JP6124880B2 - 多能性幹細胞の血管床細胞への分化のための方法 - Google Patents
多能性幹細胞の血管床細胞への分化のための方法 Download PDFInfo
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Description
(a)多能性培地中に単層の多能性幹細胞を提供する段階、
(b)β-カテニン(カドヘリン結合タンパク質、β1;ヒト遺伝子名称CTNNB1)経路および/またはWnt受容体シグナル伝達経路および/またはヘッジホッグ(HH)シグナル伝達経路を活性化する低分子を補給した刺激培地中で前記細胞をインキュベートする段階、
(c)誘導培地中で前記刺激された細胞をインキュベートすることにより、分化を誘導する段階。
(d)内皮細胞または血管平滑筋細胞の増殖に適する条件下で、段階(c)の生成物をインキュベートする段階
をさらに含む。
[本発明1001]
以下の段階を含む、多能性幹細胞を血管床細胞へ分化させるための方法:
(a)多能性培地中に単層の多能性幹細胞を提供する段階、
(b)グリコーゲン合成酵素キナーゼ3(Gsk3a-b)の低分子阻害剤、CDC様キナーゼ1(Clk1-2-4)の低分子阻害剤、マイトジェン活性化タンパク質キナーゼ15(Mapk15)の低分子阻害剤、二重特異性チロシン-(Y)-リン酸化調節キナーゼ(Dyrk1a-b 4)の低分子阻害剤、サイクリン依存性キナーゼ16(Pctk1-3 4)の低分子阻害剤、スムーズンド(Smoothened)(SMO)アクチベーター、ならびに、β-カテニン(またはγ-カテニン)とコアクチベータータンパク質であるCBP(CREB結合タンパク質)およびp300(E1A結合タンパク質p300)との間の相互作用の調節剤の群より選択される、β-カテニンおよび/またはWntシグナル伝達および/またはヘッジホッグ(HH)シグナル伝達を活性化する低分子を補給した刺激培地中で前記細胞をインキュベートする段階、
(c)誘導培地中で前記刺激された細胞をインキュベートすることにより、分化を誘導する段階。
[本発明1002]
多能性幹細胞を内皮細胞へ分化させるための、本発明1001の方法。
[本発明1003]
多能性幹細胞を血管平滑筋細胞へ分化させるための、本発明1001の方法。
[本発明1004]
段階(a)が、18時間〜30時間、多能性培地中で前記細胞をインキュベートする段階をさらに含む、本発明1001〜1003のいずれかの方法。
[本発明1005]
段階(b)が、2〜4日間、刺激培地中で前記細胞をインキュベートする段階をさらに含む、本発明1001〜1004のいずれかの方法。
[本発明1006]
段階(c)が、18時間〜48時間、誘導培地中で前記細胞をインキュベートする段階をさらに含む、本発明1001〜1005のいずれかの方法。
[本発明1007]
段階(a)の多能性培地が、タンパク質キナーゼのRho結合コイルドコイル形成タンパク質セリン/スレオニンキナーゼファミリーの阻害剤(ROCKキナーゼ阻害剤)を補給した無血清培地である、本発明1001〜1006のいずれかの方法。
[本発明1008]
ROCKキナーゼ阻害剤が、1-(5-イソキノリンスルホニル)ホモピペラジン、N-ベンジル-2-(ピリミジン-4-イルアミノ)チアゾール-4-カルボキサミド、および(+)-(R)-トランス-4-(1-アミノエチル)-N-(4-ピリジル)シクロ-ヘキサンカルボキサミドジヒドロクロリドの群から選択される、本発明1007の方法。
[本発明1009]
段階(b)の刺激培地が、インスリン、トランスフェリン、およびプロゲステロンを補給した無血清培地である、本発明1001〜1008のいずれかの方法。
[本発明1010]
段階(b)のβ-カテニンおよび/またはWntシグナル伝達および/またはヘッジホッグ(HH)シグナル伝達を活性化する低分子が、3-(3-アミノ-フェニル)-4-(1-メチル-1H-インドール-3-イル)-ピロール-2,5-ジオンである、本発明1001〜1009のいずれかの方法。
[本発明1011]
段階(b)の刺激培地が、組換え骨形成タンパク質-4(BMP4)をさらに含む、本発明1001〜1010のいずれかの方法。
[本発明1012]
誘導培地が、VEGF-A(血管内皮増殖因子)または胎盤様増殖因子1(PLGF-1)および低分子アデニラートシクラーゼアクチベーターを補給した無血清培地である、本発明1001〜1011のいずれかの方法。
[本発明1013]
低分子アデニラートアクチベーターが、フォルスコリン((3R)-(6aαH)ドデカヒドロ-6β,10α,10bα-トリヒドロキシ-3β,4aβ,7,7,10aβ-ペンタメチル-1-オキソ-3-ビニル-1H-ナフト[2,1-b]ピラン-5β-イルアセタート)、8-ブロモ-cAMP(8-ブロモアデノシン-3',5'-環状一リン酸)、およびアドレノメジュリンを含む群から選択される、本発明1012の方法。
[本発明1014]
誘導培地が、TGFβシグナル伝達およびPDGFシグナル伝達のアクチベーターを補給した血清代替培地または無血清培地である、本発明1001〜1011のいずれかの方法。
[本発明1015]
多能性幹細胞が人工多能性幹細胞である、本発明1001〜1014のいずれかの方法。
[本発明1016]
人工多能性幹細胞がヒト細胞である、本発明1015の方法。
[本発明1017]
人工多能性幹細胞が、血管合併症と関連した疾患を患う対象から得られたものである、本発明1015または1016の方法。
[本発明1018]
(d)内皮細胞または血管平滑筋細胞の増殖に適する条件下で、段階(c)の生成物をインキュベートする段階
をさらに含む、本発明1001〜1017のいずれかの方法。
[本発明1019]
本発明1001〜1018のいずれかの方法により得られた、内皮細胞または血管平滑筋細胞。
[本発明1020]
本発明1001〜1018のいずれかの方法により得られた内皮細胞または血管平滑筋細胞の、バイオバンク。
[本発明1021]
血管合併症と関連した疾患のためのインビトロモデルとしての、本発明1001〜1018のいずれかの方法により得られた、または本発明1020のバイオバンクの、内皮細胞または血管平滑筋細胞の使用。
[本発明1022]
本発明1001〜1018のいずれかの方法により得られた、または本発明1020のバイオバンクの内皮細胞を含む、治療用組成物。
[本発明1023]
本質的に本明細書において記載されるような、方法および使用。
細胞培養:
多能性培地:Y27632 ROCKキナーゼ阻害剤(市販、例えば、Tocris bioscienceのカタログ番号:1254)を補給したTeSR2。
ヒト多能性幹細胞は慣例的に、TeSR2培地(Stem cell Technologies)中でhESC-qualified Matrigel(BD Bioscience)上で培養する。培養物を、StemPro Accutase(Invitrogen)を用いて4〜6日毎に継代する。生存率の増大のために、酵素による解離の1時間前に、TeSR2培地に10μM ROCK阻害剤を補給する。
(i)PSCの付着および刺激:StemPro Accutase(Invitrogen)を用いるhPSCコロニーの酵素による解離の前に、細胞を1時間、10μM ROCK阻害剤Y27632と共にプレインキュベートする。cm2あたり35.000個の単一hPSCを、10μM ROCK阻害剤を補給したTeSR2培地中で、増殖因子を低減させMatrigel(BD bioscience)でコーティングした細胞培養プレート上にプレーティングする。1日目に、付着培地を(a)2μM化合物21(CP21R7)または(b)1μM化合物21(CP21R7)および25 ng/ml BMP4(R&D Systems)を補給したN2B27(Gibco)培地に交換する。培地交換せずにさらに3日間、細胞を培養する。
全プロトコルの間、細胞を低温に保ち、あらかじめ冷却した溶液を使用した。培地を廃棄した後、細胞を5 ml PBS(Ca2+およびMg2+を含まない)で洗浄した。次に、細胞を2〜4分間37℃で、3 mlのあらかじめ温めたStemPro Accutase(Invitrogen)中でインキュベートした。細胞を穏やかに上下へピペッティングすることにより、3 mlのStemPro-34培地(Invitrogen)に再懸濁した。細胞数および生存率を、トリパンブルーで対比染色することにより判定した。
1×106細胞を1 ml冷凍保存培地(90% FBSおよび10% DMSO)に懸濁し、冷凍バイアル中に移す。-80℃で保存する時に-1℃/分の冷却速度を達成するように、冷凍バイアルをNalgene Cryo 1℃ Freezing Containerに置く。凍結容器を-80℃で24時間保存する。長期保存のためには、細胞をその後、液体窒素中に移す。
最も広く使用される市販のGSK3β阻害剤およびCP21R7の、ヒトPA-1レポーター細胞においてβ-カテニン媒介TCF/LEF転写を活性化する可能性(DeAlmeida et al., 2007)を評定した。使用した化合物を図18に示す。用量反応アッセイにより、最も強力なGSK3阻害剤のすべて、すなわち、CP21R7(CP)、6-ブロモインジルビン-3'-オキシム(BIO)、CB36155、およびCHIR-99021(CHIR)は、協同的結合を示唆する、急勾配の活性化曲線を呈することが明らかになった(図19)。改変されていないPA-1細胞を使用して、ATPレベルを介して細胞生存率を測定し、GLI-ルシフェラーゼ応答性レポーターPA-1細胞をカウンタースクリーンとして、全般的な転写活性における変化をモニタリングする。全体的に解析したGsk3β阻害剤は、全般的な転写活性において有意でない上昇を示した(データは示されていない)。BIOの処置でGLI媒介ルシフェラーゼ活性において3倍の増加が観察され、BIOは10μMの濃度で非常に毒性であった(データは示されていない)。CB361549およびCB361556(MeBIO)ならびにフォルスコリン(陰性対照)は、ルシフェラーゼ発現を誘導しなかった。CPの濃度依存的投与により、3μMで最高のルシフェラーゼ活性を有するベル形曲線が明らかになった。566倍増加したルシフェラーゼ活性の最高値は、細胞増殖の少なめの増加および全般的な転写活性の軽微な増大のみを伴った。以前に記載されたGSK3β阻害剤SB216763の濃度依存的投与は、無視可能なβ-カテニン媒介TCF/LEFルシフェラーゼ発現を結果として生じた。β-カテニンレポーターアッセイの結果に基づき、3種のGSk3β阻害剤、すなわちBIO、CHIR、およびCPを、ヒトPSCの血管分化系列決定を駆動する可能性のため利用した。列挙した阻害剤に加えて、そのCPに対する構造類似性のために、SB216763(SB)を含める(図18を参照されたい)。多能性幹細胞を、上記の1(i)および(ii)で概説したように、(i)Rhoキナーゼ阻害剤であるY-27632を補給したmTeSR1培地上で単一細胞としてプレーティングし(Watanabe et al., 2007);(ii)刺激培地(GSK3β阻害剤を補給したN2B27培地)中でインキュベートし、かつ(iii)誘導培地(血管内皮増殖因子(VEGF;Sumi et al., 2008)を補給したStemPro SFM 34培地)中でインキュベートした。
CHIR99021 GSK3阻害剤:Merck Millipore、カタログ番号:361559
SB216763 GSK3阻害剤:Merck Millipore、カタログ番号:361566
BIO GSK3阻害剤:Merck Millipore、カタログ番号:3 361550
CP21R7:3-(3-アミノ-フェニル)-4-(1-メチル-1H-インドール-3-イル)-ピロール-2,5-ジオン(本明細書において「化合物21」とも呼ばれる;例えば、L. Gong et al; Bioorganic & Medicinal Chemistry Letters 20 (2010), 1693-1696を参照されたい)。
WNT/β-カテニンシグナル伝達は、原始線条形成を誘導する基礎となる(Tam and Loebel, 2007)。この理由で、中胚葉前駆細胞の一時的な出現を解析した。ゲノム全般にわたる遺伝子発現解析により、CPおよびBMP4の組み合わされた処置24時間で、NANOG、UTF1、およびSOX2などの多能性マーカーが下方制御されることが明らかになった(データは示されていない)。代表的なマーカー:MIXL1、T/ブラキュリ、FGF4、およびEOMESの上方制御により、細胞分化系列決定が同一の時間枠内で中胚葉に向けられていることが示された。分化の経過において、神経外胚葉および内胚葉と整合するマーカーの上昇した遺伝子発現は観察されなかった(データは示されていない)。内胚葉形成の開始についてのマーカーである遺伝子SOX17は、5日目以降に高発現した。この知見は、発生途中および成体の脈管構造におけるSOX17のインビボ発現パターンと一致する(Engert et al., 2009)。ESX1およびSOX7などの関連遺伝子が検出されなかったように、栄養外肺葉または内蔵内胚葉への分化系列決定は観察されなかった(データは示されていない)。
上記の分化プロトコルを使用した時、VEGFを補給したStemPro培地中へ選別されていない細胞を移した後に、2種の別個の細胞集団が出現した。細胞タイプは、形態およびマーカー発現により異なった。密接な細胞間接触を有する丸石様細胞は、内皮特異的マーカーであるVE-カドヘリンを発現した。平坦かつ紡錘体様形状の細胞は、平滑筋アクチン(αSMA)について陽性染色であり、血管平滑筋細胞(VSMC)同一性を示した。フローサイトメトリー解析において、細胞は、CD31+/CD140-細胞の多量分画およびCD31-/CD140b+細胞の微量分画に分離した(図10)。
Ac-LDL-取込み:細胞を、Molecular Probes/Invitrogenの2.5μg/mL Alexa Fluor 594アセチル化低密度リポタンパク質(AlexaFluor594-Ac-LDL)を含有する培地中で4時間、37℃でインキュベートした。インキュベーション後、細胞を洗浄し、4% PFAで10分間固定した。Alexaflor594-Ac-LDLの取り込みを、蛍光顕微鏡で可視化した。
炎症誘発性刺激に対する応答において、ECは、細胞内接着分子-1(ICAM1)およびE-セレクチンを含む細胞接着分子(CAM)を発現する。活性化されたECは、CAMを介して白血球を捕捉し、炎症の部位へそれらを繋ぎ止めることが可能である(Rao et al., 2007;Galkina et al., 2007)。血管炎症は、アテローム班形成の開始および進行において中心的な役割を果たす(Losis, 2000)。
Claims (18)
- 以下の段階を含む、多能性幹細胞を血管床細胞へ分化させるための方法:
(a)多能性培地中に単層の多能性幹細胞を提供する段階、
(b)β-カテニンおよび/またはWntシグナル伝達および/またはヘッジホッグ(HH)シグナル伝達を活性化する低分子を補給した刺激培地中で前記細胞をインキュベートする段階であって、ここで、該低分子が、3-(3-アミノ-フェニル)-4-(1-メチル-1H-インドール-3-イル)-ピロール-2,5-ジオンである、前記段階、
(c)誘導培地中で前記刺激された細胞をインキュベートすることにより、分化を誘導する段階。 - 多能性幹細胞を内皮細胞へ分化させるための、請求項1記載の方法。
- 多能性幹細胞を血管平滑筋細胞へ分化させるための、請求項1記載の方法。
- 段階(a)が、18時間〜30時間、多能性培地中で前記細胞をインキュベートする段階をさらに含む、請求項1〜3のいずれか一項記載の方法。
- 段階(b)が、2〜4日間、刺激培地中で前記細胞をインキュベートする段階をさらに含む、請求項1〜4のいずれか一項記載の方法。
- 段階(c)が、18時間〜48時間、誘導培地中で前記細胞をインキュベートする段階をさらに含む、請求項1〜5のいずれか一項記載の方法。
- 段階(a)の多能性培地が、タンパク質キナーゼのRho結合コイルドコイル形成タンパク質セリン/スレオニンキナーゼファミリーの阻害剤(ROCKキナーゼ阻害剤)を補給した無血清培地である、請求項1〜6のいずれか一項記載の方法。
- ROCKキナーゼ阻害剤が、1-(5-イソキノリンスルホニル)ホモピペラジン、N-ベンジル-2-(ピリミジン-4-イルアミノ)チアゾール-4-カルボキサミド、および(+)-(R)-トランス-4-(1-アミノエチル)-N-(4-ピリジル)シクロ-ヘキサンカルボキサミドジヒドロクロリドの群から選択される、請求項7記載の方法。
- 段階(b)の刺激培地が、インスリン、トランスフェリン、およびプロゲステロンを補給した無血清培地である、請求項1〜8のいずれか一項記載の方法。
- 段階(b)の刺激培地が、組換え骨形成タンパク質-4(BMP4)をさらに含む、請求項1〜9のいずれか一項記載の方法。
- 誘導培地が、VEGF-A(血管内皮増殖因子)または胎盤様増殖因子1(PLGF-1)および低分子アデニラートシクラーゼアクチベーターを補給した無血清培地である、請求項1〜10のいずれか一項記載の方法。
- 低分子アデニラートアクチベーターが、フォルスコリン((3R)-(6aαH)ドデカヒドロ-6β,10α,10bα-トリヒドロキシ-3β,4aβ,7,7,10aβ-ペンタメチル-1-オキソ-3-ビニル-1H-ナフト[2,1-b]ピラン-5β-イルアセタート)、8-ブロモ-cAMP(8-ブロモアデノシン-3',5'-環状一リン酸)、およびアドレノメジュリンを含む群から選択される、請求項11記載の方法。
- 誘導培地が、TGFβシグナル伝達およびPDGFシグナル伝達のアクチベーターを補給した血清代替培地または無血清培地である、請求項1〜10のいずれか一項記載の方法。
- 多能性幹細胞が人工多能性幹細胞である、請求項1〜13のいずれか一項記載の方法。
- 人工多能性幹細胞がヒト細胞である、請求項14記載の方法。
- 人工多能性幹細胞が、血管合併症と関連した疾患を患う対象から得られたものである、請求項14または15記載の方法。
- (d)内皮細胞または血管平滑筋細胞の増殖に適する条件下で、段階(c)の生成物をインキュベートする段階
をさらに含む、請求項1〜16のいずれか一項記載の方法。 - 内皮細胞または血管平滑筋細胞のバイオバンクを生成することをさらに含む、請求項1〜17のいずれか一項記載の方法。
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RU2323252C1 (ru) * | 2006-10-25 | 2008-04-27 | Антонина Ивановна Колесникова | Способ культивирования мезенхимальных стволовых клеток человека ex vivo |
US9175260B2 (en) * | 2007-01-30 | 2015-11-03 | TheUniversity of Georgia Research Foundation, Inc. | Early mesoderm cells, a stable population of mesendoderm cells that has utility for generation of endoderm and mesoderm lineages and multipotent migratory cells (MMC) |
FR2927633B1 (fr) * | 2008-02-19 | 2012-07-13 | Commissariat Energie Atomique | Systeme et procede de culture clonale de cellules epitheliales et leurs applications. |
US8372642B2 (en) * | 2009-02-27 | 2013-02-12 | Cellular Dynamics International, Inc. | Differentiation of pluripotent cells |
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US20150017674A1 (en) | 2015-01-15 |
US20200182861A1 (en) | 2020-06-11 |
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CA2836843A1 (en) | 2012-12-13 |
MX2013013854A (es) | 2014-01-20 |
RU2013156940A (ru) | 2015-07-20 |
EP2718425A1 (en) | 2014-04-16 |
KR20140031300A (ko) | 2014-03-12 |
RU2618871C2 (ru) | 2017-05-11 |
BR112013030166A2 (pt) | 2017-03-21 |
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