JP2006149329A - Method for preparing ciliated epithelial cell - Google Patents

Method for preparing ciliated epithelial cell Download PDF

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JP2006149329A
JP2006149329A JP2004348388A JP2004348388A JP2006149329A JP 2006149329 A JP2006149329 A JP 2006149329A JP 2004348388 A JP2004348388 A JP 2004348388A JP 2004348388 A JP2004348388 A JP 2004348388A JP 2006149329 A JP2006149329 A JP 2006149329A
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cells
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ciliated epithelial
serum
epithelial cell
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Yusuke Nishimura
佑介 西村
Tatsuo Hamazaki
辰夫 浜崎
Makoto Asajima
誠 浅島
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University of Tokyo NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a ciliated epithelial cell and to provide the ciliated epithelial cell induced from an undifferentiated cell in vitro. <P>SOLUTION: The method for preparing the ciliated epithelial cell by culturing an embryoid body (preferably the cell constituting the embryoid body is derived from a human) in a serum-free culture medium is found. The ciliated epithelial cell induced from the undifferentiated cell (preferably an embryonal stem cell derived from the human) can be obtained for the first time by the method. The resultant ciliated epithelial cell provides a material useful for diagnosis or therapy of diseases in the human, etc. The ciliated epithelial cell is useful for constructing a model or an evaluation (assay) system in developing a drug for studying or treating of abnormalities or diseases with viruses, bacteria, etc., in the respiratory system. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は、繊毛上皮細胞を調製するための方法に関する。   The present invention relates to a method for preparing ciliated epithelial cells.

胚性幹細胞（ＥＳ細胞）や組織性（体性）幹細胞などの哺乳類の未分化細胞から、腎臓や肝臓などの種々の器官や、脂肪などの種々の組織を作るための方法が開発されている。しかしながら、現在のところ、ＥＳ細胞を含むあらゆる哺乳類の幹細胞から繊毛上皮を誘導する技術は開発されていない。このような技術が存在しないことは、ヒトを含めた繊毛に関係する異常や疾病に関する研究、診断、および治療薬の開発などの過程における重大な障害となっており、繊毛上皮細胞の調製方法の開発が待たれていた。   Methods have been developed to produce various organs such as kidney and liver and various tissues such as fat from undifferentiated mammalian cells such as embryonic stem cells (ES cells) and tissue (somatic) stem cells. . However, at present, no technology has been developed to induce ciliated epithelium from any mammalian stem cells including ES cells. The absence of such technology has become a serious obstacle in the process of research, diagnosis, and development of therapeutic agents related to abnormalities and diseases related to cilia including humans. Development was awaited.

本発明は、繊毛上皮細胞を調製するための方法及びインビトロで未分化細胞から誘導された繊毛上皮細胞を提供する。   The present invention provides a method for preparing ciliated epithelial cells and ciliated epithelial cells derived from undifferentiated cells in vitro.

本発明者は、上記課題を解決すべく鋭意努力した結果、胚様体（胚様体）を無血清培地で培養すると、繊毛上皮細胞が誘導されることを見出し、本発明を完成させた。   As a result of diligent efforts to solve the above-mentioned problems, the present inventors have found that ciliary epithelial cells are induced when embryoid bodies (embryoid bodies) are cultured in a serum-free medium, and the present invention has been completed.

すなわち、本発明は、下記のとおりである。
１ 胚様体を無血清培地で培養する工程を含む、繊毛上皮細胞を調製するための方法。
２ 無血清培地が、Ｋｎｏｃｋｏｕｔ（商標）Ｓｅｒｕｍ Ｒｅｐｌａｃｅｍｅｎｔを含む培地である、１に記載の方法。
３ 胚様体を構成する細胞が、ヒトに由来する、１又は２に記載の方法。
４ 胚様体が培養容器に付着している、１〜３のいずれか１に記載の方法。
５ インビトロで未分化細胞から誘導された繊毛上皮細胞。
６ 未分化細胞が、ヒトに由来する、５に記載の細胞。
７ 未分化細胞が、胚性幹細胞である、６に記載の細胞。 That is, the present invention is as follows.
1 A method for preparing ciliated epithelial cells, comprising culturing embryoid bodies in a serum-free medium.
2. The method according to 1, wherein the serum-free medium is a medium containing Knockout ™ Serum Replacement.
3 The method according to 1 or 2, wherein the cells constituting the embryoid body are derived from a human.
4. The method according to any one of 1 to 3, wherein the embryoid body is attached to the culture vessel.
5 Ciliated epithelial cells derived from undifferentiated cells in vitro.
6. The cell according to 5, wherein the undifferentiated cell is derived from a human.
7 The cell according to 6, wherein the undifferentiated cell is an embryonic stem cell.

インビトロで未分化細胞から誘導された繊毛上皮細胞を提供する。   Ciliated epithelial cells derived from undifferentiated cells in vitro are provided.

受精卵の胎児へと成長していく過程の一つに胚盤胞と呼ばれる状態がある。胚盤胞は、球状の形をしており、外側の細胞層である栄養外胚葉と将来的に身体を形成する内部細胞塊を含む胞胚腔とから構成されている。ＥＳ細胞等の未分化細胞を浮遊状態で培養すると、周辺部が内皮様に分化した胞胚腔に類似する大きな細胞集塊を形成する。本発明では、この細胞集塊を胚様体（embryoid bodies）という。   One of the processes of growing a fertilized egg into a fetus is a state called a blastocyst. The blastocyst has a spherical shape and is composed of a trophectoderm, which is an outer cell layer, and a blastocoel containing an inner cell mass that will form the body in the future. When undifferentiated cells such as ES cells are cultured in a floating state, a large cell mass similar to the blastocoel whose peripheral portion is differentiated like an endothelium is formed. In the present invention, this cell cluster is referred to as embryoid bodies.

胚様体は、従来公知の技術にしたがって調製することができる。胚様体を構成する細胞は、胚様体を形成することができる程度に未分化な細胞であれば特に制限されることはないが、好ましくは、ＥＳ細胞又は体性幹細胞であり、特に好ましくはＥＳ細胞である。例えば、ＥＳ細胞を出発材料として、ウシ胎仔血清等を含む血清培地又は無血清培地中で浮遊培養してＥＳ細胞の集合体である胚様体を調製することができる。   The embryoid body can be prepared according to a conventionally known technique. The cells constituting the embryoid body are not particularly limited as long as they are undifferentiated cells that can form an embryoid body, but are preferably ES cells or somatic stem cells, particularly preferably. Are ES cells. For example, ES cells can be used as a starting material, and embryoid bodies that are aggregates of ES cells can be prepared by suspension culture in a serum medium or fetal serum-free medium containing fetal calf serum or the like.

本発明に用いることができる未分化細胞は、哺乳類に由来する細胞であれば特に制限されることはないが、好ましくは、げっ歯類又は霊長類に由来する細胞であり、特に好ましくは、ヒトに由来する細胞である。   The undifferentiated cells that can be used in the present invention are not particularly limited as long as they are cells derived from mammals, but are preferably cells derived from rodents or primates, particularly preferably humans. Is a cell derived from

無血清培地とは、合成培地に血清代替物としてポリペプチドホルモン類、各種ステロイドホルモン、結合タンパク質、成長因子、細胞の基質付着因子等を添加して良好な細胞増殖や生存を可能にする培養液をいう。無血清培地は、未分化細胞の増殖に適しているものであれば特に制限されないが、好ましくは、血清代替物としてインビトロジェン社より市販されているKnockout（商標） Serum Replacement（ＫＳＲ培地）を含む培地である。合成培地は、ＤＭＥＭ、ＥＭＥＭ、ＲＰＭＩ１６４０等の市販の培地を用いることができる。   A serum-free medium is a culture medium that enables good cell growth and survival by adding polypeptide hormones, various steroid hormones, binding proteins, growth factors, cell substrate adhesion factors, etc. as serum substitutes to a synthetic medium. Say. The serum-free medium is not particularly limited as long as it is suitable for growth of undifferentiated cells, but preferably a medium containing Knockout ™ Serum Replacement (KSR medium) commercially available from Invitrogen as a serum substitute. It is. As the synthetic medium, a commercially available medium such as DMEM, EMEM, RPMI1640 and the like can be used.

未分化細胞は、必要に応じ、フィーダー細胞上に播種することによって培養することができる。フィーダー細胞としては、Ｘ線、γ線、マイトマイシンＣ等で処理することによって生存はするが増殖しない状態にした細胞を用いることができる。例えば、マウス由来ＥＳ細胞のフィーダー細胞としては、マイトマイシンＣで処理したマウス胎児繊維芽細胞を用いることができる。なお、典型的には、培地は毎日交換し、かつ、継代培養を３日間おきに行う。   Undifferentiated cells can be cultured by seeding on feeder cells as necessary. As the feeder cells, cells that have survived but not proliferated by treatment with X-rays, γ-rays, mitomycin C, and the like can be used. For example, mouse fetal fibroblasts treated with mitomycin C can be used as feeder cells for mouse-derived ES cells. Typically, the medium is changed every day, and subculture is performed every 3 days.

培養した未分化細胞を、トリプシン処理によって単一細胞懸濁液とし、得られた懸濁液を、培養用の容器に播種して、ＣＯ２インキュベーター内で培養する。この操作により、フィーダー細胞は培養面に付着するが、未分化細胞は培養面に付着せずに浮遊したままとなる。そこで培養上清を回収し、遠心分離することによって、未分化細胞をフィーダー細胞と分離することができる。 The cultured undifferentiated cells are made into a single cell suspension by trypsin treatment, and the obtained suspension is seeded in a culture vessel and cultured in a CO 2 incubator. By this operation, feeder cells adhere to the culture surface, but undifferentiated cells do not adhere to the culture surface and remain floating. Therefore, undifferentiated cells can be separated from feeder cells by collecting the culture supernatant and centrifuging.

次に、得られた未分化細胞を、傾けた低付着性の培養用容器中で一晩浮遊培養し、次に容器を水平状態にして、さらに培養を２〜３日間継続して胚様体を調製する。また、ミネラルオイル上に形成させた液滴中に未分化細胞を懸垂状態とさせて、培養することによっても胚様体を調製することができる。この胚様体調製用培地としては、ウシ胎仔血清を含む培地又は無血清培地を用いることができる。   Next, the obtained undifferentiated cells are suspended and cultured overnight in a tilted, low-adhesive culture container, and then the container is placed in a horizontal state and further cultured for 2 to 3 days. To prepare. An embryoid body can also be prepared by culturing undifferentiated cells in a suspended state in droplets formed on mineral oil. As this embryoid body preparation medium, a medium containing fetal calf serum or a serum-free medium can be used.

得られた胚様体は、付着性容器を用いて血清培地又は無血清培地の中で培養することによって、培養容器の底面に付着させることができる。   The obtained embryoid body can be attached to the bottom surface of the culture container by culturing it in a serum medium or serum-free medium using an adhesive container.

得られた胚様体を無血清培地の中で培養することによって、無数の繊毛様構造を有する細胞が誘導される。   By culturing the obtained embryoid body in a serum-free medium, cells having innumerable cilia-like structures are induced.

この繊毛様構造は、ドーム様構造（管状構造）の内部に見出される。また、これらのドーム様構造の内部だけでなく、その他の領域にも見出される。繊毛様構造がドーム様構造の内部にある場合には、繊毛に特有な「あおり運動」に起因してドーム様構造の内部で液体が流動するので、容易に繊毛用構造を特定することができる。なお、この培養過程において血清培地を用いると、繊毛様構造は誘導されない。   This cilia-like structure is found inside the dome-like structure (tubular structure). It is also found not only inside these dome-like structures but also in other areas. When the cilia-like structure is inside the dome-like structure, the liquid flows inside the dome-like structure due to the “claw movement” unique to cilia, so that the structure for cilia can be easily identified. . In addition, when a serum medium is used in this culturing process, cilia-like structures are not induced.

繊毛上皮細胞とは、繊毛様構造を有する細胞であり、繊毛特有の「あおり運動」を行い、気管上皮や輸卵管などの繊毛に特有な軸糸の配列構造である９＋２構造を有し、さらに、軸糸を構成するタンパク質であるチューブリン（β−チューブリンＩＶ）や同じく繊毛に特有なタンパク質（マーカータンパク質）の遺伝子であるＦｏｘｊ１を発現する。   The ciliated epithelial cell is a cell having a cilia-like structure, performs “claw movement” peculiar to cilia, has a 9 + 2 structure which is an arrangement structure of axial yarns peculiar to cilia such as tracheal epithelium and oviduct, It expresses tubulin (β-tubulin IV), which is a protein constituting the axial thread, and Foxj1, which is also a gene specific to cilia (marker protein).

したがって、例えば、抗原染色法を用いて、軸糸の構成タンパク質であるβ−チューブリンＩＶを検出すること、また、ＲＴ−ＰＣＲ法を用いて、Ｆｏｘｊ１の発現を同定することによって、繊毛上皮細胞の誘導を確認することができる。   Therefore, for example, by detecting β-tubulin IV which is a constituent protein of the axial thread using an antigen staining method, and identifying the expression of Foxj1 using an RT-PCR method, ciliated epithelial cells Can be confirmed.

繊毛上皮細胞は、生体内においては、気管上皮や輸卵管などに存在する。   Ciliated epithelial cells are present in the tracheal epithelium, oviduct, and the like in vivo.

ＥＳ細胞の維持
マウスＥＳ細胞（例：Ｅ１４（ＡＴＣＣ−ＣＲＬ−１８２１）、Ｄ３（ＡＴＣＣ−ＣＲＬ−１１６３２））を、フィーダー細胞上で維持した。フィーダー細胞としては、あらかじめ１０μｇ／ｍＬのマイトマイシンＣで１〜３時間処理して細胞***を止めたマウス胎児繊維芽細胞を、０．１％のゼラチンをコートした培養用の容器に播種したものを用いた。ＥＳ細胞のための維持培地として用いた血清培地は、１５％の牛胎児血清（ES qualified, Invitrogen社）、１ｍＭのピルビン酸ナトリウム（Invitrogen社）、０．１ｍＭのβ−メルカプトエタノール（Sigma社）、０．１ｍＭの非必須アミノ酸（Sigma社）、１００Ｕ／ｍＬのペニシリン、１００μｇ／ｍＬのストレプトマイシン及び１，０００Ｕ／ｍＬの白血病抑制因子（Chemicon社）を含む高グルコースのＤＭＥＭ培地（Invitrogen社）であった。培養液は毎日交換し、３日に１回継代培養した。 Maintenance of ES cells Mouse ES cells (eg, E14 (ATCC-CRL-1821), D3 (ATCC-CRL-11632)) were maintained on feeder cells. As feeder cells, mouse embryonic fibroblasts that had been treated with 10 μg / mL mitomycin C for 1 to 3 hours to stop cell division were seeded in a culture vessel coated with 0.1% gelatin. Using. The serum medium used as a maintenance medium for ES cells was 15% fetal bovine serum (ES qualified, Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma). In high glucose DMEM medium (Invitrogen) containing 0.1 mM non-essential amino acids (Sigma), 100 U / mL penicillin, 100 μg / mL streptomycin and 1,000 U / mL leukemia inhibitory factor (Chemicon) there were. The culture medium was changed every day and subcultured once every 3 days.

胚様体の作成
ＥＳ細胞のコロニーを０．１％トリプシン及び０．５ｍｇ／ｍＬのＥＤＴＡを含むリン酸緩衝生理食塩水で処理し、単一細胞懸濁液を得た。得られたＥＳ細胞の懸濁液を、培養用の容器（例えば、１０ｃｍの培養用ディッシュ）に播種して、４０分間ＣＯ２インキュベーターで培養した。こうしてフィーダー細胞から分離したＥＳ細胞を、遠心分離して集めた。次に、これらのＥＳ細胞を、市販の低接着性培養用容器（ローセルバインディグディッシュ、Ｎｕｎｃ社）に移して、胚様体調製用培地中で、容器を傾けてＥＳ細胞を相互に隣接させながら１晩浮遊培養し、次に容器を水平状態にして、さらに培養を２〜３日間継続してＥＢを調製した。 Preparation of Embryoid Bodies ES cell colonies were treated with phosphate buffered saline containing 0.1% trypsin and 0.5 mg / mL EDTA to obtain a single cell suspension. The obtained suspension of ES cells was seeded in a culture vessel (for example, a 10 cm culture dish) and cultured for 40 minutes in a CO 2 incubator. The ES cells thus separated from the feeder cells were collected by centrifugation. Next, these ES cells are transferred to a commercially available container for low adhesion culture (Low cell binding dish, Nunc), and the ES cells are placed adjacent to each other by tilting the container in the medium for embryoid body preparation. Then, the suspension was cultured overnight, and then the container was placed in a horizontal state, and further culturing was continued for 2 to 3 days to prepare EB.

このＥＢ作成用培地としては、ＦＢＳを含む血清培地を用いた。また、１０％のＫＳＲ（Invitrogen社）、１００Ｕ／ｍＬのペニシリン、１００ｍｇ／ｍＬのストレプトマイシンを含む低グルコースＤＭＥＭ培地（Invitrogen社）、すなわち、無血清培地を用いてもＥＢを調製することができた。   As this EB production medium, a serum medium containing FBS was used. EB could also be prepared using low glucose DMEM medium (Invitrogen) containing 10% KSR (Invitrogen), 100 U / mL penicillin, 100 mg / mL streptomycin, ie, serum-free medium. .

繊毛上皮の誘導
０．１％のゼラチンなどでコートした細胞接着性容器（例えば、２４穴のウェルプレート）に、上述のごとくして得たＥＢを播種し、上記の血清培地の中で、又は、無血清培地の中でＥＢを培養して、ＥＢを培養プレート上に接着させた。 Induction of ciliated epithelium In a cell adhesive container (for example, a 24-well well plate) coated with 0.1% gelatin or the like, the EB obtained as described above is seeded, and the above-mentioned serum medium or The EB was cultured in a serum-free medium, and the EB was allowed to adhere on the culture plate.

次いで、無血清培地の中で、接着したＥＢの培養を継続すると、無数の繊毛様構造を有する細胞が誘導された。この繊毛様構造は、ドーム様構造（管状構造）の内部に見出された（図１）。また、これらのドーム様構造の内部だけでなく、その他の領域にも存在した。繊毛様構造がドーム様構造の内部にある場合には、繊毛に特有な「あおり運動」に起因してドーム様構造の内部で液体が流動するので、繊毛用構造を特定して観測することが容易であった。なお、この培養過程において血清培地を用いると、繊毛様構造は誘導されなかった。   Subsequently, when the culture of the adherent EB was continued in a serum-free medium, cells having innumerable cilia-like structures were induced. This cilia-like structure was found inside a dome-like structure (tubular structure) (FIG. 1). Moreover, it existed not only inside these dome-like structures but also in other areas. When the cilia-like structure is inside the dome-like structure, the liquid flows inside the dome-like structure due to the “claw movement” unique to cilia. It was easy. In addition, when a serum medium was used in this culturing process, cilia-like structures were not induced.

繊毛上皮の同定
上述のごとく、誘導された繊毛様構造は、繊毛に特有な「あおり運動」を行う。その繊毛様構造を電子顕微鏡で観察すると、その内部に管状構造（軸糸）が認められ、その軸糸の配置は、気管上皮や輸卵管などの繊毛に特有な軸糸の配列構造である９＋２構造であることが確認された（図２：感覚器などの繊毛は９＋２構造でなく９＋０構造である）。 Identification of ciliated epithelium As described above, the induced cilia-like structure performs a “claw movement” characteristic of cilia. When the cilia-like structure is observed with an electron microscope, a tubular structure (axial yarn) is observed inside, and the arrangement of the axial yarn is a 9 + 2 structure that is an arrangement structure of axial yarns peculiar to cilia such as tracheal epithelium and oviduct. (FIG. 2: cilia such as sensory organs have a 9 + 0 structure, not a 9 + 2 structure).

抗原染色法にて軸糸の構成タンパク質であるβ−チューブリンＩＶが検出され、また、ＲＴ−ＰＣＲ法にて繊毛に特有なタンパク質（マーカータンパク質）であるＦｏｘｊ１が確認された（図３）。   Β-tubulin IV, which is a constituent protein of the axial thread, was detected by the antigen staining method, and Foxj1, which is a protein peculiar to cilia (marker protein), was confirmed by the RT-PCR method (FIG. 3).

このように、本発明の方法によって誘導された細胞が、繊毛上皮細胞であることが確認された。   Thus, it was confirmed that the cells induced by the method of the present invention are ciliated epithelial cells.

本手法により、これまで実現されていなかった哺乳類の幹細胞から気管上皮や輸卵管などを構成する繊毛上皮を誘導することがはじめて実現された。これらの繊毛上皮は、繊毛上皮性のタンパク質などが特異的に欠如、あるいは異常発現しているヒトなどの疾病の診断や、痰の排出障害や妊娠障害などの繊毛上皮の欠陥や欠損などに起因するヒトなどの異常や疾病を治療する目的などに有用な材料を提供する。また、肺の気道、気管、気管上皮などを経由するウイルス、細菌などによる異常や疾病、さらには、呼吸疾病などの異常や疾病に関する研究や治療のための薬剤の開発における有用なモデルや評価系（アッセイ系）を開発することができる。   This technique was the first to induce ciliated epithelium that constitutes tracheal epithelium, oviduct, etc. from mammalian stem cells that had not been realized so far. These ciliated epithelia are caused by the diagnosis of diseases such as humans that specifically lack or abnormally express ciliated epithelial proteins, or defects or defects of ciliated epithelium such as sputum discharge disorder or pregnancy disorder Materials useful for the purpose of treating abnormalities and diseases such as human beings are provided. In addition, useful models and evaluation systems for the development of drugs for research and treatment of abnormalities and diseases such as viruses and bacteria that pass through the respiratory tract, trachea, tracheal epithelium, etc., and respiratory diseases (Assay system) can be developed.

左図は、胚葉体から誘導されたドーム様構造の光学顕微鏡写真である。右図は、１の破線で囲まれた範囲（２）を拡大して示した図である。ドーム様となった胚様体の内部に繊毛上皮様構造（３）が認められる。The left figure is an optical micrograph of a dome-like structure derived from the embryoid body. The right figure is an enlarged view of the range (2) surrounded by a broken line 1. A ciliated epithelium-like structure (3) is observed inside the dome-like embryoid body. １は、図２の一部の電子顕微鏡像である。繊毛上皮組織上に発達した繊毛が認められる（矢印２）。３は、繊毛部分の縦断面電子顕微鏡像である。繊毛の外部分と中心部分にある軸糸が認められる（矢印４）。５は、繊毛部分の横断面電子顕微鏡像である。繊毛の外部分に９対の軸糸と中心部分に１対の軸糸が認められる（矢印６）。1 is an electron microscope image of a part of FIG. Cilia developed on the ciliated epithelial tissue are observed (arrow 2). 3 is a longitudinal cross-sectional electron microscope image of a cilia part. Axial threads in the outer and central parts of the cilia are observed (arrow 4). 5 is a cross-sectional electron microscope image of the cilia portion. Nine pairs of axial threads and one pair of axial threads are recognized in the outer part of the cilia (arrow 6). １は、図１のドーム状構造を抗β−チューブリンIV抗体で染色した光学顕微鏡像である。２は、抗β−チューブリンIV抗体による染色で緑色蛍光を発する組織である（２）。３は、ドーム状構造に含まれるｍＲＮＡをＲＴ−ＰＣＲ法で増幅した試料の電気泳動像（Ｆｏｘｊ１に対する抗体で染色した）である。各レーンは、下記のとおりである。１：マーカー（標準試料）、２：培養期間（０日）、３：培養期間（５日）、４：培養期間（１０日）、５：培養期間（１５日）、６：培養期間（２０日）、７：培養期間（２５日）。1 is an optical microscope image obtained by staining the dome-shaped structure of FIG. 1 with an anti-β-tubulin IV antibody. 2 is a tissue that emits green fluorescence upon staining with an anti-β-tubulin IV antibody (2). 3 is an electrophoretic image (stained with an antibody against Foxj1) of a sample obtained by amplifying mRNA contained in the dome-shaped structure by RT-PCR. Each lane is as follows. 1: Marker (standard sample) 2: Culture period (0 days) 3: Culture period (5 days) 4: Culture period (10 days) 5: Culture period (15 days) 6: Culture period (20 Day), 7: culture period (25 days).

Claims (7)

胚様体を無血清培地で培養する工程を含む、繊毛上皮細胞を調製するための方法。   A method for preparing ciliated epithelial cells, comprising culturing an embryoid body in a serum-free medium. 無血清培地が、Ｋｎｏｃｋｏｕｔ（商標）Ｓｅｒｕｍ Ｒｅｐｌａｃｅｍｅｎｔを含む培地である、請求項１に記載の方法。   The method of claim 1, wherein the serum-free medium is a medium containing Knockout ™ Serum Replacement. 胚様体を構成する細胞が、ヒトに由来する、請求項１又は２に記載の方法。   The method according to claim 1 or 2, wherein the cells constituting the embryoid body are derived from a human. 胚様体が培養容器に付着している、請求項１〜３のいずれか１項に記載の方法。   The method according to any one of claims 1 to 3, wherein the embryoid body is attached to the culture vessel. インビトロで未分化細胞から誘導された繊毛上皮細胞。   Ciliated epithelial cells derived from undifferentiated cells in vitro. 未分化細胞が、ヒトに由来する、請求項５に記載の細胞。   The cell according to claim 5, wherein the undifferentiated cell is derived from a human. 未分化細胞が、胚性幹細胞である、請求項６に記載の細胞。   The cell according to claim 6, wherein the undifferentiated cell is an embryonic stem cell.
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JP2008178367A (en) * 2007-01-26 2008-08-07 Sumitomo Bakelite Co Ltd Culturing container for developing embryoid, method for producing the same and method for developing the embryoid
WO2009110215A1 (en) * 2008-03-03 2009-09-11 独立行政法人 科学技術振興機構 Method for induction of ciliated cell differentiation

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JP2003111588A (en) * 2000-01-11 2003-04-15 Geron Corp Technique for growth and differentiation of human pluripotent stem cell
JP2004248507A (en) * 2001-10-04 2004-09-09 National Univ Of Singapore Embryonic stem cell and neural progenitor cell derived from embryonic stem cell

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JP2003111588A (en) * 2000-01-11 2003-04-15 Geron Corp Technique for growth and differentiation of human pluripotent stem cell
JP2004248507A (en) * 2001-10-04 2004-09-09 National Univ Of Singapore Embryonic stem cell and neural progenitor cell derived from embryonic stem cell

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008178367A (en) * 2007-01-26 2008-08-07 Sumitomo Bakelite Co Ltd Culturing container for developing embryoid, method for producing the same and method for developing the embryoid
WO2009110215A1 (en) * 2008-03-03 2009-09-11 独立行政法人 科学技術振興機構 Method for induction of ciliated cell differentiation
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