JP2021090459A - 心筋細胞シート - Google Patents
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Abstract
Description
<1>多能性幹細胞由来の心筋細胞を含む細胞集団を培養して得られるシート状細胞培養物であって、前記細胞集団の全細胞数に対する多能性幹細胞由来の心筋細胞数の割合が、50%〜70%である、前記シート状細胞培養物。
<2>細胞集団が、αSMA陽性細胞、CD31陽性細胞およびTE−7抗原陽性細胞からなる群から選択される細胞をさらに含む、上記<1>に記載のシート状細胞培養物。
<3>細胞集団の全細胞数に対する多能性幹細胞由来の心筋細胞数の割合が25%以下または90%以上である、前記細胞集団を培養して得られるシート状細胞培養物よりサイトカイン産生能が高い、上記<1>または<2>に記載のシート状細胞培養物。
<4>(1)多能性幹細胞を心筋細胞誘導処理に供し、心筋細胞を含む第1の細胞集団を得るステップ、
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が50%〜70%である、上記<1>〜<3>のいずれか一つに記載のシート状細胞培養物の製造方法。
<6>心疾患を処置するための、上記<5>に記載の組成物。
<7>上記<1>〜<3>のいずれか一つに記載のシート状細胞培養物または上記<5>に記載の組成物の有効量を、それを必要とする対象に適用することを含む、前記対象における疾患を処置する方法。
<8>(1)多能性幹細胞を心筋細胞誘導処理に供し、心筋細胞を含む第1の細胞集団を得るステップ、
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が50%〜70%である、多能性幹細胞由来の心筋細胞を含むシート状細胞培養物のサイトカイン産生能を高める方法。
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が、約25%超かつ約90%未満、好ましくは約30%〜約80%、より好ましくは約40%〜約75%、特に好ましくは約50%〜約70%である、上記シート状細胞培養物の製造方法(以下、「本発明の製造方法」と称することがある)に関する。
本発明の組成物等は、本発明のシート状細胞培養物に加えて、種々の追加成分、例えば、薬学的に許容し得る担体や、シート状細胞培養物の生存性、生着性および/または機能などを高める成分、対象疾患の処置に有用な他の有効成分などを含んでいてもよい。かかる追加成分としては、既知の任意のものを使用することができ、当業者はこれらの追加成分について精通している。また、本発明の組成物等は、シート状細胞培養物の生存性、生着性および/または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などと併用することができる。
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が約25%超かつ約90%未満 、好ましくは約30%〜約80%、より好ましくは約40%〜約75%、特に好ましくは約50%〜約70%である、多能性幹細胞由来の心筋細胞を含むシート状細胞培養物のサイトカイン産生能を高める方法(以下、「サイトカイン産生能増大方法」と称することがある)に関する。
ヒトiPS細胞(253G1株)を理化学研究所バイオリソースセンターから購入し、直径10cmの培養皿内で、5ng/mLのbFGF(basic fibroblast growth factor、リプロセル社製、以下同様)を添加したPrimate ES Cell Medium(リプロセル社製)中、マイトマイシンC処理を施したマウス胎児線維芽細胞(MEF、リプロセル社製)上で維持した。細胞の継代は、3〜4日ごとに、コロニーを維持したまま(単細胞懸濁液にせずに)、細胞剥離液(CTK溶液、リプロセル社製、以下同様)を用いて行った。
例1で得た解離した細胞集団の一部を用いて、当該細胞集団における非心筋細胞の構成比を調べた。まず、細胞集団を、非心筋細胞マーカー(血管内皮細胞等のマーカーであるCD31、平滑筋細胞等のマーカーであるαSMAまたは線維芽細胞等のマーカーであるTE−7抗原)と心筋細胞マーカーであるc−TNTの両方について二重標識した。
例1で得た解離した細胞集団の一部を、心筋細胞マーカーであるCD172に特異的に結合する、PE(フィコエリスリン)結合抗ヒトCD172a/b抗体(clone SE5A5、BioLegend社製)で標識した。標識した細胞に抗PEマイクロビーズ(Miltenyi Biotec社製)を負荷し、細胞とビーズの混合物をMidiMACSTM Separator(Miltenyi Biotec社製)に設置したLS Columns(Miltenyi Biotec社製)に通して、CD172陽性の心筋細胞を含む心筋細胞集団と、CD172陰性の非心筋細胞を含む非心筋細胞集団とに分離した。
例3で分離した心筋細胞集団と非心筋細胞集団とを下表2の比率で混合し、シート状細胞培養物形成用の細胞集団A〜Dを調製した。
(1)外観
心筋細胞集団の割合が50%以上の細胞集団で形成したシート状細胞培養物B〜Dは、自発的な同期拍動を示した。また、シート状細胞培養物A〜Cは、移植に適した白色円形の形状を形成することができたが、シート状細胞培養物Dは安定したシート状の構造を形成することができなかった(図1)。
シート状細胞培養物A〜Dを、10%FBS加DMEM中、37℃、5%CO2にて2日間培養して得た培養上清に含まれるVEGFおよびSCFの濃度を、Bio-Plex(R) Suspension Array System(Bio-Rad Laboratories社製)を用い、製品マニュアルに従って測定した(n=4)。VEGFの定量にはBio-Plex ProTM ヒトサイトカイン 28-Plex アッセイを、SCFの定量にはBio-Plex ProTM ヒトサイトカイン 21-Plex アッセイをそれぞれ用いた。各群間の統計学的有意差の評価には、Non-repeated Measures ANOVAを用いた。
図2に示す結果から、シート状細胞培養物を形成する細胞集団における心筋細胞集団の割合を50%〜70%とすることで、高いサイトカイン産生量が得られることが分かる。VEGFは血管新生作用を有し、移植片の生存率を高めることなどが知られており(例えば、Suzuki et al., Circulation. 2001 Sep 18;104(12 Suppl 1):I207-12など)、SCFは、心筋細胞に分化し得る骨髄幹細胞を動員し得ることなどが知られている(例えば、Lutz et al., Cardiovasc Res. 2008 Jan;77(1):143-50など)。したがって、これらのサイトカインの産生量が高い本発明のシート状細胞培養物は、高い治療効果を有することが予測される。
シート状細胞培養物A〜DをPBSで2回洗浄後、RNeasy(R) Mini(Qiagen社製)で全RNAを抽出した。得られた全RNAを基にSuperScript(R) VILOTM cDNA Synthesis Kit(Life Technologies社製)を用いてcDNAを合成したのち、TaqMan(R) Gene Expression AssaysおよびTaqMan(R) Fast Advanced Master Mix(いずれもLife Technologies社製)を用いてリアルタイムPCRにより細胞外マトリックス構成成分の発現量を定量した。なお、TaqMan(R) Gene Expression AssaysのAssay IDおよび検出遺伝子は下表3に示すとおりである。
シート状細胞培養物A〜Dの電気伝導速度を多電極アレイ(multi-electrode array)により測定した。例4で調製した細胞集団A〜Dを、それぞれ2.1×105個/cm2の密度で、MEDプローブ(アルファメッドサイエンティフィック社製)に播種した。10%FBS加DMEM中、37℃、5%CO2にて5日間増殖培養してシート状細胞培養物を形成した後、シート状細胞培養物がプローブに付着したままの状態でMED64システム(アルファメッドサイエンティフィック社製)を用いて、細胞外電位を測定し、MED64 Mobius(WitWerx社製)を用いて各チャネルのピーク時間を検出し、伝導速度を算出した。シート状細胞培養物B〜Dの伝導速度は、それぞれ3.5±1.8cm/秒、8.6±2.8cm/秒および16±5.0cm/秒であり、心筋細胞集団の割合が多いほど大きくなった。
Claims (8)
- 多能性幹細胞由来の心筋細胞を含む細胞集団を培養して得られるシート状細胞培養物であって、前記細胞集団の全細胞数に対する多能性幹細胞由来の心筋細胞数の割合が、50%〜70%である、前記シート状細胞培養物。
- 細胞集団が、αSMA陽性細胞、CD31陽性細胞およびTE−7抗原陽性細胞からなる群から選択される細胞をさらに含む、請求項1に記載のシート状細胞培養物。
- 細胞集団の全細胞数に対する多能性幹細胞由来の心筋細胞数の割合が25%以下または90%以上である、前記細胞集団を培養して得られるシート状細胞培養物よりサイトカイン産生能が高い、請求項1または2に記載のシート状細胞培養物。
- (1)多能性幹細胞を心筋細胞誘導処理に供し、心筋細胞を含む第1の細胞集団を得るステップ、
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が50%〜70%である、請求項1〜3のいずれか一項に記載のシート状細胞培養物の製造方法。 - 請求項1〜3のいずれか一項に記載のシート状細胞培養物を含む組成物。
- 心疾患を処置するための、請求項5に記載の組成物。
- 請求項1〜3のいずれか一項に記載のシート状細胞培養物または請求項5に記載の組成物の有効量を、それを必要とする対象に適用することを含む、前記対象における疾患を処置する方法。
- (1)多能性幹細胞を心筋細胞誘導処理に供し、心筋細胞を含む第1の細胞集団を得るステップ、
(2)ステップ(1)で得た第1の細胞集団に由来する心筋細胞を含む第2の細胞集団を得るステップ、および
(3)ステップ(2)で得た第2の細胞集団を培養してシート状細胞培養物を形成するステップ
を含み、第2の細胞集団の全細胞数に対する心筋細胞数の割合が50%〜70%である、多能性幹細胞由来の心筋細胞を含むシート状細胞培養物のサイトカイン産生能を高める方法。
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