JPWO2018235899A1 - Method for culturing muscle satellite cells - Google Patents
Method for culturing muscle satellite cells Download PDFInfo
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- JPWO2018235899A1 JPWO2018235899A1 JP2019525683A JP2019525683A JPWO2018235899A1 JP WO2018235899 A1 JPWO2018235899 A1 JP WO2018235899A1 JP 2019525683 A JP2019525683 A JP 2019525683A JP 2019525683 A JP2019525683 A JP 2019525683A JP WO2018235899 A1 JPWO2018235899 A1 JP WO2018235899A1
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- laminin
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- muscle satellite
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Abstract
本発明は，筋幹細胞の未分化性を保持したまま細胞を培養する方法を提供する。より具体的には、レチノイドとラミニンを含む培地を用いて細胞を培養することにより，筋幹細胞の未分化性を保持したまま細胞を培養することを可能とする。レチノイドとしては例えば，ａｔＲＡ，ＡＭ８０などが使用できる。レチノイドとラミニンを含む培地を用いて培養することにより増殖させた筋幹細胞は，細胞移植による筋ジストロフィーの治療等に利用されうる。The present invention provides a method for culturing cells while maintaining the undifferentiated state of muscle stem cells. More specifically, by culturing cells using a medium containing retinoid and laminin, it is possible to culture cells while maintaining the undifferentiated state of muscle stem cells. As the retinoid, for example, atRA, AM80 or the like can be used. The muscle stem cells proliferated by culturing in a medium containing retinoid and laminin can be used for treatment of muscular dystrophy by cell transplantation and the like.
Description
本願は、特願２０１７−１２１７８７号（出願日：２０１７年６月２２日）の優先権を主張する出願であり、これは引用することによりその全体が本明細書に取り込まれる。 The present application is an application claiming priority of Japanese Patent Application No. 2017-121787 (filing date: June 22, 2017), which is incorporated herein by reference in its entirety.
本発明は，筋幹細胞の培養方法に関する。より詳細には，筋幹細胞の未分化性を保持したまま培養する方法に関する。また，本発明は，細胞移植などに利用可能な筋幹細胞の製造方法，およびそれに用いる組成物にも関する。 The present invention relates to a method for culturing muscle stem cells. More specifically, it relates to a method for culturing muscle stem cells while maintaining their undifferentiated state. The present invention also relates to a method for producing a muscle stem cell that can be used for cell transplantation and the like, and a composition used therefor.
骨格筋は，運動や姿勢の保持に必要であるだけでなく，最大のエネルギー利用器官として代謝調節にも重要である。また，骨格筋は再生能が高いという特徴をもつ。骨格筋は，骨格筋幹細胞（筋サテライト細胞）が活性化され，分化・融合することによって再生される（非特許文献１）。 Skeletal muscle is not only required for maintaining movement and posture, but is also important for metabolic regulation as the largest energy utilization organ. In addition, skeletal muscle is characterized by high regenerative capacity. Skeletal muscle is regenerated when skeletal muscle stem cells (muscle satellite cells) are activated and differentiated / fused (Non-patent Document 1).
筋ジストロフィーは筋線維の壊死と再生を繰り返しながら筋萎縮が進行する遺伝性の疾患であり，有効な治療法は未だに確立されていない。開発段階にある筋ジストロフィーの治療法としては，例えば，核酸医薬品を用いたエキソンスキッピング療法がある（非特許文献２）。核酸医薬品とならび，細胞移植治療の応用も期待されているが，ｉｎ ｖｉｔｒｏで筋幹細胞を培養すると，幹細胞としての未分化性が失われ，増殖を停止してしまうことが知られている（非特許文献３）。 Muscular dystrophy is a hereditary disease in which muscle atrophy progresses by repeating necrosis and regeneration of muscle fibers, and an effective treatment method has not yet been established. As a treatment method for muscular dystrophy in the development stage, for example, there is an exon skipping therapy using a nucleic acid drug (Non-patent Document 2). In addition to nucleic acid drugs, application of cell transplantation therapy is expected, but it is known that culturing muscle stem cells in vitro loses the undifferentiated nature as stem cells and stops proliferation (non- Patent Document 3).
本発明は，筋幹細胞の未分化性を保持したまま培養する方法を提供することを目的の一つとする。また，本発明は，筋幹細胞の未分化性を保持したまま培養する方法において用いる組成物を提供することを別の目的の一つとする。 An object of the present invention is to provide a method for culturing muscle stem cells while maintaining their undifferentiated state. Another object of the present invention is to provide a composition used in a method of culturing muscle stem cells while maintaining their undifferentiated state.
本発明者らは，レチノイドおよびラミニンを含む培地中で細胞を培養することによって，筋幹細胞の未分化性を保持したまま，細胞を増殖させることが可能であることを見出した。本技術は簡便かつ安全で，高い効率で筋幹細胞の未分化性を維持できることから，筋ジストロフィーをはじめとした筋疾患に対する細胞移植治療や，化合物スクリーニング系の開発，再生医療への応用が期待される。本発明はこのような知見に基づくものであり，以下の態様を包含する：
［態様１］
（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む培地を用いて細胞を培養することを特徴とする，筋サテライト細胞の培養方法。
［態様２］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様１記載の培養方法。
［態様３］
レチノイドの濃度が少なくとも１０−８Ｍである，態様１または２記載の培養方法。
［態様４］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様１〜３のいずれか記載の培養方法。
［態様５］
培地がレチノイドおよびラミニンを含む液体培地である，態様１〜４のいずれか記載の培養方法。
［態様６］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様１〜５のいずれか記載の培養方法。
［態様７］
培地がＧ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤，（Ｒ）−ＰＦＩ−２およびその混合物から成る群より選択される少なくとも１つの因子をさらに含む，態様１〜６のいずれか記載の培養方法。
［態様８］
筋サテライト細胞の未分化性が維持される，態様１〜７のいずれか記載の培養方法。
［態様９］
筋サテライト細胞がヒトの細胞である，態様１〜８のいずれか記載の培養方法。
［態様１０］
（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む液体培地を用いて細胞を培養することを特徴とする，ヒト筋サテライト細胞の培養方法であって，
レチノイドがａｔＲＡまたはＡＭ８０であり，レチノイドの濃度が少なくとも１０−８Ｍであり，
ラミニンがヒトラミニン５１１Ｅ８フラグメントであり，ラミニンが液体培地中に含められており，ラミニンの濃度が少なくとも１．５μｇ／ｍｌであり，そして
ヒト筋サテライト細胞の未分化性が維持される，培養方法。
［態様１１］
（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む，筋サテライト細胞の筋分化を抑制するための組成物。
［態様１２］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様１１記載の組成物。
［態様１３］
レチノイドの濃度が少なくとも１０−８Ｍである，態様１１または１２記載の組成物。
［態様１４］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様１１〜１３のいずれか記載の組成物。
［態様１５］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様１１〜１４のいずれか記載の組成物。
［態様１６］
筋サテライト細胞の未分化性が維持される，態様１１〜１５のいずれか記載の組成物。
［態様１７］
筋サテライト細胞がヒトの細胞である，態様１１〜１６のいずれか記載の組成物。
［態様１８］
（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む，細胞培養培地。
［態様１９］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様１８記載の細胞培養培地。
［態様２０］
レチノイドの濃度が少なくとも１０−８Ｍである，態様１８または１９記載の細胞培養培地。
［態様２１］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様１８〜２０のいずれか記載の細胞培養培地。
［態様２２］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様１８〜２１のいずれか記載の細胞培養培地。
［態様２３］
培地がＧ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤，（Ｒ）−ＰＦＩ−２およびその混合物から成る群より選択される少なくとも１つの因子をさらに含む，態様１８〜２２のいずれか記載の細胞培養培地。
［態様２４］
筋サテライト細胞の培養に用いるための態様１８〜２３のいずれか記載の細胞培養培地。
［態様２５］
筋サテライト細胞の未分化性を維持するための態様１８〜２４のいずれか記載の細胞培養培地。
［態様２６］
筋サテライト細胞がヒトの細胞である，態様１８〜２５のいずれか記載の細胞培養培地。
［態様２７］
（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む，細胞培養培地サプリメント。
［態様２８］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様２７記載の細胞培養培地サプリメント。
［態様２９］
細胞培養培地に加えられた際のレチノイドの濃度が少なくとも１０−８Ｍである，態様２７または２８記載の細胞培養培地サプリメント。
［態様３０］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様２７〜２９のいずれか記載の細胞培養培地サプリメント。
［態様３１］
細胞培養培地に加えられた際のラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様２７〜３０のいずれか記載の細胞培養培地サプリメント。
［態様３２］
Ｇ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤，（Ｒ）−ＰＦＩ−２およびその混合物から成る群より選択される少なくとも１つの因子をさらに含む，態様２７〜３１のいずれか記載の細胞培養培地サプリメント。
［態様３３］
筋サテライト細胞の培養に用いるための態様２７〜３２のいずれか記載の細胞培養培地サプリメント。
［態様３４］
筋サテライト細胞の未分化性を維持するための態様２７〜３３のいずれか記載の細胞培養培地サプリメント。
［態様３５］
筋サテライト細胞がヒトの細胞である，態様２７〜３４のいずれか記載の細胞培養培地サプリメント。
［態様３６］
細胞移植に用いるための細胞の製造方法であって，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む培地を用いて筋サテライト細胞を培養する工程を含む，方法。
［態様３７］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様３６記載の製造方法。
［態様３８］
レチノイドの濃度が少なくとも１０−８Ｍである，態様３６または３７記載の製造方法。
［態様３９］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様３６〜３８のいずれか記載の製造方法。
［態様４０］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様３６〜３９のいずれか記載の製造方法。
［態様４１］
培地がＧ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤，（Ｒ）−ＰＦＩ−２およびその混合物から成る群より選択される少なくとも１つの因子をさらに含む，態様３６〜４０のいずれか記載の製造方法。
［態様４２］
筋サテライト細胞がヒトの細胞である，態様３６〜４１のいずれか記載の製造方法。
［態様４３］
筋サテライト細胞がｉＰＳ細胞由来の細胞である，態様３６〜４２のいずれか記載の製造方法。
［態様４４］
筋サテライト細胞が筋ジストロフィー患者由来の細胞である，態様３６〜４３のいずれか記載の製造方法。
［態様４５］
筋サテライト細胞が遺伝子改変細胞である，態様３６〜４４のいずれか記載の製造方法。
［態様４６］
細胞が糖尿病の治療に用いるための細胞である，態様３６〜４５のいずれか記載の製造方法。
［態様４７］
患者における疾患または傷害の治療方法であって，（ｉ）レチノイド，および（ｉｉ）ラミニンを含む培地を用いて筋サテライト細胞を培養する工程，および培養した筋サテライト細胞またはそれに由来する細胞を患者に移植する工程を含む，方法。
［態様４８］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様４７記載の治療方法。
［態様４９］
レチノイドの濃度が少なくとも１０−８Ｍである，態様４７または４８記載の治療方法。
［態様５０］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様４７〜４９のいずれか記載の治療方法。
［態様５１］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様４７〜５０のいずれか記載の治療方法。
［態様５２］
培地がＧ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤，（Ｒ）−ＰＦＩ−２およびその混合物から成る群より選択される少なくとも１つの因子をさらに含む，態様４７〜５１のいずれか記載の治療方法。
［態様５３］
患者がヒトである，態様４７〜５２のいずれか記載の治療方法。
［態様５４］
疾患が筋ジストロフィー，先天性ミオパチー，遠位型ミオパチー，筋強直性疾患，炎症性筋疾患，周期性四肢麻痺，代謝性筋疾患，重症筋無力症，先天性筋無力症候群，ミトコンドリア病，サルコペニアおよび糖尿病から成る群より選択される，態様４７〜５３のいずれか記載の治療方法。
［態様５５］
筋サテライト細胞がｉＰＳ細胞由来の細胞である，態様４７〜５４のいずれか記載の治療方法。
［態様５６］
筋サテライト細胞が遺伝子改変細胞である，態様４７〜５５のいずれか記載の治療方法。
［態様５７］
少なくとも５×１０５個の筋サテライト細胞を含む，細胞組成物。
［態様５８］
（ｉ）レチノイドおよび（ｉｉ）ラミニンをさらに含む，態様５７記載の細胞組成物。
［態様５９］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様５７または５８記載の細胞組成物。
［態様６０］
レチノイドの濃度が少なくとも１０−８Ｍである，態様５７〜５９のいずれか記載の細胞組成物。
［態様６１］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様５７〜６０のいずれか記載の細胞組成物。
［態様６２］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様５７〜６１のいずれか記載の細胞組成物。
［態様６３］
筋サテライト細胞の純度が少なくとも８５％である，態様５７〜６２のいずれか記載の細胞組成物。
［態様６４］
筋サテライト細胞がヒトの細胞である，態様５７〜６３のいずれか記載の細胞組成物。
［態様６５］
筋サテライト細胞がｉＰＳ細胞由来の細胞である，態様５７〜６４のいずれか記載の細胞組成物。
［態様６６］
筋サテライト細胞が筋ジストロフィー患者由来の細胞である，態様５７〜６５のいずれか記載の細胞組成物。
［態様６７］
筋サテライト細胞が遺伝子改変細胞である，態様５７〜６６のいずれか記載の細胞組成物。
［態様６８］
凍結されている，態様５７〜６７のいずれか記載の細胞組成物。
［態様６９］
１つの容器に納められている，態様５７〜６８のいずれか記載の細胞組成物。
［態様７０］
筋サテライト細胞またはそれに由来する細胞の分化，増殖または生存に影響する物質のスクリーニング方法であって，
（ｉ）レチノイドおよび（ｉｉ）ラミニンの存在下で筋サテライト細胞を培養する工程，
培養した筋サテライト細胞またはそれに由来する細胞に試験物質を接触させる工程，
試験物質が筋サテライト細胞またはそれに由来する細胞の分化，増殖または生存に影響するか否かを評価する工程
を含む，方法。
［態様７１］
レチノイドがａｔＲＡ，ＡＭ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３，およびその混合物から成る群より選択される，態様７０記載のスクリーニング方法。
［態様７２］
レチノイドの濃度が少なくとも１０−８Ｍである，態様７０または７１記載のスクリーニング方法。
［態様７３］
ラミニンがラミニン５１１，ラミニン５２１，ラミニン５２２，ラミニン５２３，およびその混合物から成る群より選択される，態様７０〜７２のいずれか記載のスクリーニング方法。
［態様７４］
ラミニンの濃度が少なくとも１．５μｇ／ｍｌである，態様７０〜７３のいずれか記載のスクリーニング方法。
［態様７５］
筋サテライト細胞がヒトの細胞である，態様７０〜７４のいずれか記載のスクリーニング方法。
［態様７６］
筋サテライト細胞がｉＰＳ細胞由来の細胞である，態様７０〜７５のいずれか記載のスクリーニング方法。
［態様７７］
筋サテライト細胞が筋ジストロフィー患者由来の細胞である，態様７０〜７６のいずれか記載のスクリーニング方法。
［態様７８］
筋サテライト細胞が遺伝子改変細胞である，態様７０〜７７のいずれか記載のスクリーニング方法。The present inventors have found that by culturing cells in a medium containing retinoid and laminin, it is possible to proliferate the cells while maintaining the undifferentiated state of muscle stem cells. Since this technology is simple and safe, and can maintain the undifferentiated state of muscle stem cells with high efficiency, it is expected to be applied to cell transplantation therapy for muscle diseases such as muscular dystrophy, development of compound screening system, and regenerative medicine. . The present invention is based on such findings and includes the following aspects:
[Aspect 1]
A method for culturing muscle satellite cells, which comprises culturing cells using a medium containing (i) retinoid and (ii) laminin.
[Aspect 2]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. The culture method according to
[Aspect 3]
The culture method according to
[Mode 4]
The culture method according to any one of
[Aspect 5]
The culture method according to any one of
[Aspect 6]
The culture method according to any one of
[Aspect 7]
The medium is selected from the group consisting of G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor, (R) -PFI-2 and mixtures thereof. 7. The culture method according to any one of
[Aspect 8]
The culture method according to any one of
[Aspect 9]
9. The culture method according to any one of
[Aspect 10]
A method for culturing human muscle satellite cells, which comprises culturing cells using a liquid medium containing (i) retinoid and (ii) laminin
The retinoid is atRA or AM80, and the retinoid concentration is at least 10 −8 M,
A culture method, wherein laminin is human laminin 511E8 fragment, laminin is contained in a liquid medium, the concentration of laminin is at least 1.5 μg / ml, and the undifferentiated state of human muscle satellite cells is maintained.
[Aspect 11]
A composition for suppressing muscle differentiation of muscle satellite cells, comprising (i) a retinoid and (ii) laminin.
[Aspect 12]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A composition according to aspect 11, which comprises:
[Aspect 13]
13. The composition according to
[Aspect 14]
The composition of any of embodiments 11-13, wherein the laminin is selected from the group consisting of
[Aspect 15]
The composition according to any of embodiments 11-14, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 16]
16. The composition according to any one of aspects 11 to 15, wherein the undifferentiated state of muscle satellite cells is maintained.
[Aspect 17]
17. The composition according to any of aspects 11-16, wherein the muscle satellite cells are human cells.
[Aspect 18]
A cell culture medium comprising (i) a retinoid and (ii) laminin.
[Aspect 19]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A cell culture medium according to aspect 18.
[Aspect 20]
20. The cell culture medium of embodiment 18 or 19, wherein the concentration of retinoid is at least 10-8M .
[Aspect 21]
21. The cell culture medium of any of aspects 18-20, wherein the laminin is selected from the group consisting of
[Aspect 22]
The cell culture medium of any of aspects 18-21, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 23]
The medium is selected from the group consisting of G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor, (R) -PFI-2 and mixtures thereof. 23. The cell culture medium of any of aspects 18-22, further comprising at least one factor that
[Aspect 24]
The cell culture medium according to any one of aspects 18 to 23, for use in culturing muscle satellite cells.
[Aspect 25]
25. The cell culture medium according to any one of aspects 18 to 24, for maintaining the undifferentiated state of muscle satellite cells.
[Aspect 26]
The cell culture medium according to any one of aspects 18 to 25, wherein the muscle satellite cells are human cells.
[Aspect 27]
A cell culture medium supplement comprising (i) a retinoid and (ii) laminin.
[Aspect 28]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A cell culture medium supplement according to aspect 27.
[Aspect 29]
The cell culture medium supplement of embodiment 27 or 28, wherein the concentration of retinoid when added to the cell culture medium is at least 10 −8 M.
[Aspect 30]
30. The cell culture medium supplement of any of aspects 27-29, wherein the laminin is selected from the group consisting of
[Aspect 31]
31. The cell culture medium supplement of any of embodiments 27-30, wherein the concentration of laminin when added to the cell culture medium is at least 1.5 μg / ml.
[Aspect 32]
At least selected from the group consisting of G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor, (R) -PFI-2 and mixtures thereof. 32. The cell culture medium supplement of any of aspects 27-31, further comprising one factor.
[Aspect 33]
A cell culture medium supplement according to any of aspects 27-32 for use in culturing muscle satellite cells.
[Aspect 34]
The cell culture medium supplement according to any one of aspects 27 to 33, for maintaining the undifferentiated state of muscle satellite cells.
[Aspect 35]
The cell culture medium supplement of any of aspects 27-34, wherein the muscle satellite cells are human cells.
[Aspect 36]
A method for producing cells for use in cell transplantation, which comprises the step of culturing muscle satellite cells using a medium containing (i) retinoid and (ii) laminin.
[Aspect 37]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A manufacturing method according to Aspect 36.
[Aspect 38]
38. The production method according to embodiment 36 or 37, wherein the concentration of retinoid is at least 10 −8 M.
[Aspect 39]
39. The method of any of aspects 36-38, wherein the laminin is selected from the group consisting of
[Aspect 40]
40. The production method according to any one of aspects 36 to 39, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 41]
The medium is selected from the group consisting of G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor, (R) -PFI-2 and mixtures thereof. 41. The manufacturing method according to any one of aspects 36 to 40, further comprising at least one factor
[Aspect 42]
42. The production method according to any of aspects 36 to 41, wherein the muscle satellite cells are human cells.
[Aspect 43]
The production method according to any one of aspects 36 to 42, wherein the muscle satellite cells are cells derived from iPS cells.
[Aspect 44]
44. The production method according to any one of aspects 36 to 43, wherein the muscle satellite cells are cells derived from a muscular dystrophy patient.
[Aspect 45]
The production method according to any one of aspects 36 to 44, wherein the muscle satellite cells are genetically modified cells.
[Aspect 46]
46. The production method according to any of aspects 36 to 45, wherein the cells are cells for use in treating diabetes.
[Aspect 47]
A method for treating a disease or injury in a patient, comprising culturing muscle satellite cells using a medium containing (i) retinoid and (ii) laminin, and culturing the muscle satellite cells or cells derived therefrom to the patient. A method comprising the step of transplanting.
[Aspect 48]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. The treatment method according to aspect 47, which comprises:
[Aspect 49]
49. The method of treatment according to embodiment 47 or 48, wherein the concentration of retinoid is at least 10-8M .
[Aspect 50]
50. The method of treatment according to any of aspects 47-49, wherein the laminin is selected from the group consisting of
[Aspect 51]
The method of treatment according to any of aspects 47-50, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 52]
The medium is selected from the group consisting of G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor, (R) -PFI-2 and mixtures thereof. 52. The method of treatment according to any of aspects 47-51, further comprising at least one factor
[Aspect 53]
53. The method of treatment according to any of aspects 47-52, wherein the patient is a human.
[Aspect 54]
Diseases are muscular dystrophy, congenital myopathy, distal myopathy, myotonic disease, inflammatory myopathy, periodic quadriplegia, metabolic myopathy, myasthenia gravis, congenital myasthenic syndrome, mitochondrial disease, sarcopenia and diabetes The method of treatment according to any of aspects 47-53, selected from the group consisting of:
[Aspect 55]
55. The therapeutic method according to any one of aspects 47 to 54, wherein the muscle satellite cells are cells derived from iPS cells.
[Mode 56]
The treatment method according to any one of aspects 47 to 55, wherein the muscle satellite cells are genetically modified cells.
[Aspect 57]
A cell composition comprising at least 5 × 10 5 muscle satellite cells.
[Aspect 58]
The cell composition of embodiment 57, further comprising (i) a retinoid and (ii) laminin.
[Aspect 59]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A cell composition according to embodiment 57 or 58.
[Aspect 60]
The concentration of retinoid that is at least 10 -8 M, cell composition according to any of embodiments 57-59.
[Aspect 61]
61. The cell composition of any of aspects 57-60, wherein the laminin is selected from the group consisting of
[Aspect 62]
62. The cell composition of any of aspects 57-61, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 63]
63. The cell composition of any of aspects 57-62, wherein the muscle satellite cells have a purity of at least 85%.
[Aspect 64]
64. The cell composition of any of aspects 57-63, wherein the muscle satellite cells are human cells.
[Aspect 65]
65. The cell composition of any of aspects 57-64, wherein the muscle satellite cells are iPS cell-derived cells.
[Aspect 66]
66. The cell composition of any of aspects 57-65, wherein the muscle satellite cells are cells from a muscular dystrophy patient.
[Aspect 67]
67. The cell composition of any of aspects 57-66, wherein the muscle satellite cells are genetically modified cells.
[Aspect 68]
68. The cell composition of any of aspects 57-67, which is frozen.
[Aspect 69]
69. The cell composition of any of aspects 57-68, contained in a single container.
[Aspect 70]
A method for screening a substance that affects the differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom, comprising:
Culturing muscle satellite cells in the presence of (i) retinoid and (ii) laminin,
A step of bringing a test substance into contact with cultured muscle satellite cells or cells derived therefrom,
A method comprising the step of assessing whether a test substance affects the differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom.
[Aspect 71]
The retinoid is selected from the group consisting of atRA, AM80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80, fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003, and mixtures thereof. A screening method according to embodiment 70, which comprises:
[Mode 72]
The screening method according to embodiment 70 or 71, wherein the retinoid concentration is at least 10 −8 M.
[Aspect 73]
73. The screening method of any of aspects 70-72, wherein the laminin is selected from the group consisting of
[Aspect 74]
The screening method according to any of aspects 70 to 73, wherein the concentration of laminin is at least 1.5 μg / ml.
[Aspect 75]
The screening method according to any of aspects 70 to 74, wherein the muscle satellite cells are human cells.
[Aspect 76]
The screening method according to any one of aspects 70 to 75, wherein the muscle satellite cells are cells derived from iPS cells.
[Mode 77]
77. The screening method according to any of aspects 70 to 76, wherein the muscle satellite cells are cells derived from a muscular dystrophy patient.
[Aspect 78]
The screening method according to any of aspects 70 to 77, wherein the muscle satellite cells are genetically modified cells.
上述のとおり，レチノイドとラミニンを含む培地中で細胞を培養することによって，筋幹細胞の未分化性を保持したまま，細胞を増殖させることが可能であることが見出された。以下に，本発明を詳細に説明する。 As described above, it was found that by culturing the cells in a medium containing retinoid and laminin, the cells can be proliferated while maintaining the undifferentiated state of the muscle stem cells. The present invention will be described in detail below.
筋幹細胞（筋サテライト細胞）
骨格筋の組織幹細胞は，筋幹細胞あるいは筋サテライト細胞と呼ばれる小さな多能性の細胞であり，基底膜と筋鞘の間に存在している。筋サテライト細胞は，通常は細胞周期の静止期にとどまり未分化状態を維持しているが，骨格筋に何らかの損傷を受けると活性化して分裂し，筋芽細胞へと分化し，筋管を形成して筋線維を再生させる。 Muscle stem cells (muscle satellite cells)
Tissue stem cells of skeletal muscle are small pluripotent cells called muscle stem cells or muscle satellite cells, and are located between the basement membrane and the sarcolemma. Muscle satellite cells normally remain in the resting phase of the cell cycle and maintain an undifferentiated state, but when damaged by some kind of skeletal muscle, they are activated and divide, differentiate into myoblasts, and form myotubes. To regenerate muscle fibers.
静止期の未分化の筋サテライト細胞は，Ｐａｘ７の発現を指標として同定することができる。筋サテライト細胞の活性化により筋芽細胞が分化するにつれて，Ｐａｘ７の発現は減少し，ＭｙｏＤの発現が上昇する。さらに，筋芽細胞が筋管を形成するのに伴い，ミオゲニンの発現が上昇する。また，筋細胞が融合した筋管は，抗ミオシン重鎖抗体による免疫染色により可視化することができる。 The undifferentiated muscle satellite cells in the stationary phase can be identified by using the expression of Pax7 as an index. The expression of Pax7 decreases and the expression of MyoD increases as myoblasts differentiate by the activation of muscle satellite cells. Moreover, myogenin expression increases as myoblasts form myotubes. In addition, the myotube fused with myocytes can be visualized by immunostaining with an anti-myosin heavy chain antibody.
細胞の形態に関しては，未分化の筋サテライト細胞は丸い形態を示すのに対し，分化の進んだ細胞は扁平で巨大な形態を示すことから，未分化の筋サテライト細胞を同定することができる。 Regarding the cell morphology, undifferentiated muscle satellite cells have a round morphology, whereas the more differentiated cells have a flat and huge morphology, so that undifferentiated muscle satellite cells can be identified.
本発明において用いる筋サテライト細胞は，動物の筋サテライト細胞であることが好ましく，哺乳動物の筋サテライト細胞であることがより好ましく，マウス，ラット，ブタ，ウサギ，サルまたはヒトの筋サテライト細胞であることがさらに好ましく，ヒトまたはマウスの筋サテライト細胞であることがさらに好ましく，ヒトの筋サテライト細胞であることが特に好ましい。 The muscle satellite cells used in the present invention are preferably animal muscle satellite cells, more preferably mammalian muscle satellite cells, and more preferably mouse, rat, pig, rabbit, monkey or human muscle satellite cells. More preferably, human or mouse muscle satellite cells are more preferable, and human muscle mouse satellite cells are particularly preferable.
本発明において用いる筋サテライト細胞は，動物の組織から単離することができる。単離方法としては，（ｉ）０．１４％プロテアーゼ（Sigma-Aldrich社）を含むＤＭＥＭ培地で３７°Ｃ，３０分間処理し，ＤＭＥＭ培地による洗浄を行った後に播種する方法，（ｉｉ）酵素消化後にフローサイトメーター（ＢＤ社，AriaII）により単離する方法（Exp Cell Res. 10, 245-255, 2004; Nat Med. 20, 255-264,2014）などが挙げられるが，これらに限定はされない。組織から筋サテライト細胞を分離する方法は，当業者には公知であり，例えば，Fukada et al. Exp Cell Res2004;296:245-255.を参照してもよい。 The muscle satellite cells used in the present invention can be isolated from animal tissues. As an isolation method, (i) a method of treating with a DMEM medium containing 0.14% protease (Sigma-Aldrich) at 37 ° C for 30 minutes, washing with the DMEM medium, and then seeding, (ii) enzyme Examples include, but are not limited to, a method of isolation by a flow cytometer (BD, AriaII) after digestion (Exp Cell Res. 10, 245-255, 2004; Nat Med. 20, 255-264, 2014). Not done. Methods of separating muscle satellite cells from tissues are known to those of skill in the art and may be referred to, for example, Fukada et al. Exp Cell Res 2004; 296: 245-255.
本発明において用いる筋サテライト細胞は，動物のＥＳ細胞，ｉＰＳ細胞や，他の幹細胞から作製してもよい。なお，ｉＰＳ細胞等から誘導された細胞は，「サテライト細胞様」筋幹細胞であり得るが，本願においては，これも筋サテライト細胞に含まれるものとみなす。 The muscle satellite cells used in the present invention may be produced from animal ES cells, iPS cells, or other stem cells. Note that cells derived from iPS cells and the like may be “satellite cell-like” muscle stem cells, but in the present application, these are also considered to be included in muscle satellite cells.
レチノイド
レチノイドとは，ビタミンＡまたはそれに化学的に関連する一群の化合物のことをいう。ビタミンＡとは，レチノール，レチナール，レチノイン酸（これらをビタミンＡ１と呼ぶ）およびこれらの３−デヒドロ体（ビタミンＡ２と呼ぶ）と，その誘導体の総称で，ビタミンの中の脂溶性ビタミンに分類される。レチノイドは，上皮細胞の増殖を調節する医薬として使用されている。レチノイドは，視力における役割，細胞増殖および分化の調節，骨組織の増殖，免疫機能，および腫瘍抑制遺伝子の活性化など，全身において多くの重要な機能を有している。本発明者らは，レチノイドとラミニンを含む培地中で細胞を培養することによって，筋幹細胞の未分化性を保持したまま，細胞を増殖させることが可能であることを見出した。本発明において使用されうるレチノイドには，トレチノイン，タミバロテンなどが含まれるが，これらに限定はされない。 Retinoid Retinoid refers to vitamin A or a group of compounds chemically related thereto. Vitamin A is a generic term for retinol, retinal, retinoic acid (these are called vitamin A1) and their 3-dehydro forms (called vitamin A2), and their derivatives, which are classified as fat-soluble vitamins in vitamins. It Retinoids are used as drugs that regulate the growth of epithelial cells. Retinoids have many important functions throughout the body, including a role in vision, regulation of cell proliferation and differentiation, proliferation of bone tissue, immune function, and activation of tumor suppressor genes. The present inventors have found that by culturing cells in a medium containing retinoid and laminin, it is possible to proliferate the cells while maintaining the undifferentiated state of muscle stem cells. Retinoids that can be used in the present invention include, but are not limited to, tretinoin, tamibarotene and the like.
トレチノイン（オールトランスレチノイン酸（ａｔＲＡ）ともいう）は，ビタミンＡ誘導体の一種であり，レチノイン酸のうち，二重結合がすべてトランス型をとった，オール・トランス異性体である。 Tretinoin (also referred to as all-trans retinoic acid (atRA)) is a kind of vitamin A derivative, and is an all-trans isomer of retinoic acid in which all double bonds are in trans form.
タミバロテン（ＡＭ８０，レチノ安息香酸ともいう）は，合成レチノイドであり，トレチノイン（ＡＴＲＡ）耐性急性前骨髄球性白血病（ＡＰＬ）に対する化学療法剤として用いられる経口剤である。 Tamibarotene (AM80, also called retinobenzoic acid) is a synthetic retinoid and is an oral agent used as a chemotherapeutic agent for tretinoin (ATRA) -resistant acute promyelocytic leukemia (APL).
本発明において使用されうるレチノイドには，ＲＡＲアゴニストが含まれ，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０などの他のＲＡＲアゴニストも含まれる。本発明において使用されうるレチノイドとしては他に，フェンレチニド，ＣＤ４３７，ＢＭＳ４５３，タザロテン，ドコサヘキサエン酸，Ａ１１２０，Ｃｈ５５，ＥＣ２３，Ａ７４０００３なども挙げられる。 Retinoids that can be used in the present invention include RAR agonists and also other RAR agonists such as AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80. Other retinoids that can be used in the present invention also include fenretinide, CD437, BMS453, tazarotene, docosahexaenoic acid, A1120, Ch55, EC23, A740003 and the like.
ラミニン
ラミニンは，細胞外マトリクスの高分子量タンパク質（およそ４００〜９００ｋＤａ）であり，基底膜の主要な構成要素となっている。ラミニンは，基底膜の重要な成分であり，細胞の分化，遊走，および接着に影響を及ぼす。ラミニンは，α鎖，β鎖およびγ鎖を含むヘテロ三量体タンパク質であり，１５種類のアイソフォームを形成しうる５種のα鎖，３種のβ鎖，および３種のγ鎖が特定されている。 Laminin Laminin is a high molecular weight protein (approximately 400 to 900 kDa) in the extracellular matrix and is a major constituent of the basement membrane. Laminin is an important component of the basement membrane and affects cell differentiation, migration, and adhesion. Laminin is a heterotrimeric protein containing α-, β-, and γ-chains, and is characterized by 5 types of α-chain, 3 types of β-chain, and 3 types of γ-chain that can form 15 types of isoforms. Has been done.
ラミニン分子は各鎖の組成に従って命名される。本明細書においては，例えば，α５鎖，β１鎖及びγ１鎖から構成されるラミニンを「ラミニンα５β１γ１」または「ラミニン５１１」と記載する。その他の１４種類のアイソフォームは，ラミニン１１１，ラミニン２１１，ラミニン１２１，ラミニン２２１，ラミニン３３２，ラミニン３１１，ラミニン３２１，ラミニン４１１，ラミニン４２１，ラミニン５２１，ラミニン２１３，ラミニン４２３，ラミニン５２２，ラミニン５２３と記載される。ラミニンは，全長であってもよいしその一部であってもよく，例えば，ラミニン５１１は，そのα５鎖，β１鎖及びγ１鎖のそれぞれが，互いに独立に，全長であってもよいしその一部であってもよい。ラミニンは，組換えタンパク質の製造および精製が容易であることからＥ８フラグメントであることが好ましく，実用化の観点からヒトラミニンのＥ８フラグメントであることがさらに好ましい。
Laminin molecules are named according to the composition of each chain. In the present specification, for example, laminin composed of α5 chain, β1 chain and γ1 chain is referred to as “laminin α5β1γ1” or “
本明細書においては，ラミニンのＥ８フラグメントのことを単に「ラミニンＥ８」と記載することもあり，また，例えば，ラミニン５１１のＥ８フラグメントのことを「ラミニン５１１Ｅ８」とも記載する（他のラミニンのアイソフォームについても同様）。なお，単に「ラミニンＥ８」と記載したときは，ラミニンのアイソフォームは限定されず，いずれのアイソフォームのラミニンのＥ８フラグメントであってもよいものとする。
In the present specification, the E8 fragment of laminin may be simply referred to as “laminin E8”, and for example, the E8 fragment of
ヒトラミニン５１１タンパク質のＥ８フラグメントは，ＥＳ細胞およびｉＰＳ細胞を含む幹細胞の培養用基質として，スライドガラスやシャーレなどの培養容器の表面をコーティングすることにより使用されている。本発明者らは，予想外なことに，ラミニンを培養容器底面のコーティングとして用いるのではなく，液体培養培地中にレチノイドと共に含めることによって，筋幹細胞の未分化性を保持したまま，効率的に細胞を増殖させることが可能であることを見出した。このようなレチノイドとラミニンとの組み合わせにより，予想を超える相乗的な効果を得ることができる。なお，ラミニンの液体培地への添加と培養容器表面のコーティングとの併用は，排除されない。
The E8 fragment of
ラミニンのＥ８フラグメントは，マウスラミニンα１β１γ１（以下，「マウスラミニン１１１」とも記す。）をエラスターゼで消化して得られたフラグメントの中で，強い細胞接着活性をもつフラグメントとして同定されたものである（Edgar D., Timpl R., Thoe nen H. The heparin-binding domainof laminin is responsible for its effects on ne urite outgrowth and neuronal survival. EMBO J., 3:1463-1468,1984.，Goodman SL.,
Deutzmann R., von der Mark K.Two distinct cell-binding domains in laminin canin dependently promote nonneuronal cell adhesion and spreading. J. Cell Biol., 105: 589-598,1987.）。例えば，ヒトラミニンについてもエラスターゼで消化した際にマウスラミニン１１１のＥ８フラグメント（マウスラミニン１１１Ｅ８）に相当するフラグメントの存在が推定されるが，これらを分離・同定したことは報告されていない。したがって，本発明に用いられるヒトラミニンのＥ８フラグメントは，対応するヒトラミニンのエラスターゼ消化産物であることを要するものではなく，マウスラミニン１１１Ｅ８と同様の細胞接着活性を有し，同様の構造を有し，同程度の分子量を有するヒトラミニンのフラグメントであればよい。The laminin E8 fragment was identified as a fragment having a strong cell adhesion activity among fragments obtained by digesting mouse laminin α1β1γ1 (hereinafter also referred to as “mouse laminin 111”) with elastase ( Edgar D., Timpl R., Thoe nen H. The heparin-binding domainof laminin is responsible for its effects on neurite outgrowth and neuronal survival.EMBO J., 3: 1463-1468,1984., Goodman SL.,
Deutzmann R., von der Mark K. Two distinct cell-binding domains in laminin canin dependently promote nonneuronal cell adhesion and spreading. J. Cell Biol., 105: 589-598, 1987.). For example, human laminin is also presumed to have a fragment corresponding to the E8 fragment of mouse laminin 111 (mouse laminin 111E8) when digested with elastase, but it has not been reported that these fragments were isolated or identified. Therefore, the human laminin E8 fragment used in the present invention does not need to be a corresponding human laminin elastase digestion product, has the same cell adhesion activity as mouse laminin 111E8, and has the same structure, Any fragment of human laminin having a molecular weight of the order may be used.
ラミニンのＥ８フラグメントの製造方法は特に限定されず，例えば，ラミニンの所望のアイソフォームの全長をエラスターゼ等のタンパク質分解酵素で消化し，目的のフラグメントを分取，精製する方法や，組換えタンパク質として製造する方法などが挙げられる。製造量，品質の均一性，製造コスト等の観点から，組換えタンパク質として製造することが好ましい。 The method for producing the E8 fragment of laminin is not particularly limited. For example, a method of digesting the entire length of the desired isoform of laminin with a proteolytic enzyme such as elastase, collecting and purifying the desired fragment, or a recombinant protein The method of manufacturing may be mentioned. From the viewpoint of production amount, uniformity of quality, production cost, etc., it is preferable to produce it as a recombinant protein.
組換えヒトラミニン（全長またはその一部）は，公知の配列情報と，公知の遺伝子組換え技術を適宜用いることにより製造することができる。組換えヒトラミニン５１１Ｅ８の製造方法を例として以下説明する。組換えヒトラミニン５１１Ｅ８の製造方法としては，例えば，ヒトラミニン５１１Ｅ８のα鎖，β鎖およびγ鎖の各タンパク質をコードするＤＮＡをそれぞれ取得し，これをそれぞれ発現ベクターに挿入し，得られた３種類の発現ベクターを適切な宿主細胞に共導入して発現させ，３量体を形成しているタンパク質を公知の方法で精製することにより製造できる。例えば，Ｉｄｏら（Hiroyuki Ido, Aya Nakamu ra, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Se kiguchi, “The requirement of the glutamic acid residue at the third position fr om the carboxyl termini of the laminin γ chains in integrin binding by laminins ” The Journal of Biological Chemistry, 282, 11144-11154,2007.）に記載の方法に従って作製することができるが，これに限定されるものではない。例えばヒトラミニン５１１を構成するα５鎖，β１鎖，γ１鎖をそれぞれコードする遺伝子（ｃＤＮＡ）の塩基配列情報および各鎖のアミノ酸配列情報は，公知のデータベース（ＧｅｎＢａｎｋ等）から取得することができる。ラミニンは例えば，これら特定のアミノ酸配列において，少なくとも１個のアミノ酸が欠失，置換及び／又は付加されたタンパク質であってもよい。
Recombinant human laminin (full length or a part thereof) can be produced by appropriately using known sequence information and known gene recombination technology. The method for producing recombinant human laminin 511E8 will be described below as an example. As a method for producing the recombinant human laminin 511E8, for example, DNAs encoding the α-chain, β-chain and γ-chain proteins of human laminin 511E8 are respectively obtained, and each is inserted into an expression vector, and the resulting three types of It can be produced by co-introducing the expression vector into an appropriate host cell for expression and purifying the protein forming a trimer by a known method. For example, Ido et al. (Hiroyuki Ido, Aya Nakamu ra, Reiko Kobayashi, Shunsuke Ito, Shaoliang Li, Sugiko Futaki, and Kiyotoshi Se kiguchi, “The requirement of the glutamic acid residue at the third position fr om the carboxyl termini of the laminin γ Chains in integrin binding by laminins ”can be prepared according to the method described in The Journal of Biological Chemistry, 282, 11144-11154, 2007.), but is not limited thereto. For example, the nucleotide sequence information of the gene (cDNA) encoding the α5 chain, β1 chain, and γ1 chain constituting
ラミニンは，例えば，株式会社ニッピ（東京，日本）から市販されている培養基質ｉＭａｔｒｉｘ５１１−Ｅ８またはｉＭａｔｒｉｘ５１１−Ｅ８−ｓｉｌｋを用いることができる。ｉＭａｔｒｉｘ５１１−Ｅ８は，ヒトラミニン５１１−Ｅ８フラグメントと同一の配列を有する，遺伝子組換えＣＨＯ−Ｓ細胞由来の組換えタンパク質である。ｉＭａｔｒｉｘ５１１−Ｅ８−ｓｉｌｋは，ヒトラミニン５１１−Ｅ８フラグメントと同一の配列を有する，遺伝子組換えカイコ生産系由来の組換えタンパク質である。 As the laminin, for example, a culture substrate iMatrix511-E8 or iMatrix511-E8-silk commercially available from Nippi Co., Ltd. (Tokyo, Japan) can be used. iMatrix511-E8 is a recombinant protein derived from genetically modified CHO-S cells having the same sequence as the human laminin 511-E8 fragment. iMatrix511-E8-silk is a recombinant protein derived from a recombinant silkworm production system having the same sequence as the human laminin 511-E8 fragment.
細胞培養方法
本発明の一つの態様は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む培地を用いて細胞を培養することを特徴とする，筋サテライト細胞の培養方法に関する。細胞の培養は，例えば，増殖培地（２０％ウシ胎児血清，ペニシリン・ストレプトマイシン，２〜１０ｎｇ／ｍｌ最終濃度のｂＦＧＦを添加したＤＭＥＭ）を用いて，５％ＣＯ２インキュベーター内で３７℃にて行うことができる。培地の組成，培養条件（温度，期間等）は，当業者であれば適宜調整することができる。培地には，Ｇ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤（例えば，Stem cell reports, 2015, 5, p621-632に記載のＳＢ２０３５８０），または（Ｒ）−ＰＦＩ−２（Cell stem cell 22, 177-190, 2018）などの他の因子がさらに含まれていてもよい。培養容器としては，例えば，シャーレ，細胞培養皿，細胞培養フラスコなどを使用することができる。 Cell Culture Method One embodiment of the present invention relates to a method for culturing muscle satellite cells, which comprises culturing cells using a medium containing (i) retinoid and (ii) laminin. Culturing of cells is performed at 37 ° C. in a 5% CO 2 incubator using, for example, a growth medium (20% fetal bovine serum, penicillin streptomycin, DMEM supplemented with 2 to 10 ng / ml final concentration of bFGF). be able to. Those skilled in the art can appropriately adjust the composition of the medium and the culture conditions (temperature, period, etc.). The medium contains G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor (for example, Stem cell reports, 2015, 5, p621-632). Other factors such as the described SB203580) or (R) -PFI-2 (Cell stem cell 22, 177-190, 2018) may be further included. As the culture container, for example, a petri dish, a cell culture dish, a cell culture flask, or the like can be used.
また，筋サテライト細胞の培養時には，酸素濃度を適宜調整することができる。例えば，Ｏ２の濃度は，少なくとも０．５％以上，１％以上，２％以上，３％以上，４％以上，５％以上，６％以上のいずれかの濃度とすることができる。また，Ｏ２は１０％以下，８％以下，６％以下，５％以下，４％以下，３％以下とすることができる。Ｏ２濃度は，上記の上限および下限の任意の組み合わせ，例えば，１％〜５％の範囲，好ましくは２％〜４％の範囲とすることができる。ヒト細胞の培養の場合，好適なＯ２濃度は例えば２．５〜３．５％，より具体的には３％である。よって，ヒト細胞の培養は，例えば，増殖培地（２０％ウシ胎児血清，ペニシリン・ストレプトマイシン・グルタミン酸，２．５ｎｇ／ｍｌ最終濃度のｂＦＧＦを添加したＤＭＥＭ，Ｈｉｇｈ Ｇｌｕｃｏｓｅを用いて３７℃，３％Ｏ２， ５％ＣＯ２インキュベーター内で行うことができる。このように，本発明の一つの態様は，２〜４％の酸素濃度，好ましくは３％Ｏ２の存在下で細胞を培養することを特徴とする，筋サテライト細胞の培養方法に関する。Also, during the culture of muscle satellite cells, the oxygen concentration can be adjusted appropriately. For example, the concentration of O 2 can be at least 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more. Further, O 2 can be 10% or less, 8% or less, 6% or less, 5% or less, 4% or less, or 3% or less. The O 2 concentration can be any combination of the above upper and lower limits, for example, in the range of 1% to 5%, preferably in the range of 2% to 4%. For the human cell cultures, suitable O 2 concentration, for example 2.5 to 3.5%, more specifically 3%. Therefore, culture of human cells can be performed, for example, by using a growth medium (20% fetal bovine serum, penicillin / streptomycin / glutamic acid, DMEM and High Glucose added with 2.5 ng / ml final concentration of bFGF) at 37 ° C. and 3
レチノイドは，少なくとも１０−１３Ｍ以上，１０−１２Ｍ以上，１０−１１Ｍ以上，１０−１０Ｍ以上，１０−９Ｍ以上，１０−８Ｍ以上，１０−７Ｍ以上，１０−６Ｍ以上，１０−５Ｍ以上，１０−４Ｍ以上，１０−３Ｍ以上のいずれかの濃度で培地に添加することができる。また，レチノイドは，１０−２Ｍ未満，１０−３Ｍ未満，１０−４Ｍ未満，１０−５Ｍ未満，１０−６Ｍ未満，１０−７Ｍ未満，１０−８Ｍ未満，１０−９Ｍ未満，１０−１０Ｍ未満，１０−１１Ｍ未満，１０−１２Ｍ未満のいずれかの濃度で添加することができる。よって，レチノイドは，上記の上限および下限の任意の組み合わせ，例えば，１０−１１Ｍ〜１０−５Ｍの濃度範囲，好ましくは１０−１０Ｍ〜１０−６Ｍの濃度範囲，１０−９Ｍ〜１０−７Ｍの濃度範囲のいずれかの濃度で添加することができる。好適なレチノイドの添加濃度は例えば，１０−８Ｍまたは１０−７Ｍである。Retinoid is at least 10 −13 M or more, 10 −12 M or more, 10 −11 M or more, 10 −10 M or more, 10 −9 M or more, 10 −8 M or more, 10 −7 M or more, 10 −6 M. As described above, it can be added to the medium at any concentration of 10 −5 M or higher, 10 −4 M or higher, 10 −3 M or higher. Moreover, retinoid is less than 10 −2 M, less than 10 −3 M, less than 10 −4 M, less than 10 −5 M, less than 10 −6 M, less than 10 −7 M, less than 10 −8 M, 10 −9. It can be added at any concentration of less than M, less than 10 −10 M, less than 10 −11 M, and less than 10 −12 M. Therefore, the retinoid may be any combination of the above upper and lower limits, for example, a concentration range of 10 −11 M to 10 −5 M, preferably 10 −10 M to 10 −6 M, a concentration range of 10 −9 M to. It can be added at any concentration in the concentration range of 10 −7 M. A suitable retinoid addition concentration is, for example, 10 −8 M or 10 −7 M.
本発明の一部の態様では，１０−８Ｍまたは１０−７ＭのａｔＲＡまたはＡＭ８０を使用することができる。In some aspects of the invention, 10 −8 M or 10 −7 M of atRA or AM80 can be used.
ラミニンは，培養容器の底面積に基づき，少なくとも０．０１μｇ／ｃｍ２以上，０．０５μｇ／ｃｍ２以上，０．１μｇ／ｃｍ２以上，０．２μｇ／ｃｍ２以上，０．３μｇ／ｃｍ２以上，０．４μｇ／ｃｍ２以上，０．５μｇ／ｃｍ２以上，０．６μｇ／ｃｍ２以上，０．７μｇ／ｃｍ２以上，０．８μｇ／ｃｍ２以上，０．９μｇ／ｃｍ２以上，１．０μｇ／ｃｍ２以上，１．５μｇ／ｃｍ２以上，２．０μｇ／ｃｍ２以上のいずれかの濃度で培地に添加あるいは培養容器にコーティングすることができる。また，レチノイドは，５．０μｇ／ｃｍ２未満，２．０μｇ／ｃｍ２未満，１．５μｇ／ｃｍ２未満，１．０μｇ／ｃｍ２未満，０．９μｇ／ｃｍ２未満，０．８μｇ／ｃｍ２未満，０．７μｇ／ｃｍ２未満，０．６μｇ／ｃｍ２未満，０．５μｇ／ｃｍ２未満，０．４μｇ／ｃｍ２未満，０．３μｇ／ｃｍ２未満，０．２μｇ／ｃｍ２未満，０．１μｇ／ｃｍ２未満，０．０５μｇ／ｃｍ２未満のいずれかの濃度で培地に添加あるいは培養容器にコーティングすることができる。よって，レチノイドは，上記の上限および下限の任意の組み合わせ，例えば，０．０１μｇ／ｃｍ２〜５．０μｇ／ｃｍ２の濃度範囲，好ましくは０．１μｇ／ｃｍ２〜１．０μｇ／ｃｍ２の濃度範囲，０．２μｇ／ｃｍ２〜０．５μｇ／ｃｍ２の濃度範囲のいずれかの濃度で培地に添加あるいは培養容器にコーティングすることができる。好適なラミニンの使用濃度は例えば，０．２μｇ／ｃｍ２または０．５μｇ／ｃｍ２である。Laminin is at least 0.01 μg / cm 2 or more, 0.05 μg / cm 2 or more, 0.1 μg / cm 2 or more, 0.2 μg / cm 2 or more, 0.3 μg / cm 2 based on the bottom area of the culture vessel. above, 0.4 [mu] g / cm 2 or more, 0.5 [mu] g / cm 2 or more, 0.6 [mu] g / cm 2 or more, 0.7 [mu] g / cm 2 or more, 0.8 [mu] g / cm 2 or more, 0.9 [mu] g / cm 2 or more, It can be added to the medium or coated on the culture container at any concentration of 1.0 μg / cm 2 or more, 1.5 μg / cm 2 or more, and 2.0 μg / cm 2 or more. Further, retinoids, less than 5.0 [mu] g / cm 2, less than 2.0 [mu] g / cm 2, less than 1.5 [mu] g / cm 2, less than 1.0 [mu] g / cm 2, less than 0.9μg / cm 2, 0.8μg / cm less than 2, less than 0.7 [mu] g / cm 2, less than 0.6 [mu] g / cm 2, less than 0.5 [mu] g / cm 2, less than 0.4μg / cm 2, 0.3μg / cm less than 2, less than 0.2 [mu] g / cm 2 , Less than 0.1 μg / cm 2, or less than 0.05 μg / cm 2 can be added to the medium or coated on the culture vessel. Therefore, retinoids, any of the upper and lower limits of the combination, for example, 0.01μg / cm 2 ~5.0μg / cm 2 in a concentration range, preferably 0.1μg / cm 2 ~1.0μg /
ラミニンは，液体培地中における濃度の観点では，少なくとも０．１μｇ／ｍｌ以上，０．５μｇ／ｍｌ以上，１．０μｇ／ｍｌ以上，１．５μｇ／ｍｌ以上，２．０μｇ／ｍｌ以上，３．０μｇ／ｍｌ以上，４．０μｇ／ｍｌ以上，５．０μｇ／ｍｌ以上，６．０μｇ／ｍｌ以上，７．０μｇ／ｍｌ以上，８．０μｇ／ｍｌ以上，９．０μｇ／ｍｌ以上，１０．０μｇ／ｍｌ以上のいずれかの濃度で培地に添加することができる。また，レチノイドは，１５．０μｇ／ｍｌ未満，１０．０μｇ／ｍｌ未満，９．０μｇ／ｍｌ未満，８．０μｇ／ｍｌ未満，７．０μｇ／ｍｌ未満，６．０μｇ／ｍｌ未満，５．０μｇ／ｍｌ未満，４．０μｇ／ｍｌ未満，３．０μｇ／ｍｌ未満，２．０μｇ／ｍｌ未満，１．５μｇ／ｍｌ未満，１．０μｇ／ｍｌ未満，０．５μｇ／ｍｌ未満，０．１μｇ／ｍｌ未満，０．０５μｇ／ｍｌ未満のいずれかの濃度で培地に添加することができる。よって，レチノイドは，上記の上限および下限の任意の組み合わせ，例えば，０．０５μｇ／ｍｌ〜１５．０μｇ／ｍｌの濃度範囲，好ましくは１．０μｇ／ｍｌ〜１０．０μｇ／ｍｌの濃度範囲，１．５μｇ／ｍｌ〜５．０μｇ／ｍｌの濃度範囲のいずれかの濃度で添加することができる。本発明において０．２μｇ／ｃｍ２のラミニン濃度は，１．５〜２μｇ／ｍｌに相当する。よって，好適なラミニンの添加濃度は例えば，１．５μｇ／ｍｌ，２．０μｇ／ｍｌまたは５．０μｇ／ｍｌである。From the viewpoint of the concentration in the liquid medium, laminin is at least 0.1 μg / ml or more, 0.5 μg / ml or more, 1.0 μg / ml or more, 1.5 μg / ml or more, 2.0 μg / ml or more, 3. 0 μg / ml or more, 4.0 μg / ml or more, 5.0 μg / ml or more, 6.0 μg / ml or more, 7.0 μg / ml or more, 8.0 μg / ml or more, 9.0 μg / ml or more, 10.0 μg It can be added to the medium at any concentration of 1 / ml or more. In addition, retinoid is less than 15.0 μg / ml, less than 10.0 μg / ml, less than 9.0 μg / ml, less than 8.0 μg / ml, less than 7.0 μg / ml, less than 6.0 μg / ml, 5.0 μg / Ml, less than 4.0 μg / ml, less than 3.0 μg / ml, less than 2.0 μg / ml, less than 1.5 μg / ml, less than 1.0 μg / ml, less than 0.5 μg / ml, 0.1 μg / ml It can be added to the medium at any concentration of less than ml or less than 0.05 μg / ml. Therefore, the retinoid may be any combination of the above upper and lower limits, for example, a concentration range of 0.05 μg / ml to 15.0 μg / ml, preferably a concentration range of 1.0 μg / ml to 10.0 μg / ml, 1 It can be added at any concentration in the concentration range of 0.5 μg / ml to 5.0 μg / ml. In the present invention, a laminin concentration of 0.2 μg / cm 2 corresponds to 1.5 to 2 μg / ml. Therefore, a suitable addition concentration of laminin is, for example, 1.5 μg / ml, 2.0 μg / ml or 5.0 μg / ml.
本発明の一部の態様では，０．２μｇ／ｃｍ２または０．５μｇ／ｃｍ２のヒトラミニン５１１Ｅ８フラグメントを使用することができる。本発明の一部の態様では，１．５μｇ／ｍｌ，２．０μｇ／ｍｌまたは５．０μｇ／ｍｌのヒトラミニン５１１Ｅ８フラグメントを使用することができる。In some aspects of the invention, 0.2 μg / cm 2 or 0.5 μg / cm 2 of human laminin 511E8 fragment can be used. In some aspects of the invention, 1.5 μg / ml, 2.0 μg / ml or 5.0 μg / ml of human laminin 511E8 fragment can be used.
分化抑制剤
本発明の一つの態様は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含むことを特徴とする，筋サテライト細胞の筋分化を抑制するための組成物に関する。このような組成物を培地に加えて筋サテライト細胞，好ましくはヒトの筋サテライト細胞を培養することにより，細胞の筋分化を抑制しながら細胞を増殖させることができる。このような組成物は，分化抑制剤と呼ぶこともできる。使用するレチノイド，ラミニンおよび組成物中の他の成分の種類，濃度等は，本明細書の開示および技術常識に基づき適宜決定することができる。 Differentiation inhibitor One aspect of the present invention relates to a composition for inhibiting muscle differentiation of muscle satellite cells, which comprises (i) a retinoid and (ii) laminin. By culturing muscle satellite cells, preferably human muscle satellite cells, by adding such a composition to a medium, the cells can be grown while suppressing muscle differentiation of the cells. Such a composition can also be called a differentiation inhibitor. The type, concentration, etc. of the retinoid, laminin and other components in the composition to be used can be appropriately determined based on the disclosure of the present specification and common technical knowledge.
分化誘導
本発明に係る分化抑制剤を含む培地を用いて増殖させた筋サテライト細胞をｉｎ ｖｉｔｒｏで筋肉細胞に分化させるためには，培地を本発明に係る分化抑制剤を含まない分化培地（例えば，２％ウマ血清，ペニシリン・ストレプトマイシンを含むＤＭＥＭ）に交換して，細胞を培養することができる。 Induction of Differentiation In order to differentiate muscle satellite cells proliferated using the medium containing the differentiation inhibitor of the present invention into muscle cells in vitro, the medium is a differentiation medium containing no differentiation inhibitor of the present invention (for example, , DMEM containing 2% horse serum, penicillin-streptomycin) can be used to culture the cells.
培地組成物
本発明の一つの態様は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含むことを特徴とする，細胞培養培地に関する。このような培地を用いて筋サテライト細胞，好ましくはヒトの筋サテライト細胞を培養することにより，筋サテライト細胞の未分化性を維持しながら細胞を増殖させることができる。使用するレチノイド，ラミニンおよび培地組成物中の他の成分の種類，濃度等は，本明細書の開示および技術常識に基づき適宜決定することができる。本発明の一つの態様は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む，ヒト筋サテライト細胞を培養するための液体細胞培養培地であって，レチノイドがａｔＲＡまたはＡＭ８０であり，レチノイドの濃度が少なくとも１０−８Ｍであり，ラミニンがヒトラミニン５１１Ｅ８フラグメントであり，ラミニンが液体培地中に含められており，ラミニンの濃度が少なくとも１．５μｇ／ｍｌであり，そして，培地中で培養されたヒト筋サテライト細胞の未分化性が維持される，細胞培養培地に関する。 Medium Composition One aspect of the present invention relates to a cell culture medium characterized in that it comprises (i) a retinoid and (ii) a laminin. By culturing muscle satellite cells, preferably human muscle satellite cells, using such a medium, the cells can be grown while maintaining the undifferentiated state of the muscle satellite cells. The types and concentrations of the retinoid, laminin and other components in the medium composition to be used can be appropriately determined based on the disclosure of the present specification and common general knowledge. One embodiment of the present invention is a liquid cell culture medium for culturing human muscle satellite cells comprising (i) retinoid and (ii) laminin, wherein the retinoid is atRA or AM80 and the concentration of retinoid is at least. Human muscle satellites at 10 −8 M, laminin is human laminin 511E8 fragment, laminin is included in liquid medium, laminin concentration is at least 1.5 μg / ml, and cultured in medium. It relates to a cell culture medium in which the undifferentiation of cells is maintained.
培地サプリメント
本発明の一つの態様は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含むことを特徴とする，細胞培養培地サプリメントに関する。このようなサプリメントを培養培地に加えて筋サテライト細胞，好ましくはヒトの筋サテライト細胞を培養することにより，細胞の筋分化を抑制しながら細胞を増殖させることができる。使用するレチノイド，ラミニンおよびサプリメント中の他の成分の種類，濃度等は，本明細書の開示および技術常識に基づき適宜決定することができる。本サプリメントは，液体の形態であっても，粉末の形態であってもよい。本培地サプリメントは，筋サテライト細胞の培養に使用するキットに要素の一つとして含められてもよい。 Medium supplement One aspect of the invention relates to a cell culture medium supplement, characterized in that it comprises (i) a retinoid and (ii) laminin. By culturing muscle satellite cells, preferably human muscle satellite cells, by adding such a supplement to the culture medium, the cells can be grown while suppressing muscle differentiation of the cells. The types, concentrations, etc. of the retinoids, laminin, and other components in the supplement to be used can be appropriately determined based on the disclosure of the present specification and common general knowledge. The supplement may be in liquid or powder form. The present medium supplement may be included as one of the components in the kit used for culturing muscle satellite cells.
本細胞培養培地サプリメントは，例えば，細胞培養培地に加えられた際のレチノイドの濃度が少なくとも１０−８Ｍとなるように調製されうる。また，本細胞培養培地サプリメントは，例えば，細胞培養培地に加えられた際のラミニンの濃度が少なくとも１．５μｇ／ｍｌとなるように調製されうる。本細胞培養培地サプリメントは，Ｇ−ＣＳＦ，ｂＦＧＦ，ＩＬ−１α，ＩＬ−１３，ＩＦＮ−γ，ＴＮＦ−α，ＧＳＫ３阻害剤，ｓａｌ００３，ｐ３８阻害剤（例えば，Stem cell reports, 2015, 5, p621-632に記載のＳＢ２０３５８０），（Ｒ）−ＰＦＩ−２（Cell stem cell 22, 177-190, 2018）およびその混合物から成る群より選択される少なくとも１つの因子をさらに含んでいてもよい。The cell culture medium supplement can be prepared, for example, so that the concentration of retinoid when added to the cell culture medium will be at least 10 −8 M. Further, the present cell culture medium supplement can be prepared, for example, so that the concentration of laminin when added to the cell culture medium is at least 1.5 μg / ml. This cell culture medium supplement contains G-CSF, bFGF, IL-1α, IL-13, IFN-γ, TNF-α, GSK3 inhibitor, sal003, p38 inhibitor (for example, Stem cell reports, 2015, 5, p621). SB203580), (R) -PFI-2 (Cell stem cell 22, 177-190, 2018) and a mixture thereof, which may further include at least one factor.
細胞製造方法
本発明の一つの態様は，細胞移植に用いるための細胞の製造方法であって，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む培地を用いて筋サテライト細胞を培養する工程を含むことを特徴とする，細胞の製造方法に関する。培養する筋サテライト細胞は，当業者に公知の任意の方法を用いて調製することができる。使用する培地の組成，培養条件（温度，期間等）は，当業者であれば適宜調整することができる。 Method for Producing Cells One embodiment of the present invention is a method for producing cells for use in cell transplantation, comprising the step of culturing muscle satellite cells using a medium containing (i) retinoid and (ii) laminin. And a method for producing cells. The muscle satellite cells to be cultured can be prepared using any method known to those skilled in the art. Those skilled in the art can appropriately adjust the composition of the medium used and the culture conditions (temperature, period, etc.).
細胞の移植は，例えば，損傷した部位の再生を目的として，あるいは機能の低下した臓器・組織の再生を目的として行うことができる。移植の対象は，好ましくはヒトであるが，特に限定はされない。ヒトに対して移植を行う場合は，細胞の培養にはＧＭＰグレードの試薬が用いられる。本製造方法において用いられる筋サテライト細胞は，ＣＲＩＳＰＲ／Ｃａｓ系などのゲノム編集技術等によって遺伝子を改変した細胞であってもよい。 The transplantation of cells can be performed, for example, for the purpose of regenerating a damaged site, or for the purpose of regenerating an organ / tissue having a lowered function. The subject of transplantation is preferably a human, but is not particularly limited. When transplanting to humans, GMP grade reagents are used for cell culture. The muscle satellite cells used in the present production method may be cells whose genes have been modified by a genome editing technique such as CRISPR / Cas system.
細胞組成物
本発明の一つの態様は，少なくとも１０５個の筋サテライト細胞を含む細胞組成物に関する。従来，筋サテライト細胞を未分化状態を維持したまま増殖させて大量に得ることは困難であったが，このような細胞組成物は，（ｉ）レチノイドおよび（ｉｉ）ラミニンを含む培地を用いて筋サテライト細胞を培養することにより調製されうる。本細胞組成物は，少なくとも１０５個，例えば，１０６個以上，好ましくは１０７個以上，より好ましくは１０８個以上，さらに好ましくは１０９個以上の筋サテライト細胞を含む。なお，本細胞組成物には筋サテライト細胞以外の細胞が混入していても良いが，筋サテライト細胞の純度が高いことが好ましい。本細胞組成物における筋サテライト細胞の純度は，少なくとも５０％以上，好ましくは８０％以上，より好ましくは８５％以上，９０％以上，または９５％以上である。本細胞組成物に含まれる細胞は，好ましくはヒトの細胞であるが，特に限定はされない。本発明の一つの態様では，筋サテライト細胞を含む細胞組成物は１つの容器に納められている。 Cellular Compositions One aspect of the invention pertains to cellular compositions that include at least 10 5 muscle satellite cells. Conventionally, it has been difficult to grow muscle satellite cells while maintaining an undifferentiated state and obtain a large amount thereof. Such a cell composition is prepared by using a medium containing (i) retinoid and (ii) laminin. It can be prepared by culturing muscle satellite cells. The cell composition comprises at least 10 5 , for example 10 6 or more, preferably 10 7 or more, more preferably 10 8 or more, even more preferably 10 9 or more muscle satellite cells. Although cells other than muscle satellite cells may be mixed in the present cell composition, it is preferable that the muscle satellite cells have high purity. The purity of the muscle satellite cells in the present cell composition is at least 50% or higher, preferably 80% or higher, more preferably 85% or higher, 90% or higher, or 95% or higher. The cells contained in the present cell composition are preferably human cells, but are not particularly limited. In one aspect of the invention, the cell composition comprising muscle satellite cells is contained in a single container.
本細胞組成物は，例えば，治療を目的とした移植に用いることができる。細胞の移植は，例えば，損傷した部位の再生を目的として，あるいは機能の低下した組織の再生を目的として行うことができる。移植の対象は，好ましくはヒトであるが，特に限定はされない。本発明の一つの態様は，疾患または傷害の治療に用いるための，少なくとも１０５個，例えば，１０６個以上，好ましくは１０７個以上，より好ましくは１０８個以上，さらに好ましくは１０９個以上の筋サテライト細胞を含む細胞組成物に関する。治療の対象となる疾患は例えば，筋ジストロフィー，先天性ミオパチー，遠位型ミオパチー，筋強直性疾患，炎症性筋疾患，周期性四肢麻痺，代謝性筋疾患，重症筋無力症，先天性筋無力症候群，ミトコンドリア病,およびサルコペニアであるが，これらに限定はされない。The cell composition can be used, for example, in transplantation for therapeutic purposes. The transplantation of cells can be performed, for example, for the purpose of regenerating a damaged site or for regenerating a tissue having a reduced function. The subject of transplantation is preferably a human, but is not particularly limited. One aspect of the invention is at least 10 5 , eg 10 6 or more, preferably 10 7 or more, more preferably 10 8 or more, even more preferably 10 9 for use in the treatment of a disease or injury. A cell composition comprising one or more muscle satellite cells. Diseases to be treated include, for example, muscular dystrophy, congenital myopathy, distal myopathy, myotonic disease, inflammatory myopathy, periodic quadriplegia, metabolic myopathy, myasthenia gravis, congenital myasthenic syndrome. , Mitochondrial disease, and sarcopenia, but are not limited thereto.
本細胞組成物に含まれる筋サテライト細胞は，何らかの疾患を有する患者，または疾患を発症する遺伝的素因を有する被験者に由来する細胞であってもよい。疾患は例えば，筋ジストロフィーである。このような疾患に関連する細胞組成物は，医薬品の開発を目的としたスクリーニングやアッセイに用いることができる。 The muscle satellite cells included in the cell composition may be cells derived from a patient having some disease or a subject having a genetic predisposition to develop the disease. The disease is, for example, muscular dystrophy. The cell composition associated with such a disease can be used for screening and assay for the purpose of drug development.
筋サテライト細胞は，ＣＲＩＳＰＲ／Ｃａｓ系などのゲノム編集技術等によって遺伝子を改変した細胞であってもよい。筋サテライト細胞はｉＰＳ細胞由来の細胞であってもよい。 The muscle satellite cell may be a cell whose gene has been modified by a genome editing technique such as CRISPR / Cas system. The muscle satellite cells may be cells derived from iPS cells.
また，本発明の一つの態様は，少なくとも５×１０５個，例えば，１０６個以上，好ましくは１０７個以上，より好ましくは１０８個以上，さらに好ましくは１０９個以上の筋サテライト細胞の封入されたバイアルまたは容器に関する。バイアルまたは容器は，ガラス製またはプラチック製でありうる。バイアルまたは容器中の細胞は，凍結状態または反凍結状態であってもよい。Moreover, one embodiment of the present invention is at least 5 × 10 5 , for example, 10 6 or more, preferably 10 7 or more, more preferably 10 8 or more, more preferably 10 9 or more muscle satellite cells. Of encapsulated vials or containers. The vial or container can be made of glass or plastic. The cells in the vial or container may be frozen or anti-frozen.
治療方法
本発明の一つの態様は，患者における疾患または傷害の治療方法であって，（ｉ）レチノイド，および（ｉｉ）ラミニンを含む培地を用いて筋サテライト細胞を培養する工程，および，培養した筋サテライト細胞またはそれに由来する細胞を患者に移植する工程を含む，治療方法に関する。 Method of Treatment One aspect of the present invention is a method of treating a disease or injury in a patient, which comprises culturing muscle satellite cells using a medium containing (i) retinoid and (ii) laminin, and culturing. The present invention relates to a therapeutic method including a step of transplanting muscle satellite cells or cells derived therefrom into a patient.
培養する筋サテライト細胞は，当業者に公知の任意の方法を用いて調製することができる。筋サテライト細胞は，ＣＲＩＳＰＲ／Ｃａｓ系などのゲノム編集技術等によって遺伝子を改変した細胞であってもよい。筋サテライト細胞はｉＰＳ細胞由来の細胞であってもよい。筋サテライト細胞に由来する細胞とは，筋サテライト細胞から分化した細胞を含む。使用する培地の組成，培養条件（温度，期間等）は，当業者であれば適宜調整することができる。 The muscle satellite cells to be cultured can be prepared using any method known to those skilled in the art. The muscle satellite cell may be a cell whose gene has been modified by a genome editing technique such as CRISPR / Cas system. The muscle satellite cells may be cells derived from iPS cells. The cells derived from muscle satellite cells include cells differentiated from muscle satellite cells. Those skilled in the art can appropriately adjust the composition of the medium used and the culture conditions (temperature, period, etc.).
細胞の移植は，例えば，損傷した部位の再生を目的として，あるいは機能の低下した臓器・組織の再生を目的として行うことができる。移植の対象となる患者は，好ましくはヒトであるが，特に限定はされない。治療の対象は，イヌ，ネコ，サルなどを含む非ヒト哺乳動物であってもよい。治療の対象となる疾患は例えば，筋ジストロフィー，先天性ミオパチー，遠位型ミオパチー，筋強直性疾患，炎症性筋疾患，周期性四肢麻痺，代謝性筋疾患，重症筋無力症，先天性筋無力症候群，ミトコンドリア病,およびサルコペニアであるが，これらに限定はされない。治療の対象となる疾患には例えば，糖尿病も含まれる。疾患または傷害の治療に有効な物質を分泌するように遺伝子改変した筋サテライト細胞またはそれに由来する細胞を体内に埋め込むことにより，疾患を治療することも可能である。 The transplantation of cells can be performed, for example, for the purpose of regenerating a damaged site, or for the purpose of regenerating an organ / tissue having a lowered function. The patient to be transplanted is preferably a human, but is not particularly limited. The subject of treatment may be a non-human mammal, including dogs, cats, monkeys and the like. Diseases to be treated include, for example, muscular dystrophy, congenital myopathy, distal myopathy, myotonic disease, inflammatory myopathy, periodic quadriplegia, metabolic myopathy, myasthenia gravis, congenital myasthenic syndrome. , Mitochondrial disease, and sarcopenia, but are not limited thereto. Diseases to be treated also include, for example, diabetes. The disease can also be treated by implanting muscle satellite cells or cells derived therefrom that have been genetically modified so as to secrete a substance effective for treating the disease or injury.
移植は，例えば，移植部位に細胞を直接注入することにより行うことができる。あるいは，細胞を例えばスキャフォールド上で培養し，スキャフォールドと細胞を共に体内に移植してもよい。 Transplantation can be performed, for example, by directly injecting cells into the transplant site. Alternatively, the cells may be cultured on a scaffold, for example, and the scaffold and cells may be transplanted into the body together.
スクリーニング方法
本発明の一つの態様は，筋サテライト細胞またはそれに由来する細胞の分化，増殖または生存に影響する物質のスクリーニング方法であって，（ｉ）レチノイドおよび（ｉｉ）ラミニンの存在下で筋サテライト細胞を培養する工程，培養した筋サテライト細胞またはそれに由来する細胞に試験物質を接触させる工程，試験物質が筋サテライト細胞またはそれに由来する細胞の分化，増殖または生存に影響するか否かを評価する工程を含むことを特徴とする方法に関する。 Screening Method One embodiment of the present invention is a method for screening a substance that affects the differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom, the muscle satellite being present in the presence of (i) retinoid and (ii) laminin. Culturing cells, contacting test substance with cultured muscle satellite cells or cells derived therefrom, and assessing whether the test substance affects differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom A method comprising the steps of:
筋サテライト細胞の単離は，当業者に公知の任意の方法により行うことができる。筋サテライト細胞はｉＰＳ細胞由来の細胞であってもよい。 The isolation of muscle satellite cells can be performed by any method known to those skilled in the art. The muscle satellite cells may be cells derived from iPS cells.
筋サテライト細胞は，何らかの疾患を有する患者，または疾患を発症する遺伝的素因を有する被験者に由来する細胞であってもよい。疾患は例えば，筋ジストロフィーである。例えば，筋ジストロフィー患者の筋サテライト細胞またはそれに由来する細胞の増殖または生存を改善する物質は，筋ジストロフィーの治療に有用となりうると考えられる。「細胞の分化に影響する」とは，細胞の分化を促進または抑制することを含む。「細胞の増殖に影響する」とは，細胞の増殖を促進または抑制することを含む。「細胞の生存に影響する」とは，細胞の生存期間もしくは生存率を改善する，または細胞をアポトーシスやネクローシス等により死滅させることを含む。本スクリーニング方法において用いられる筋サテライト細胞は，ＣＲＩＳＰＲ／Ｃａｓ系などのゲノム編集技術等によって遺伝子を改変した細胞であってもよい。試験物質には，低分子化合物，ペプチド，タンパク質，核酸などが含まれるが，特に限定はない。 The muscle satellite cells may be cells derived from a patient having some disease or a subject having a genetic predisposition to develop the disease. The disease is, for example, muscular dystrophy. For example, substances that improve the proliferation or survival of muscle satellite cells or cells derived therefrom in patients with muscular dystrophy could be useful in the treatment of muscular dystrophy. “Affecting cell differentiation” includes promoting or suppressing cell differentiation. “Affecting cell growth” includes promoting or suppressing cell growth. “Affecting cell survival” includes improving the survival time or survival rate of cells, or killing cells by apoptosis, necrosis, or the like. The muscle satellite cell used in the present screening method may be a cell whose gene has been modified by a genome editing technique such as CRISPR / Cas system. Test substances include, but are not limited to, low molecular weight compounds, peptides, proteins, nucleic acids and the like.
以下に，実施例を示して本発明を具体的に説明するが，これらにより本発明は何ら制限を受けるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
実施例１：骨格筋幹細胞の調製
Ｃ５７ＢＬ／６Ｊマウス（３〜１０週齢）の後肢筋（大.四頭筋，前頸骨筋，腓腹筋等）より骨格筋幹細胞を単離した。単離方法としては，（ｉ）０．１４％プロテアーゼ（Sigma-Aldrich社）を含むＤＭＥＭ培地で３７℃，３０分間処理し，ＤＭＥＭ培地による洗浄を行った後に播種する方法，あるいは（ｉｉ）酵素消化後にフローサイトメーター（ＢＤ社，AriaII）により単離する方法（Exp Cell Res. 10, 245-255, 2004; Nat Med. 20 , 255-264,2014）を用いた。得られた骨格筋幹細胞は増殖培地（２０％ウシ胎児血清，ペニシリン・ストレプトマイシン，２〜１０ｎｇ／ｍｌ最終濃度のｂＦＧＦ（Peprotec社）を添加したDMEM, High Glucose, GlutaMAX,Pyruvate（Thermo FisherScientific社）で３７℃，５％ＣＯ２インキュベーター内で培養した。 Example 1: Preparation of skeletal muscle stem cells Skeletal muscle stem cells were isolated from the hindlimb muscles (large quadriceps, tibialis anterior, gastrocnemius, etc.) of C57BL / 6J mice (3 to 10 weeks old). As the isolation method, (i) a method of treating with DMEM medium containing 0.14% protease (Sigma-Aldrich) at 37 ° C. for 30 minutes, washing with DMEM medium, and then seeding, or (ii) enzyme A method (Exp Cell Res. 10, 245-255, 2004; Nat Med. 20, 255-264, 2014) for isolation by a flow cytometer (BD, AriaII) after digestion was used. The obtained skeletal muscle stem cells were grown in a growth medium (20% fetal bovine serum, penicillin-streptomycin, DMEM, High Glucose, GlutaMAX, Pyruvate (Thermo Fisher Scientific) supplemented with bFGF (Peprotec) at a final concentration of 2 to 10 ng / ml). The cells were cultured at 37 ° C in a 5% CO 2 incubator.
実施例２：レチノイド化合物の骨格筋幹細胞の未分化性の維持に対する作用
実施例１で調製した骨格筋幹細胞培養系において，オールトランスレチノイン酸（all-trans-Retinoicacid：以下ａｔＲＡ）あるいは合成レチノイド化合物Ａｍ８０を増殖培地に１０−１１Ｍ〜１０−５Ｍの濃度で添加し，３７℃，５％ＣＯ２インキュベーター内で３日間培養した。骨格筋幹細胞の未分化性は，以下に示す免疫染色法によって解析した。まず細胞を４％パラホルムアルデヒドにより室温で１０分間固定した後，３回ＰＢＳによって洗浄した。次に０．５％ＴｒｉｔｏｎＸ−１００／ＰＢＳによって室温，５分間処理し，再度ＰＢＳによって３回洗浄を行った。５％ＢＳＡ／ＰＢＳで室温，１時間ブロッキング処理を行った後，抗Ｐａｘ７抗体（SantaCruz社）と抗ＭｙｏＤ抗体（Ｄａｋｏ社）を用いて４℃，overnightで処理した。ＰＢＳによって３回洗浄を行った後，二次抗体（抗マウスＩｇＧ−Alexa Fluor594と抗ウサギＩｇＧ−Alexa Fluor488）で室温，１時間処理し，再度ＰＢＳで３回洗浄した後，Hoechst33342を用いて核染色を行い，蛍光顕微鏡（オリンパス社）によって観察した。 Example 2: Effect of retinoid compound on maintaining undifferentiated state of skeletal muscle stem cells In the skeletal muscle stem cell culture system prepared in Example 1, all-trans-retinoic acid (hereinafter referred to as atRA) or synthetic retinoid compound Am80. Was added to the growth medium at a concentration of 10 −11 M to 10 −5 M, and the mixture was cultured at 37 ° C. in a 5% CO 2 incubator for 3 days. The undifferentiation of skeletal muscle stem cells was analyzed by the immunostaining method described below. First, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then washed 3 times with PBS. Next, it was treated with 0.5% Triton X-100 / PBS at room temperature for 5 minutes, and washed again with PBS three times. After blocking with 5% BSA / PBS at room temperature for 1 hour, it was treated with anti-Pax7 antibody (Santa Cruz) and anti-MyoD antibody (Dako) at 4 ° C. overnight. After washing three times with PBS, the cells were treated with a secondary antibody (anti-mouse IgG-Alexa Fluor 594 and anti-rabbit IgG-Alexa Fluor 488) at room temperature for 1 hour, washed again with PBS three times, and then nucleated using Hoechst 33342. It was stained and observed with a fluorescence microscope (Olympus).
まず全細胞数中のＭｙｏＤ陽性細胞を算出し，実験に用いた筋細胞培養系の純度を解析した。その結果，１００％筋系譜のＭｙｏＤ陽性細胞であることを確認した。次にＭｙｏＤ陽性細胞中の，未分化性マーカーであるＰａｘ７陽性の細胞（骨格筋幹細胞）割合を解析した。その結果，ａｔＲＡおよびＡｍ８０ともに１０−７Ｍ〜１０−５Ｍの濃度でＰａｘ７陽性細胞割合が有意に増加した（図１ａおよびｂ）。First, MyoD-positive cells in the total number of cells were calculated, and the purity of the muscle cell culture system used in the experiment was analyzed. As a result, it was confirmed that the cells were MyoD-positive cells with 100% muscle lineage. Next, the ratio of Pax7-positive cells (skeletal muscle stem cells) that are undifferentiated markers in MyoD-positive cells was analyzed. As a result, the ratio of Pax7-positive cells increased significantly at a concentration of 10 −7 M to 10 −5 M for both atRA and Am80 (FIGS. 1a and b).
実施例３：レチノイド化合物の骨格筋幹細胞の筋管形成能に対する作用
実施例１で調製した骨格筋幹細胞培養系を分化培地（２％ウマ血清，ペニシリン・ストレプトマイシンを含むＤＭＥＭ）で培養し，筋分化を誘導した。さらに分化培地にａｔＲＡあるいはＡｍ８０を１０−１１Ｍ〜１０−５Ｍの濃度で添加し，レチノイド化合物の分化能に対する作用を解析した。具体的には抗ミオシン重鎖抗体（Developmental Studies HybridomaBank）による免疫染色を実施例２と同様に行い，筋細胞が融合した筋管を可視化した。 Example 3: Effects of retinoid compounds on myotube- forming ability of skeletal muscle stem cells The skeletal muscle stem cell culture system prepared in Example 1 was cultured in a differentiation medium (2% horse serum, DMEM containing penicillin / streptomycin) to perform muscle differentiation. Was induced. Further, atRA or Am80 was added to the differentiation medium at a concentration of 10 −11 M to 10 −5 M, and the effect of the retinoid compound on the differentiation ability was analyzed. Specifically, immunostaining with an anti-myosin heavy chain antibody (Developmental Studies Hybridoma Bank) was performed in the same manner as in Example 2 to visualize the myotube fused with myocytes.
結果として，ａｔＲＡ，Ａｍ８０ともに１０−７Ｍおよび１０−５Ｍの濃度で著しく筋管の形成を抑制した（図２）。As a result, both atRA and Am80 markedly suppressed myotube formation at concentrations of 10 −7 M and 10 −5 M (FIG. 2).
実施例４：レチノイド化合物の骨格筋幹細胞の増殖能に対する作用
実施例１で調製した骨格筋幹細胞培養系の増殖培地にａｔＲＡを１０−７Ｍの濃度で添加し，レチノイド化合物の筋細胞の増殖能に対する作用を解析した。解析にはClick-iT(登録商標) Plus EdU AlexaFluor(登録商標) 594 Imaging Kit（Thermo Fisher SCIENTIFIC社）と抗ＭｙｏＤ抗体を用いて，定法に従って検出した。また，免疫染色は実施例２と同様の方法で行った。 Example 4 Effect of Retinoid Compound on Proliferation Ability of Skeletal Muscle Stem Cells AtRA was added to the growth medium of the skeletal muscle stem cell culture system prepared in Example 1 at a concentration of 10 −7 M, and the retinoid compound proliferative ability of muscle cells. Was analyzed. For analysis, Click-iT (registered trademark) Plus EdU AlexaFluor (registered trademark) 594 Imaging Kit (Thermo Fisher SCIENTIFIC) and anti-MyoD antibody were used for detection according to a standard method. The immunostaining was performed in the same manner as in Example 2.
結果として，１０−７ＭのａｔＲＡの添加により，ＭｙｏＤ陽性の骨格筋細胞の増殖能（ＥｄＵを取り込んだ細胞割合）が有意に増加した（図３）。As a result, the proliferation ability of MyoD-positive skeletal muscle cells (proportion of cells incorporating EdU) was significantly increased by the addition of 10 −7 M atRA (FIG. 3).
実施例５：レチノイド化合物添加による骨格筋幹細胞の長期的な維持
実施例１で調製した骨格筋幹細胞培養を１０−７ＭのａｔＲＡを添加あるいは無添加の増殖培地で培養し，５回経代した。これらの細胞におけるＰａｘ７，ミオシン重鎖および内在性コントロールとしてベータチューブリン（β-Tubulin）を定法に従いウェスタンブロット法によって解析した。さらに１０−７ＭのａｔＲＡを添加した増殖培地で１６回経代した骨格筋筋幹細胞培養系における未分化性を抗Ｐａｘ７抗体と抗ＭｙｏＤ抗体を用いて，実施例２と同様に解析した。 Example 5: Long-term maintenance of skeletal muscle stem cells by addition of retinoid compound The skeletal muscle stem cell culture prepared in Example 1 was cultured in a growth medium with or without addition of 10 −7 M atRA, and the cells were passaged 5 times. . Pax7, myosin heavy chain and beta tubulin as an endogenous control in these cells were analyzed by Western blotting according to a standard method. Furthermore, the undifferentiation in the skeletal muscle stem cell culture system that had been passaged 16 times in the growth medium supplemented with 10 −7 M atRA was analyzed in the same manner as in Example 2 using the anti-Pax7 antibody and the anti-MyoD antibody.
結果として，５回の経代によってａｔＲＡ無添加の培地で培養した骨格筋幹細胞は多くの細胞が扁平で巨大な形態を示すのに対し，ａｔＲＡ添加区の細胞はその多くが経代をしていない初代の骨格筋細胞と同様に丸い形態を示した（図４ａおよびｂ）。また，ａｔＲＡ添加区の骨格筋細胞は，無添加区の細胞と比較して幹細胞マーカーであるＰａｘ７を高く発現し，分化マーカーであるミオシン重鎖（ＭｙＨＣ）の発現が減弱した（図４ｃ）。さらに１６回経代し，長期的に維持した骨格筋幹細胞においても８８％の細胞が未分化マーカーであるＰａｘ７を発現し，１０−７ＭのａｔＲＡの添加することで未分化性を長期に維持できることが明らかとなった。（図５）。また，この細胞を分化培地によって分化誘導したところ，正常にミオシン重鎖を発現する筋管を形成した（図６）。As a result, many of the skeletal muscle stem cells cultured in the medium without addition of atRA showed a flat and huge morphology after 5 passages, whereas most of the cells in the addition of atRA had been passaged. It exhibited a rounded morphology similar to that of primary skeletal muscle cells (Fig. 4a and b). In addition, skeletal muscle cells in the atRA-added group expressed Pax7, which is a stem cell marker, higher than cells in the non-added group, and expression of myosin heavy chain (MyHC), which is a differentiation marker, was weakened (Fig. 4c). Further, in skeletal muscle stem cells that had been maintained 16 times and maintained for a long time, 88% of cells expressed the undifferentiation marker Pax7, and the undifferentiation was maintained for a long time by adding 10 −7 M of atRA. It became clear that it was possible. (Fig. 5). When the cells were induced to differentiate with a differentiation medium, myotubes that normally express myosin heavy chain were formed (Fig. 6).
実施例６：他のレチノイド化合物の筋分化抑制に対する作用
実施例１で調製した骨格筋幹細胞を分化培地にＲＡＲアゴニストであるａｔＲＡ，Ａｍ８０，ＡＭ５８０，Ａｍ５５５Ｓ，Ｅｓ８０，Ｅｓ５８０，Ｒｅ８０，Ｆｖ８０，Ｃｈ５５，Ａｚ８０，さらにＲＸＲアゴニストであるＰＡ０２４，ＨＸ６３０，ＬＧ２６８を１０−６Ｍ〜１０−８Ｍの濃度で添加して９６時間培養した。その後，筋管形成能を比較することによって筋分化抑制に対するレチノイド化合物の作用を解析した。 Example 6 Action of Other Retinoid Compounds on Inhibition of Muscle Differentiation The skeletal muscle stem cells prepared in Example 1 were used as a RAR agonist in the differentiation medium atRA, Am80, AM580, Am555S, Es80, Es580, Re80, Fv80, Ch55, Az80. Further, RX024 agonist PA024, HX630, and LG268 were added at a concentration of 10 −6 M to 10 −8 M, and cultured for 96 hours. Then, the effects of retinoid compounds on the suppression of muscle differentiation were analyzed by comparing the myotube-forming ability.
結果として，ＲＡＲアゴニストは全て筋管の形成を抑制した。一方，ＲＸＲアゴニストは筋管の形成を抑制しなかった（図７）。 As a result, all RAR agonists suppressed myotube formation. On the other hand, RXR agonist did not suppress myotube formation (FIG. 7).
実施例７：培養基質ｉＭａｔｒｉｘ−５１１の骨格筋幹細胞の未分化性維持・分化抑制に対する作用
株式会社ニッピ（東京，日本）から購入した培養基質ｉＭａｔｒｉｘ５１１−Ｅ８−ｓｉｌｋを用いて，骨格筋幹細胞の分化抑制効果を検討した。これまで，ｉＭａｔｒｉｘ-５１１によってヒトＥＳ細胞とｉＰＳ細胞の樹立から拡大まで行えることが報告されている（Sci Rep 4:3594）。また，ｉＭａｔｒｉｘ−５１１は継代時の細胞懸濁液に添加することで，培養皿にプレコーティング（ｐｒｅ−ｃｏａｔｉｎｇ）した時と同様の効果が得られることが報告されている（Sci Rep 7:41165）。そこで，実施例１で調製した骨格筋幹細胞をＭａｔｒｉｇｅｌでＰｒｅ−ｃｏａｔｉｎｇした培養皿，０．５μｇ／ｃｍ２となるようｉＭａｔｒｉｘ−５１１でＰｒｅ−ｃｏａｔｉｎｇした培養皿，継代時に細胞懸濁液に０．２μｇ／ｃｍ２，あるいは０．５μｇ／ｃｍ２となるよう添加し，コーティング無しの培養皿に播種し，４８時間培養した。その後，MACHEREY-NAGEL社のNucleoSpin RNAを用いてＲＮＡを抽出し，ＴＯＹＯＢＯ社のReverTra Ace qPCR RT Master Mix with gDNA Removerを用いて逆転写反応を行い，ｃＤＮＡを調製した。得られたｃＤＮＡをKAPABiosystems社のKAPA SYBR FAST qPCR MasterMixを用いて，Ｐａｘ７，Ｍｙｏｇｅｎｉｎ（Ｍｙｏｇ），さらにMuscle CreatineKinase（ＭＣＫ）の発現量をｑＲＴ−ＰＣＲ法によって解析した。 Example 7: Effect of culture substrate iMatrix-511 on maintenance of undifferentiated state and suppression of differentiation of skeletal muscle stem cells Using a culture substrate iMatrix511-E8-silk purchased from Nippi Co., Ltd. (Tokyo, Japan), differentiation of skeletal muscle stem cells The suppression effect was examined. Until now, it has been reported that iMatrix-511 can be used for the establishment and expansion of human ES cells and iPS cells (Sci Rep 4: 3594). In addition, it is reported that by adding iMatrix-511 to the cell suspension at the time of passage, the same effect as when pre-coating the culture dish can be obtained (Sci Rep 7: 41165). Therefore, the skeletal muscle stem cells prepared in Example 1 were pre-coated with Matrigel, the culture dish pre-coated with iMatrix-511 so as to have a concentration of 0.5 μg / cm 2, and 0 in the cell suspension at the time of passage. It was added .2μg / cm 2, or 0.5 [mu] g / cm 2 and so as, seeded in culture dishes without coating, and cultured for 48 hours. Then, RNA was extracted using NucleoSpin RNA from MACHEREY-NAGEL, and reverse transcription reaction was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover from TOYOBO to prepare cDNA. The obtained cDNA was analyzed for expression levels of Pax7, Myogenin (Myog), and Muscle Creatine Kinase (MCK) by qRT-PCR method using KAPA SYBR FAST qPCR MasterMix manufactured by KAPA Biosystems.
結果として，ｉＭａｔｒｉｘ−５１１をＰｒｅ−ｃｏａｔｉｎｇした実験区とｉＭａｔｒｉｘ−５１１を継代時に細胞懸濁液に添加した実験区（０．２μｇ／ｃｍ２および０．５μｇ／ｃｍ２）において，Ｍａｔｒｉｇｅｌをｐｒｅ−ｃｏａｔｉｎｇした実験区と比較してＭｙｏｇとＭＣＫの発現が有意に減少した。一方，Ｐａｘ７の発現量には影響しなかった。（図８）As a result, in the experiment group in which iMatrix-511 was pre-coated and the experiment group in which iMatrix-511 was added to the cell suspension at the time of passage (0.2 μg / cm 2 and 0.5 μg / cm 2 ), the Matrigel was pre-coated. Expression of Myog and MCK was significantly reduced as compared with the experimental group subjected to coating. On the other hand, it did not affect the expression level of Pax7. (Figure 8)
実施例８：レチノイド化合物とｉＭａｔｒｉｘ−５１１の骨格筋幹細胞の未分化性維持・分化抑制に対する相乗効果
実施例６と同様に，骨格筋幹細胞を下記の３通りの方法で培養した：
（１）ＭａｔｒｉｇｅｌでＰｒｅ−ｃｏａｔｉｎｇした培養皿に播種し，増殖培地で培養する方法；
（２）ｉＭａｔｒｉｘ−５１１を継代時に細胞懸濁液に０．２μｇ／ｃｍ２となるように添加し，コーティング無しの培養皿に播種し，増殖培地で培養する方法；
（３）ｉＭａｔｒｉｘ−５１１を継代時に細胞懸濁液に０．２μｇ／ｃｍ２となるように添加し，コーティング無しの培養皿に播種し，１０−７ＭのａｔＲＡを添加した増殖培地で培養する方法。 Example 8: Synergistic effect of retinoid compound and iMatrix-511 on maintenance of undifferentiated state / suppression of differentiation of skeletal muscle stem cells As in Example 6, skeletal muscle stem cells were cultured by the following three methods:
(1) A method of seeding in a culture dish pre-coated with Matrigel and culturing in a growth medium;
(2) A method in which iMatrix-511 is added to the cell suspension at the time of subculturing so that the concentration becomes 0.2 μg / cm 2 , seeded in an uncoated culture dish, and cultured in a growth medium;
(3) iMatrix-511 was added to the cell suspension at the time of passage at 0.2 μg / cm 2 , seeded on an uncoated culture dish, and cultured in a growth medium containing 10 −7 M atRA. how to.
その後，実施例２と同様の方法でＰａｘ７陽性細胞率およびＭｙｏｇ陽性細胞率から骨格筋幹細胞の未分化性・分化抑制に対するｉＭａｔｒｉｘ−５１１とａｔＲＡの相乗効果を解析した。 Then, in the same manner as in Example 2, the synergistic effect of iMatrix-511 and atRA on the undifferentiated / suppressed differentiation of skeletal muscle stem cells was analyzed from the Pax7-positive cell rate and the Myog-positive cell rate.
結果として，ｉＭａｔｒｉｘ−５１１とａｔＲＡを併用した実験区が最もＰａｘ７の陽性細胞率が高く，Ｍｙｏｇ陽性細胞率が低くなり，筋分化を抑制して未分化性が維持されていることが明らかとなった（図９）。 As a result, it was clarified that the experimental group in which iMatrix-511 and atRA were used together had the highest Pax7-positive cell rate and the lowest Myog-positive cell rate, suppressing muscle differentiation and maintaining undifferentiation. (Fig. 9).
実施例９：培養基質ｉＭａｔｒｉｘ−５１１とレチノイド化合物添加による骨格筋幹細胞の未分化性維持・分化抑制に対する作用
株式会社ニッピ（東京，日本）から購入した培養基質ｉＭａｔｒｉｘ５１１−Ｅ８−ｓｉｌｋを用いて，骨格筋幹細胞の分化抑制効果を検討した。これまで，ｉＭａｔｒｉｘ−５１１によってヒトＥＳ細胞とｉＰＳ細胞の樹立から拡大まで行えることが報告されている（Sci Rep 4:3594）。また，ｉＭａｔｒｉｘ−５１１は継代時の細胞懸濁液に添加することで，培養皿にプレコーティング（ｐｒｅ−ｃｏａｔｉｎｇ）した時と同様の効果が得られることが報告されている（Sci Rep 7:41165）。そこで，下記６通りの実験区に実施例１で調製した骨格筋幹細胞を播種し，培養４８時間後に解析した。
（１）Ｍａｔｒｉｇｅｌをｐｒｅ−ｃｏａｔｉｎｇした培養皿に播種し，増殖培地で培養する方法
（２）Ｍａｔｒｉｇｅｌをｐｒｅ−ｃｏａｔｉｎｇした培養皿に播種し，１０−７Ｍのオールトランス型レチノイン酸（ａｔＲＡ）を添加した増殖培地で培養する方法
（３）０．２μｇ／ｃｍ２となるようｉＭａｔｒｉｘ−５１１でＰｒｅ−ｃｏａｔｉｎｇした培養皿に播種し、増殖培地で培養する方法
（４）０．２μｇ／ｃｍ２となるようｉＭａｔｒｉｘ−５１１でＰｒｅ−ｃｏａｔｉｎｇした培養皿に播種し、１０−７ＭのａｔＲＡを添加した増殖培地で培養する方法
（５）継代時に細胞懸濁液に０．２μｇ／ｃｍ２となるよう添加し，コーティング無しの培養皿に播種し，増殖培地で培養する方法
（６）継代時に細胞懸濁液に０．２μｇ／ｃｍ２となるよう添加し，コーティング無しの培養皿に播種し，１０−７ＭのａｔＲＡを添加した増殖培地で培養する方法 Example 9: Effect of culture substrate iMatrix-511 and addition of retinoid compound on maintenance of undifferentiated state and suppression of differentiation of skeletal muscle stem cells Using culture substrate iMatrix511-E8-silk purchased from Nippi Co., Ltd. (Tokyo, Japan), skeleton The effect of suppressing the differentiation of muscle stem cells was examined. Up to now, it has been reported that iMatrix-511 can perform from establishment to expansion of human ES cells and iPS cells (Sci Rep 4: 3594). In addition, it is reported that by adding iMatrix-511 to the cell suspension at the time of passage, the same effect as when pre-coating the culture dish can be obtained (Sci Rep 7: 41165). Therefore, the skeletal muscle stem cells prepared in Example 1 were seeded in the following 6 experimental groups and analyzed after 48 hours of culture.
(1) Method of seeding Matrigel on a culture dish pre-coated and culturing in a growth medium (2) Seeding Matrigel on a culture dish pre-coated and adding 10 −7 M of all-trans retinoic acid (atRA) Method of culturing with added growth medium (3) Seeding in a culture dish pre-coated with iMatrix-511 to obtain 0.2 μg / cm 2, and culturing with growth medium (4) 0.2 μg / cm 2 A method of seeding in a culture dish pre-coated with iMatrix-511 and culturing in a growth medium containing 10 −7 M of atRA (5) 0.2 μg / cm 2 in cell suspension at passage As described above, seeding on an uncoated culture dish, and culturing in a growth medium (6) 0.2 μ in the cell suspension at the time of passage How / cm 2 and so as added, seeded in culture dishes without coating are cultured in growth medium supplemented with atRA of 10 -7 M
培養４８時間後に細胞を４％パラホルムアルデヒドにより室温で１０分間固定した後，３回ＰＢＳによって洗浄した。次に０．５％ＴｒｉｔｏｎＸ−１００／ＰＢＳによって室温，５分間処理し，再度ＰＢＳによって３回洗浄を行った。５％ＢＳＡ／ＰＢＳで室温，１時間ブロッキング処理を行った後，抗Ｐａｘ７抗体（Santa Cruz社）と抗ＭｙｏＤ抗体（Ｄａｋｏ社）を用いて４℃，overnightで処理した。ＰＢＳによって３回洗浄を行った後，二次抗体（抗マウスＩｇＧ−Alexa Fluor 594と抗ウサギＩｇＧ−Alexa Fluor 488）で室温，１時間処理し，再度ＰＢＳで３回洗浄した後，Hoechst 33342を用いて核染色を行い，蛍光顕微鏡（オリンパス社）によって観察した。Ｐａｘ７陽性細胞率およびＭｙｏｇ陽性細胞率を解析し，ｉＭａｔｒｉｘ−５１１とレチノイド化合物の相乗効果を検討した。 After 48 hours of culture, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then washed 3 times with PBS. Next, it was treated with 0.5% Triton X-100 / PBS at room temperature for 5 minutes, and washed again with PBS three times. After blocking with 5% BSA / PBS at room temperature for 1 hour, it was treated with an anti-Pax7 antibody (Santa Cruz) and an anti-MyoD antibody (Dako) at 4 ° C. overnight. After washing three times with PBS, the cells were treated with a secondary antibody (anti-mouse IgG-Alexa Fluor 594 and anti-rabbit IgG-Alexa Fluor 488) at room temperature for 1 hour, and washed again with PBS three times, and then Hoechst 33342 was added. It was used for nuclear staining and observed with a fluorescence microscope (Olympus). The Pax7 positive cell rate and the Myog positive cell rate were analyzed, and the synergistic effect of iMatrix-511 and a retinoid compound was examined.
結果として，ｉＭａｔｒｉｘ−５１１とａｔＲＡを併用した実験区が最もＰａｘ７の陽性細胞率が高く，Ｍｙｏｇ陽性細胞率が低くなり，筋分化を抑制して未分化性が維持されていることが明らかとなった（図１０）。 As a result, it was clarified that the experimental group in which iMatrix-511 and atRA were used together had the highest Pax7-positive cell rate and the lowest Myog-positive cell rate, suppressing muscle differentiation and maintaining undifferentiation. (Fig. 10).
実施例１０：ヒト骨格筋幹細胞の調製
ヒト骨格筋検体（中臀筋）より骨格筋幹細胞を単離した。単離方法としては，筋検体を酵素消化し単離された細胞を培養後にフローサイトメーター（ＢＤ社，AriaII）により純化する方法（Methods Mol Biol. 1460:241-253, 2016）を用いた。得られた骨格筋幹細胞は増殖培地（２０％ウシ胎児血清，ペニシリン・ストレプトマイシン・グルタミン酸，２.５ｎｇ／ｍｌ最終濃度のｂＦＧＦ（Peprotec社）を添加したＤＭＥＭ，Ｈｉｇｈ Ｇｌｕｃｏｓｅ（Thermo Fisher Scientific社）を用いて３７℃，３％Ｏ２, ５％ＣＯ２インキュベーター内で培養した。 Example 10: Preparation of human skeletal muscle stem cells Skeletal muscle stem cells were isolated from a human skeletal muscle sample (gluteus medius). As an isolation method, a method (Methods Mol Biol. 1460: 241-253, 2016) in which a muscle sample was enzymatically digested and the isolated cells were cultured and purified by a flow cytometer (BD, AriaII) was used. The obtained skeletal muscle stem cells used a growth medium (20% fetal bovine serum, penicillin / streptomycin / glutamic acid, 2.5 ng / ml final concentration of bFGF (Peprotec) -added DMEM, High Glucose (Thermo Fisher Scientific)). The cells were cultured at 37 ° C. in a 3% O 2 and 5% CO 2 incubator.
実施例１１：レチノイド化合物のヒト骨格筋幹細胞の増殖に対する効果
実施例１０で調製したヒト骨格筋幹細胞培養系において，ａｔＲＡあるいはＡｍ８０を増殖培地に１０−８Ｍ〜１０−６Ｍの濃度で添加し，３７℃，３％Ｏ２, ５％ＣＯ２インキュベーター内で３日間培養した。骨格筋幹細胞の増殖をＷＳＴ−８アッセイ（Dojindo社）により評価した。アッセイ溶液添加３時間後，Multiskan JX（Thermo Fisher Scientific社）を用いて４５０ｎｍの吸光度を測定することで細胞増殖の定量を行った。 Example 11: Effect of retinoid compound on proliferation of human skeletal muscle stem cells In the human skeletal muscle stem cell culture system prepared in Example 10, atRA or Am80 was added to a proliferation medium at a concentration of 10 -8 M to 10 -6 M. , 37 ° C, 3% O 2 , 5% CO 2 incubator for 3 days. Proliferation of skeletal muscle stem cells was evaluated by WST-8 assay (Dojindo). After 3 hours from the addition of the assay solution, cell proliferation was quantified by measuring the absorbance at 450 nm using Multiskan JX (Thermo Fisher Scientific).
その結果，１０−６ＭのＡｍ８０添加によりヒト骨格筋幹細胞の増殖が抑制された（図１１）。As a result, the growth of human skeletal muscle stem cells was suppressed by the addition of 10 −6 M Am80 (FIG. 11).
実施例１２：レチノイド化合物のヒト骨格筋幹細胞の筋管形成能に対する作用
実施例１０で調製したヒト骨格筋幹細胞培養系を分化培地（5％ウマ血清，ペニシリン・ストレプトマイシン・グルタミン酸を含むＤＭＥＭ）で培養し，筋分化を誘導した。さらに分化培地にａｔＲＡあるいはＡｍ８０を１０−８Ｍ〜１０−６Ｍの濃度で添加し，レチノイド化合物の分化能に対する作用を解析した。具体的には抗ミオシン重鎖抗体（Developmental Studies Hybridoma Bank）による免疫染色を実施例２と同様に行い，筋細胞が融合した筋管を可視化した。 Example 12: Effect of retinoid compound on myotube- forming ability of human skeletal muscle stem cells The human skeletal muscle stem cell culture system prepared in Example 10 was cultured in a differentiation medium (5% horse serum, DMEM containing penicillin / streptomycin / glutamic acid). And induced muscle differentiation. Further, atRA or Am80 was added to the differentiation medium at a concentration of 10 −8 M to 10 −6 M, and the effect of the retinoid compound on the differentiation ability was analyzed. Specifically, immunostaining with an anti-myosin heavy chain antibody (Developmental Studies Hybridoma Bank) was performed in the same manner as in Example 2 to visualize the myotube fused with myocytes.
結果として，ａｔＲＡ，Ａｍ８０ともに１０−７Ｍおよび１０−６Ｍの濃度で筋管の形成を抑制したが、分化抑制効果はａｔＲＡの方が強かった（図１２）。As a result, both atRA and Am80 suppressed myotube formation at a concentration of 10 −7 M and 10 −6 M, but atRA had a stronger differentiation suppressing effect (FIG. 12).
実施例１３：ａｔＲＡのヒト骨格筋幹細胞における筋分化制御因子の発現に及ぼす影響
実施例１０で調製したヒト骨格筋幹細胞培養系の増殖培地にａｔＲＡを１０−７Ｍ〜１０−６Ｍの濃度で添加し，ａｔＲＡの筋分化制御因子の発現に及ぼす影響を解析した。３日間の培養後、RNeasy Micro Kit（Qiagen社）を用いてＲＮＡを単離し、QuantiTect RT Kit（Qiagen社）を用いて逆転写反応を行った。得られたｃＤＮＡを用いてＭＹＯＤ１，ＭＹＯＧの遺伝子発現をリアルタイムＰＣＲ法（Thermal Cycler Dice: Takara社）で相対定量解析した。内部コントロール遺伝子としてＮＤＵＦＡ１３を用いた。 Example 13: Effect of atRA on expression of muscle differentiation regulator in human skeletal muscle stem cells AtRA was added to the growth medium of the human skeletal muscle stem cell culture system prepared in Example 10 at a concentration of 10 -7 M to 10 -6 M. Then, the effect of atRA on the expression of the muscle differentiation regulator was analyzed. After culturing for 3 days, RNA was isolated using RNeasy Micro Kit (Qiagen) and reverse transcription reaction was performed using QuantiTect RT Kit (Qiagen). Using the obtained cDNA, the gene expression of MYOD1 and MYOG was subjected to relative quantitative analysis by a real-time PCR method (Thermal Cycler Dice: Takara). NDUFA13 was used as an internal control gene.
結果として，１０−７Ｍ〜１０−６ＭのａｔＲＡの添加により，ＭＹＯＤ１，ＭＹＯＧの遺伝子発現は抑制されたが、１０−６Ｍの方がより強く抑制された（図１３）。As a result, the gene expression of MYOD1 and MYOG was suppressed by the addition of 10 −7 M to 10 −6 M of atRA, but 10 −6 M was more strongly suppressed (FIG. 13).
実施例１４：ａｔＲＡとｉＭａｔｒｉｘ−５１１がヒト骨格筋幹細胞における筋分化制御因子の発現に及ぼす相乗効果
実施例１０で調製したヒト骨格筋幹細胞培養系の増殖培地にａｔＲＡは１０−６Ｍの濃度で，また，ｉＭａｔｒｉｘ−５１１は０．１〜０．５μｇ／ｃｍ２となるように添加し，３日間培養した。実施例１３と同様の方法で，ＭＹＯＤ１，ＭＹＯＧの遺伝子発現をリアルタイムＰＣＲ法で相対定量解析した。 Example 14: Synergistic Effect of atRA and iMatrix-511 on Expression of Muscle Differentiation Regulator in Human Skeletal Muscle Stem Cells atRA at a concentration of 10 −6 M in the growth medium of the human skeletal muscle stem cell culture system prepared in Example 10. In addition, iMatrix-511 was added at 0.1 to 0.5 μg / cm 2 and cultured for 3 days. In the same manner as in Example 13, the relative quantitative analysis of the gene expression of MYOD1 and MYOG was carried out by the real-time PCR method.
その結果，１０−６ＭのａｔＲＡと０．１μｇ／ｃｍ２のｉＭａｔｒｉｘ−５１１を添加した場合において，ＭＹＯＤ１，ＭＹＯＧの遺伝子発現が最も抑制された（図１４）。As a result, the gene expression of MYOD1 and MYOG was most suppressed when 10 −6 M atRA and 0.1 μg / cm 2 iMatrix-511 were added (FIG. 14).
実施例１５：ａｔＲＡとｉＭａｔｒｉｘ−５１１がヒト骨格筋幹細胞の増殖に及ぼす相乗効果
実施例１１と同様の方法で，ヒト骨格筋幹細胞を１０−６ＭのａｔＲＡと０．１μｇ／ｃｍ２のｉＭａｔｒｉｘ−５１１の存在下で３日間培養した。実施例１１と同様の方法で，細胞増殖の定量を行った。 Example 15: Synergistic effect of atRA and iMatrix-511 on proliferation of human skeletal muscle stem cells In the same manner as in Example 11, human skeletal muscle stem cells were treated with 10-6 M atRA and 0.1 μg / cm 2 of iMatrix-. Cultured in the presence of 511 for 3 days. The cell proliferation was quantified in the same manner as in Example 11.
その結果，１０−６ＭのａｔＲＡと０．１μｇ／ｃｍ２のｉＭａｔｒｉｘ−５１１の添加によりヒト骨格筋幹細胞の増殖が促進された（図１５）。As a result, the proliferation of human skeletal muscle stem cells was promoted by the addition of 10 −6 M atRA and 0.1 μg / cm 2 of iMatrix-511 (FIG. 15).
実施例１６：ａｔＲＡとｉＭａｔｒｉｘ−５１１がヒト骨格筋幹細胞の移植効率に及ぼす影響
実施例１１と同様の方法で，ヒト骨格筋幹細胞を１０−６ＭのａｔＲＡと０．１μｇ／ｃｍ２のｉＭａｔｒｉｘ−５１１の存在下で培養した。５週齢のＮＯＧ−ｍｄｘマウスの前脛骨筋に，培養された細胞を移植した（細胞数４ｘ１０５個）。移植４週間後，前脛骨筋の迅速凍結切片を作成し，ウサギ抗ジストロフィン抗体（Abcam社）とマウス抗ヒトスペクトリン抗体（Leica社），ラット抗ラミニンα２鎖抗体（Santa Cruz社）を用いて染色した。二次抗体（抗ウサギＩｇＧ−Alexa Fluor 488と抗マウスＩｇＧ−Ｃｙ３，抗ラットＩｇＧ−Alexa Fluor 647）による二次染色の後，蛍光顕微鏡（Leica社）によって染色像を取得した。前脛骨筋の断面の全筋線維に対するヒトスペクトリン陽性筋線維の割合を算出し，移植効率とした。 Example 16: Effect of atRA and iMatrix-511 on the transplantation efficiency of human skeletal muscle stem cells In the same manner as in Example 11, human skeletal muscle stem cells were treated with 10-6 M atRA and 0.1 μg / cm 2 of iMatrix-. Cultured in the presence of 511. Cultured cells were transplanted into the tibialis anterior muscle of 5-week-old NOG-mdx mice (the number of cells was 4 × 10 5 ). 4 weeks after transplantation, a quick frozen section of tibialis anterior muscle was prepared, and rabbit anti-dystrophin antibody (Abcam), mouse anti-human spectrin antibody (Leica), and rat anti-laminin α2 chain antibody (Santa Cruz) were used. Stained. After secondary staining with a secondary antibody (anti-rabbit IgG-Alexa Fluor 488 and anti-mouse IgG-Cy3, anti-rat IgG-Alexa Fluor 647), a stained image was obtained with a fluorescence microscope (Leica). The ratio of human spectrin-positive muscle fibers to all muscle fibers in the tibialis anterior section was calculated and used as the transplantation efficiency.
その結果，１０−６ＭのａｔＲＡと０．１μｇ／ｃｍ２のｉＭａｔｒｉｘ−５１１を添加し培養されたヒト骨格筋幹細胞が最も高い移植効率を示し（図１６ａ）、その値は細胞移植の治療効果が見込める１０％を超えた（図１６ｂ）。As a result, human skeletal muscle stem cells cultured with addition of 10 −6 M of atRA and 0.1 μg / cm 2 of iMatrix-511 showed the highest transplantation efficiency (FIG. 16 a), which is the therapeutic effect of cell transplantation. Exceeded the expected 10% (Fig. 16b).
本明細書には，本発明の好ましい実施態様を示してあるが，そのような実施態様が単に例示の目的で提供されていることは，当業者には明らかであり，当業者であれば，本発明から逸脱することなく，様々な変形，変更，置換を加えることが可能であろう。本明細書に記載されている発明の様々な代替的実施形態が，本発明を実施する際に使用されうることが理解されるべきである。また，本明細書中において参照している特許および特許出願書類を含む，全ての刊行物に記載の内容は，その引用によって，本明細書中に明記された内容と同様に取り込まれていると解釈すべきである。 While preferred embodiments of the invention are provided herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only, Various modifications, changes and substitutions may be made without departing from the invention. It should be understood that various alternative embodiments of the invention described herein can be used in carrying out the invention. Also, the contents described in all publications, including the patents and patent application documents referred to in this specification, are incorporated by reference in the same manner as the contents specified in this specification. It should be interpreted.
本発明者らは，レチノイドとラミニンを含む培地中で細胞を培養することによって，筋幹細胞の未分化性を保持したまま，細胞を増殖させることが可能であることを見出した。本技術は簡便かつ安全で，高い効率で筋幹細胞の未分化性を維持できることから，筋ジストロフィーをはじめとした筋疾患に対する細胞移植治療や，化合物スクリーニング系の開発，再生医療への応用が期待される。
The present inventors have found that by culturing cells in a medium containing retinoid and laminin, it is possible to proliferate the cells while maintaining the undifferentiated state of muscle stem cells. Since this technology is simple and safe, and can maintain the undifferentiated state of muscle stem cells with high efficiency, it is expected to be applied to cell transplantation therapy for muscle diseases such as muscular dystrophy, development of compound screening system, and regenerative medicine. .
Claims (21)
レチノイドがａｔＲＡまたはＡＭ８０であり，レチノイドの濃度が少なくとも１０−８Ｍであり，ラミニンがヒトラミニン５１１Ｅ８フラグメントであり，
ラミニンが液体培地中に含められており，ラミニンの濃度が少なくとも１．５μｇ／ｍｌであり，そして
ヒト筋サテライト細胞の未分化性が維持される，培養方法。A method for culturing human muscle satellite cells, which comprises culturing cells using a liquid medium containing (i) retinoid and (ii) laminin
The retinoid is atRA or AM80, the retinoid concentration is at least 10 −8 M, the laminin is the human laminin 511E8 fragment,
A culture method, wherein laminin is contained in a liquid medium, the concentration of laminin is at least 1.5 μg / ml, and the undifferentiated state of human muscle satellite cells is maintained.
レチノイドがａｔＲＡまたはＡＭ８０であり，レチノイドの濃度が少なくとも１０−８Ｍであり，
ラミニンがヒトラミニン５１１Ｅ８フラグメントであり，ラミニンが液体培地中に含められており，ラミニンの濃度が少なくとも１．５μｇ／ｍｌであり，そして
培地中で培養されたヒト筋サテライト細胞の未分化性が維持される，細胞培養培地。A liquid cell culture medium for culturing human muscle satellite cells comprising (i) a retinoid and (ii) laminin, comprising:
The retinoid is atRA or AM80, and the retinoid concentration is at least 10 −8 M,
Laminin is the human laminin 511E8 fragment, laminin is contained in the liquid medium, the concentration of laminin is at least 1.5 μg / ml, and the human muscle satellite cells cultured in the medium maintain the undifferentiated state. Cell culture medium.
（ｉ）レチノイドおよび（ｉｉ）ラミニンの存在下で筋サテライト細胞を培養する工程，培養した筋サテライト細胞またはそれに由来する細胞に試験物質を接触させる工程，
試験物質が筋サテライト細胞またはそれに由来する細胞の分化，増殖または生存に影響するか否かを評価する工程
を含む，方法。A method for screening a substance that affects the differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom, comprising:
(I) culturing muscle satellite cells in the presence of retinoid and (ii) laminin, contacting the cultured muscle satellite cells or cells derived therefrom with a test substance,
A method comprising the step of assessing whether a test substance affects the differentiation, proliferation or survival of muscle satellite cells or cells derived therefrom.
Further comprising (i) retinoid and (ii) laminin, the retinoid is atRA or AM80, the retinoid concentration is at least 10 −8 M, the laminin is human laminin 511E8 fragment, and the laminin concentration is at least 1.5 μg / 21. The cell composition of claim 20, wherein the muscle satellite cells are at least 85% pure and the muscle satellite cells are human cells.
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| WO2018235899A1 (en) | 2018-12-27 |
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