JP2022069552A - Rnaでの幹細胞分化による肝細胞の誘導 - Google Patents
Rnaでの幹細胞分化による肝細胞の誘導 Download PDFInfo
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Abstract
Description
本出願は、2016年11月16日に出願された米国仮特許出願番号第62/423,113号の利益を主張しており、この仮出願は、参考として本明細書中に援用される。
本開示は、細胞密度、試薬濃度、およびmRNAの特異的組み合わせの特異的組み合わせおよび範囲を利用する動態的に制御された細胞成長プロセスを通じて、多能性幹細胞からの肝細胞の誘導を方向付けることに関する。
ヒト幹細胞の生成および結果としての分化における近年の努力は、細胞運命の可塑性、ヒト疾患のモデル、および臨床上の治療剤に関するパラダイムを変化させてきた。胚性幹細胞(ESC)および体細胞から作製される人工多能性幹細胞(iPSC)はともに、それらの相当する初代細胞から区別不能な特異的細胞タイプの増大しつつあるリストへと分化され得る。結果として、幹細胞は、新たなヒト細胞療法の開発にとって極めて有望である。iPSCは、制限のない細胞利用可能性、その細胞を得る手順の非侵襲性、および免疫抑制薬からの解放を許して、各処置を個々の患者に免疫を合わせる潜在的能力のために、個別化医療の分野において特別な潜在的能力を示す。
本開示は、細胞成長動態および関連パラメーターを調節する(それによって、細胞密度、試薬濃度、およびmRNAの組み合わせの特異的組み合わせが、分化/誘導の方向付けを制御するために使用される)ことによって、幹細胞分化を誘導するための方法を提供する。
本発明は、例えば、以下の項目を提供する。
(項目1)
肝細胞への幹細胞の分化を誘導するための方法であって、該方法は、
(a)人工多能性幹細胞を出発細胞として、分化のための条件下で培養する工程;
(b)多能性状態から中内胚葉系統に向かって出るように、該出発細胞を誘導する工程;
(c)有効用量においてかつ特定の時間域内でmRNAの第1の組み合わせでの培養細胞トランスフェクションを通じて、該分化している細胞を内胚葉細胞に向かって方向付ける工程;
(d)mRNAの第2の組み合わせでのトランスフェクションを通じて、該内胚葉細胞を肝前駆細胞に向かってさらに方向付ける工程;
(e)mRNAの第3の組み合わせで該肝前駆細胞を肝細胞へとさらに成熟させる工程;および
(f)前駆細胞クラスターを単層へと継代するか、または該肝前駆細胞から形成されるクラスターを集め、単層へと再度プレートすることによって、該肝細胞を得る工程、
を包含する方法。
(項目2)
前記mRNAの第1の組み合わせは、FoxA2 mRNAを含む、項目1に記載の方法。
(項目3)
前記mRNAの第1の組み合わせは、Sox17 mRNAを含む、項目1に記載の方法。
(項目4)
前記mRNAの第1の組み合わせは、FoxA2およびSox17 mRNAを含む、項目1に記載の方法。
(項目5)
前記mRNAの第1の組み合わせは、FoxA2、Sox17、GATA4、およびGATA6 mRNAを含む、項目1に記載の方法。
(項目6)
前記mRNAの第2の組み合わせは、Hex mRNAを含む、項目1に記載の方法。
(項目7)
前記mRNAの第2の組み合わせは、Tbx3 mRNAを含む、項目1に記載の方法。(項目8)
前記mRNAの第2の組み合わせは、Tbx3およびHex mRNAを含む、項目1に記載の方法。
(項目9)
前記mRNAの第2の組み合わせは、Tbx3、GATA4、GATA6、およびHex
mRNAを含む、項目1に記載の方法。
(項目10)
前記mRNAの第3の組み合わせは、HNF1a mRNAを含む、項目1に記載の方法。
(項目11)
前記mRNAの第3の組み合わせは、HNF4a mRNAを含む、項目1に記載の方法。
(項目12)
前記mRNAの第3の組み合わせは、HNF4a、HNF1a、HNF6、CEB/Pa、およびCEB/Pb mRNAを含む、項目1に記載の方法。
(項目13)
前記出発細胞は、被験体の体液または組織から採取される、項目1に記載の方法。
(項目14)
項目1に記載の方法によって得られる細胞。
(項目15)
項目14に記載の細胞を含む、疾患、障害、または形成異常を処置するための組成物。
(項目16)
疾患、障害、または形成異常を処置するための方法であって、該方法は、項目14に記載の細胞および項目15に記載の組成物のうちの少なくとも一方をその必要性のある被験体に投与する工程を包含する、方法。
(項目17)
前記細胞は、レシピエント被験体に由来する、項目16に記載の方法。
(項目18)
前記出発細胞は、レシピエントから採取される、項目16に記載の方法。
(項目19)
人工多能性幹細胞から、分化した肝細胞を生成するための方法であって、該方法は、
(a)該人工多能性幹細胞を出発細胞として、分化のための条件下で培養する工程;
(b)多能性状態から中内胚葉系統に向かって出るように該出発細胞を誘導する工程;
(c)有効用量においてかつ特定の時間域内でmRNAの第1の組み合わせでの培養細胞トランスフェクションを通じて、該分化している細胞を内胚葉細胞に向かって方向付ける工程;
(d)mRNAの第2の組み合わせでのトランスフェクションを通じて、該内胚葉細胞を肝前駆細胞に向かってさらに方向付ける工程;
(e)mRNAの第3の組み合わせで該肝前駆細胞を肝細胞へとさらに成熟させる工程;および
(f)前駆細胞クラスターを単層へと継代するか、または該肝前駆細胞から形成されるクラスターを集め、単層へと再度プレートすることによって、該肝細胞を得る工程、
を包含する方法。
(項目20)
人工多能性幹細胞から内胚葉細胞を生成するための方法であって、該方法は、
(a)該人工多能性幹細胞を出発細胞として、分化のための条件下で培養する工程;
(b)多能性状態から中内胚葉系統に向かって出るように、該出発細胞を誘導する工程;および
(c)有効用量においてかつ特定の時間域内で内胚葉特異的mRNAでの培養細胞トランスフェクションを通じて、該分化している細胞を内胚葉に向かって方向付ける工程、
を包含する方法。
本発明を記載する場合に、本明細書で定義されない全ての用語は、当該分野で認識される一般的な意味を有する。以下の説明が本発明の具体的実施形態または特定の使用のものである程度まで、その説明は例証的であるに過ぎず、本願発明の限定ではない。以下の説明は、本発明の趣旨および範囲の中に包含される全ての選択肢、改変および均等物を網羅することが意図される。
本発明の理解を容易にするために、多くの用語が以下に定義される。本明細書で定義される用語は、本発明に直接関連する分野の当業者によって一般に理解されるとおりの意味を有する。「1つの、ある(a)」、「1つの、ある(an)」および「上記、この、その(the)」のような用語は、単数形の実体のみに言及することを意図するのではなく、具体例が例証のために使用され得る包括的なクラスを含む。本明細書中の用語法は、本発明の具体的実施形態を記載するために使用されるが、それらの使用法は、請求項の中で概説されるものを除いて、本発明の範囲を定めない。
例示的な細胞タイプとしては、例えば、内胚葉細胞、肝前駆細胞、および肝細胞が挙げられ得る。
iPSCを、標準サイズの6ウェル細胞培養プレート(約9.5cm2成長面積/ウェル)または標準サイズ12ウェル細胞培養プレート(約3.8cm2成長面積/ウェル)へとプレートして、分化を開始した。他のサイズの培養容器は、必要に応じて同様に適用可能であり、ときおり、試薬および時間の使用のより高い効率のために、6ウェルまたは12ウェルのプレートを超えるものがより好ましい場合がある。
内胚葉細胞を、市販の細胞培養容器上にプレートする。6ウェルプレートを、図2に示される実験において使用したが、他のウェルサイズも適用可能である。プレートをMatrigel(BD Biosciences)で予め被覆し、次いで、1×105~1×106 細胞を、8mM D-グルコースを補充したDMEM/F12またはMCDB131中にプレートした。ときおり、このステージにおいて1% DMSOを添加することは、肝前駆細胞を生成する効率の増大に有用であることが観察された。
肝前駆細胞を、DMEM/F12、MEMa、またはDMEM B27中のMatrigel(BD Biosciences)またはコラーゲンI(Sigma)で予め被覆した6ウェルプレートまたは他のプレートの中で培養した。他の類似の付着細胞培養培地はまた、使用に適している。
解離および再度プレートする代わりに、肝前駆細胞を、1週間~2ヶ月間、またはさらにより長期間成長し続けさせた。その期間に、クラスター化した前駆細胞は、継続して3次元(3D)スフェアを形成し、懸濁物へと移した(図4)。
本発明に従って生成した成熟肝細胞の機能をさらに試験するために、iPSC由来肝細胞の肝機能を、以下のような肝臓疾患または傷害マウスモデルにおいて試験する:1)胆汁うっ滞性肝傷害の外科的胆管結紮(BDL)マウスモデル、2)胆汁うっ滞性肝傷害のMDR2/Tgfbr2/Il2ra遺伝的改変マウスモデル、3)胆汁うっ滞性肝傷害の代替としてのDDC修正食(DDC-modified diet)、ANIT修正食もしくはd-ガラクトサミン誘導性マウスモデル、4)NASH肝傷害の高カロリー食誘導性マウスモデル、5)NASH肝傷害のob/ob, nSREBP-1cもしくはPTEN遺伝的改変マウスモデル、6)NASH肝傷害の代替としてのMCDD/CDAAマウスモデル、または7)毒性肝傷害のCCl4/TAA/DEN/DMN誘導性マウスモデル。
cGMP手順の下で適するように適合された開示されたプロトコルを使用してヒトiPSC由来肝細胞を使用する臨床試験を、他の細胞療法を参照しながら動物研究に従って投与する。その製造された肝臓細胞または3D肝臓細胞に基づくミニ臓器を、肝臓またはヒト身体の他の部分(例えば、筋肉、結合組織、または他の器官のある種の部位)に送達して、有効性を達成する。
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