WO2015178496A1 - Method for producing lung progenitor cell - Google Patents

Method for producing lung progenitor cell Download PDF

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WO2015178496A1
WO2015178496A1 PCT/JP2015/064838 JP2015064838W WO2015178496A1 WO 2015178496 A1 WO2015178496 A1 WO 2015178496A1 JP 2015064838 W JP2015064838 W JP 2015064838W WO 2015178496 A1 WO2015178496 A1 WO 2015178496A1
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直登 二宮
俊喜 横須賀
浅島　誠
晃 栗崎
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国立研究開発法人産業技術総合研究所
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Abstract

The present invention provides: a method for directly producing a lung progenitor cell, and also a mature lung cell, from a somatic cell; and a lung progenitor cell or a mature lung cell produced according to this method. More specifically, a lung progenitor cell is directly produced by introducing, to a somatic cell: genes of four factors including the transcription factors Foxa2, Gata6, Irx3, Sox21, and NMyc, and preferably five factors additionally including the transcription factor Irx3; a protein; and a low-molecular-weight compound and/or a miRNA having the activity of inducing the expression of these factors. It has also been found that, in the case of a human somatic cell, direct differentiation into a lung progenitor cell can be achieved efficiently by also adding Sox9 in addition to the aforementioned five factors. The lung progenitor cells produced as above and various mature lung cells differentiated therefrom, such as lung epithelial cells, can be used as an in vitro and in vivo evaluative cell material or kit for evaluating differentiation into functional lung cells and for evaluating toxicity.

Description

肺前駆細胞の作製方法Method for producing lung progenitor cells
　本発明は、肺の前駆細胞を特異的に作製する方法に関する。 The present invention relates to a method for specifically producing lung progenitor cells.
　再生医療を安全・確実に遂行するためには、治療対象となる組織細胞やその前駆細胞などを安定して調製することが必要となる。例えば肺の組織に関して、喫煙などと関わりが深い慢性閉塞性肺疾患は肺胞が不可逆的に破壊される慢性疾患であり、現在のところ気管支拡張薬、去痰薬、鎮咳薬などを使用して症状の進行を遅らせることは可能であるが、肺機能を組織レベルで回復させる方法が存在しない。近年、ｉＰＳ細胞などの多能性幹細胞を利用して特定の組織細胞への特異的分化を目的とした研究が進んでいるが、目的組織の細胞のみを選択的に分化させ、移植部位に適した細胞集団を調製する技術開発が進められている。
　例えば肺前駆細胞の分化方法については、幹細胞を内胚葉細胞に分化させ、そしてさらに肺を構成する組織細胞へと分化する手法が周知である。例えば、ｉｎ　ｖｉｔｒｏでＥＳ細胞を培養し、凝集させて胚様体と呼ばれる細胞塊を形成させる方法や平面培養で分化誘導し、内胚葉細胞を形成する手法が従来から広く行われている（非特許文献９、特許文献３）。特に高濃度のアクチビンなどを添加して内胚葉細胞を効率的に作成させる方法が利用されている（非特許文献１０、１２）。また、ｉＰＳ細胞から分化させた内胚葉細胞を元に分化制御する作製方法の検討も進んでいる（非特許文献１）。さらに、内胚葉細胞を前方内胚葉に分化誘導するためには、ＤｏｒｓｏｍｏｒｐｈｉｎとＳＢ４３１５４２などのＢＭＰシグナル阻害剤とＴＧＦ−ｂｅｔａ／ａｃｔｉｖｉｎシグナル阻害剤を添加して培養した後、ＢＭＰやＦＧＦを含む培地で培養する方法（非特許文献１１、１６）で肺前駆細胞を作製することも可能である。しかし、未だＥＳ／ｉＰＳ細胞を使用した分化方法は、奇形腫などの腫瘍化を排除する方法が確立しておらず、また、非常に未分化なｉＰＳ細胞を作製した後、多くのステップを踏んで再び目的組織細胞へと厳密なコントロールにより再分化させるため、細胞の調製に長い時間と労力がかかるとともに、目的外の細胞の混入などいくつもの課題が残っている。そのため、ｉＰＳ細胞から分化させた細胞を利用した臨床応用は、特にその安全性の検証や分化細胞の信頼性、分化効率の安定性などの検証に時間を要している。
　一方、腫瘍化の危険性を回避する目的でＥＳ／ｉＰＳ細胞を使用しない細胞治療法も検討されている。例えば、間葉系幹細胞を移植することにより、主にその分泌因子を介したパラクライン作用により、過剰な炎症反応を抑制し、血管新生を促すことで組織の正常化を促進することが報告されている（非特許文献２）。また、成体の肺組織には幹細胞が存在することを示唆する報告があり、これらの細胞を利用して肺の組織再生を促すという考え方もあるが（特許文献１~４、非特許文献３）、まだ実用的な方法は確立していない。
　また、ＥＳ／ｉＰＳ細胞を使用しないで目的細胞を作製する方法として、近年新たな方法が開発されつつある。ｉＰＳ細胞はＯｃｔ４、Ｓｏｘ２、Ｋｌｆ４、ｃＭｙｃの４つの遺伝子を導入することにより、ＥＳ細胞に非常によく似た未分化状態の多能性幹細胞を、分化細胞である線維芽細胞から転換させる方法で作られたが（非特許文献４）、目的とする分化細胞毎に適切な遺伝子を選択して導入することで、線維芽細胞からｉＰＳ細胞を経由することなく直接的に神経（非特許文献５）、肝臓（非特許文献６、７）、軟骨（非特許文献８）などの細胞が作製できることが報告されてきており、これらの新しい分化方法は「ダイレクトリプログラミング法」と呼ばれている。この方法では、遺伝子を導入して細胞の運命を転換する過程で、ｉＰＳ細胞様の多能性幹細胞は経由しないことから、奇形腫などへの癌化の危険性がない安全な細胞であると期待されている。しかしながら、肺組織細胞に関しては、これまでこのような直接分化させる方法は存在しなかった。慢性閉塞性肺疾患患者や肺切除後の患者のＱＯＬを高めるためにも、安全な肺細胞の作製方法が望まれており、肺組織細胞の直接分化法の開発が急務であった。 In order to perform regenerative medicine safely and reliably, it is necessary to stably prepare tissue cells or precursor cells to be treated. For example, chronic obstructive pulmonary disease, which is closely related to smoking in lung tissue, is a chronic disease in which the alveoli are irreversibly destroyed. At present, symptoms using bronchodilators, expectorants, antitussives, etc. Although it is possible to slow the progression of lung cancer, there is no way to restore lung function at the tissue level. In recent years, research aimed at specific differentiation into specific tissue cells using pluripotent stem cells such as iPS cells has progressed, but only the cells of the target tissue are selectively differentiated and suitable for transplantation sites. Development of technology to prepare cell populations is ongoing.
For example, as a method for differentiating lung progenitor cells, a technique for differentiating stem cells into endoderm cells and further differentiating into tissue cells constituting the lung is well known. For example, a method of culturing ES cells in vitro and aggregating them to form a cell mass called an embryoid body, or a method of inducing differentiation by planar culture to form endoderm cells has been widely used (non-contained). Patent Document 9 and Patent Document 3). In particular, a method for efficiently producing endoderm cells by adding a high concentration of activin or the like is used (Non-patent Documents 10 and 12). In addition, a production method for controlling differentiation based on endoderm cells differentiated from iPS cells has been studied (Non-patent Document 1). Furthermore, in order to induce differentiation of endoderm cells into anterior endoderm, a BMP signal inhibitor such as Dorsomorphin and SB431542 and a TGF-beta / activin signal inhibitor are added and cultured in a medium containing BMP and FGF. It is also possible to produce lung progenitor cells by the culture method (Non-patent Documents 11 and 16). However, the differentiation method using ES / iPS cells has not yet established a method for eliminating tumorigenesis such as teratoma, and many steps have been taken after producing very undifferentiated iPS cells. In order to re-differentiate cells into target tissue cells under strict control, it takes a long time and labor to prepare cells, and there are still a number of problems such as contamination of cells other than the target cells. Therefore, clinical application using cells differentiated from iPS cells requires time especially for verification of safety, reliability of differentiated cells, stability of differentiation efficiency, and the like.
On the other hand, cell therapy methods that do not use ES / iPS cells are being studied for the purpose of avoiding the risk of tumorigenesis. For example, it has been reported that transplanting mesenchymal stem cells promotes normalization of tissues by suppressing excessive inflammatory reactions and promoting angiogenesis mainly by paracrine action via secretory factors. (Non-Patent Document 2). In addition, there is a report suggesting that stem cells are present in adult lung tissue, and there is a concept of using these cells to promote lung tissue regeneration (Patent Documents 1 to 4, Non-Patent Document 3). A practical method has not been established yet.
Further, in recent years, new methods have been developed as methods for producing target cells without using ES / iPS cells. An iPS cell is a method in which undifferentiated pluripotent stem cells very similar to ES cells are transformed from differentiated fibroblasts by introducing four genes, Oct4, Sox2, Klf4, and cMyc. Although it was made (Non-Patent Document 4), by selecting and introducing an appropriate gene for each target differentiated cell, the nerve is directly transmitted from the fibroblast without going through the iPS cell (Non-Patent Document 5). ), Liver (Non-Patent Documents 6 and 7), cartilage (Non-Patent Document 8) and the like have been reported to be prepared, and these new differentiation methods are called “direct reprogramming methods”. In this method, since iPS cell-like pluripotent stem cells do not pass through the process of introducing a gene to change the fate of cells, the cells are safe without risk of canceration such as teratomas. Expected. However, there has never been such a direct differentiation method for lung tissue cells. In order to increase the QOL of patients with chronic obstructive pulmonary disease and patients after lung resection, a safe method for producing lung cells is desired, and development of a direct differentiation method for lung tissue cells has been urgently required.
国際公開２０１０−１１９８１９（ＷＯ２０１０１１９８１９Ａ１）International Publication 2010-119819 (WO20101011981 A1) 国際公開２０１３−６６８０２（ＷＯ２０１３０６６８０２Ａ２）International Publication 2013-66802 (WO201306682A2) 米国公開公報２０１４−１７６９１（ＵＳ２０１４００１７６９１Ａ１）US Publication No. 2014-17691 (US20140017691A1) 特表２００５−５２０５１９Special table 2005-520519
　上記のように、肺の再生医療において、幹細胞から肺組織細胞を分化させる安全で精度の高い方法はこれまで提供されてきたとは言い難い。特に、ＥＳ細胞やｉＰＳ細胞を用いる場合、多段階の分化段階を経て正確に肺組織に分化させることが必要であるが、その分化効率や安定性が不十分である上に、腫瘍化の問題が未解決である。本発明は、このような技術背景を踏まえ、新たな肺前駆細胞の作製方法として、体細胞から直接肺前駆細胞を作製する、肺細胞における「ダイレクトリプログラミング法」を提供しようとするものである。 As described above, it is difficult to say that a safe and highly accurate method for differentiating lung tissue cells from stem cells has been provided so far in lung regenerative medicine. In particular, when ES cells or iPS cells are used, it is necessary to differentiate into lung tissue accurately through multiple stages of differentiation, but the differentiation efficiency and stability are insufficient, and there is a problem of tumorigenesis. Is unresolved. In light of such a technical background, the present invention intends to provide a “direct reprogramming method” for lung cells, in which lung precursor cells are directly produced from somatic cells, as a new method for producing lung precursor cells. .
　本発明者らは、肺の発生過程で特異的に発現する転写因子に注目し、肺の組織が形成され始める胎児期の肺前駆体組織で、その時期に特異的に発現する遺伝子について文献情報やデータベースの情報を総合的に検討した。その結果、２０００種類以上存在する転写因子の中から、将来肺組織を構成する様々な細胞に分化する肺細胞の元となる前駆細胞中で高発現している遺伝子を絞り込み、特にＦｏｘａ２、Ｇａｔａ６、Ｓｏｘ２１及びＮＭｙｃの４種類の転写因子、さらにＩｒｘ３を加えた５種類の転写因子を選択した。そして線維芽細胞で当該４転写因子又は５転写因子を発現させることで、線維芽細胞を肺前駆細胞へと分化転換することに成功した。
　これらの因子は、マウス胎児由来の線維芽細胞や成体マウスの表皮の線維芽細胞に遺伝子導入することにより、２~３週間で肺前駆細胞特異的に発現する転写因子Ｎｘｋ２．１陽性の肺前駆細胞へと直接分化させることができる。これら４つの転写因子から一つの因子でも抜くと、Ｎｋｘ２．１タンパク質を発現する肺前駆細胞が全く出現しないことから、Ｆｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃの転写因子はいずれも必須因子であると考えられる。さらにＩｒｘ３転写因子を加えた５転写因子が効率よく肺前駆細胞への直接分化を促すことも確認できたことから、Ｉｒｘ３転写因子は、肺前駆細胞への直接分化促進には欠かせない因子であると考えられる。Ｉｒｘ３転写因子を加えたこれら５つの転写因子で直接分化させた肺前駆細胞はｉｎ　ｖｉｔｒｏでの培養により、肺胞上皮Ｉ型細胞などを含む肺組織細胞へと分化する能力を持っていることを確認できた。このような知見をもとに、これまでにない肺胞上皮細胞などへの分化能を持つ肺前駆細胞作製方法に係る本発明を完成させた。
　さらに、ヒト細胞の場合には、上記５因子に加えてＳｏｘ９を加えることによって、効率的に肺前駆細胞へ直接分化させることができることを見出した。
　すなわち、本発明は以下の通りである。
〔１〕　下記の（ａ）~（ｄ）の転写因子ファミリー内の転写因子からそれぞれ１つ以上選択された４種の転写因子を含む、体細胞からのＮｋｘ２．１を発現する肺前駆細胞作製用試薬；
（ａ）　Ｆｏｘファミリー、
（ｂ）　Ｇａｔａファミリー、
（ｃ）　Ｓｏｘファミリー、及び
（ｄ）　Ｍｙｃファミリー。
〔２〕　（ａ）　Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
　前記〔１〕に記載の試薬。
〔３〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ転写因子を含む、前記〔１〕又は〔２〕に記載の試薬。
〔４〕　さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上選択された転写因子を含む、前記〔１〕~〔３〕のいずれかに記載の試薬。
〔５〕　（ｅ）Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、前記〔１〕~〔４〕のいずれかに記載の試薬。
〔６〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３転写因子を含む、前記〔１〕~〔５〕のいずれかに記載の試薬。
〔７〕　（ｃ）ＳｏｘファミリーからＳｏｘ９を含む２つ以上の転写因子を含む、前記〔１〕~〔６〕のいずれかの試薬。
〔８〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含む、前記〔６〕又は〔７〕に記載の試薬。
〔９〕　体細胞からＮｋｘ２．１を発現する肺前駆細胞を作製する方法であって、下記の（１）~（３）の工程を含むことを特徴とする、方法；
（１）下記の（ａ）~（ｄ）の転写因子ファミリーからそれぞれ１つ以上の転写因子を選択する工程、
（ａ）　Ｆｏｘファミリー、
（ｂ）　Ｇａｔａファミリー、
（ｃ）　Ｓｏｘファミリー、及び
（ｄ）　Ｍｙｃファミリー、
（２）標的体細胞内で、前記４種類の転写因子を含む転写因子群を作用させる工程、
（３）転写因子作用後の細胞でＮｋｘ２．１遺伝子を発現していることを確認する工程。
〔１０−１〕　工程（２）が、前記転写因子をコードする核酸を標的体細胞内に導入する工程を含むことを特徴とする、前記〔９〕に記載の方法。
　具体的には、転写遺伝子をウイルスベクターなどで単離した体細胞内に、又は直接肺組織の体細胞にウイルスベクターや遺伝子導入剤を利用して導入することができる（Ｌａｕｂｅ　ＢＬ，Ｔｒａｎｓｌ　Ｒｅｓｐｉｒ　Ｍｅｄ．２０１４　２，３．参照）。その際、転写因子遺伝子としては、ＤＮＡに限定されることなく、転写因子のｍＲＮＡをＲＮＡ導入試薬やセンダイウイルスなどのＲＮＡウイルスベクターで直接ヒト肺組織に導入することもできる。
〔１０−２〕　工程（２）が、前記転写因子を標的体細胞培養培地中に添加する工程、又は標的体細胞を含む肺組織内に単独若しくは薬理学的に許容される担体と共に投与される工程を含むことを特徴とする、前記〔９〕に記載の方法。
　具体的には、脂質ベースのタンパク質導入試薬で直接肺組織に導入することにできるし、転写因子タンパク質に膜透過ペプチドを付加した融合タンパク質を直接肺組織細胞に導入することも可能である。
〔１１〕　前記体細胞が、単離された体細胞である前記〔９〕又は〔１０−１〕もしくは〔１０−２〕に記載の方法。
〔１２〕　（ａ）　Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ３，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
前記〔９〕~〔１１〕のいずれかに記載の方法。
〔１３〕　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃを含むことを特徴とする、前記〔９〕~〔１２〕のいずれかに記載の方法。
〔１４〕　工程（１）において、さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上の転写因子を選択する工程を含み、工程（２）において、当該転写因子も含めた５種類の転写因子を含む転写因子群を作用させることを特徴とする、前記〔９〕~〔１３〕のいずれかに記載の方法。
〔１５〕　（ｅ）Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、前記〔１４〕に記載の方法。
〔１６〕　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３転写因子を含むことを特徴とする、前記〔１４〕又は〔１５〕に記載の方法。
〔１７〕　体細胞がヒト由来体細胞であって、工程（１）において、（ｃ）ＳｏｘファミリーからＳｏｘ９を含む２以上の転写因子を選択する工程を含み、工程（２）において、当該転写因子も含めた６種類の転写因子を含む転写因子群を作用させることを特徴とする、前記〔１４〕~〔１６〕のいずれかに記載の方法。
〔１８〕　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含むことを特徴とする、前記〔１７〕に記載の方法。
〔１９〕　単離された体細胞に対して前記〔１１〕~〔１８〕のいずれかに記載の方法を適用してＮｋｘ２．１を発現する肺前駆細胞を作製し、次いで、得られた肺前駆細胞を所望の肺組織細胞への分化誘導用培地で培養することを特徴とする、分化された肺組織細胞の製造方法。
〔２０〕　分化誘導用培地が、低血清又は無血清の上皮細胞用培地に肺組織細胞分化誘導因子を添加した培地である前記〔１９〕に記載の方法。
〔２１〕　Ｎｋｘ２．１を発現する肺前駆細胞から所望の肺組織細胞への分化誘導促進剤のスクリーニング方法であって、下記の（１）~（４）の工程を含むことを特徴とする、方法；
（１）単離された体細胞に対して前記〔１１〕~〔１８〕のいずれかに記載の方法を適用してＮｋｘ２．１を発現する肺前駆細胞を作製する工程、
（２）得られた肺前駆細胞の１部を、所望の肺組織細胞への分化誘導用培地で培養する工程、
（３）肺前駆細胞の他の１部を、工程（２）で用いた分化誘導用培地に被検物質を添加した培地で培養するか、又は肺前駆細胞内に被検物質を導入後に工程（２）で用いた分化誘導用培地で培養する工程、
（４）工程（２）及び工程（３）で用いた肺前駆細胞の所望の肺組織細胞特異的マーカーの発現量を一定時間経過後に測定して比較し、後者の発現量が高い場合に、被検物質を肺前駆細胞から所望の肺組織細胞への分化誘導促進剤の候補物質となると評価して、選択する工程。
〔２２〕　Ｎｋｘ２．１を発現する肺前駆細胞から所望の肺組織細胞への分化誘導促進剤のスクリーニング用キットであって、前記〔１１〕~〔１８〕のいずれかに記載の方法により作製した肺前駆細胞を含むことを特徴とするキット。
〔２３〕　Ｎｋｘ２．１を発現する肺前駆細胞又は肺組織細胞を補充する方法に使用するための組成物であって、
前記組成物は、下記の（ａ）~（ｄ）の転写因子ファミリーからそれぞれ１つ以上選択された転写因子を含むものであり、
前記方法は体細胞からＮｋｘ２．１を発現する肺前駆細胞への直接分化を誘導することを特徴とする方法；
（ａ）　Ｆｏｘファミリー、
（ｂ）　Ｇａｔａファミリー、
（ｃ）　Ｓｏｘファミリー、及び
（ｄ）　Ｍｙｃファミリー。
〔２４〕　（ａ）Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
　前記〔２３〕に記載の方法。
〔２５〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ転写因子を含む、前記〔２３〕又は〔２４〕に記載の方法。
〔２６〕　さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上選択された転写因子を含む、前記〔２３〕~〔２５〕のいずれかに記載の方法。
〔２７〕　（ｅ）Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、前記〔２６〕に記載の方法。
〔２８〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３の転写因子を含む、前記〔２６〕又は〔２７〕に記載の方法。
〔２９〕　（ｃ）ＳｏｘファミリーからＳｏｘ９を含む２つ以上の転写因子を含む、前記〔２３〕~〔２８〕のいずれか１項に記載の方法。
〔３０〕　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含む、前記〔２９〕に記載の方法。 The present inventors focused on transcription factors that are specifically expressed in the developmental process of the lung, and literature information on genes that are specifically expressed at that time in the fetal progenitor tissue, where lung tissue begins to form. And database information were comprehensively examined. As a result, genes that are highly expressed in the progenitor cells that are the source of lung cells that will differentiate into various cells that will form lung tissue in the future are selected from among 2000 or more types of transcription factors, particularly Foxa2, Gata6, Four types of transcription factors, Sox21 and NMyc, and five types of transcription factors including Irx3 were selected. Then, fibroblasts were successfully transformed into lung progenitor cells by expressing the 4 transcription factor or 5 transcription factor in fibroblasts.
These factors are gene transcription into mouse embryonic fibroblasts and adult mouse epidermal fibroblasts, which are expressed in lung progenitor cells in 2 to 3 weeks. Differentiate directly into cells. If one of these four transcription factors is removed, no lung progenitor cells that express Nkx2.1 protein will appear at all. Therefore, the transcription factors of Foxa2, Gata6, Sox21 and NMyc are considered to be essential factors. . Furthermore, it was confirmed that 5 transcription factors including Irx3 transcription factor efficiently promoted direct differentiation into lung progenitor cells. Therefore, Irx3 transcription factor is an indispensable factor for promoting direct differentiation into lung progenitor cells. It is believed that there is. Lung progenitor cells directly differentiated with these five transcription factors plus Irx3 transcription factor have the ability to differentiate into lung tissue cells, including alveolar epithelial type I cells, by in vitro culture. It could be confirmed. Based on these findings, the present invention relating to a method for producing lung progenitor cells capable of differentiating into alveolar epithelial cells and the like, which has never been achieved, has been completed.
Furthermore, in the case of human cells, it was found that by adding Sox9 in addition to the above five factors, it can be efficiently differentiated directly into lung progenitor cells.
That is, the present invention is as follows.
[1] Preparation of lung progenitor cells expressing Nkx2.1 from somatic cells, each of which includes four transcription factors selected from one or more of the transcription factors in the transcription factor family of (a) to (d) below: Reagents;
(A) Fox family,
(B) Gata family,
(C) Sox family, and (d) Myc family.
[2] (a) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd8, Fox3, Foxd8 , Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox1, Fox3 Including transcription factors,
(B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
(C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
The reagent according to [1] above.
[3] The reagent according to [1] or [2] above, which contains at least Foxa2, Gata6, Sox21 and NMyc transcription factor.
[4] The reagent according to any one of [1] to [3], further comprising (e) one or more transcription factors selected from transcription factors in the transcription factor family belonging to the Irx family.
[5] (e) The reagent according to any one of [1] to [4] above, wherein the transcription factor selected from the Irx family includes any of the transcription factors Irx1, Irx2, Irx3, Irx4, Irx5 or Irx6 .
[6] The reagent according to any one of [1] to [5], which contains at least Foxa2, Gata6, Sox21, NMyc and Irx3 transcription factors.
[7] (c) The reagent according to any one of [1] to [6] above, which comprises two or more transcription factors including Sox9 from the Sox family.
[8] The reagent according to [6] or [7] above, which contains at least the transcription factors of Foxa2, Gata6, Sox21, NMyc, Irx3 and Sox9.
[9] A method for producing lung progenitor cells expressing Nkx2.1 from somatic cells, which comprises the following steps (1) to (3):
(1) a step of selecting one or more transcription factors from the following transcription factor families (a) to (d):
(A) Fox family,
(B) Gata family,
(C) Sox family, and (d) Myc family,
(2) a step of causing a group of transcription factors including the four types of transcription factors to act in a target somatic cell;
(3) A step of confirming that the Nkx2.1 gene is expressed in the cell after the action of the transcription factor.
[10-1] The method according to [9] above, wherein the step (2) includes a step of introducing a nucleic acid encoding the transcription factor into a target somatic cell.
Specifically, a transcriptional gene can be introduced into a somatic cell isolated by a viral vector or directly into a somatic cell of a lung tissue using a viral vector or a gene introduction agent (Laube BL, Transl Respir Med). .2014 2, 3). In this case, the transcription factor gene is not limited to DNA, and mRNA of the transcription factor can be directly introduced into human lung tissue using an RNA introduction reagent or an RNA virus vector such as Sendai virus.
[10-2] The step (2) is administered in the step of adding the transcription factor to the target somatic cell culture medium, or in the lung tissue containing the target somatic cell alone or with a pharmacologically acceptable carrier The method according to [9] above, comprising a step.
Specifically, it can be directly introduced into lung tissue with a lipid-based protein introduction reagent, or a fusion protein obtained by adding a membrane-permeable peptide to a transcription factor protein can be directly introduced into lung tissue cells.
[11] The method according to [9], [10-1] or [10-2] above, wherein the somatic cell is an isolated somatic cell.
[12] (a) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd3, Foxd8 , Foxe3, Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn5, Foxn1, Fox3, Foxn3 Including some transcription factors,
(B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
(C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
The method according to any one of [9] to [11].
[13] The method according to any one of [9] to [12], wherein the transcription factor group includes at least Foxa2, Gata6, Sox21, and NMyc.
[14] In the step (1), the method further includes (e) selecting one or more transcription factors from transcription factors in the transcription factor family belonging to the Irx family, and the transcription factor is also included in the step (2). The method according to any one of [9] to [13] above, wherein a group of transcription factors including 5 types of transcription factors is allowed to act.
[15] The method according to [14], wherein the transcription factor selected from (e) Irx family comprises any one of Irx1, Irx2, Irx3, Irx4, Irx5, and Irx6.
[16] The method according to [14] or [15], wherein the transcription factor group includes at least Foxa2, Gata6, Sox21, NMyc, and Irx3 transcription factors.
[17] The somatic cell is a human-derived somatic cell, and in step (1), the method includes (c) selecting two or more transcription factors including Sox9 from the Sox family, and in step (2), the transcription factor The method according to any one of [14] to [16] above, wherein a group of transcription factors including 6 types of transcription factors including the above is allowed to act.
[18] The method according to [17], wherein the transcription factor group includes at least Foxa2, Gata6, Sox21, NMyc, Irx3, and Sox9 transcription factors.
[19] Applying the method according to any one of [11] to [18] to isolated somatic cells to produce lung progenitor cells expressing Nkx2.1, and then the obtained lung A method for producing differentiated lung tissue cells, comprising culturing precursor cells in a medium for inducing differentiation into desired lung tissue cells.
[20] The method according to [19], wherein the differentiation-inducing medium is a medium obtained by adding a lung tissue cell differentiation-inducing factor to a low-serum or serum-free medium for epithelial cells.
[21] A screening method for an agent for inducing differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell, comprising the following steps (1) to (4): Method;
(1) A step of producing lung progenitor cells expressing Nkx2.1 by applying the method according to any of [11] to [18] to isolated somatic cells,
(2) culturing a part of the obtained lung progenitor cells in a medium for inducing differentiation into desired lung tissue cells,
(3) Another part of lung progenitor cells is cultured in a medium obtained by adding a test substance to the differentiation-inducing medium used in step (2), or after introducing the test substance into lung progenitor cells Culturing in the differentiation-inducing medium used in (2),
(4) When the expression level of a desired lung tissue cell-specific marker of the lung progenitor cells used in step (2) and step (3) is measured after a certain period of time and compared, and the latter expression level is high, A step of evaluating and selecting a test substance as a candidate substance for an agent for promoting differentiation induction from lung progenitor cells to desired lung tissue cells.
[22] A screening kit for an agent for promoting differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell, which is prepared by the method according to any one of [11] to [18] A kit comprising lung progenitor cells.
[23] A composition for use in a method of supplementing lung progenitor cells or lung tissue cells expressing Nkx2.1,
The composition comprises one or more transcription factors selected from the following transcription factor families (a) to (d):
Said method induces direct differentiation of somatic cells into lung progenitor cells expressing Nkx2.1;
(A) Fox family,
(B) Gata family,
(C) Sox family, and (d) Myc family.
[24] (a) The transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd8, Fox3, Foxd8, Fox3 , Foxf1, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox1, Fox3 Including transcription factors,
(B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
(C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
The method according to [23] above.
[25] The method according to [23] or [24] above, which comprises at least Foxa2, Gata6, Sox21 and NMyc transcription factor.
[26] The method according to any one of [23] to [25], further comprising (e) one or more transcription factors selected from transcription factors in the transcription factor family belonging to the Irx family.
[27] The method according to [26], wherein the transcription factor selected from the Irx family comprises any of the transcription factors Irx1, Irx2, Irx3, Irx4, Irx5, and Irx6.
[28] The method according to [26] or [27] above, comprising at least Foxa2, Gata6, Sox21, NMyc, and Irx3 transcription factors.
[29] The method according to any one of [23] to [28] above, comprising (c) two or more transcription factors including Sox9 from the Sox family.
[30] The method according to [29] above, comprising at least Foxa2, Gata6, Sox21, NMyc, Irx3 and Sox9 transcription factors.
　本発明により、体細胞から直接肺前駆細胞を作製することができる。この方法で作製した肺前駆細胞は肺胞上皮細胞など肺組織の様々な細胞に分化する能力を有しているので、ｉｎ　ｖｉｔｒｏで分化させた肺胞上皮細胞などを、肺組織に対する毒性試験用の細胞キットに応用できる。また、肺前駆細胞をより成熟した肺組織細胞へと分化させる際の分化薬剤のスクリーニング用細胞としても利用できる。さらに肺癌など遺伝子変異を伴う疾患の原因遺伝子を保持した細胞から肺前駆細胞を作製することにより、肺癌などの遺伝子変異を伴う疾患の治療薬候補をスクリーニングするための細胞キットとしても利用できる。 According to the present invention, lung progenitor cells can be produced directly from somatic cells. Since lung progenitor cells produced by this method have the ability to differentiate into various cells of lung tissue such as alveolar epithelial cells, alveolar epithelial cells differentiated in vitro can be used for toxicity tests on lung tissue. It can be applied to cell kits. It can also be used as a screening cell for a differentiation agent when differentiating lung progenitor cells into more mature lung tissue cells. Furthermore, by producing lung progenitor cells from cells retaining a causative gene of a disease associated with a gene mutation such as lung cancer, it can also be used as a cell kit for screening therapeutic drug candidates for diseases associated with a gene mutation such as lung cancer.
転写因子を線維芽細胞に導入することにより肺前駆細胞を作製する方法の作製条件検討。ａ．マウス胎児線維芽細胞に胎児肺組織で発現する転写因子１３種類をレトロウイルスで感染させ、肺前駆細胞を作製する基本条件。ｂ．マウス胎児（１１．５日胚）の肺前駆組織で特異的に発現するＮｋｘ２．１遺伝子は、線維芽細胞に１３種類の転写因子を導入すると発現開始し、線維芽細胞（ＭＥＦ）を肺前駆細胞へと直接分化可能であることが確認された。ｃ．マウス線維芽細胞から直接分化させた肺前駆細胞のコロニー状の形態。ｄ．線維芽細胞から直接分化させたコロニー状の肺前駆細胞集団は転写因子Ｎｋｘ２．１を発現した。ｅ．１３個の転写因子から１因子ずつ除去した転写因子の組合せで、肺前駆細胞を作製する必須因子の絞り込みを行った際の肺前駆細胞特異的転写因子Ｎｋｘ２．１遺伝子の発現。ｆ．ｅのサンプルにおけるＥ−ｃａｄｈｅｒｉｎの発現。ｇ．上記ｅ‐ｆで絞り込んだ６因子から、再び必須因子の洗い出しをＮｋｘ２．１遺伝子の発現を指標に行った。ｈ．ｇのサンプルにおけるＥ−ｃａｄｈｅｒｉｎの発現。ｉ．上記ｇ‐ｈで絞り込んだ５因子からさらに必須因子の洗い出しをＮｋｘ２．１遺伝子の発現を指標に行った。ｊ．ｉのサンプルにおけるＥ−ｃａｄｈｅｒｉｎの発現。Examination of production conditions for a method of producing lung progenitor cells by introducing transcription factors into fibroblasts. a. Basic conditions for producing lung progenitor cells by infecting mouse fetal fibroblasts with 13 kinds of transcription factors expressed in fetal lung tissue with retrovirus. b. The Nkx2.1 gene that is specifically expressed in the lung progenitor tissue of the mouse embryo (11.5 day embryo) starts when 13 types of transcription factors are introduced into fibroblasts. It was confirmed that it can be directly differentiated into cells. c. Colony morphology of lung progenitor cells differentiated directly from mouse fibroblasts. d. A colony-like lung progenitor cell population directly differentiated from fibroblasts expressed the transcription factor Nkx2.1. e. Expression of a lung progenitor cell-specific transcription factor Nkx2.1 gene when narrowing down essential factors for producing lung progenitor cells by combining transcription factors obtained by removing one factor from thirteen transcription factors. f. Expression of E-cadherin in sample e. g. From the 6 factors narrowed down by ef, the essential factors were again identified using the expression of the Nkx2.1 gene as an index. h. Expression of E-cadherin in g samples. i. The essential factors were further identified from the 5 factors narrowed down by the above gh, and the expression of Nkx2.1 gene was used as an index. j. Expression of E-cadherin in i samples. マウス胎児の背側表皮由来の線維芽細胞から直接作製した肺前駆細胞が、肺特異的前駆細胞であることの検証。ａ．５種類の転写因子で作製した肺前駆細胞の位相差顕微鏡像。ｂ．５種類の転写因子で作製した肺前駆細胞におけるＮｋｘ２．１の発現。ｃ．マウス胎児におけるＮｋｘ２．１陽性細胞の局在部位。ｄ．肺前駆細胞特異的マーカータンパク質の発現の特異性。ｅ．作製した肺前駆細胞で脳や甲状腺特異的遺伝子発現が見られないことの定量ＲＴ−ＰＣＲによる検証。ｆ．各種線維芽細胞からのＮｋｘ２．１陽性の肺前駆細胞コロニーの出現頻度。なお、Ｄｏｒｓａｌ　ｓｋｉｎ：マウス胎児の背側表皮由来線維芽細胞、Ｌｉｍｂ　ｓｋｉｎ：マウス胎児四肢由来線維芽細胞、ＭＤＦ：成体マウス頭部表皮線維芽細胞。Verification that lung progenitor cells made directly from mouse embryonic dorsal epidermis fibroblasts are lung-specific progenitor cells. a. Phase contrast microscopic images of lung progenitor cells prepared with 5 types of transcription factors. b. Expression of Nkx2.1 in lung progenitor cells made with 5 types of transcription factors. c. Localization site of Nkx2.1 positive cells in mouse fetus. d. Specificity of expression of lung progenitor cell specific marker protein. e. Verification by quantitative RT-PCR that no brain or thyroid-specific gene expression is seen in the prepared lung progenitor cells. f. Frequency of appearance of Nkx2.1 positive lung progenitor colonies from various fibroblasts. Dorsal skin: dorsal epidermis fibroblasts of mouse fetus, Limb skin: mouse embryo limb-derived fibroblasts, MDF: adult mouse head epidermis fibroblasts. （図２）のデータに関してさらに検証を行い、図２ｄのマウス胎児におけるＮｋｘ２．１陽性細胞の局在部位について、上皮細胞マーカーであるＥｐＣＡＭや肺前駆細胞で発現する転写因子Ｓｏｘとの共染色を行い（図３ｂ）、この結果を定量した（図３ｃ）。なお、図３ｂ及び図３ｃのうち、赤枠以外はそれぞれ図２ｄ及び図２ｅと同じ。Further verification was performed on the data of (FIG. 2), and the co-staining with EpCAM as an epithelial cell marker and transcription factor Sox expressed in lung progenitor cells was performed on the localization site of Nkx2.1 positive cells in the mouse fetus in FIG. The results were quantified (Figure 3c). 3B and 3C are the same as FIGS. 2D and 2E, respectively, except for the red frame. マウス胎児四肢由来の線維芽細胞から直接作製した肺前駆細胞が、肺特異的前駆細胞であることの検証。ａ．マウス胎児四肢由来線維芽細胞に胎児肺組織で発現する転写因子５種類をレトロウイルスで感染させ、肺前駆細胞を作製する条件。ｂ．５種類の転写因子で作製した肺前駆細胞の位相差顕微鏡像。ｃ．５種類の転写因子で作製した肺前駆細胞におけるＮｋｘ２．１の発現。ｄ．肺前駆細胞特異的マーカータンパク質の発現の特異性。ｅ．作製した肺前駆細胞で脳や甲状腺特異的遺伝子発現が見られないことの定量ＲＴ−ＰＣＲによる検証。Verification that lung progenitor cells directly produced from mouse embryonic limb-derived fibroblasts are lung-specific progenitor cells. a. Conditions for producing lung progenitor cells by infecting mouse fetal limb-derived fibroblasts with 5 types of transcription factors expressed in fetal lung tissue with retrovirus. b. Phase contrast microscopic images of lung progenitor cells prepared with 5 types of transcription factors. c. Expression of Nkx2.1 in lung progenitor cells made with 5 types of transcription factors. d. Specificity of expression of lung progenitor cell specific marker protein. e. Verification by quantitative RT-PCR that no brain or thyroid-specific gene expression is seen in the prepared lung progenitor cells. 成体マウスの表皮由来線維芽細胞から作製した肺前駆細胞の分化能力の検証。ａ．成体マウス表皮由来線維芽細胞に胎児肺組織で発現する転写因子５種類をレトロウイルスで感染させ、肺前駆細胞を作製する条件。ｂ．５種類の転写因子で作製した肺前駆細胞の位相差顕微鏡像。ｃ．５種類の転写因子で作製した肺前駆細胞におけるＮｋｘ２．１の発現。ｄ．肺前駆細胞特異的マーカータンパク質の発現の特異性。ｅ．作製した肺前駆細胞で脳や甲状腺特異的遺伝子発現が見られないことの定量ＲＴ−ＰＣＲによる検証。Verification of differentiation ability of lung progenitor cells prepared from adult mouse epidermis-derived fibroblasts. a. Conditions for producing lung progenitor cells by infecting adult mouse epidermis-derived fibroblasts with 5 types of transcription factors expressed in fetal lung tissue with retroviruses. b. Phase contrast microscopic images of lung progenitor cells prepared with 5 types of transcription factors. c. Expression of Nkx2.1 in lung progenitor cells made with 5 types of transcription factors. d. Specificity of expression of lung progenitor cell specific marker protein. e. Verification by quantitative RT-PCR that no brain or thyroid-specific gene expression is seen in the prepared lung progenitor cells. 線維芽細胞からＮｋｘ２．１陽性の上皮細胞へと転換していることの検証。ａ．線維芽細胞と線維芽細胞から直接作製した肺前駆細胞の上皮細胞マーカーと間葉マーカーの発現の定量ＲＴ−ＰＣＲ。ｂ．作製した肺前駆細胞における上皮・間葉マーカーの免疫蛍光染色。Verification of conversion from fibroblasts to Nkx2.1 positive epithelial cells. a. Quantitative RT-PCR of expression of epithelial cell markers and mesenchymal markers in lung progenitor cells made directly from fibroblasts and fibroblasts. b. Immunofluorescence staining of epithelial / mesenchymal markers in the prepared lung progenitor cells. 作製した肺前駆細胞のマイクロアレイによる網羅的遺伝子発現解析。ａ．Ｅ１１．５胎児由来肺、繊維芽細胞及び作製した肺前駆細胞の遺伝子発現パターンを階層クラスタリング解析法により比較。ｂ．ａの結果の内、各サンプルにおける肺前駆細胞マーカー遺伝子Ｎｋｘ２．１，ＳＰ−Ｃ，Ｆｏｘａ１，Ｆｏｘａ２，Ｇａｔａ６、上皮細胞マーカー遺伝子Ｅ−ｃａｄｈｅｒｉｎ，ＥｐＣＡＭ、繊維芽細胞マーカー遺伝子Ｔｇｆｂ１ｉ１，Ｔｈｙ１，Ｔｗｉｓｔ２，Ｚｅｂ１，Ｚｅｎ２の遺伝子発現パターン。Comprehensive gene expression analysis by microarray of prepared lung progenitor cells. a. Comparison of gene expression patterns of E11.5 fetal lung, fibroblasts and prepared lung progenitor cells by hierarchical clustering analysis. b. Among the results of a, lung progenitor cell marker genes Nkx2.1, SP-C, Foxa1, Foxa2, Gata6, epithelial cell marker genes E-cadherin, EpCAM, fibroblast marker genes Tgfb1i1, Thy1, Twist2, Zeb1 in each sample , Zen2 gene expression pattern. 作製した肺前駆細胞のマイクロアレイによる網羅的遺伝子発現解析の続き。Ｅ１１．５胎児由来肺、作製した肺前駆細胞及び成体由来気管支、肺、胃、腸、肝臓、心臓、腎臓、大脳皮質、骨格筋、脂肪の遺伝子発現パターンを階層クラスタリング解析により比較。Continuation of comprehensive gene expression analysis by microarray of prepared lung progenitor cells. Comparison of gene expression patterns of E11.5 fetal lung, prepared lung progenitor cells and adult bronchus, lung, stomach, intestine, liver, heart, kidney, cerebral cortex, skeletal muscle, and fat by hierarchical clustering analysis. （図８ａ）のデータに関してさらに検証を行うため、３種類の線維芽細胞（Ｄｏｒｓａｌ　ＭＥＦ，Ｌｉｍｂ　ＭＥＦ，ＭＤＦ）から作製した肺前駆細胞のｎ数を増やしても類似の遺伝子発現パターンを示し、Ｅ１１．５胎児由来肺組織と同じクラスターに分類されたが、成体のすい臓、肺、気管支、胃、腸、肝臓、心臓、腎臓、大脳皮質、骨格筋、脂肪とは全く違う遺伝子発現パターン示した。In order to further verify the data of (Fig. 8a), even if the number of lung progenitor cells prepared from three types of fibroblasts (Dorsal MEF, Limb MEF, MDF) is increased, a similar gene expression pattern is shown, E11 .5 Although it was classified into the same cluster as the fetal lung tissue, the gene expression pattern was completely different from that of adult pancreas, lung, bronchi, stomach, intestine, liver, heart, kidney, cerebral cortex, skeletal muscle, and fat. マウス線維芽細胞から作製した肺前駆細胞の分化能力の検証。ａ．肺前駆細胞の自発的な分化誘導。ｂ．ｉｎ　ｖｉｔｒｏで自発的に分化させた肺細胞の免疫蛍光染色によるマーカー発現の検証。１５日間直接分化誘導した肺前駆細胞をさらに５１日まで培養した後、肺細胞分化能を免疫蛍光染色により検証した。粘液分泌細胞マーカー（Ｍｕｃ５ＡＣ）、肺胞上皮Ｉ型細胞（ＡＱＰ５）。Verification of differentiation ability of lung progenitor cells prepared from mouse fibroblasts. a. Spontaneous differentiation induction of lung progenitor cells. b. Verification of marker expression by immunofluorescence staining of lung cells spontaneously differentiated in vitro. The lung progenitor cells directly induced to differentiate for 15 days were further cultured for up to 51 days, and then the lung cell differentiation ability was verified by immunofluorescence staining. Mucus secreting cell marker (Muc5AC), alveolar epithelial type I cells (AQP5). マウス線維芽細胞から作製した肺前駆細胞は自発的に立体構造を形成する。ａ．自発的に形成した肺前駆細胞の立体構造とＮｋｘ２．１の発現。ｂ．ａの拡大図。Lung progenitor cells prepared from mouse fibroblasts spontaneously form a three-dimensional structure. a. Spontaneous formation of lung progenitor cells and Nkx2.1 expression. b. The enlarged view of a. マウス線維芽細胞から作製した肺前駆細胞の分化能力の検証のつづき。ａ．肺前駆細胞のｉｎ　ｖｉｔｒｏ分化誘導条件の検討。右表の培養条件で培養を続けた。ｂ．各培地でｉｎ　ｖｉｔｒｏで分化させた肺細胞の定量ＲＴ−ＰＣＲによる分化マーカー発現。ｃ．ｂのＮｏ．６のサンプルの培養上清中に分泌されたＳＰ−Ａのタンパク量。図中、ＷＦＫはＷｎｔ３ａ＋ＦＧＦ３＋ＫＧＦ。ｄ．Ｎｏ．６培地を用いて分化させた肺細胞の免疫蛍光染色による分化マーカー発現の検証。Verification of the differentiation potential of lung progenitor cells prepared from mouse fibroblasts. a. Examination of in vitro differentiation induction conditions of lung progenitor cells. The culture was continued under the culture conditions shown in the right table. b. Expression of differentiation markers by quantitative RT-PCR of lung cells differentiated in vitro in each medium. c. b No. The amount of SP-A protein secreted into the culture supernatant of 6 samples. In the figure, WFK is Wnt3a + FGF3 + KGF. d. No. Verification of differentiation marker expression by immunofluorescence staining of lung cells differentiated using 6 medium. ヒト細胞から作製した肺前駆細胞。ａ．ヒト線維芽細胞に６因子を遺伝子導入して作製した肺前駆細胞におけるＮＫＸ２．１の遺伝子発現の定量ＲＴ−ＰＣＲ解析。ｂ．ヒト線維芽細胞に６因子を遺伝子導入して作製した肺前駆細胞におけるＮＫＸ２．１タンパク質の免疫蛍光染色。ｃ．ヒト脂肪組織由来繊維芽細胞に６因子を遺伝子導入して作製した肺前駆細胞におけるＮＫＸ２．１タンパク質の免疫蛍光染色。Lung progenitor cells made from human cells. a. Quantitative RT-PCR analysis of NKX2.1 gene expression in lung progenitor cells prepared by introducing 6 factors into human fibroblasts. b. Immunofluorescence staining of NKX2.1 protein in lung progenitor cells prepared by introducing 6 factors into human fibroblasts. c. Immunofluorescence staining of NKX2.1 protein in lung progenitor cells prepared by introducing 6 factors into human adipose tissue-derived fibroblasts.
１．本発明の肺前駆細胞を作製する特異的な転写因子
　本発明の肺前駆細胞を作製する特異的な転写因子は、少なくとも、Ｆｏｘファミリー転写因子、Ｇａｔａファミリー転写因子、Ｓｏｘファミリー転写因子、及びＭｙｃファミリー転写因子に属する、それぞれの転写因子遺伝子又はその発現産物としてのタンパク質を含み、好ましくはさらにＩｒｘファミリー転写因子又はその発現産物としてのタンパク質を含んでいる。また、特にヒト肺前駆細胞を作製する場合には、Ｓｏｘファミリーから２種類の転写因子又はその発現産物が含まれることが好ましい。
　Ｆｏｘファミリー転写因子としては、Ｆｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ３，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３及びＦｏｘｑ１を挙げることができ、好ましくはその機能ドメインが高度に保存されたＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３転写因子である。これら転写因子のアミノ酸配列及びその遺伝子の塩基配列は、公共データベースから入手でき、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）のｈｕｍａｎ−Ｆｏｘａ２（Ｐｒｏｔｅｉｎ：ＮＰ＿０６８５５６）、ｍｏｕｓｅ−Ｆｏｘａ２（Ｐｒｏｔｅｉｎ：ＮＰ＿０３４５７６）を用いることができる。
　Ｇａｔａファミリー転写因子としては、Ｇａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，及びＧａｔａ６を挙げることができ、好ましくはその機能ドメインが高度に保存されたＧａｔａ６，Ｇａｔａ５，Ｇａｔａ４転写因子であり、例えばｈｕｍａｎ−Ｇａｔａ６（Ｐｒｏｔｅｉｎ：ＮＰ＿００５２４８）、ｍｏｕｓｅ−Ｇａｔａ６（Ｐｒｏｔｅｉｎ：ＮＰ＿０３４３８８）を用いることができる。
　Ｓｏｘファミリー転写因子としては、Ｓｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０及びＳｒｙを挙げることができ、好ましくはその機能ドメインが高度に保存されたＳｏｘ２１，Ｓｏｘ３，Ｓｏｘ１，Ｓｏｘ１５，Ｓｏｘ２及びＳｏｘ９転写因子であり、例えばｈｕｍａｎ−Ｓｏｘ２１（Ｐｒｏｔｅｉｎ：ＮＰ＿００９０１５）、ｍｏｕｓｅ−Ｓｏｘ２１（Ｐｒｏｔｅｉｎ：ＮＰ＿８０８４２１）及びｈｕｍａｎ−Ｓｏｘ９（Ｐｒｏｔｅｉｎ：ＮＰ＿０００３３７）を用いることができる。Ｍｙｃファミリー転写因子としては、その機能ドメインが高度に保存されたｃＭｙｃ，ＬＭｙｃ，及びＮＭｙｃ挙げることができ、好ましくはＮＭｙｃ転写因子であり、例えばｈｕｍａｎＮＭｙｃｇｅｎｅ（ｍＲＮＡ：ＮＭ＿００５３７８）、ｍｏｕｓｅＮＭｙｃ　ｇｅｎｅ（ｍＲＮＡ：ＮＭ＿００８７０９）を用いることができる。特に、ヒト肺前駆細胞を作製する場合には、Ｓｏｘ９を含む２種類の転写因子を存在させることが好ましい。
　また、Ｉｒｘファミリー転写因子としては、Ｉｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５及びＩｒｘ６を挙げることができ、好ましくはその機能ドメインが高度に保存されたＩｒｘ３，Ｉｒｘ１，Ｉｒｘ２，Ｉｒｘ４及びＩｒｘ６転写因子であり、例えば
ｈｕｍａｎ−Ｉｒｘ３（Ｐｒｏｔｅｉｎ：ＮＰ＿０７７３１２）、ｍｏｕｓｅ−Ｉｒｘ３（Ｐｒｏｔｅｉｎ：ＮＰ＿０３２４１９）を用いることができる。
　これらの転写因子群、すなわちＦｏｘファミリー転写因子、Ｇａｔａファミリー転写因子、Ｓｏｘファミリー転写因子、及びＭｙｃファミリー転写因子、さらにはＩｒｘファミリー転写因子を含む転写因子群、好ましくはＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ転写因子、より好ましくはＦｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１及びＮＭｙｃ転写因子、特に好ましくはＦｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１，ＮＭｙｃ及びＳｏｘ９転写因子を含む転写因子群を遺伝子導入などによって線維芽細胞などの体細胞（組織細胞）内で発現させてネットワークを再構築させることで、体細胞を肺前駆細胞へと直接分化させることができる。
　これら４、５又は６種類を含む転写因子は、典型的には、それぞれの遺伝子をウイルスベクターもしくは非ウイルスベクターを用いて、又は用いずに線維芽細胞などの体細胞内に導入して同時に発現させる。
　ここで、これら４、５又は６種類の転写因子のうち、いずれか１つ以上を遺伝子導入に代えて遺伝子の発現産物のタンパク質として体細胞内に導入してもよく、さらにはこれら転写因子を発現誘導する活性のある低分子化合物（例えばＦｏｘａ２の発現誘導活性があるメラトニン（非特許文献１３））やｍｉＲＮＡ及びその阻害剤（例えばＦｏｘａ２の発現誘導活性があるａｎｔｉ−ｍｉＲ１２４ａ２やＮＭｙｃの発現誘導活性があるａｎｔｉ−ｍｉＲ−３４ａ（非特許文献１４、１５））を体細胞に作用させることで代替することもできる。
　Ｆｏｘａ２転写因子、またはそれをコードする遺伝子は、上述のように公共データベース（ＧｅｎＢａｎｋ（ＮＣＢＩ）など）からアミノ酸配列、塩基配列を入手できる。例えば、Ｆｏｘａ２アミノ酸配列は、ｈｕｍａｎ−Ｆｏｘａ２（Ｐｒｏｔｅｉｎ：ＮＰ＿０６８５５６）、ｍｏｕｓｅ−Ｆｏｘａ２（Ｐｒｏｔｅｉｎ：ＮＰ＿０３４５７６）であり、Ｆｏｘａ２遺伝子は当該アミノ酸配列をコードする塩基配列を用いることができる。また、その全長を用いる必要はなくＦｏｘａ２転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、Ｆｏｘａ２転写因子として用いることができる。ここで、ヒトＦｏｘａ２及びマウスＦｏｘａ２のアミノ酸配列レベルのホモロジーは約９７％であるのに対して、Ｆｏｘａ２と同じＦｏｘａファミリー内でＦｏｘａ２ときわめてよく似た性質を有するＦｏｘａ１及びＦｏｘａ３についてみると、ヒトＦｏｘａ２及びヒトＦｏｘａ１は約５３％、ヒトＦｏｘａ２及びヒトＦｏｘａ３は約４２％、ヒトＦｏｘａ２及びヒトＦｏｘｂ１は約４８％、ヒトＦｏｘａ２及びヒトＦｏｘｂ２は約４３％である。したがって、例えばヒトＦｏｘａ１、ヒトＦｏｘａ２、ヒトＦｏｘａ３、ヒトＦｏｘｂ１及びヒトＦｏｘｂ２それぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＦｏｘａ２と同様に用いることができる。
　Ｇａｔａ６転写因子、またはそれをコードする遺伝子についても同様であり、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）などから得られるｈｕｍａｎ−Ｇａｔａ６（Ｐｒｏｔｅｉｎ：ＮＰ＿００５２４８）もしくはｍｏｕｓｅ−Ｇａｔａ６（Ｐｒｏｔｅｉｎ：ＮＰ＿０３４３８８）のアミノ酸配列からなるＧａｔａ６又は当該アミノ酸配列をコードするＧａｔａ６遺伝子を用いることができる。また、その全長を用いる必要はなくＧａｔａ６転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、Ｇａｔａ６転写因子として用いることができる。ここで、ヒトＧａｔａ６及びマウスＧａｔａ６のアミノ酸配列レベルのホモロジーは約８８％であるのに対して、Ｇａｔａ６と同じＧａｔａファミリー内でＧａｔａ６ときわめてよく似た性質を有するＧａｔａ４及びＧａｔａ５についてみると、ヒトＧａｔａ６及びヒトＧａｔａ４は約５０％、ヒトＧａｔａ６及びヒトＧａｔａ５は約４４％である。したがって、例えばヒトＧａｔａ６、ヒトＧａｔａ４及びヒトＧａｔａ５それぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＧａｔａ６と同様に用いることができる。
　Ｓｏｘ２１転写因子、またはそれをコードする遺伝子についても同様であり、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）などから得られるｈｕｍａｎ−Ｓｏｘ２１（Ｐｒｏｔｅｉｎ：ＮＰ＿００９０１５）、ｍｏｕｓｅ−Ｓｏｘ２１（Ｐｒｏｔｅｉｎ：ＮＰ＿８０８４２１）のアミノ酸配列からなるＳｏｘ２１又は当該アミノ酸配列をコードするＳｏｘ２１遺伝子を用いることができる。また、その全長を用いる必要はなくＳｏｘ２１転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、Ｓｏｘ２１転写因子として用いることができる。ここで、ヒトＳｏｘ２１（配列番号７）及びマウスＳｏｘ２１（配列番号８）のアミノ酸配列レベルのホモロジーは約９９％であるのに対して、Ｓｏｘ２１と同じＳｏｘファミリー内でＳｏｘ２１ときわめてよく似た性質を有するＳｏｘ１４、Ｓｏｘ３、Ｓｏｘ１、Ｓｏｘ１５、Ｓｏｘ２及びについてみると、ヒトＳｏｘ２１及びヒトＳｏｘ１４は約５８％、ヒトＳｏｘ２１及びヒトＳｏｘ３は約４８％、ヒトＳｏｘ２１及びヒトＳｏｘ１は約４７％、ヒトＳｏｘ２１及びヒトＳｏｘ１５は約４４％、ヒトＳｏｘ２１及びヒトＳｏｘ２は約４３％である。したがって、例えばヒトＳｏｘ２１、ヒトＳｏｘ１４及びヒトＳｏｘ２それぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＳｏｘ２１と同様に用いることができる。
　Ｓｏｘ９転写因子、またはそれをコードする遺伝子についても同様であり、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）などから得られるｈｕｍａｎ−Ｓｏｘ９（Ｐｒｏｔｅｉｎ：ＮＰ＿０００３３７）、ｍｏｕｓｅ−Ｓｏｘ９（Ｐｒｏｔｅｉｎ：ＮＰ＿０３５５７８）のアミノ酸配列からなるＳｏｘ９又は当該アミノ酸配列をコードするＳｏｘ９遺伝子を用いることができる。また、その全長を用いる必要はなくＳｏｘ９転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、Ｓｏｘ９転写因子として用いることができる。ここで、ヒトＳｏｘ９（配列番号ＮＰ＿０００３３７）及びマウスＳｏｘ９（配列番号ＮＰ＿０３５５７）のアミノ酸配列レベルのホモロジーは約９７％であるのに対して、Ｓｏｘ９と同じＳｏｘファミリー内でＳｏｘ９ときわめてよく似た性質を有するＳｏｘ１０とＳｏｘ８についてみると、ヒトＳｏｘ９及びヒトＳｏｘ１０は約５８％、ヒトＳｏｘ９及びヒトＳｏｘ８は約４８％である。したがって、例えばヒトＳｏｘ９、ヒトＳｏｘ８及びヒトＳｏｘ１０それぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＳｏｘ９と同様に用いることができる。
　ＮＭｙｃ転写因子、またはそれをコードする遺伝子についても同様であり、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）などから得られるｈｕｍａｎＮＭｙｃｇｅｎｅ（ｍＲＮＡ：ＮＭ＿００５３７８）、ｍｏｕｓｅＮＭｙｃ　ｇｅｎｅ（ｍＲＮＡ：ＮＭ＿００８７０９）の塩基配列からなるＮＭｙｃ遺伝子又は当該塩基配列がコードするＮＭｙｃを用いることができる。また、その全長を用いる必要はなくＮＭｙｃ転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、ＮＭｙｃ転写因子として用いることができる。ここで、ヒトＮＭｙｃ及びマウスＮＭｙｃのアミノ酸配列レベルのホモロジーは約８５％であるのに対してＮＭｙｃと同じＭｙｃファミリー内でＮＭｙｃときわめてよく似た性質を有するＬＭｙｃ及びｃＭｙｃについてみると、ヒトＮＭｙｃ及びヒトＬＭｙｃは約３７％、ヒトＮＭｙｃ及びヒトｃＭｙｃは約３２％である。したがって、例えばヒトＮＭｙｃ、ヒトＬＭｙｃ及びヒトｃＭｙｃそれぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＮＭｙｃと同様に用いることができる。
　Ｉｒｘ３転写因子、またはそれをコードする遺伝子についても同様であり、例えば、ＧｅｎＢａｎｋ（ＮＣＢＩ）などから得られるｈｕｍａｎ−Ｉｒｘ３（Ｐｒｏｔｅｉｎ：ＮＰ＿０７７３１２）、ｍｏｕｓｅ−Ｉｒｘ３（Ｐｒｏｔｅｉｎ：ＮＰ＿０３２４１９）のアミノ酸配列からなるＩｒｘ３又は当該アミノ酸配列をコードするＩｒｘ３遺伝子を用いることができる。また、その全長を用いる必要はなくＩｒｘ３転写因子としての活性が保持されていれば、その部分長であっても、アミノ酸レベルで１個~数個程度（なお、数個とは１~２０個、好ましくは１~１０個、より好ましくは１~５個を表す。）改変（欠失・置換・付加・挿入）されている場合であっても、Ｉｒｘ３転写因子として用いることができる。ここで、ヒトＩｒｘ３及びマウスＩｒｘ３のアミノ酸配列レベルのホモロジーは約８６％であるのに対して、Ｉｒｘ３と同じＩｒｘファミリー内でＩｒｘ３ときわめてよく似た性質を有するＩｒｘ１及びＩｒｘ２についてみると、ヒトＩｒｘ３及びヒトＩｒｘ１は約４５％、ヒトＩｒｘ３及びヒトＩｒｘ２は約３８％である。したがって、例えばヒトＩｒｘ３、ヒトＩｒｘ１及びヒトＩｒｘ２それぞれの共通配列が保存され、かついずれかのアミノ酸配列と４０％以上、好ましくは５０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上のホモロジー（同一性）のあるアミノ酸配列もＩｒｘ３と同様に用いることができる。
２．肺前駆細胞の作製方法
（２−１）肺前駆細胞について
　本発明において、「肺前駆細胞」というとき、肺を構成する様々な組織細胞への分化多能性を有する前駆細胞であって、肺前駆細胞マーカーであるＮｋｘ２．１陽性の細胞を指す。典型的には、初期発生期の消化管から肺の原基が出芽して形態形成が始まる分化段階（マウスでは、受精後１０日前後、ヒトでは受精後３０日前後）で、肺の予定領域で出現する胎児性の肺前駆細胞（肺幹細胞）が含まれる。
（２−２）肺前駆細胞への分化方法
　本発明では、線維芽細胞などの体細胞（組織細胞）から１ステップで肺前駆細胞を作製する方法を開発した。Ｆｏｘファミリー転写因子、Ｇａｔａファミリー転写因子、Ｓｏｘファミリー転写因子、及びＭｙｃファミリー転写因子、さらにはＩｒｘファミリー転写因子を含む転写因子群を線維芽細胞などに導入する。例えば、Ｆｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃの４つの転写因子、好ましくはＦｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１及びＮＭｙｃの５つの転写因子、特にヒトの場合にはＦｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１及びＮＭｙｃにＳｏｘ９を加えた６つの転写因子を含む転写因子群を線維芽細胞などの体細胞、又は脂肪細胞由来線維芽細胞に遺伝子導入し、２~３週間培養することで肺前駆細胞マーカー陽性の細胞を容易に作製することができる。線維芽細胞などの体細胞内で遺伝子導入などによって外来遺伝子を発現させて肺前駆細胞特異的な転写因子ネットワークを作って働かせることで、体細胞を肺前駆細胞へと直接分化させることができる。
　本発明において「体細胞」とは、ヒト、マウスを含む哺乳動物由来の体細胞であり、胎児由来の体細胞、成人由来の体細胞、患者由来の体細胞の場合を含む。好ましくは、ヒト又はマウスなどの哺乳動物由来の線維芽細胞である。
　また、一般的に生体組織では上皮組織は間質組織によって覆われているが、このような成体の様々な間質組織に線維芽細胞が存在しており、採取可能である。
　例えば、ヒト成体由来の線維芽細胞として、表皮由来線維芽細胞又は脂肪組織由来の線維芽細胞を好ましく用いることができる。
　さらに、本発明の体細胞は、単離された体細胞のみならず、ヒトを含む肺組織中の体細胞も包含する。
　前記遺伝子を体細胞へ導入する手段としては、レトロウイルスを用いる他に、レンチウイルスやアデノウイルス、アデノ随伴ウイルス、さらにはセンダイウイルスなど任意のウイルスを用いることができる。また、通常のプラスミドベクターや染色体外で独立に維持されるエピソーマルベクターなど任意のベクターを用いることも可能である。さらに、遺伝子導入方法として、リポフェクション法やエレクトロポレーション、ハイドロダイナミック法など様々な方法が可能である。
　また、転写遺伝子を直接肺組織の体細胞にウイルスベクターや遺伝子導入剤を利用して導入することができる（Ｌａｕｂｅ　ＢＬ，Ｔｒａｎｓｌ　Ｒｅｓｐｉｒ　Ｍｅｄ．２０１４　２，３．参照）。転写因子のｍＲＮＡを用いる場合は、ＲＮＡ導入試薬やセンダイウイルスなどのＲＮＡウイルスベクターを用いることで、直接ヒト肺組織に導入することができる。また、転写因子タンパク質を投与する場合は、脂質ベースのタンパク質導入試薬で直接肺組織に導入するか、又は転写因子タンパク質に膜透過ペプチドを付加した融合タンパク質を直接肺組織細胞に導入することも可能である。
（２−３）培養方法
　遺伝子導入後の体細胞は、２~３週間培養することで肺前駆細胞マーカー陽性の細胞を容易に作製することができる。また、遺伝子導入の際に、薬剤選択マーカー遺伝子も同時に導入することで、前記４又は５種類の転写因子遺伝子が導入された細胞のみを容易に選択できる。
　本発明において用いる培地は、遺伝子導入をする体細胞の培養に適した培地を用いることが好ましい。例えば、線維芽細胞の場合は、１０％ウシ胎児血清含有ＤＭＥＭ培地、又は市販の線維芽細胞培養培地などが適している。
　このようにして作製した肺前駆細胞はある程度の増殖性があり、必要なだけ増殖させて使用することができる。
（２−４）肺前駆細胞の検出方法
　本発明において、前記４又は５種類の転写因子遺伝子導入後に培養工程を経た体細胞が、ｉＰＳ細胞様の未分化細胞を経由せずに、直接肺前駆細胞に分化したことは、もとの体細胞特有のマーカー遺伝子、例えば線維芽細胞の場合はＴｈｙ１などの発現がみられず、かつＥＳ／ｉＰＳ細胞特有の各種マーカー、例えばＯｃｔ４、Ｎａｎｏｇ及びＫｌｆ４などが発現せずに、肺前駆細胞特有のマーカーであるＮｋｘ２．１が発現していることを確認すればよい。さらに、肺組織特異性マーカーであるＳＰＣに加えてＥ−ｃａｄｈｅｒｉｎなどの発現を確認することが好ましく、他の組織特異性マーカーが発現していないことを確認することがより好ましい。
　各マーカーの発現を確認するためには、それぞれの抗体、例えばＮｋｘ２．１やＥ−ｃａｄｈｅｒｉｎ特異的抗体を用いることができ、他にウエスタンブロッティング法、免疫蛍光染色法、免疫組織化学染色、フローサイトメトリーなどの方法などを用いることができる。
３．肺組織細胞への分化方法
　本発明の肺前駆細胞は、肺を構成する様々な組織細胞への分化多能性を有している。したがって、それぞれの肺組織細胞への既知の分化誘導法を適用することで、望みの肺組織細胞に分化させることができる。
　例えば、肺上皮細胞を得ようとする場合は、本発明で得られた肺前駆細胞を、代表的な内胚葉由来上皮細胞用培地のうちの低血清培地として知られる０．２％ウシ胎児血清を添加したＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０培地で培養することで、増殖培養が可能であり、肺上皮細胞や粘液分泌細胞を得ようとする場合は、本発明で得られた肺前駆細胞を、０．２％ウシ胎児血清を添加したＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０培地でさらに長期間培養すればよい。また、培地に各種細胞増殖因子を添加した分化誘導により、繊毛細胞、基底細胞やクララ細胞等に分化するという報告もある（非特許文献１、非特許文献１１）。
　これらの分化誘導工程は、ｉｎ　ｖｉｔｒｏのみならず、マウスなど実験動物を用いたｉｎ　ｖｉｖｏ条件下でも様々な肺組織細胞へと分化させることができる（非特許文献１１）。例えば毛細血管が発達し、移植した細胞の生着効率が高い腎皮膜下に移植することで、本肺前駆細胞を肺胞上皮細胞などへ分化させることができる。
　代表的な上皮細胞用培地のうちの低血清培地又は無血清培地を分化誘導培地として用いることで、各種肺成熟細胞への分化が起こり、培地中に増殖因子（例えば、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦの混合物）を添加することで、さらに粘液分泌細胞、肺胞上皮Ｉ型細胞や肺胞上皮ＩＩ型細胞及びクララ細胞などの成熟肺細胞への分化効率が高まる。
　特に低血清ＲＰＭＩ１６４０培地培地、例えば０．１~０．５％のウシ胎児血清を含有させたＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０培地に、増殖因子（例えば、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を添加した場合には、特異的に粘液分泌細胞への分化能が高まる。一方、無血清培地、例えばＤＭＥＭ＋ＫＳＲ培地に増殖因子（例えば、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を添加した場合には、粘液分泌細胞のみならず、肺胞上皮Ｉ型細胞や肺胞上皮ＩＩ型細胞及びクララ細胞などの各種成熟肺細胞いずれに対しても高い分化能を示す。
４．本発明で作製した肺前駆細胞のその他の用途
　本発明の肺前駆細胞は、成熟した肺組織細胞へと分化させる工程で用いる分化薬剤のスクリーニング用細胞としても利用できる。例えば、望みの肺組織細胞への分化工程で被検物質、被検薬剤を添加し、分化状態を観察することで、被検薬剤を評価できる。ここで、被検物質が被検遺伝子など核酸の場合は、肺前駆細胞内に発現ベクターを用いるなどの既知の核酸導入法を適用して細胞内に導入しておくことが好ましいが、低分子化合物、タンパク質、ペプチド、糖類など他の被検物質、被検薬剤の場合は分化誘導培地中に添加する。
　その際、肺前駆細胞を２つのグループに分け、片方に被検物質を投与し、他方には投与せずに分化誘導処理を施し、両者の分化状態を比較する手法を採ることが好ましい。
　また、本発明の肺前駆細胞は、通常の細胞移植技術を応用することで、肺組織の損傷部位などに対する肺組織細胞の移植治療に供することができる。
　一方、本発明の肺前駆細胞をｉｎ　ｖｉｔｒｏで分化させた肺胞上皮細胞など肺組織細胞は、肺組織に対する毒性試験用の細胞キットに応用できる。例えば、これら細胞をマルチウエルディッシュに播種した多検体サンプルを、薬剤の毒性試験、安全性試験に用いることができる。
　さらに、本発明の肺前駆細胞製造方法によって、各種肺癌の原因遺伝子を導入した線維芽細胞から肺前駆細胞を作製することにより、得られた肺癌原因遺伝子を保持した肺前駆細胞は各種抗癌剤効果の評価系として、すなわち肺癌治療薬候補をスクリーニングするための細胞キットとして用いることができる。
　また、同様に、他の肺疾患の原因遺伝子を導入した線維芽細胞から肺前駆細胞を作製することにより、肺疾患治療薬候補をスクリーニングするための細胞キットとして用いることもできる。
　以下、実施例に則して本発明を更に詳しく説明するが、本発明の技術的範囲はこれらの記載によって何等制限されるものではない。
　本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
　また、本明細書中で引用される技術文献の内容は、本明細書の開示内容の一部と見なされる。 1. Specific Transcription Factor for Producing Lung Progenitor Cells of the Present Invention Specific transcription factors for producing pulmonary progenitor cells of the present invention include at least a Fox family transcription factor, a Gata family transcription factor, a Sox family transcription factor, and a Myc family. It includes each transcription factor gene belonging to the transcription factor or a protein as its expression product, and preferably further contains an Irx family transcription factor or a protein as its expression product. In particular, when producing human lung progenitor cells, it is preferable that two types of transcription factors or their expression products are included from the Sox family.
The Fox family transcription factors include Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd2, Fox1, Fox1, Fox1, Fox1, Fox1 , Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Foxo1, Foxo3a, Foxo4, Foxp1, Fox3, Foxp2, Foxp1, Fox3 The domain is a highly conserved Foxa1, Foxa2, Foxa3 transcription factor. The amino acid sequences of these transcription factors and the base sequences of the genes thereof can be obtained from public databases. For example, GenBank (NCBI) human-Foxa2 (Protein: NP — 068556) and mouse-Foxa2 (Protein: NP — 034576) can be used.
Examples of the Gata family transcription factor include Gata1, Gata2, Gata3, Gata4, Gata5, and Gata6, and preferably Gata6, Gata5, Gata4 transcription factors whose functional domains are highly conserved, such as human-Gata6. (Protein: NP_005248), mouse-Gata6 (Protein: NP_034388) can be used.
As Sox family transcription factors, Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox21, Sox21, Sox21, Sox30, Sox21, Sox Preferably, the functional domains of which are highly conserved Sox21, Sox3, Sox1, Sox15, Sox2 and Sox9 transcription factors such as human-Sox21 (Protein: NP_009015), mouse-Sox21 (Protein: NP_808421) and human- Sox9 (Protein: NP_000337) can be used. Examples of the Myc family transcription factor include cMyc, LMyc, and NMyc whose functional domains are highly conserved, and are preferably NMyc transcription factors such as humanNMycgene (mRNA: NM_005378), mouseNMyc gene (mRNA: NM_008709). Can be used. In particular, when producing human lung progenitor cells, it is preferable to have two types of transcription factors including Sox9.
In addition, examples of the Irx family transcription factor include Irx1, Irx2, Irx3, Irx4, Irx5 and Irx6, preferably Irx3, Irx1, Irx2, Irx4 and Irx6 transcription factors whose functional domains are highly conserved. For example, human-Irx3 (Protein: NP — 077312) and mouse-Irx3 (Protein: NP — 032419) can be used.
These transcription factors, ie, Fox family transcription factors, Gata family transcription factors, Sox family transcription factors, and Myc family transcription factors, as well as transcription factors comprising Irx family transcription factors, preferably Foxa2, Gata6, Sox21 and NMyc transcription Somatic cells such as fibroblasts by gene transfer of factors, more preferably Foxa2, Gata6, Irx3, Sox21 and NMyc transcription factors, particularly preferably Transcription factors including Foxa2, Gata6, Irx3, Sox21, NMyc and Sox9 transcription factors By expressing in (tissue cells) and restructuring the network, somatic cells can be directly differentiated into lung progenitor cells.
These 4, 5, or 6 transcription factors are typically expressed simultaneously by introducing each gene into a somatic cell such as a fibroblast with or without a viral or non-viral vector. Let
Here, any one or more of these 4, 5 or 6 types of transcription factors may be introduced into a somatic cell as a protein of a gene expression product instead of gene introduction. Expression-inducing activity of low-molecular-weight compounds having an activity to induce expression (for example, melatonin having an expression-inducing activity of Foxa2 (Non-patent Document 13)) and miRNA and inhibitors thereof (for example, anti-miR124a2 having an activity of inducing expression of Foxa2 and NMyc) Alternatively, anti-miR-34a (Non-patent Documents 14 and 15)) can be replaced by acting on somatic cells.
The Foxa2 transcription factor or the gene encoding it can be obtained from the public database (such as GenBank (NCBI)) as described above. For example, the Foxa2 amino acid sequence is human-Foxa2 (Protein: NP — 068556) and mouse-Foxa2 (Protein: NP — 034576), and the Foxa2 gene can use a base sequence encoding the amino acid sequence. In addition, if the activity as a Foxa2 transcription factor is maintained, it is not necessary to use the full length, and even if it is a partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Foxa2 transcription factor. Here, the homology at the amino acid sequence level of human Foxa2 and mouse Foxa2 is about 97%, whereas Foxa2 and Foxa3 having properties very similar to Foxa2 within the same Foxa family as Foxa2 are considered to be human Foxa2. Human Foxa1 is about 53%, human Foxa2 and human Foxa3 are about 42%, human Foxa2 and human Foxb1 are about 48%, and human Foxa2 and human Foxb2 are about 43%. Therefore, for example, human Foxa1, human Foxa2, human Foxa3, human Foxb1 and human Foxb2 have conserved consensus sequences, and any amino acid sequence is 40% or more, preferably 50% or more, more preferably 80% or more, and further Preferably, an amino acid sequence having 90% or more homology (identity) can be used similarly to Foxa2.
The same applies to the Gata6 transcription factor or the gene encoding the same. For example, Gata6 consisting of an amino acid sequence of human-Gata6 (Protein: NP_005248) or mouse-Gata6 (Protein: NP — 034388) obtained from GenBank (NCBI) or the like. A Gata6 gene encoding the amino acid sequence can be used. Moreover, it is not necessary to use the full length, and if the activity as a Gata6 transcription factor is maintained, even if it is the partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Gata6 transcription factor. Here, the homology at the amino acid sequence level of human Gata6 and mouse Gata6 is about 88%, whereas Gata4 and Gata5 having properties very similar to Gata6 in the same Gata family as Gata6 are seen as human Gata6. And human Gata4 is about 50%, and human Gata6 and human Gata5 are about 44%. Therefore, for example, the common sequences of human Gata6, human Gata4 and human Gata5 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more with any amino acid sequence. An amino acid sequence having homology (identity) can also be used in the same manner as Gata6.
The same applies to the Sox21 transcription factor or a gene encoding the same. For example, human-Sox21 (Protein: NP_009015) obtained from GenBank (NCBI) or the like, Sox21 consisting of an amino acid sequence of mouse-Sox21 (Protein: NP_808421) or A Sox21 gene encoding the amino acid sequence can be used. In addition, if the activity as a Sox21 transcription factor is maintained, it is not necessary to use the full length, and even if its partial length is about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Sox21 transcription factor. Here, the homology at the amino acid sequence level of human Sox21 (SEQ ID NO: 7) and mouse Sox21 (SEQ ID NO: 8) is about 99%, while it has very similar properties to Sox21 within the same Sox family as Sox21. As for Sox14, Sox3, Sox1, Sox15, Sox2 and so on, human Sox21 and human Sox14 are about 58%, human Sox21 and human Sox3 are about 48%, human Sox21 and human Sox1 are about 47%, human Sox21 and human Sox15. Is approximately 44%, human Sox21 and human Sox2 are approximately 43%. Therefore, for example, the consensus sequences of human Sox21, human Sox14 and human Sox2 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, more preferably 90% or more with any amino acid sequence. An amino acid sequence having homology (identity) can also be used in the same manner as Sox21.
The same applies to the Sox9 transcription factor or the gene encoding it, for example, human-Sox9 (Protein: NP_000337) obtained from GenBank (NCBI) or the like, Sox9 consisting of the amino acid sequence of mouse-Sox9 (Protein: NP_0357578) or A Sox9 gene encoding the amino acid sequence can be used. Moreover, it is not necessary to use the full length, and if the activity as a Sox9 transcription factor is maintained, even if it is a partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as a Sox9 transcription factor. Here, the homology at the amino acid sequence level of human Sox9 (SEQ ID NO: NP — 000337) and mouse Sox9 (SEQ ID NO: NP — 03557) is about 97%, while it has properties very similar to Sox9 within the same Sox family as Sox9. As for Sox10 and Sox8, the human Sox9 and human Sox10 are about 58%, and the human Sox9 and human Sox8 are about 48%. Thus, for example, the consensus sequences of human Sox9, human Sox8 and human Sox10 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, more preferably 90% or more with any amino acid sequence. An amino acid sequence having homology (identity) can also be used in the same manner as Sox9.
The same applies to the NMyc transcription factor or the gene encoding the same. For example, the human NMycgene (mRNA: NM_005378) obtained from GenBank (NCBI) or the like, the NMyc gene consisting of the base sequence of mouse NMyc gene (mRNA: NM_008709) or the base NMyc encoded by the sequence can be used. Moreover, it is not necessary to use the full length, and if the activity as an NMyc transcription factor is maintained, even if it is the partial length, about 1 to several at the amino acid level (note that several is 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as an NMyc transcription factor. Here, the homology at the amino acid sequence level of human NMyc and mouse NMyc is about 85%, whereas LMyc and cMyc having properties very similar to NMyc within the same Myc family as NMyc Human LMyc is about 37%, human NMyc and human cMyc are about 32%. Therefore, for example, the common sequences of human NMyc, human LMyc, and human cMyc are conserved, and any amino acid sequence is 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more. An amino acid sequence having homology (identity) can also be used similarly to NMyc.
The same applies to the Irx3 transcription factor or a gene encoding the same. For example, Irx3 consisting of an amino acid sequence of human-Irx3 (Protein: NP — 077312), mouse-Irx3 (Protein: NP — 032419) obtained from GenBank (NCBI) or the like The Irx3 gene encoding the amino acid sequence can be used. In addition, if the activity as an Irx3 transcription factor is maintained without using the full length, even if it is the partial length, about 1 to several at the amino acid level (note that several are 1 to 20) It preferably represents 1 to 10, more preferably 1 to 5.) Even if it is modified (deletion / substitution / addition / insertion), it can be used as an Irx3 transcription factor. Here, while the homology at the amino acid sequence level of human Irx3 and mouse Irx3 is about 86%, Irx1 and Irx2 having properties very similar to Irx3 in the same Irx family as Irx3, human Irx3 And human Irx1 is about 45%, human Irx3 and human Irx2 are about 38%. Therefore, for example, the common sequences of human Irx3, human Irx1 and human Irx2 are conserved, and 40% or more, preferably 50% or more, more preferably 80% or more, and still more preferably 90% or more with any amino acid sequence. An amino acid sequence having homology (identity) can also be used in the same manner as Irx3.
2. Method for Producing Lung Progenitor Cell (2-1) Lung Progenitor Cell In the present invention, the term “lung progenitor cell” refers to a progenitor cell that has pluripotency into various tissue cells constituting the lung, It refers to cells that are positive for Nkx2.1, a progenitor cell marker. Typically, at the stage of differentiation (approximately 10 days after fertilization in mice and approximately 30 days after fertilization in humans) at which the lung primordium emerges from the early developmental gastrointestinal tract and begins morphogenesis, the expected lung region Fetal lung progenitor cells (pulmonary stem cells) appearing in
(2-2) Method for Differentiating into Lung Progenitor Cells In the present invention, a method for producing lung progenitor cells from somatic cells (tissue cells) such as fibroblasts in one step was developed. A transcription factor group including a Fox family transcription factor, a Gata family transcription factor, a Sox family transcription factor, a Myc family transcription factor, and an Irx family transcription factor is introduced into fibroblasts and the like. For example, four transcription factors of Foxa2, Gata6, Sox21 and NMyc, preferably Foxa2, Gata6, Irx3, Sox21 and NMyc, five transcription factors, especially in the case of humans, Sox9 in Foxa2, Gata6, Irx3, Sox21 and NMyc. Genes of transcription factors including the added 6 transcription factors are introduced into somatic cells such as fibroblasts, or adipocyte-derived fibroblasts, and cultured for 2 to 3 weeks to facilitate pulmonary progenitor cell marker positive cells Can be produced. Somatic cells can be directly differentiated into lung progenitor cells by expressing foreign genes in somatic cells such as fibroblasts by gene transfer, etc. to create and act on a lung progenitor cell-specific transcription factor network.
In the present invention, “somatic cells” are somatic cells derived from mammals including humans and mice, and include cases of somatic cells derived from fetuses, somatic cells derived from adults, and somatic cells derived from patients. Preferably, it is a fibroblast derived from a mammal such as a human or a mouse.
In general, epithelial tissues are covered with stromal tissues in living tissues, but fibroblasts are present in various stromal tissues of such adults and can be collected.
For example, epidermis-derived fibroblasts or adipose tissue-derived fibroblasts can be preferably used as human adult-derived fibroblasts.
Furthermore, the somatic cells of the present invention include not only isolated somatic cells but also somatic cells in lung tissues including humans.
As a means for introducing the gene into somatic cells, any virus such as lentivirus, adenovirus, adeno-associated virus, and Sendai virus can be used in addition to retrovirus. Moreover, it is also possible to use arbitrary vectors, such as a normal plasmid vector and the episomal vector maintained independently outside a chromosome. Furthermore, various methods such as lipofection, electroporation, and hydrodynamic methods can be used as gene transfer methods.
In addition, a transcription gene can be directly introduced into somatic cells of lung tissue using a viral vector or a gene introduction agent (see Laube BL, Transl Respir Med. 2014 2, 3.). When using mRNA of a transcription factor, it can be directly introduced into human lung tissue by using an RNA introduction reagent or an RNA virus vector such as Sendai virus. In addition, when administering transcription factor protein, it is possible to introduce it directly into lung tissue with a lipid-based protein introduction reagent, or to introduce a fusion protein in which a transmembrane peptide is added to transcription factor protein directly into lung tissue cells. It is.
(2-3) Culture method The somatic cells after gene introduction can be easily produced as a lung progenitor cell marker positive cell by culturing for 2 to 3 weeks. In addition, by introducing a drug selection marker gene at the time of gene introduction, it is possible to easily select only cells into which the 4 or 5 types of transcription factor genes have been introduced.
The medium used in the present invention is preferably a medium suitable for culturing somatic cells into which genes are introduced. For example, in the case of fibroblasts, a DMEM medium containing 10% fetal bovine serum or a commercially available fibroblast culture medium is suitable.
The lung progenitor cells produced in this way have a certain degree of proliferative ability and can be used after being proliferated as much as necessary.
(2-4) Method for detecting lung progenitor cells In the present invention, somatic cells that have undergone the culture process after introduction of the four or five types of transcription factor genes are directly transmitted to the lung progenitors without going through iPS cell-like undifferentiated cells. The differentiation into cells means that the expression of marker genes specific to the original somatic cells, such as Thy1 in the case of fibroblasts, and various markers specific to ES / iPS cells, such as Oct4, Nanog and Klf4, etc. What is necessary is just to confirm that Nkx2.1 which is a marker peculiar to a lung progenitor cell is expressing, without expressing. Furthermore, it is preferable to confirm the expression of E-cadherin and the like in addition to SPC, which is a lung tissue specific marker, and more preferably to confirm that other tissue specific markers are not expressed.
In order to confirm the expression of each marker, each antibody, for example, Nkx2.1 or E-cadherin specific antibody can be used. Besides, Western blotting method, immunofluorescence staining method, immunohistochemical staining, flow site A method such as a meter can be used.
3. Method for Differentiating into Lung Tissue Cells The lung progenitor cells of the present invention have pluripotency into various tissue cells constituting the lung. Therefore, it is possible to differentiate into desired lung tissue cells by applying a known differentiation induction method to each lung tissue cell.
For example, when pulmonary epithelial cells are to be obtained, the lung progenitor cells obtained in the present invention are 0.2% fetal bovine serum known as a low serum medium among typical endoderm-derived epithelial cell culture media. When cultured in an Advanced RPMI1640 medium supplemented with a medium, proliferation culture is possible, and when pulmonary epithelial cells and mucus-secreting cells are to be obtained, the lung progenitor cells obtained in the present invention are 0.2% bovine. What is necessary is just to culture | cultivate for a long period more in Advanced RPMI1640 culture medium which added fetal serum. There are also reports of differentiation into ciliated cells, basal cells, Clara cells, and the like by differentiation induction by adding various cell growth factors to the medium (Non-patent Documents 1 and 11).
These differentiation induction processes can be differentiated into various lung tissue cells not only in vitro but also in vivo conditions using experimental animals such as mice (Non-patent Document 11). For example, the lung progenitor cells can be differentiated into alveolar epithelial cells or the like by transplanting under a renal capsule where capillary vessels develop and the engraftment efficiency of the transplanted cells is high.
By using a low serum medium or a serum-free medium among typical epithelial cell culture media as differentiation induction media, differentiation into various lung mature cells occurs, and growth factors (for example, Wnt3a, FGF10, KGF Addition of the mixture) further increases the efficiency of differentiation into mature lung cells such as mucus-secreting cells, alveolar epithelial type I cells, alveolar epithelial type II cells, and Clara cells.
In particular, when a growth factor (for example, Wnt3a, FGF10, KGF) is added to a low serum RPMI1640 medium, for example, an Advanced RPMI1640 medium containing 0.1 to 0.5% fetal bovine serum, Increases the ability to differentiate into mucus-secreting cells. On the other hand, when growth factors (for example, Wnt3a, FGF10, KGF) are added to a serum-free medium, for example, DMEM + KSR medium, not only mucus-secreting cells but also alveolar epithelial type I cells, alveolar epithelial type II cells, and Clara. It shows high differentiation ability for all types of mature lung cells such as cells.
4). Other Uses of Lung Progenitor Cells Produced in the Present Invention The lung progenitor cells of the present invention can also be used as screening cells for differentiation drugs used in the process of differentiation into mature lung tissue cells. For example, the test drug can be evaluated by adding a test substance and a test drug in the desired differentiation process into lung tissue cells and observing the differentiation state. Here, when the test substance is a nucleic acid such as a test gene, it is preferably introduced into the cell by applying a known nucleic acid introduction method such as using an expression vector in lung progenitor cells. In the case of other test substances and test drugs such as compounds, proteins, peptides, and sugars, they are added to the differentiation induction medium.
At that time, it is preferable to divide the lung progenitor cells into two groups, administer the test substance to one of them, perform the differentiation induction treatment without administration to the other, and compare the differentiation states of the two.
In addition, the lung progenitor cells of the present invention can be used for transplantation treatment of lung tissue cells to damaged sites of lung tissue by applying a normal cell transplantation technique.
On the other hand, lung tissue cells such as alveolar epithelial cells obtained by differentiating lung progenitor cells of the present invention in vitro can be applied to cell kits for toxicity tests on lung tissues. For example, a multi-sample sample in which these cells are seeded in a multi-well dish can be used for drug toxicity tests and safety tests.
Further, by producing lung progenitor cells from fibroblasts introduced with various causative genes for lung cancer by the method for producing lung progenitor cells of the present invention, the obtained lung progenitor cells have the effect of various anticancer agents. It can be used as an evaluation system, that is, as a cell kit for screening a lung cancer therapeutic drug candidate.
Similarly, by producing lung progenitor cells from fibroblasts into which another causative gene for lung disease has been introduced, it can also be used as a cell kit for screening lung disease therapeutic drug candidates.
EXAMPLES Hereinafter, although this invention is demonstrated in more detail based on an Example, the technical scope of this invention is not restrict | limited at all by these description.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
The contents of technical documents cited in the present specification are considered to be part of the disclosure of the present specification.
（実施例１）転写因子の組合せを利用した線維芽細胞から肺前駆細胞への直接分化法
　初期肺の肺形成期に特異的に発現する転写因子１３種類を、既知の文献やデータベースから選び出し、ｐＭＹｓレトロウイルスベクターにｃＤＮＡを挿入後、レトロウイルス作製用細胞であるＰｌａｔＥ細胞でレトロウイルスを作製しマウス胎児線維芽細胞（マウス胎児の背側表皮由来線維芽細胞：Ｄｏｒｓａｌ　ｓｋｉｎ　ＭＥＦ又は単にＭＥＦともいう。）に感染させた。１週間後、一般的な内胚葉由来上皮細胞用培地であるＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０に０．２％ウシ胎児血清を添加した培地に変えてさらに培養を継続した。肺前駆細胞への直接分化を肺前駆細胞特異的マーカー遺伝子であるＮｋｘ２．１と上皮マーカーのひとつであるＥ−ｃａｄｈｅｒｉｎの発現が陽性であるコロニーの出現を指標に評価した（図１ａ）。その結果、２~３週間後に上皮細胞様のコロニー状に増殖する細胞を検出し、それらはＮｋｘ２．１陽性の細胞集団であることを確認できた（図１ｂ‐ｄ）。次に１３種類の転写因子の中から、肺前駆細胞への直接分化に必須の転写因子を洗い出すため、１３因子の組合せから１因子ずつ順番に除いた組合せを検討した。転写因子を除去すると特にＮｋｘ２．１の発現が上昇する、抑制作用が見られた転写因子７個を捨て、その他の６個の転写因子を選択した（図１ｅ‐ｆ）。次にこれら６因子からさらに１因子ずつ除去して不要因子を探索したところ、Ｓｏｘ９を除去してもＮｋｘ２．１の発現が減少しないことからＦｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１及びＮＭｙｃの５因子に絞り込んだ（図１ｇ‐ｈ）。さらに５因子から１因子ずつ差し引いて必須因子を探索したところ、Ｆｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ因子の場合は、これら何れの因子を除去した場合でもＮｋｘ２．１の発現がほぼ消失した。一方、Ｉｒｘ３因子を除去した場合は、Ｎｋｘ２．１の発現が大幅に減少するものの、５０％以上発現している様子が観察された（図１ｉ‐ｊ）。また、免疫染色によるＮｋｘ２．１陽性コロニー解析においても、他の因子のいずれを除去した場合ではコロニーが全く観察できなかったのに対し、Ｉｒｘ３を除去した場合には小さなコロニーであるが、少数のコロニーが明確に観察された（データは示していない）。
　これらの結果からみて、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃの４因子は必須因子であり、Ｉｒｘ３因子については必須因子ではないが、肺前駆細胞への直接分化を促進するためには欠かせない因子であり、効率よくＮｋｘ２．１陽性の肺前駆細胞を作製するには、上記４因子にＩｒｘ３を加えた５因子を用いることが好ましいという結論が得られた。
　以上のことから、本発明の転写因子としては、Ｆｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃの４因子の組み合わせが必要遺伝子の最少の組合せであるが、以下の実施例では、これら４因子にＩｒｘ３因子を加えた５因子を含む組み合わせを用いたＮｋｘ２．１陽性の肺前駆細胞についての検討を行った。
（実施例２）転写因子の遺伝子導入により作製した肺前駆細胞の特異性の検証
（２−１）マウス胎児線維芽細胞（ＭＥＦ）から分化した肺前駆細胞
　上記（実施例１）で選定した５つの転写因子により作製した肺前駆細胞の肺組織特異性について免疫蛍光染色により検証を行った。５つの転写因子を実施例１と同じマウス胎児由来線維芽細胞に遺伝子導入し、一般的な内胚葉由来上皮細胞用培地（Ａｄｖａｎｃｅｄ　ＲＰＭＩ１６４０＋０．２％ウシ胎児血清）で培養していると２~３週間後に上皮様の細胞からなるコロニーが出現し始め（図２ａ）、それらは肺前駆細胞マーカーＮｋｘ２．１陽性の細胞であることが確認された（図２ｂ）。しかし、Ｎｋｘ２．１は、胎生期に肺前駆細胞以外にも同じく前方内胚葉から発生する甲状腺組織や脳の間脳でも発現することが知られている（図２ｃ）。そこで、転写因子により作製した肺前駆細胞の肺組織特異性についてそれぞれ、甲状腺組織で特異的に発現するＰａｘ８や間脳で発現するＴｕｊ１、上皮細胞で発現するＥ−ｃａｄｈｅｒｉｎなどを指標に免疫蛍光染色により検証を行ったところ、Ｎｋｘ２．１陽性で、かつ、Ｅ−ｃａｄｈｅｒｉｎ（＋）、Ｔｕｊ１（−）、Ｐａｘ８（−）であり、甲状腺や間脳などの細胞ではなく、肺前駆細胞であることが示唆された（図２ｄ‐ｅ）。
（２−２）他の組織由来線維芽細胞による肺前駆細胞の作製
　マウス胎児の背側表皮由来の線維芽細胞（Ｄｏｒｓａｌ　ｓｋｉｎ　ＭＥＦ）以外に、さらにマウス胎児の手足由来の線維芽細胞（Ｌｉｍｂ　ＭＥＦ）、及び成体マウスの表皮由来線維芽細胞（ＭＤＦ）を用いて、上記（２−１）と同様に上記５つの転写因子を導入し、同様の分化培地で培養した。その結果、いずれの線維芽細胞でもＮｋｘ２．１陽性の肺前駆細胞コロニーが直接分化されることが確認された（図２ｆ）。
（実施例３）マウス線維芽細胞から作製した肺前駆細胞の特異性の検証
（３−１）マウス胎児線維芽細胞から分化した肺前駆細胞
　上記（２−１）で得られたＮｋｘ２．１陽性のマウス胎児線維芽細胞（マウス胎児背側表皮由来線維芽細胞）からの分化細胞に対して、さらにマウス胎児の肺前駆細胞で発現するＳｏｘ及び上皮細胞マーカーのＥｐＣＡＭとの共染色を行い（図３ｂ）、その結果を定量して（図３ｃ）検証を行った。その結果、上記（２−１）で得られたマウス胎児線維芽細胞からの分化細胞は、Ｎｋｘ２．１陽性で、かつ上皮細胞マーカーのＥ−ｃａｄｈｅｒｉｎ及びＥｐＣＡＭが陽性で、甲状腺組織マーカーのＰａｘ８及び間脳組織マーカーのＴｕｊ１はいずれも陰性であり、さらに、肺前駆細胞でＮｋｘ２．１と共に発現するＳｏｘ２も陽性であることが確認された（図２、３）。この結果、マウス胎児線維芽細胞が上記５つの転写因子により肺前駆細胞に直接分化することの確証が得られた。
（３−２）マウス胎児の手足由来線維芽細胞から分化した肺前駆細胞
　上記（２−２）で得られたＮｋｘ２．１陽性のマウス胎児の手足由来線維芽細胞からの分化細胞に対して、上記（２−１）及び（３−１）で用いた各種組織マーカーを指標に免疫蛍光染色により検証を行った結果、上記（３−１）で確認されたマウス胎児の背側表皮由来の線維芽細胞の場合と同様に、Ｎｋｘ２．１陽性で、かつＥ−ｃａｄｈｅｒｉｎ（＋）、Ｔｕｊ１（−）、Ｐａｘ８（−）、及びＥｐＣＡＭ（＋）、及びＳｏｘ２（＋）であることが確認された（図４）。この結果、確かに肺前駆細胞に分化したことが確認でき、マウス胎児の手足由来の線維芽細胞を用いても、上記５つの転写因子により同様に肺前駆細胞に直接分化することの確証が得られた。
（３−３）成体マウス由来線維芽細胞から分化した肺前駆細胞
　さらに上記（２−２）で得られたＮｋｘ２．１陽性の成体マウスの表皮由来線維芽細胞を用いても５つの転写因子により同様に作製できることを確認した。
　上記（２−２）の成体マウス由来線維芽細胞からのＮｋｘ２．１陽性分化細胞は、成体（１２週齢）マウスの耳の後ろの表皮から切り出した線維芽細胞を１０％ウシ胎児血清含有ＤＭＥＭ培地で培養して調製した。当該成体マウス由来線維芽細胞に対し、実施例１と同様の手法で５つの転写因子を導入し、同様の分化培地で２~３週間培養したところ、上記（２−２）で述べたように、Ｎｋｘ２．１陽性のコロニーが作製された（図２ｆ）。
　当該成体マウス由来線維芽細胞からのＮｋｘ２．１陽性分化細胞に対して、さらに上記（２−１）及び（３−１）で用いた各種組織マーカーを指標に免疫蛍光染色により検証を行った結果、マウス胎児に由来する線維芽細胞と同様に、Ｎｋｘ２．１陽性で、かつＥ−ｃａｄｈｅｒｉｎ（＋）、Ｔｕｊ１（−）、Ｐａｘ８（−）、及びＥｐＣＡＭ（＋）、及びＳｏｘ２（＋）である肺前駆細胞に分化していることが確認された（図５ａ‐ｅ）。
　すなわち、成体マウス由来線維芽細胞においても、上記５つの転写因子により、同様に肺前駆細胞に直接分化することの確証が得られた。
（３−４）線維芽細胞からＮｋｘ２．１陽性上皮細胞へと転換していることの検証
　上記（２−１）でＮｋｘ２．１陽性のマウス胎児線維芽細胞（間葉系細胞）から得られた肺前駆細胞に対して、上皮細胞マーカー（Ｅ−ｃａｄｈｅｒｉｎ）と間葉系細胞マーカー（Ｔｗｉｓｔ２，Ｔｈｙ１，Ｆｓｐ１）の発現状態を検証した。
　その結果、上記５転写因子による直接分化過程でＴｈｙ１陽性の線維芽細胞からＥ−ｃａｄｈｅｒｉｎ陽性の上皮細胞へと転換しＮｋｘ２．１陽性の肺前駆細胞になったことが確認できた（図６）。
（実施例４）５つの転写因子を導入して作製した肺前駆細胞のマイクロアレイによる遺伝子発現解析
（４−１）マイクロアレイによる網羅的遺伝子解析−１
　本実施例では、マウス線維芽細胞から作製した肺前駆細胞の特異性を検証するため、実施例２で得られた３種類のマウス線維芽細胞（Ｄｏｒｓａｌ　ｓｋｉｎ　ＭＥＦ，Ｌｉｍｂ　ＭＥＦ，ＭＤＦ）由来の肺前駆細胞についてマイクロアレイで網羅的遺伝子解析を行った。
　その結果、マウス線維芽細胞から作製した肺前駆細胞の遺伝子発現は、３種類とも分化前のマウス繊維芽細胞と比較してマウス胎児肺組織細胞に類似した遺伝子発現パターンを示すことが明らかとなった（図７ａ‐ｂ）。
　また作製した肺前駆細胞と成体気管支、肺、胃、腸、肝臓、心臓、腎臓、視床下部、骨格筋、脂肪といった他の組織との比較を行った。結果作製した肺前駆細胞の遺伝子発現パターンは、Ｅ１１．５胎児由来の肺に最もよく類似してり、成体の気管支、肺及び他の組織とは似ていないことが明らかになった（図８ａ）。
（４−２）マイクロアレイによる網羅的遺伝子解析−２
　さらに、新たな３種類のマウス線維芽細胞（Ｄｏｒｓａｌ　ｓｋｉｎ　ＭＥＦ，Ｌｉｍｂ　ＭＥＦ，ＭＤＦ）を用い、実施例（２−１）と同様の方法で５つの転写因子を導入して肺前駆細胞を樹立した後、サンプル数を増やして更なるマイクロアレイ解析した。その結果においても類似の結果が得られたことから、本細胞がＥ１１．５胎児由来の肺に最もよく類似しており、成体の気管支、肺及び他の組織とは似ていないことがさらに確実となった（図８ｂ）。
（実施例５）５つの転写因子を導入して作製した肺前駆細胞の分化能の解析
（５−１）低血清内胚葉維持培地による分化能の検討−１
　上記（実施例１）で選定した５つの転写因子を用いて（実施例２−１）で作製した肺前駆細胞の分化能を検討した。ｉｎ　ｖｉｔｒｏの分化は、マウス胎児線維芽細胞に５因子を導入後、内胚葉細胞を維持できる代表的な低血清含有ＲＰＭＩ１６４０培地（Ａｄｖａｎｃｅｄ　ＲＰＭＩ１６４０＋０．２％血清培地）を利用して５１日間培養し、自発的な分化能を評価した（図９ａ）。その結果、Ｎｋｘ２．１陰性の細胞でＭｕｃ５ａｃ陽性の粘液分泌細胞やＡＱＰ５陽性の肺胞上皮Ｉ型細胞が分化することを見出した（図９ｂ）。
　このことから、一般的な内胚葉維持培地、特に低血清含有ＲＰＭＩ１６４０培地を用いることで、粘液分泌細胞や肺胞上皮Ｉ型細胞にまで直接分化できることが強く示唆された。
（５−２）低血清内胚葉維持培地による分化能の検討−２
　上記（５−１）と同様に、マウス胎児線維芽細胞に、５つの転写因子を導入しＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０＋０．２％血清添加培地で分化させた肺前駆細胞を５３日以上培養していると、自発的に肺前駆細胞で構成された管腔様の三次元構造を形成することがしばしば観察された（図１０）。
（５−３）肺前駆細胞分化培地による分化能の検討
　上記（実施例２−１）などで得られた肺前駆細胞の分化能をさらに検討するため、図１１ａ右表に表示したいくつかの代表的な分化培地として知られる基礎培地を用いてそれぞれの分化能の検討を行った（図１１ａ）。
　具体的には、上記（５−１）と同様に、マウス胎児線維芽細胞に５因子を導入しＡｄｖａｎｃｅｄ　ＲＰＭＩ１６４０＋０．２％血清添加培地で１７日間分化誘導して肺前駆細胞を得た後、図１１ａ右表に記載の分化培地１~６をそれぞれ用いてさらに５３日まで培養して分化誘導した。得られた分化細胞が、どのような成熟肺細胞へ分化したかを定量ＲＴ−ＰＣＲによる分化マーカー測定により検証した。
　その結果、例えば代表的な低血清含有ＲＰＭＩ１６４０培地であるＮｏ．３培地（Ａｄｖａｎｃｅｄ　ＲＰＭＩ　１６４０）に増殖因子（Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を加えたＮｏ．４培地で分化させた場合は粘液分泌細胞マーカー（Ｍｕｃ５ＡＣ、Ｍｕｃ５ｂ）が高発現していることから、主として粘液分泌肺細胞に分化したと考えられる。このことから、上記実施例（５−１）で示唆されたとおり低血清含有ＲＰＭＩ１６４０培地を用いることで、粘液分泌細胞や肺胞上皮Ｉ型細胞が分化してくることか確認でき、さらに増殖因子（例えばＷｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を添加することで粘液分泌細胞への特異性が高まることが示された。
　一方、代表的な動物細胞用無血清培地であるＮｏ．５培地（ＤＭＥＭ＋１０％ＫＳＲ）に増殖因子を加えたＮｏ．６培地で分化させた場合は、クララ細胞マーカー（ＣＣ１０）、肺胞上皮Ｉ型細胞マーカー（ＡＱＰ５）、及び肺胞上皮ＩＩ型細胞マーカー（ＳＰ−Ｂ及びＳＰ−Ｃ）のいずれも強く発現していることから、クララ細胞、肺胞上皮Ｉ型細胞及び肺胞上皮ＩＩ型細胞に分化した肺細胞が混在していると考えられる。このように、分化培地の種類を変えることにより分化方向性が変化することが確認されると共に、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦなど増殖因子の添加により肺組織細胞への分化が促進される様子が定量的ＲＴ−ＰＣＲにより観察された（図１１ｂ）。
　また、上記肺前駆細胞をＮｏ．５培地（無血清培地）に増殖因子（ＷＦＫ：Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を加えたＮｏ．６培地で分化誘導を行った細胞について、培養上清に対するＥＬＩＳＡアッセイを行ったところ、肺特異的タンパク質であるＳＰ−Ａが分泌されていることが確認された（図１１ｃ）。さらに、当該細胞について、各種成熟肺組織マーカーによる免疫蛍光染色を行ったところ、Ｍｕｃ２，Ｍｕｃ５ａｃ，Ｍｕｃ５ｂ陽性の粘液分泌細胞，ＣＣ１０陽性のクララ細胞，ＳＰ−Ｂ，ＳＰ−Ｃ陽性の肺胞上皮ＩＩ型細胞やＡＱＰ５陽性の肺胞上皮Ｉ型細胞の存在が確認された（図１１ｄ）。
　このことからみて、代表的な無血清培地を分化誘導培地として選択し、増殖因子（例えば、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を添加することで、粘液分泌細胞のみならず、肺胞上皮Ｉ型細胞や肺胞上皮ＩＩ型細胞及びクララ細胞などの成熟肺細胞に効率的に分化することが示された。
　以上の結果から、肺細胞の分化誘導培地としては、無血清又は低血清培地を選択することが重要であり、増殖因子（例えば、Ｗｎｔ３ａ，ＦＧＦ１０，ＫＧＦ）を添加すると、さらに分化誘導効率が向上する。つまり、これら増殖因子は、「肺組織細胞分化誘導因子」として働くといえる。特に、低血清含有ＲＰＭＩ１６４０培地又は無血清のＤＭＥＭ＋ＫＳＲ培地に肺組織細胞分化誘導因子を添加した培地が好ましい。
（実施例８）ヒト線維芽細胞からのヒト肺前駆細胞の作製
（８−１）ヒト新生児皮膚由来繊維芽細胞からのヒト肺前駆細胞の作製
　Ｆｏｘａ２，Ｇａｔａ６，Ｉｒｘ３，Ｓｏｘ２１及びＮＭｙｃの５因子をｐＭＹｓレトロウイルスベクターに挿入したレトロウイルス発現ベクターを用いて、シュードレンチウイルス作製用細胞であるＰｌａｔＧＰ細胞でウイルスを作製しヒト新生児の皮膚由来繊維芽細胞（ＡＴＣＣ細胞バンクより入手）に感染させた。１週間後、代表的な無血清肺細胞用培地の市販のＳＡＧＭ培地で培養したところ、ＮＫＸ２．１の遺伝子発現に若干の上昇が見られ、かつヒト肺前駆細胞様のコロニー形成がわずかに認められた（図示せず）。
　そこで、上記５因子にさらにＳｏｘ９を加えた６因子を遺伝子導入して、ＳＡＧＭ培地で５０日間培養したところ、ＮＫＸ２．１の遺伝子発現が著しく発現上昇し（図１２ａ）、ＮＫＸ２．１タンパク質陽性のヒト肺前駆細胞の生成が確認された（図１２ｂ）。
（８−２）ヒト成体の脂肪組織由来線維芽細胞からのヒト肺前駆細胞の作製
　次いで、上記（８−１）と同様に、５因子にさらにＳｏｘ９を加えた６因子を用い、ヒト成体の脂肪組織から調製した線維芽細胞に対して遺伝子導入し、同様にＳＡＧＭ培地で６０日間培養したところ、同様にＮＫＸ２．１タンパク質陽性のヒト肺前駆細胞の生成が確認できた（図１２ｃ）。 (Example 1) Direct differentiation method from fibroblasts to lung progenitor cells using a combination of transcription factors 13 types of transcription factors that are specifically expressed in the lung formation stage of the initial lung are selected from known literatures and databases, After inserting the cDNA into the pMYs retrovirus vector, a retrovirus is prepared using PlatE cells, which are cells for retrovirus production, and mouse fetal fibroblasts (mouse fetal dorsal epidermis-derived fibroblasts: also called Dorsal skin MEF or simply MEF) .) One week later, the culture was continued by changing to a medium in which 0.2% fetal calf serum was added to Advanced RPMI1640, which is a general medium for endoderm-derived epithelial cells. Direct differentiation into lung progenitor cells was evaluated using as an index the appearance of colonies that were positive for the expression of lung progenitor cell-specific marker gene Nkx2.1 and one of epithelial markers, E-cadherin (FIG. 1a). As a result, after 2 to 3 weeks, cells proliferating into epithelial cell-like colonies were detected, and they were confirmed to be Nkx2.1-positive cell populations (FIG. 1b-d). Next, in order to identify transcription factors essential for direct differentiation into lung progenitor cells from among the 13 types of transcription factors, combinations in which one factor was sequentially removed from the combination of 13 factors were examined. When the transcription factor was removed, the expression of Nkx2.1 was increased in particular, and 7 transcription factors showing a suppressive action were discarded, and the other 6 transcription factors were selected (FIGS. 1e-f). Next, when one additional factor was removed from these 6 factors and an unnecessary factor was searched, the expression of Nkx2.1 was not decreased even if Sox9 was removed, so it was narrowed down to 5 factors of Foxa2, Gata6, Irx3, Sox21 and NMyc. (Fig. 1g-h). Further, when the essential factors were searched by subtracting one factor from five factors, the expression of Nkx2.1 almost disappeared in the case of Foxa2, Gata6, Sox21 and NMyc factors even when any of these factors was removed. On the other hand, when the Irx3 factor was removed, the expression of Nkx2.1 was significantly reduced, but it was observed that the expression was 50% or more (FIG. 1i-j). In addition, in the Nkx2.1 positive colony analysis by immunostaining, no colonies could be observed when any of the other factors was removed, whereas small colonies were observed when Irx3 was removed. Colonies were clearly observed (data not shown).
From these results, at least four factors of Foxa2, Gata6, Sox21 and NMyc are essential factors, and Irx3 factor is not an essential factor, but is an essential factor for promoting direct differentiation into lung progenitor cells. Thus, in order to efficiently produce Nkx2.1-positive lung progenitor cells, it was concluded that it is preferable to use 5 factors obtained by adding Irx3 to the above 4 factors.
From the above, as the transcription factor of the present invention, the combination of 4 factors of Foxa2, Gata6, Sox21 and NMyc is the minimum combination of necessary genes. In the following examples, Irx3 factor is added to these 4 factors. Nkx2.1 positive lung progenitor cells using a combination containing 5 factors were examined.
(Example 2) Verification of specificity of lung progenitor cells prepared by gene transfer of transcription factors (2-1) Lung progenitor cells differentiated from mouse fetal fibroblasts (MEF) 5 selected in (Example 1) above The lung tissue specificity of lung progenitor cells prepared with two transcription factors was verified by immunofluorescence staining. When 5 transcription factors are transfected into the same mouse embryo-derived fibroblasts as in Example 1 and cultured in a general endoderm-derived epithelial cell culture medium (Advanced RPMI 1640 + 0.2% fetal bovine serum), 2 to 3 Colonies consisting of epithelial-like cells began to appear after a week (FIG. 2a), and they were confirmed to be cells that were positive for the lung progenitor cell marker Nkx2.1 (FIG. 2b). However, Nkx2.1 is known to be expressed not only in pulmonary progenitor cells but also in thyroid tissue and brain diencephalon that develop from the anterior endoderm during embryonic period (FIG. 2c). Therefore, with regard to lung tissue specificity of lung progenitor cells prepared with transcription factors, immunofluorescence staining is performed using Pax8 specifically expressed in thyroid tissue, Tuj1 expressed in diencephalon, E-cadherin expressed in epithelial cells, etc. as indicators. As a result of the verification, Nkx2.1 is positive and E-cadherin (+), Tuj1 (-), Pax8 (-) and not thyroid gland or diencephalon cells but lung progenitor cells Was suggested (FIGS. 2d-e).
(2-2) Preparation of lung progenitor cells from other tissue-derived fibroblasts In addition to mouse fetal dorsal epidermis fibroblasts (Dorsal skin MEF), mouse fetal limb fibrocytes (Libb MEF) ), And adult mouse epidermis-derived fibroblasts (MDF), the above five transcription factors were introduced in the same manner as in (2-1) above, and cultured in the same differentiation medium. As a result, it was confirmed that Nkx2.1 positive lung progenitor colonies were directly differentiated in any fibroblasts (FIG. 2f).
(Example 3) Verification of specificity of lung progenitor cells prepared from mouse fibroblasts (3-1) Lung progenitor cells differentiated from mouse fetal fibroblasts Nkx2.1 positive obtained in (2-1) above Differentiated cells from mouse fetal fibroblasts (fibroblasts derived from mouse fetal dorsal epidermis) were further co-stained with Sox expressed in mouse fetal lung progenitor cells and EpCAM, an epithelial cell marker (Fig. 3b) and the results were quantified (FIG. 3c) and verified. As a result, the differentiated cells from the mouse fetal fibroblasts obtained in (2-1) above were Nkx2.1 positive, the epithelial cell markers E-cadherin and EpCAM were positive, and the thyroid tissue markers Pax8 and It was confirmed that Tuj1 as a diencephalic tissue marker was negative, and that Sox2 expressed together with Nkx2.1 in lung progenitor cells was also positive (FIGS. 2 and 3). As a result, it was confirmed that mouse embryo fibroblasts were directly differentiated into lung progenitor cells by the above five transcription factors.
(3-2) Lung progenitor cells differentiated from mouse fetal limb-derived fibroblasts With respect to differentiated cells from Nkx2.1-positive mouse fetal limb-derived fibroblasts obtained in (2-2) above, As a result of verification by immunofluorescence staining using the various tissue markers used in (2-1) and (3-1) as an index, fibers derived from the dorsal epidermis of the mouse fetus confirmed in (3-1) above As in the case of blast cells, Nkx2.1 positive and E-cadherin (+), Tuj1 (−), Pax8 (−), EpCAM (+), and Sox2 (+) were confirmed. (FIG. 4). As a result, it was confirmed that the cells had differentiated into lung progenitor cells, and even when fibroblasts derived from mouse fetal limbs were used, confirmation of direct differentiation into lung progenitor cells by the above five transcription factors was obtained. It was.
(3-3) Pulmonary progenitor cells differentiated from adult mouse-derived fibroblasts Furthermore, using the Nkx2.1-positive adult mouse epidermis-derived fibroblasts obtained in (2-2) above, there are five transcription factors. It was confirmed that it could be produced similarly.
The Nkx2.1-positive differentiated cells from the adult mouse-derived fibroblasts of (2-2) above are obtained by using 10% fetal bovine serum-containing DMEM from fibroblasts excised from the epidermis behind the ears of adult (12-week-old) mice. Prepared by culturing in medium. Five transcription factors were introduced into the adult mouse-derived fibroblasts in the same manner as in Example 1, and cultured in the same differentiation medium for 2 to 3 weeks. As described in (2-2) above, , Nkx2.1 positive colonies were generated (FIG. 2f).
Results of verification by immunofluorescence staining of Nkx2.1-positive differentiated cells from the adult mouse-derived fibroblasts using the various tissue markers used in (2-1) and (3-1) above as indices. , As well as fibroblasts derived from mouse embryos, are Nkx2.1 positive and E-cadherin (+), Tuj1 (−), Pax8 (−), EpCAM (+), and Sox2 (+) It was confirmed that the cells differentiated into lung progenitor cells (FIGS. 5a to 5e).
That is, it was confirmed that adult mouse-derived fibroblasts were also directly differentiated into lung progenitor cells by the above five transcription factors.
(3-4) Verification of conversion from fibroblasts to Nkx2.1-positive epithelial cells Obtained from Nkx2.1-positive mouse fetal fibroblasts (mesenchymal cells) in (2-1) above. The expression state of epithelial cell marker (E-cadherin) and mesenchymal cell marker (Twist2, Thy1, Fsp1) was verified with respect to lung progenitor cells.
As a result, it was confirmed that Thy1-positive fibroblasts were converted to E-cadherin-positive epithelial cells in the direct differentiation process with the above 5 transcription factors to become Nkx2.1-positive lung progenitor cells (FIG. 6). .
(Example 4) Gene expression analysis by microarray of lung progenitor cells prepared by introducing five transcription factors (4-1) Comprehensive gene analysis by microarray-1
In this example, in order to verify the specificity of lung progenitor cells prepared from mouse fibroblasts, lungs derived from the three types of mouse fibroblasts (Dorsal skin MEF, Limb MEF, MDF) obtained in Example 2 were used. Exhaustive gene analysis was performed on the progenitor cells using a microarray.
As a result, it was clarified that the gene expression of lung progenitor cells prepared from mouse fibroblasts showed a gene expression pattern similar to that of mouse fetal lung tissue cells compared to mouse fibroblasts before differentiation. (FIGS. 7a-b).
The prepared lung progenitor cells were compared with other tissues such as adult bronchi, lung, stomach, intestine, liver, heart, kidney, hypothalamus, skeletal muscle, and fat. Results The gene expression pattern of the prepared lung progenitor cells was found to be most similar to the lung derived from E11.5 fetus, and not similar to adult bronchi, lung and other tissues (FIG. 8a). ).
(4-2) Comprehensive gene analysis by microarray-2
Furthermore, using three new types of mouse fibroblasts (Dorsal skin MEF, Limb MEF, MDF), five transcription factors were introduced in the same manner as in Example (2-1) to establish lung progenitor cells. Later, the number of samples was increased for further microarray analysis. Similar results were obtained, further confirming that the cells are most similar to E11.5 fetal lungs and not similar to adult bronchi, lungs and other tissues. (Fig. 8b).
(Example 5) Analysis of differentiation ability of lung progenitor cells prepared by introducing five transcription factors (5-1) Examination of differentiation ability using low serum endoderm maintenance medium-1
The differentiation ability of lung progenitor cells prepared in (Example 2-1) was examined using the five transcription factors selected in (Example 1) above. For in vitro differentiation, 5 factors were introduced into mouse embryonic fibroblasts, and then cultured for 51 days using a typical low-serum RPMI1640 medium (Advanced RPMI1640 + 0.2% serum medium) capable of maintaining endoderm cells, Spontaneous differentiation ability was evaluated (FIG. 9a). As a result, it was found that Muc5ac-positive mucus-secreting cells and AQP5-positive alveolar epithelial type I cells were differentiated in Nkx2.1-negative cells (FIG. 9b).
This strongly suggested that it was possible to directly differentiate into mucous secretory cells and alveolar epithelial type I cells by using a general endoderm maintenance medium, particularly a low serum-containing RPMI 1640 medium.
(5-2) Examination of differentiation ability with low serum endoderm maintenance medium-2
As in (5-1) above, when lung progenitor cells obtained by introducing five transcription factors into mouse fetal fibroblasts and differentiated in a medium supplemented with RPMI1640 + 0.2% serum are cultured for 53 days or more, It was often observed to form a lumen-like three-dimensional structure composed entirely of lung progenitor cells (FIG. 10).
(5-3) Examination of differentiation ability by lung precursor cell differentiation medium In order to further examine the differentiation ability of lung precursor cells obtained in the above (Example 2-1), several Each differentiation ability was examined using a basal medium known as a typical differentiation medium (FIG. 11a).
Specifically, after 5 factors were introduced into mouse fetal fibroblasts and induced to differentiate for 17 days in a medium supplemented with RPMI1640 + 0.2% serum as in (5-1) above, lung progenitor cells were obtained. Differentiation mediums 1 to 6 described in the right table of 11a were further cultured until 53 days to induce differentiation. It was verified by differentiation marker measurement by quantitative RT-PCR how the differentiated cells obtained differentiated into mature lung cells.
As a result, for example, No. 1 which is a typical low serum-containing RPMI1640 medium. No. 3 medium (Advanced RPMI 1640) with growth factors (Wnt3a, FGF10, KGF) added. When differentiated in 4 media, the mucus-secreting cell markers (Muc5AC, Muc5b) are highly expressed, so it is considered that they differentiated mainly into mucus-secreting lung cells. From this, it was confirmed that mucus-secreting cells and alveolar epithelial type I cells were differentiated by using RPMI1640 medium containing low serum as suggested in the above Example (5-1). Addition of (for example, Wnt3a, FGF10, KGF) was shown to increase the specificity to mucus-secreting cells.
On the other hand, No. which is a typical serum-free medium for animal cells. No. 5 medium (DMEM + 10% KSR) with growth factors added. When differentiated in 6 medium, all of Clara cell marker (CC10), alveolar epithelial type I cell marker (AQP5), and alveolar epithelial type II cell marker (SP-B and SP-C) are strongly expressed. Therefore, it is considered that differentiated lung cells are mixed into Clara cells, alveolar epithelial type I cells, and alveolar epithelial type II cells. Thus, it is confirmed that the direction of differentiation is changed by changing the type of differentiation medium, and the manner in which differentiation into lung tissue cells is promoted by the addition of growth factors such as Wnt3a, FGF10, KGF is quantitative. Observed by RT-PCR (Figure 11b).
The lung progenitor cells are designated as No. No. 5 medium (serum-free medium) with growth factors (WFK: Wnt3a, FGF10, KGF) added. When cells subjected to differentiation induction in 6 media were subjected to ELISA assay on the culture supernatant, it was confirmed that SP-A, a lung-specific protein, was secreted (FIG. 11c). Further, the cells were subjected to immunofluorescence staining with various mature lung tissue markers. As a result, Muc2, Muc5ac, Muc5b positive mucus secreting cells, CC10 positive Clara cells, SP-B, SP-C positive alveolar epithelium II. Type cells and AQP5-positive alveolar epithelial type I cells were confirmed (FIG. 11d).
In view of this, by selecting a representative serum-free medium as a differentiation-inducing medium and adding growth factors (eg, Wnt3a, FGF10, KGF), not only mucus-secreting cells but also alveolar epithelial type I cells and It has been shown to efficiently differentiate into mature lung cells such as alveolar epithelial type II cells and Clara cells.
From the above results, it is important to select a serum-free or low-serum medium as a differentiation induction medium for lung cells. When growth factors (for example, Wnt3a, FGF10, KGF) are added, differentiation induction efficiency is further improved. To do. That is, it can be said that these growth factors function as “pulmonary tissue cell differentiation inducing factors”. In particular, a low serum-containing RPMI1640 medium or a medium prepared by adding a lung tissue cell differentiation inducing factor to serum-free DMEM + KSR medium is preferable.
(Example 8) Preparation of human lung progenitor cells from human fibroblasts (8-1) Preparation of human lung progenitor cells from human neonatal skin-derived fibroblasts Five factors of Foxa2, Gata6, Irx3, Sox21 and NMyc Using a retrovirus expression vector inserted into the pMYs retrovirus vector, a virus was prepared with PlatGP cells, which are pseudolentivirus-producing cells, and infected with human neonatal skin-derived fibroblasts (obtained from ATCC cell bank). One week later, when cultured in a typical serum-free lung cell culture medium, SAGM medium, NKX2.1 gene expression was slightly increased, and human lung progenitor cell-like colony formation was slightly observed. (Not shown).
Therefore, when 6 factors obtained by adding Sox9 to 5 factors were introduced and cultured in SAGM medium for 50 days, the gene expression of NKX2.1 was remarkably increased (FIG. 12a), and NKX2.1 protein positive. Generation of human lung progenitor cells was confirmed (FIG. 12b).
(8-2) Production of human lung progenitor cells from human adult adipose tissue-derived fibroblasts Next, as in (8-1) above, using 6 factors obtained by adding Sox9 to 5 factors, When the gene was introduced into fibroblasts prepared from adipose tissue and cultured in SAGM medium for 60 days, the production of NKX2.1 protein-positive human lung progenitor cells was confirmed (FIG. 12c).

Claims (30)

  1. 　下記の（ａ）~（ｄ）の転写因子ファミリー内の転写因子からそれぞれ１つ以上選択された４種の転写因子を含む、体細胞からのＮｋｘ２．１を発現する肺前駆細胞作製用試薬；
    （ａ）　Ｆｏｘファミリー、
    （ｂ）　Ｇａｔａファミリー、
    （ｃ）　Ｓｏｘファミリー、及び
    （ｄ）　Ｍｙｃファミリー。     A reagent for producing pulmonary progenitor cells expressing Nkx2.1 from somatic cells, comprising four transcription factors each selected from one or more of the transcription factors in the transcription factor family of (a) to (d) below:
    (A) Fox family,
    (B) Gata family,
    (C) Sox family, and (d) Myc family.
  2. 　（ａ）　Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
    　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
    　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
    　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
    　請求項１に記載の試薬。     (A) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd1, Foxe2, Foxd1, Foxe2, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox3FoxFox1, Fox3Fox Including
    (B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
    (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
    The reagent according to claim 1.
  3. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ転写因子を含む、請求項１又は２に記載の試薬。 The reagent according to claim 1 or 2, comprising at least Foxa2, Gata6, Sox21 and NMyc transcription factor.
  4. 　さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上選択された転写因子を含む、請求項１~３のいずれか１項に記載の試薬。 The reagent according to any one of claims 1 to 3, further comprising (e) a transcription factor selected from one or more transcription factors in a transcription factor family belonging to the Irx family.
  5. 　（ｅ）　Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、請求項４に記載の試薬。 (E) The reagent according to claim 4, wherein the transcription factor selected from the Irx family comprises any of the transcription factors Irx1, Irx2, Irx3, Irx4, Irx5 or Irx6.
  6. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３の転写因子を含む、請求項４又は５に記載の試薬。 The reagent according to claim 4 or 5, comprising at least Foxa2, Gata6, Sox21, NMyc and Irx3 transcription factors.
  7. 　（ｃ）　ＳｏｘファミリーからＳｏｘ９を含む２つ以上の転写因子を含む、請求項１~６のいずれか１項に記載の試薬。 (C) The reagent according to any one of claims 1 to 6, comprising two or more transcription factors including Sox9 from the Sox family.
  8. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含む、請求項６又は７に記載の試薬。 The reagent according to claim 6 or 7, comprising at least the transcription factors of Foxa2, Gata6, Sox21, NMyc, Irx3 and Sox9.
  9. 　体細胞からＮｋｘ２．１を発現する肺前駆細胞を作製する方法であって、下記の（１）~（３）の工程を含むことを特徴とする、方法；
    （１）下記の（ａ）~（ｄ）の転写因子ファミリーからそれぞれ１つ以上の転写因子を選択する工程、
    （ａ）　Ｆｏｘファミリー、
    （ｂ）　Ｇａｔａファミリー、
    （ｃ）　Ｓｏｘファミリー、及び
    （ｄ）　Ｍｙｃファミリー、
    （２）標的体細胞内で、前記４種類の転写因子を含む転写因子群を作用させる工程、
    （３）転写因子作用後の細胞でＮｋｘ２．１遺伝子を発現していることを確認する工程。     A method for producing lung progenitor cells expressing Nkx2.1 from somatic cells, comprising the following steps (1) to (3):
    (1) a step of selecting one or more transcription factors from the following transcription factor families (a) to (d):
    (A) Fox family,
    (B) Gata family,
    (C) Sox family, and (d) Myc family,
    (2) a step of causing a group of transcription factors including the four types of transcription factors to act in a target somatic cell;
    (3) A step of confirming that the Nkx2.1 gene is expressed in the cell after the action of the transcription factor.
  10. 　工程（２）が、前記転写因子をコードする遺伝子を標的体細胞内に導入する工程を含むことを特徴とする、請求項９に記載の方法。 The method according to claim 9, wherein the step (2) includes a step of introducing a gene encoding the transcription factor into a target somatic cell.
  11. 　前記体細胞が、単離された体細胞である請求項９又は１０に記載の方法。 The method according to claim 9 or 10, wherein the somatic cell is an isolated somatic cell.
  12. 　（ａ）　Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ３，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
    　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
    　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
    　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
    請求項９~１１のいずれか一項に記載の方法。     (A) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd3, Foxd3, Foxd3, Foxd3, Foxd3, Foxd3, Foxd3, Foxd3 Foxf1, Foxf2, Foxg1, Fox1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox3Fox1, Fox3Fox1, Fox3 Including factors,
    (B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
    (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
    The method according to any one of claims 9 to 11.
  13. 　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃを含むことを特徴とする、請求項９~１２のいずれか一項に記載の方法。 The method according to any one of claims 9 to 12, wherein the transcription factor group includes at least Foxa2, Gata6, Sox21 and NMyc.
  14. 　工程（１）において、さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上の転写因子を選択する工程を含み、工程（２）において、当該転写因子も含めた５種類の転写因子を含む転写因子群を作用させることを特徴とする、請求項９~１３のいずれか一項に記載の方法。 In step (1), the method further comprises (e) selecting one or more transcription factors from transcription factors in the transcription factor family belonging to the Irx family. In step (2), five types including the transcription factor are also included. The method according to any one of claims 9 to 13, wherein a transcription factor group containing a transcription factor is allowed to act.
  15. 　（ｅ）　Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、請求項１４に記載の方法。 (E) The method according to claim 14, wherein the transcription factor selected from the Irx family comprises any of the transcription factors Irx1, Irx2, Irx3, Irx4, Irx5 or Irx6.
  16. 　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３の転写因子を含むことを特徴とする、請求項１４又は１５に記載の方法。 The method according to claim 14 or 15, wherein the transcription factor group includes at least the transcription factors of Foxa2, Gata6, Sox21, NMyc, and Irx3.
  17. 　体細胞がヒト由来体細胞であって、工程（１）において、（ｃ）ＳｏｘファミリーからＳｏｘ９を含む２以上の転写因子を選択する工程を含み、工程（２）において、当該転写因子も含めた６種類の転写因子を含む転写因子群を作用させることを特徴とする、請求項１４~１６のいずれか一項に記載の方法。 The somatic cell is a human somatic cell, and in step (1), the method includes (c) selecting two or more transcription factors including Sox9 from the Sox family, and in step (2), the transcription factor is also included. The method according to any one of claims 14 to 16, wherein a group of transcription factors including 6 types of transcription factors is allowed to act.
  18. 　前記転写因子群が、少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含むことを特徴とする、請求項１７に記載の方法。 The method according to claim 17, wherein the transcription factor group includes at least the transcription factors of Foxa2, Gata6, Sox21, NMyc, Irx3, and Sox9.
  19. 　体細胞に対して請求項９~１８のいずれか１項に記載の方法を適用してＮｋｘ２．１を発現する肺前駆細胞を作製し、次いで、得られた肺前駆細胞を所望の肺組織細胞への分化誘導用培地で培養することを特徴とする、分化された肺組織細胞の製造方法。 A lung progenitor cell expressing Nkx2.1 is produced by applying the method according to any one of claims 9 to 18 to a somatic cell, and then the obtained lung progenitor cell is a desired lung tissue cell A method for producing differentiated lung tissue cells, which comprises culturing in a medium for inducing differentiation into cells.
  20. 　分化誘導用培地が、低血清又は無血清の上皮細胞用培地に肺組織細胞分化誘導因子を添加した培地である請求項１９に記載の方法。 The method according to claim 19, wherein the differentiation-inducing medium is a medium obtained by adding a lung tissue cell differentiation-inducing factor to a low-serum or serum-free medium for epithelial cells.
  21. 　Ｎｋｘ２．１を発現する肺前駆細胞から所望の肺組織細胞への分化誘導促進剤のスクリーニング方法であって、下記の（１）~（４）の工程を含むことを特徴とする、方法；
    （１）体細胞に対して請求項９~１８のいずれか一項に記載の方法を適用してＮｋｘ２．１を発現する肺前駆細胞を作製する工程、
    （２）得られた肺前駆細胞の１部を、所望の肺組織細胞への分化誘導用培地で培養する工程、
    （３）肺前駆細胞の他の１部を、工程（２）で用いた分化誘導用培地に被検物質を添加した培地で培養するか、又は肺前駆細胞内に被検物質を導入後に工程（２）で用いた分化誘導用培地で培養する工程、
    （４）工程（２）及び工程（３）で用いた肺前駆細胞の所望の肺組織細胞特異的マーカーの発現量を一定時間経過後に測定して比較し、後者の発現量が高い場合に、被検物質を肺前駆細胞から所望の肺組織細胞への分化誘導促進剤の候補物質となると評価して、選択する工程。     A method for screening for an agent for inducing differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell, comprising the following steps (1) to (4):
    (1) A step of producing lung progenitor cells expressing Nkx2.1 by applying the method according to any one of claims 9 to 18 to somatic cells,
    (2) culturing a part of the obtained lung progenitor cells in a medium for inducing differentiation into desired lung tissue cells,
    (3) Another part of lung progenitor cells is cultured in a medium obtained by adding a test substance to the differentiation-inducing medium used in step (2), or after introducing the test substance into lung progenitor cells Culturing in the differentiation-inducing medium used in (2),
    (4) When the expression level of a desired lung tissue cell-specific marker of the lung progenitor cells used in step (2) and step (3) is measured after a certain period of time and compared, and the latter expression level is high, A step of evaluating and selecting a test substance as a candidate substance for an agent for promoting differentiation induction from lung progenitor cells to desired lung tissue cells.
  22. 　Ｎｋｘ２．１を発現する肺前駆細胞から所望の肺組織細胞への分化誘導促進剤のスクリーニング用キットであって、請求項９~１８のいずれか一項に記載の方法により作製した肺前駆細胞を含むことを特徴とするキット。 A kit for screening for an agent for inducing differentiation from a lung progenitor cell expressing Nkx2.1 into a desired lung tissue cell, wherein the lung progenitor cell produced by the method according to any one of claims 9 to 18 is used. A kit comprising:
  23. 　Ｎｋｘ２．１を発現する肺前駆細胞又は肺組織細胞を補充する方法に使用するための組成物であって、
    前記組成物は、下記の（ａ）~（ｄ）の転写因子ファミリーからそれぞれ１つ以上選択された転写因子を含むものであり、
    前記方法は体細胞からＮｋｘ２．１を発現する肺前駆細胞への直接分化を誘導することを特徴とする方法；
    （ａ）　Ｆｏｘファミリー、
    （ｂ）　Ｇａｔａファミリー、
    （ｃ）　Ｓｏｘファミリー、及び
    （ｄ）　Ｍｙｃファミリー。     A composition for use in a method of supplementing lung progenitor cells or lung tissue cells expressing Nkx2.1 comprising:
    The composition comprises one or more transcription factors selected from the following transcription factor families (a) to (d):
    Said method induces direct differentiation of somatic cells into lung progenitor cells expressing Nkx2.1;
    (A) Fox family,
    (B) Gata family,
    (C) Sox family, and (d) Myc family.
  24. 　（ａ）　Ｆｏｘファミリーから選択された転写因子がＦｏｘａ１，Ｆｏｘａ２，Ｆｏｘａ３，Ｆｏｘｂ１，Ｆｏｘｂ２，Ｆｏｘｃ１，Ｆｏｘｃ２，Ｆｏｘｄ１，Ｆｏｘｄ２，Ｆｏｘｄ３，Ｆｏｘｄ４，Ｆｏｘｄ５，Ｆｏｘｄ６，Ｆｏｘｄ７，Ｆｏｘｄ８，Ｆｏｘｅ１，Ｆｏｘｅ２，Ｆｏｘｅ３，Ｆｏｘｆ１，Ｆｏｘｆ２，Ｆｏｘｇ１，Ｆｏｘｈ１，Ｆｏｘｉ１，Ｆｏｘｊ１，Ｆｏｘｊ２，Ｆｏｘｊ３，Ｆｏｘｋ１，Ｆｏｘｍ１，Ｆｏｘｎ１，Ｆｏｘｎ２，Ｆｏｘｎ３，Ｆｏｘｎ４，Ｆｏｘｎ５，Ｆｏｘｎ６，Ｆｏｘｏ１，Ｆｏｘｏ３ａ，Ｆｏｘｏ４，Ｆｏｘｐ１，Ｆｏｘｐ２，Ｆｏｘｐ３又はＦｏｘｑ１のいずれかの転写因子を含み、
    　（ｂ）　Ｇａｔａファミリーから選択された転写因子がＧａｔａ１，Ｇａｔａ２，Ｇａｔａ３，Ｇａｔａ４，Ｇａｔａ５，又はＧａｔａ６のいずれかの転写因子を含み、
    　（ｃ）　Ｓｏｘファミリーから選択された転写因子がＳｏｘ１，Ｓｏｘ２，Ｓｏｘ３，Ｓｏｘ４，Ｓｏｘ５，Ｓｏｘ６，Ｓｏｘ７，Ｓｏｘ８，Ｓｏｘ９，Ｓｏｘ１０，Ｓｏｘ１１，Ｓｏｘ１２，Ｓｏｘ１３，Ｓｏｘ１４，Ｓｏｘ１５，Ｓｏｘ１７，Ｓｏｘ１８，Ｓｏｘ２１，Ｓｏｘ３０又はＳｒｙのいずれかの転写因子を含み、そして
    　（ｄ）　Ｍｙｃファミリーから選択された転写因子がｃＭｙｃ，ＬＭｙｃ又はＮＭｙｃのいずれかの転写因子を含む、
    　請求項２３に記載の方法。     (A) Transcription factors selected from the Fox family are Foxa1, Foxa2, Foxa3, Foxb1, Foxb2, Foxc1, Foxc2, Foxd1, Foxd2, Foxd3, Foxd4, Foxd5, Foxd6, Foxd7, Foxd1, Foxe2, Foxd1, Foxe2, Foxf2, Foxg1, Foxh1, Foxi1, Foxj1, Foxj2, Foxj3, Foxk1, Foxm1, Foxn1, Foxn2, Foxn3, Foxn4, Foxn5, Foxn6, Fox1, Fox3FoxFox1, Fox3Fox Including
    (B) a transcription factor selected from the Gata family includes any transcription factor of Gata1, Gata2, Gata3, Gata4, Gata5, or Gata6;
    (C) Transcription factors selected from the Sox family are Sox1, Sox2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox18, Sox21, Sox21, Sox21, Sox21, Sox21, (D) a transcription factor selected from the Myc family comprises any of the cMyc, LMyc, or NMyc transcription factors,
    24. The method of claim 23.
  25. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１及びＮＭｙｃ転写因子を含む、請求項２３又は２４に記載の方法。 The method according to claim 23 or 24, comprising at least Foxa2, Gata6, Sox21 and NMyc transcription factor.
  26. 　さらに（ｅ）Ｉｒｘファミリーに属する転写因子ファミリー内の転写因子から１つ以上選択された転写因子を含む、請求項２３~２５のいずれか一項に記載の方法。 The method according to any one of claims 23 to 25, further comprising (e) one or more transcription factors selected from transcription factors in the transcription factor family belonging to the Irx family.
  27. 　（ｅ）　Ｉｒｘファミリーから選択された転写因子がＩｒｘ１，Ｉｒｘ２，Ｉｒｘ３，Ｉｒｘ４，Ｉｒｘ５又はＩｒｘ６のいずれかの転写因子を含む、請求項２６に記載の方法。 (E) The method according to claim 26, wherein the transcription factor selected from the Irx family comprises any of the transcription factors Irx1, Irx2, Irx3, Irx4, Irx5 or Irx6.
  28. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ及びＩｒｘ３の転写因子を含む、請求項２６又は２７に記載の方法。 28. The method according to claim 26 or 27, comprising at least transcription factors of Foxa2, Gata6, Sox21, NMyc and Irx3.
  29. 　（ｃ）　ＳｏｘファミリーからＳｏｘ９を含む２つ以上の転写因子を含む、請求項２３~２８のいずれか一項に記載の方法。 (C) The method according to any one of claims 23 to 28, comprising two or more transcription factors including Sox9 from the Sox family.
  30. 　少なくともＦｏｘａ２，Ｇａｔａ６，Ｓｏｘ２１，ＮＭｙｃ，Ｉｒｘ３及びＳｏｘ９の転写因子を含む、請求項２９に記載の方法。 30. The method of claim 29, comprising at least Foxa2, Gata6, Sox21, NMyc, Irx3 and Sox9 transcription factors.
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