JP2015517311A - 合成メッセンジャーrnaを使用したヒト人工多能性幹細胞のフィーダーフリー誘導 - Google Patents
合成メッセンジャーrnaを使用したヒト人工多能性幹細胞のフィーダーフリー誘導 Download PDFInfo
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Abstract
Description
この出願は、2012年5月13日に出願された米国仮出願第61/646,292号に対する優先権の利益を主張する。この内容は、全体が本明細書に援用される。
本明細書において、本方法による使用に適した細胞として、初代細胞および樹立細胞株、胚性細胞、免疫細胞、幹細胞および分化細胞が挙げられるがこれらに限定されず、その例として、線維芽細胞、実質細胞、造血細胞および上皮細胞を含む、外胚葉、内胚葉および中胚葉に由来する細胞等が挙げられるがこれらに限定されない。本明細書において、幹細胞は、造血幹細胞、間葉系幹細胞、上皮幹細胞および筋サテライト細胞等、単能性細胞、複能性細胞および多能性細胞;胚性幹細胞および成体幹細胞を含む。一実施形態において、体細胞は、脱分化または再プログラム化される。いかなる適した体細胞を使用してもよい。代表的な体細胞として、線維芽細胞、ケラチノサイト、アディポサイト(adipocyte)、筋細胞、臓器および組織細胞、ならびに造血幹細胞を含む造血細胞および短期または長期造血生着をもたらす細胞等が挙げられるがこれらに限定されない様々な血液細胞が挙げられる。最も好ましい細胞型として、ヒト線維芽細胞、ケラチノサイトおよび造血幹細胞が挙げられるがこれらに限定されない。本方法は、脱分化および任意選択で再分化細胞に特に有用であり、細胞ゲノムに永続的な変更を生じない。
本方法を細胞の再分化または再プログラム化に広く使用して、例えば、造血幹細胞、間葉系幹細胞、上皮幹細胞および筋サテライト細胞、または赤血球細胞、リンパ球を含む白血球細胞、血小板、ストローマ細胞、脂肪細胞、破骨細胞を含む骨細胞、皮膚細胞を含む上皮組織、平滑筋、骨格筋および心筋を含む筋組織、内皮細胞を含む血管組織、肝細胞を含む肝臓組織ならびにニューロンを含む神経組織等が挙げられるがこれらに限定されないヒト組織の分化細胞を形成するようさらにモジュレートすることのできるiPS細胞を産生することもできる。心筋細胞、造血幹細胞、破骨細胞等の骨細胞、肝細胞、網膜細胞およびニューロン等が挙げられるがこれらに限定されない様々な分化細胞型へとiPS細胞の分化を誘導する方法。単離された胚性幹細胞、造血幹細胞および人工多能性幹細胞等が挙げられるがこれらに限定されない幹細胞は、分化を誘導するRNAの一過性トランスフェクションにより、分化するよう誘導することができる。その上またはその代わりに、細胞型特異的条件下で細胞を培養することにより、細胞を再分化させることができる。例えば、iPS細胞は、CF−1フィーダーにおいて維持し、その後フィーダーフリー条件に適応させることができる。ノギン、Dkk−1およびIGF−1の存在下で細胞を培養することにより、iPS細胞を誘導して分化した網膜細胞を形成させることができる。
ライゲーション非依存性クローニング(Ligation Independent Cloning)(LIC)を使用して、直鎖状PCR産物in vitro転写(IVT)鋳型を生成するためのプラスミド構築物を構築した。本発明者らは先ず、対象とするタンパク質をコードするオープンリーディングフレーム(ORF)インサートを受け入れるよう設計された挿入部位に隣接する5’および3’非翻訳領域(UTR)を組み入れた、親プラスミド(pIVT)を構築した。ORF隣接配列は、Warrenら、Cell Stem Cell、2010年に記載されている通りのものであり、低二次構造リーダーおよび強力なコザック(Kozak)部位(5’UTR)ならびにマウスα−グロビン3’UTRを含む。テイルド(tailed)プライマーを使用してプラスミドから増幅された2種のPCR産物の再アニーリングにより、5’オーバーハングを有する直鎖化バージョンのpIVTベクターを産生した。相補的オーバーハングを有するORF PCR産物を類似の手順により産生し、ベクターPCR産物と共にプールし、熱ショックによりDH5α細菌に形質転換して、遺伝子特異的構築物(pIVT−KLF4等)をクローニングした。得られたプラスミドを鋳型PCR反応に使用して、Warrenら、Cell Stem Cell、2010年に記載されている通り、T7プロモーター、UTR隣接ORF、およびポリAテイルの付加を駆動するためのT120テイルを組み入れた直鎖状IVT鋳型を作製した。テイルドリバースプライマー(T120CTTCCTACTCAGGCTTTATTCAAAGACCA)の使用により、T120テイル領域を導入した。M3OおよびVPx3融合構築物に関して、トランス活性化ドメインをコードする配列を、テイルドプライマーを使用したPCRによりORFに付け加えた。PCR産物鋳型ストックをほぼ100ng/uLの濃度で維持した。
mRNA合成プロセスを図5にまとめる。4:1比のARCAキャップアナログ対GTPを使用したIVT反応において合成mRNAを生成して、高いパーセンテージのキャッピングされた転写物を生成した。CTPを5m−CTPで、UTPをプソイド−UTPで完全置換したヌクレオチド三リン酸(NTP)ミックスを用いて、RNA産物の免疫原性を低下させた。キャップアナログおよび修飾NTPは、Trilink Biotechnologiesから購入した。2.5×NTPミックスを調製して(ARCA:ATP:5m−CTP:GTP:プソイド−UTP、15:15:3.75:3.75:3.75mM)、IVT反応の実施に使用されるMEGAscript T7キット(Ambion)により提供された標準NTPを置き換えた。各40uLのIVT反応液は、16uLのNTPミックス、4uLの10×T7バッファー、16uLのDNA鋳型および4uLのT7酵素を含んだ。反応液を37℃で4〜6時間インキュベートし、次に2uLのTURBO DNaseでさらに37℃で15分間処理し、その後、MEGAclear(Ambion)スピンカラムにおいて精製し、100uLの容量においてRNA産物を溶出した。キャッピングしていない転写物から免疫原性5’三リン酸部分を除去するため、10uLのAntarcticホスファターゼ反応バッファーおよび3uLのAntarcticホスファターゼ(NEB)を各プレップに加えた。ホスファターゼ反応液を37℃で30分間インキュベートし、IVT産物を再精製した。Nanodrop(Thermo Scientific)によりRNA収量を定量化し、その結果、TE pH7.0(Ambion)の添加によりプレップを標準化作業濃度100ng/uLに調整した。所望の化学量論比において様々なRFを表すプレップをプールすることにより、RNAカクテルを組み立てた。使用した各RFの割合は、それぞれの転写物の予測される分子量を考慮し、3×モル濃度で含まれたOct4およびその誘導体以外の全RFは、等モル濃度であった。短命の核化(nuclearized)単量体LanYFP蛍光タンパク質をコードするmRNAの10%スパイクをカクテルに加えて、再プログラム化試行におけるトランスフェクション効力のモニターを容易にした。
再プログラム化に標的化された細胞は、BJ新生児線維芽細胞(ATCC)、HDF−f胎児線維芽細胞、HDF−n新生児線維芽細胞およびHDF−a成体線維芽細胞(ScienCell)およびXFF異種フリー新生児線維芽細胞(Millipore)を含んだ。それぞれBJ、HDFおよびXFF細胞のために、BJ培地(DMEM+10%FBS)、ScienCell線維芽細胞培地およびFibroGRO異種フリーヒト線維芽細胞増殖培地(Millipore)において増殖培養を行った。使用したフィーダー細胞は、3001G照射済み新生児ヒト包皮線維芽細胞(GlobalStem)およびFibroGROマイトマイシンC不活性化異種フリーヒト新生児線維芽細胞(Millipore)であった。異種フリーフィーダーに基づくおよびフィーダーフリー再プログラム化試行に該当する細胞継代ステップは、TrypLE Select(Gibco)、動物性製品フリー細胞解離試薬を使用して行った。
メーカーの指示書に従って、CELLstart(Gibco)異種フリー培養基でコーティングされた6ウェル組織培養プレートにおいて、記載されている全再プログラム化実験を行った。最初のBJ再プログラム化実験において、FBS含有BJ培地を使用して、GlobalStemフィーダーをウェル当たり250Kで蒔いた。後のフィーダーに基づく試行の一部において、播種密度を増加させ、新規RFカクテルを使用して直面する高い損耗率に応じてほとんどコンフルエントなフィーダー層を持続する試みにおいて、培地交換の際に臨機応変にフィーダーを補充した。異種フリーフィーダーは、使用するのであれば、血清を含まないPluritonに基づく再プログラム化培地に蒔いた。Pluriton無血清培地(Stemgent)、ならびに抗生物質、Pluritonサプリメントおよび200ng/ml B18Rインターフェロン阻害剤(eBioscience)に標的細胞を蒔いた。再プログラム化の間とその後に、培地を毎日交換し、最終トランスフェクション翌日にB18R補給を中断した。再プログラム化の間に新鮮なフィーダーにおいて細胞を分割した実験において、再プレーティングに使用される培地に10uM Y27632(Stemgent)を含めた。標的細胞播種の翌日にトランスフェクションを開始し、これを本文に示す持続時間において24時間間隔で反復した。他に注記されている場合を除き、毎日の培地交換の4時間前にRNAiMAX(Invitrogen)を使用して、1200ngのRNA用量を各ウェルに送達した。100ng/uLのRNAをカルシウム/マグネシウム不含DPBSに5×希釈し、1ugのRNA当たり5uLのRNAiMAXを同じ希釈液に10×希釈し、これらをプールして10ng/uLのRNA/ビヒクル懸濁液を産生し、15分間の室温インキュベーション後に培養培地に分注することにより、RNAiMAXに基づくトランスフェクションカクテルを作製した。Stemfect試薬(Stemgent)を使用するトランスフェクションに関して、RNAおよびStemfect(1ugのRNA当たり4uL)をStemfectバッファーにおいて混合して、10ng/uLのRNA濃度を得た。混合物を15分間インキュベートし、次に培養培地に送達した、あるいは後の使用のために冷蔵した。
iPSCコロニー生産性を評価するために、DPBS(カルシウム/マグネシウム含有)中4%パラホルムアルデヒドを使用して再プログラム化培養物を固定し、DPBS(カルシウム/マグネシウム含有)に100×希釈したStainAlive TRA−1−60 Alexa488抗体(Stemgent)で免疫染色した。コロニーを採取し、増殖し、その後に免疫染色して多能性の分子および機能検証のための三血球系分化アッセイを行った。DNAフィンガープリント法および核型解析を行った。奇形腫形成を行い、2セット以上のマウスモデルにおいて確認した。これにより、幹細胞の多能性を実証した。
Claims (20)
- 体細胞を脱分化または再プログラム化するための方法であって、
Oct4、Sox2、Klf4、cMyc、NanogおよびLin28から選択される再プログラム化因子の合成mRNAのうちいずれか1種または複数とトランス活性化ドメインとの融合産物を有効量含む組成物を、単離された前記体細胞にトランスフェクトし、これにより前記体細胞を再プログラム化または脱分化するステップ
を含む、方法。 - 前記組成物が、N末端MyoDトランス活性化ドメインと融合したOct4を含む、請求項1に記載の方法。
- Oct4が、タンデムに3連でN末端MyoDトランス活性化ドメインと融合している、請求項2に記載の方法。
- 請求項1に記載の再プログラム化因子の合成mRNAのうちいずれか1種または複数を使用することにより哺乳動物細胞を再プログラム化するための方法であって、
a)フィーダーフリー表面において標準6ウェルプレートのウェルあたり細胞25k〜250kの密度で、または他の表面領域のウェルにおいてウェル当たり比例して減らした数の細胞で、標的細胞を成長させるステップと、
b)再プログラム化の間に各回50ng〜800ng/mlで変動する用量のmRNAを細胞にトランスフェクトするステップと
を含む、方法。 - 標的細胞を、フィーダーフリー表面において標準6ウェルプレートのウェルあたり細胞50k、75k、100kまたは150kの密度で成長させ、
a)再プログラム化の間に各回50ng〜800ng/mlで変動する用量のmRNAを細胞にトランスフェクトするステップであって、より初期の時点においてより後期の時点よりも低い用量を使用する、ステップと、
b)継代せずにiPSCを獲得するステップと
を含む、請求項4に記載の方法。 - 標的細胞を、フィーダーフリー表面において標準6ウェルプレートのウェルあたり細胞50k、75k、100kまたは150kの密度で成長させるが、各ウェルの容量を、0.5ml〜5mlの適切な培地となるよう調整し、
a)再プログラム化の間に各回50ng〜800ng/mlで変動する用量のmRNAを細胞にトランスフェクトするステップであって、より初期の時点においてより後期の時点よりも低い用量を使用する、ステップと、
b)継代せずにiPSCを獲得するステップと
を含む、請求項4に記載の方法。 - 前記哺乳動物細胞が、ヒト細胞である、請求項4に記載の方法。
- 異種フリーである、請求項4に記載の方法。
- 前記1種または複数の因子が、mRNA、制御性RNA、siRNA、miRNAおよびこれらの組合せからなる群より選択される、請求項1に記載の方法。
- 前記体細胞に、少なくとも2種の異なるRNAをトランスフェクトする、請求項1に記載の方法。
- 前記体細胞が、単能性細胞、複能性細胞、多能性細胞および分化細胞からなる群より選択される、請求項1に記載の方法。
- 前記1種または複数のRNAが、前記体細胞から単能性細胞、複能性細胞または多能性細胞への脱分化を誘導する、請求項1に記載の方法。
- 前記因子のうち少なくとも1種が、OCT4、SOX2、NANOG、LIN28、KLF4およびMYC mRNAからなる群より選択される、請求項4に記載の方法。
- OCT4、SOX2、NANOGおよびLIN28 mRNAを組み合わせて投与する、請求項4に記載の方法。
- OCT4、SOX2、KLF4およびMYC mRNAを組み合わせて投与する、請求項4に記載の方法。
- 前記トランスフェクトされた細胞が、培養において人工多能性幹(iPS)細胞として維持される、請求項1に記載の方法。
- 前記トランスフェクトされた細胞が、人工多能性幹細胞を形成し、分化細胞を形成するように前記iPS細胞を誘導するステップをさらに含む、請求項1に記載の方法。
- 患者における疾患または障害の1種または複数の症状を処置または阻害するための方法であって、請求項1に記載の方法に従って細胞をin vitroで脱分化させるステップと、前記細胞を前記患者に投与するステップとを含む、方法。
- 前記組成物が、RargおよびLrH−1トランス活性化ドメインをさらに含む、請求項2に記載の方法。
- 前記組成物が、VP16トランス活性化ドメインと融合したOct4を含む、請求項1に記載の方法。
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AU2013263101B2 (en) | 2019-02-14 |
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CA2872688C (en) | 2023-09-19 |
HK1204005A1 (en) | 2015-11-06 |
WO2013173248A3 (en) | 2014-12-18 |
EP2850179A2 (en) | 2015-03-25 |
KR102215413B1 (ko) | 2021-02-16 |
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JP6448531B2 (ja) | 2019-01-09 |
AU2019203335B2 (en) | 2021-10-21 |
CN110241087A (zh) | 2019-09-17 |
KR20150035594A (ko) | 2015-04-06 |
KR102081811B1 (ko) | 2020-02-26 |
EP2850179A4 (en) | 2016-02-24 |
AU2013263101A1 (en) | 2014-11-27 |
AU2019203335A1 (en) | 2019-05-30 |
EP3561054B1 (en) | 2023-07-05 |
JP6530452B2 (ja) | 2019-06-12 |
ES2951822T3 (es) | 2023-10-25 |
ES2729563T3 (es) | 2019-11-04 |
WO2013173248A2 (en) | 2013-11-21 |
DK2850179T3 (da) | 2019-06-17 |
EP2850179B1 (en) | 2019-03-20 |
CN104520424B (zh) | 2019-06-28 |
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EP3561054C0 (en) | 2023-07-05 |
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