JP6744223B2 - 多能性幹細胞の心筋細胞への分化のための方法 - Google Patents
多能性幹細胞の心筋細胞への分化のための方法 Download PDFInfo
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Description
本願は、多能性幹細胞(PSC)を心筋細胞に分化させるための方法に関する。さらに、本願は、化学的に定義された培地誘導の一連の段階に基づき、ヒト胚性幹細胞(hESC)および人工多能性幹細胞(iPSC)を増殖性心筋細胞に分化させるための方法にも関する。
長年、様々な細胞培養系が前臨床医薬品開発において用いられている。しかしながら、株化細胞モデルは腫瘍原性組織または形質転換不死化細胞のいずれかに由来することから、株化細胞モデルは、薬学的に関連する疾患に特異的な生理機能を一部しか反映しない。具体的には、最終分化した心筋細胞は限定された増殖能を有していることが示されているため、それらは、医薬品開発のための細胞モデルを効果的に生じさせる能力を有しない。したがって、研究および医薬品開発において信頼性の高い細胞モデルとして用いることができるより疾患に関連するヒト細胞型が必要とされている。
(a)3〜7×105/cm2の密度で多能性細胞を準備する段階
(b)式
の化合物を含むインスリン不含培地で前記細胞をインキュベートする段階。
[本発明1001]
(a)3〜7×10 5 /cm 2 の密度で多能性細胞を準備する段階、
(b)式
の化合物を含むインスリン不含培地中で前記細胞をインキュベートする段階
を含む、多能性幹細胞を心筋細胞に分化させるための方法。
[本発明1002]
前記細胞が、0.3〜10μMの前記化合物を含むインスリン不含培地中でインキュベートされる、本発明1001の方法。
[本発明1003]
段階(b)が前記細胞を12〜48時間インキュベートする段階を含む、本発明1001または1002の方法。
[本発明1004]
Wnt-C59を含むインスリン不含培地中で前記細胞をインキュベートする段階(c)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(c)が、1〜10μMのWnt-C59を含むインスリン不含培地中で前記細胞をインキュベートする段階を含む、本発明1004の方法。
[本発明1006]
段階(c)が、前記細胞を24〜72時間インキュベートする段階を含む、本発明1004または1005の方法。
[本発明1007]
前記細胞が、段階と段階の間にインスリン不含培地中で24〜48時間インキュベートされる、本発明1001〜1006のいずれかの方法。
[本発明1008]
インスリンを含む培地中で前記細胞をインキュベートする段階(d)をさらに含む、本発明1001〜1007のいずれかの方法。
[本発明1009]
段階(b)、(c)および(d)の培地がアスコルビン酸を含む、本発明1001〜1008のいずれかの方法。
[本発明1010]
前記多能性幹細胞が人工多能性幹細胞である、本発明1001〜1009のいずれかの方法。
[本発明1011]
前記人工多能性幹細胞がヒト細胞である、本発明1010の方法。
[本発明1012]
前記人工多能性幹細胞が、心臓細胞の機能不全に起因する疾患に罹患する対象から入手される、本発明1010または1011の方法。
[本発明1013]
本発明1001〜1012のいずれかの方法により入手された心筋細胞。
[本発明1014]
本発明1001〜1012のいずれかの方法により入手された心筋細胞のバイオバンク。
[本発明1015]
心臓細胞の機能不全に起因する疾患についてのインビトロモデルとしての、本発明1001〜1012のいずれかの方法により入手された心筋細胞または本発明1014のバイオバンクの心筋細胞の使用。
[本発明1016]
本発明1001〜1012のいずれかの方法により入手された心筋細胞または本発明1014のバイオバンクの心筋細胞を含む治療用組成物。
[本発明1017]
本明細書に本質的に記載された方法および使用。
本発明は、先行技術のプロトコールと比較して、より短時間でかつ増殖性心筋細胞の収量の有意な増加を示す、多能性幹細胞を心筋細胞に分化させるための改善された方法を提供する。
(a)3〜7×105細胞/cm2の密度で多能性細胞を準備する段階
(b)式
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(CP21)
の化合物を含むインスリン不含培地中で前記細胞をインキュベートする段階。
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」または「CP21」とも呼ばれる;L. Gong et al; Bioorganic& Medicinal Chemistry Letters 20 (2010), 1693-1696を参照)。
Matrigel (BD Bioscience、Cat.354277)
mTeSR1培地 (Stemcell Technologies、Cat.05850)
Accutase (Innovative Cell Technologies、Cat.AT-104)
Rock阻害剤、Y-27632 (Millipore、Cat.SCM075)
RPMI培地 (Gibco by Life Technologies、Cat.61870)
アスコルビン酸(Sigma、Cat.A4544)
50×B-27(登録商標)Supplement Minus Insulin(Gibco by Life Technologies、Cat.0050129SA)
ペニシリン-ストレプトマイシン(Gibco by Life Technologies、Cat.15070)
50×B27 インスリン含、ビタミンA不含(Gibco by Life Technologies、Cat.12587)
0.05%トリプシン/EDTA、1×(Gibco by Life Technologies、Cat.25300)
autoMACS Running Buffer (Miltenyi、Cat.130-091-221)
Inside Perm+InsideFix (Miltenyi、Inside Stain Kit、Cat.130-090-477)
0.1%ゼラチン(Millipore、Cat.ES-006-B)
クライオジェニックバイアル(Corning#430659)
ミスターフロスティー凍結容器 (Thermo Scientific#5100-0001)
DMSO (Sigma#D2438)
ウシ胎仔血清(Invitrogen#16000044)
Falcon細胞培養用ディッシュ35×10 mm (BD#353001)
Falcon細胞培養用ディッシュ100×20 mm (BD#353003)
6ウェルプレートCorning Costar (Sigma#CLS3516)
抗サルコメアαアクチニン[EA-53]抗体(Abcam、Cat.ab9465)
抗心臓トロポニンT抗体 (Abcam、Cat.ab45932)
Alexa Fluor(登録商標) 488およびロバ抗マウスIgG (H+L) (Invitrogen、Cat.A21202)
Alexa Fluor(登録商標) 647ロバ抗ウサギIgG (H+L) (Invitrogen、Cat.A31573)
Alexa Fluor(登録商標) 555ロバ抗ウサギIgG (H+L) (Invitrogen、Cat.A31572)
Hoechst 33258、五水和物(ビスベンズイミド) (Molecular Probes、Cat.H3569)
ヒト胚性幹細胞(hESC)または人工多能性幹細胞(iPSC)を、マトリゲル(BD Bioscience, Cat.354277)でコーティングした56 cm2ディッシュにおいて37℃および5%CO2にて10 ml mTeSR1培地(Stemcell Technologies, Cat.05850)中で培養した。
分化プロセスの効率を試験するために、分化の14日目に、心筋細胞に特異的な抗原を用いて細胞免疫組織化学的検査および蛍光活性化細胞選別(FACS)により、心筋細胞を特徴づけた。
細胞を180 μl/cm2 PBS-/-で洗い、37℃および5%CO2での100 μl/cm2 0.05%1×トリプリン/EDTA(Gibco by Life Technologies, Cat.25300)によって5〜10分間解離させた。
上記に記載のプロトコールを種々のCP21濃度で繰り返した。結果を下記の表に示す:(-)心筋細胞は得られなかった、(+)〜(+++):得られた心筋細胞の量。
心筋細胞の純度を向上させるために、濃縮段階を開発した。
14日目に、精製方法で記載しているように心筋細胞を再プレーティングした。18日目に、上記で概説しているように細胞を解離させ、続いて、そのαアクチニン発現およびトロポニンT発現についてFACSにより分析した。80%以上の心筋細胞を有する培養物を凍結プロトコールに供した。80%未満の心筋細胞を有する培養物は廃棄した。
イソプロテレノールは、心臓β1受容体を刺激することにより心拍数および心筋収縮性を向上させる。幹細胞由来の心筋細胞におけるこの催不整脈作用を検出するために、37℃にて1時間130 μl/cm2の0.1%ゼラチンでコーティングした特別なE-Plate Cardio 96(Roche, Cat. No. 05232368001)上に7×104/cm2の細胞をプレーティングした。上記のように細胞を2日間プレートに付着させ回収した後に、培地をiCell Cardiomyocytes Maintenance Medium(Cellular Dynamics, Cat. No.CMM-100-120-005)に交換した。xCELLigence RTCA Cardio System(Roche Applied Science)を用いて、細胞を測定した。プレーティングの7日後、細胞を3μM イソプロテレノールで処理し、直接測定した。各96ウェルプレートを12,9 msの解像度で測定した。最初の3分間は中断なしに測定され、その後の24時間にわたって、15分毎に1分間、細胞を測定した。
免疫蛍光染色のために、室温で15分間、4%パラホルムアルデヒドによって、細胞を固定した。
分化後、細胞をその心筋細胞含量について分析した。図1は分化14日目の心筋細胞を定量化するFACS分析を図示する。
Claims (7)
- 前記細胞が、段階と段階の間にインスリン不含培地中で24〜48時間インキュベートされる、請求項1に記載の方法。
- インスリンを含む培地中で前記細胞をインキュベートする段階(d)をさらに含む、請求項1または2のいずれか一項に記載の方法。
- 段階(b)、(c)および(d)の培地がアスコルビン酸を含む、請求項1〜3のいずれか一項に記載の方法。
- 前記多能性幹細胞が人工多能性幹細胞である、請求項1〜4のいずれか一項に記載の方法。
- 前記人工多能性幹細胞がヒト細胞である、請求項5記載の方法。
- 前記人工多能性幹細胞が、心臓細胞の機能不全に起因する疾患に罹患する対象から入手される、請求項5または6記載の方法。
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EP3569693A4 (en) * | 2017-06-05 | 2020-11-18 | Terumo Kabushiki Kaisha | METHOD OF PREPARING A CELL CULTURE |
WO2020247957A2 (en) * | 2019-06-06 | 2020-12-10 | President And Fellows Of Harvard College | Cardiomyocytes and compositions and methods for producing the same |
CA3171960A1 (en) * | 2020-02-28 | 2021-09-02 | Purdue Research Foundation | Generating aorta-gonad-mesonephros-like hematopoietic cells from human pluripotent stem cells under a defined condition |
CN113249310B (zh) * | 2021-05-17 | 2022-10-11 | 湖北大学 | 一种拓展性多能干细胞诱导分化为心肌细胞的方法及应用 |
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JP2008523823A (ja) * | 2004-12-22 | 2008-07-10 | イーエス・セル・インターナショナル・プライヴェート・リミテッド | ヒト胚性幹細胞の分化ならびにそれに由来する心筋細胞および心筋前駆細胞 |
WO2007002136A2 (en) * | 2005-06-22 | 2007-01-04 | Geron Corporation | Differentiation of primate pluripotent stem cells to cardiomyocyte-lineage cells |
UA103918C2 (en) * | 2009-03-02 | 2013-12-10 | Айерем Элелси | N-(hetero)aryl, 2-(hetero)aryl-substituted acetamides for use as wnt signaling modulators |
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