様細胞が、開始細胞あたり少なくとも約2001個の細胞を生成する能力を有する、請求項11に記載の方法。
- 前記HPP−ECFC様細胞が、自己再生能力を有する、請求項11または12に記載の方法。
- 以下のステップd)をさらに含む、請求項1〜13のいずれか一項に記載の方法。
d)前記単離したECFCsが、内皮増殖培地を含む組成物内で増殖するステップ。 - 以下のステップe)をさらに含む、請求項14に記載の方法。
e)前記増殖細胞を18回まで継代するステップ。 - 前記単離した細胞が、約3か月未満のうちに少なくとも約1兆個の細胞集団へ増殖する、請求項14または15に記載の方法。
- ヒトNRP−1+CD31+内皮コロニー形成細胞様細胞(ECFC様細胞)の単離集団であって、前記単離ECFC様細胞が、哺乳動物に同時移植細胞なしに移植した場合に、血管形成能力を有し、かつ前記単離ECFC様細胞が、インビトロでのヒト多能性細胞由来であり、
ここで仮想的タンパク質LOC100132288、CUB及びSushiマルチプルドメイン1、リンパ限定膜タンパク質、アリルアセトアミドデアセチラーゼ(エステラーゼ)、フォリスタチン様5、ENSG00000215262、仮想的LOC84856、グアニル酸シクラーゼ活性剤2B(ウログアニリン)、ケラチン75、繊維芽細胞活性化タンパク質、アルファ(FAP)、22番染色体オープンリーディングフレーム34、ガスダーミンC、ENSG00000222954、ヒドロキシステロイド(11−ベータ)デヒドロゲナーゼ1、インドールアミン2,3−ジオキシゲナーゼ2、及びZicファミリーメンバー4の遺伝子の1つ以上が、臍帯血ECFCsに比べて前期単離されたECFC様細胞において高発現し、及び/または、
レセプター(化学感覚)トランスポータータンパク質4、染色体Xオープンリーディングフレーム61、アシル−CoA シンセターゼ中鎖ファミリーメンバー2A、セルピンペプチダーゼインヒビター、クレードA(アルファ−1 アンチプロテイナーゼ、アンチトリプシン)、メンバー3、ENSG00000218052、ケモカイン(C−Cモチーフ)リガンド23、48含有コイルドコイルドメイン、及びRAS(RAD及びGEM)−様GTPバインディング1の遺伝子の1つ以上が臍帯血ECFCsに比べて前期単離されたECFC様細胞において低発現している単離集団。 - 前記ECFC様細胞の少なくとも約35%が、高増殖可能な(HPP)ECFC様細胞である、請求項17に記載の単離集団。
- 前記ECFC様細胞の少なくとも約50%が、HPP−ECFC様細胞である、請求項18に記載の単離集団。
- 前記HPP−ECFC様細胞が、開始細胞あたり少なくとも約2001個の細胞を生成する能力を有し、かつ自己再生能力を有する、請求項17に記載の単離集団。
- 前記単離集団中のHPP−ECFC様細胞が、さらに、CD144+、KDR+、及びα−SMA−のうちの1つ以上により特徴付けられる、請求項17〜20のいずれか一項に記載の単離集団。
- 前記単離集団中の前記HPP−ECFC様細胞が、コブルストーン形態を示す、請求項17〜21のいずれか一項に記載の単離集団。
- 前記単離集団中の前記HPP−ECFC様細胞が、マトリゲルで培養した時にキャピラリー様ネットワークを形成し得る、請求項17〜22のいずれか一項に記載の単離集団。
- 前記単離集団中の前記HPP−ECFC様細胞が、インビトロで18回まで継代され得る、請求項17〜23のいずれか一項に記載の単離集団。
- 請求項1〜16のいずれか一項に記載の方法によって得られる、ヒトNRP − 1 + CD31 + 内皮コロニー形成細胞様細胞(ECFC様細胞)の単離集団。
- 前記集団の中の、少なくとも約95%の細胞が、ECFC様細胞であり、かつNRP − 1 + CD31 + である、請求項25に記載の単離集団。
- 請求項1〜16のいずれか一項に記載の方法により得られた内皮コロニー形成細胞様細胞(ECFC様細胞)を含む、医薬品組成物。
- 以下のステップを含む、被験物質の細胞活動修正能力を調べる方法。
請求項17〜26のいずれか一項に記載の前記細胞集団の少なくとも1つを被験物質に露出するステップ、及び
細胞増殖及び細胞生存能のうち1つ以上において、前記被験物質の前記効果を観察するステップ。
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