JP2009538826A - 若返り用の細胞および細胞抽出物の投与 - Google Patents
若返り用の細胞および細胞抽出物の投与 Download PDFInfo
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Abstract
Description
本発明は、皮膚、毛髪、皮下脂肪、軟骨、筋肉、骨格構造を含むが、これらに限定されない、美容上の外観に直接または間接的に寄与する、人の目に見える部分を改善するための方法、ならびに細胞および組織、好ましくは皮膚の健康および損傷を改善する、より好ましくは、老化した皮膚を若々しい外観に回復させるための方法に関する。いくつかの態様では、本発明は、i)分化可能細胞、ii)細胞もしくは卵の抽出物、またはiii)抽出物に含まれる精製核酸配列もしくは合成核酸配列、ポリペプチド、または天然物を含む成分の分化および抽出を誘導可能な、これらの成分を含む組成物に関する。いくつかの態様では、細胞は、分化可能細胞、好ましくは幹細胞である。いくつかの態様では、組成物は、外表面層の除去後における、皮膚および/または創傷への組成物の塗布を含む方法に使用される。いくつかの態様では、本発明は、細胞の脱分化の方法、および/または再分化後における脱分化の方法に関する。いくつかの態様では、本発明は、皮膚疾患の管理、予防、および処置に関する。
皮膚は、外界からの攻撃に対抗する最初の防護壁であり、ならびに物理的および化学的な防御の両方を司る。ビタミンDは、太陽熱の放射の作用によって表皮で産生される。このビタミンは、カルシウムが腸から吸収されて骨に固定され、人体の発生および成長を可能とするために必要である。しかしながら、日光を過剰に浴びすぎると、皮膚の損傷、および潜在的には癌に至る。さらに皮膚の細胞は、物理的手段によって損なわれる、すなわち傷害を負うか、または加齢に伴って損なわれる可能性がある。したがって、皮膚の健康状態を管理および改善し、ならびに皮膚の状態および疾患を予防および処置し、ならびに正常な皮膚の外観を維持し、ならびに老化した皮膚を若々しい外観に回復させるための組成物および方法を同定する必要がある。
本発明は、皮膚、毛髪、皮下脂肪、軟骨、筋肉、骨格構造を含むが、これらに限定されない、美容上の外観に直接または間接的に寄与する、人の外から見える部分の改善、ならびに細胞および組織、好ましくは皮膚の健康および損傷の改善、ならびに、より好ましくは、老化した皮膚の若々しい外観への修復に関する。いくつかの態様では、本発明は、抽出物に含まれる精製核酸配列もしくは合成核酸配列、ポリペプチド、または天然物を含むが、これらに限定されない、脱分化を誘導可能な、細胞の組成物、細胞もしくは卵の抽出物、および抽出物の成分に関する。いくつかの態様では、細胞は、分化可能細胞、好ましくは幹細胞である。いくつかの態様では、組成物は、外表面層の除去後における、皮膚および/または創傷への組成物の塗布を含む方法に使用される。いくつかの態様では、本発明は、細胞の脱分化の方法、および/または再分化後における脱分化の方法に関する。いくつかの態様では、本発明は、皮膚疾患の管理、予防、および処置に関する。いくつかの態様では、本発明は、失われたか、または損傷した組織、器官、および四肢の修復または新規形成に関する。
「抗感染剤」とは、ベンジルペニシリン、ペニシリン、ペニシリンG、6-フェニルアセチルペニシリン、ペニシリンV、ミクロノマイシン、クラブラン酸、オキサシリン、デカリニウム、クロキサシリン、スルベニシリン、アンピシリン、シレラール(cilleral)、およびプリンシペン(principen)、ならびにこれらの組み合わせを含むが、これらに限定されない。
本発明は、細胞および組織、好ましくは皮膚の健康および損傷の改善、およびより好ましくは、老化した皮膚もしくは損傷した皮膚の、若々しく健康な外観への修復に関する。いくつかの態様では、本発明は、細胞、細胞または卵の抽出物、および該抽出物に含まれる精製核酸配列もしくは合成核酸配列、ポリペプチド、または天然物を含むが、これらに限定されない、分化を誘導可能な抽出物成分の組成物に関する。いくつかの態様では、細胞は、分化可能細胞、好ましくは幹細胞または卵である。いくつかの好ましい態様では、抽出物は水性抽出物である。いくつかの態様では、抽出物は鳥類供給源以外に由来する。いくつかの態様では、組成物は、外表面層の除去後における皮膚および/または創傷への組成物の塗布を含む方法で使用される。いくつかの態様では、本発明は、細胞の脱分化、および/または再分化後における脱分化の方法に関する。いくつかの態様では、本発明は、皮膚疾患の管理、予防、および処置に関する。
いくつかの態様では、本発明は、胚性幹細胞、または胚性幹細胞から調製された抽出物を含む組成物を提供する。いくつかの好ましい態様では、細胞または抽出物は、以下に詳述するように、局所適用用に製剤化される。本発明は、任意の特定の種類の胚性幹細胞の使用に限定されない。実際には、無脊椎動物および脊椎動物を含むが、これらに限定されない、動物界のあらゆる種を含む、脊索動物門の種を含む、全ての霊長類、齧歯類、肉食動物、ウサギ目、および偶蹄目の動物を含むが、これらに限定されない哺乳類網のあらゆる種、および重要な点としてあらゆる目を含む、いくつかの動物種に由来する胚性幹細胞の使用が想定される。サル、マウス、ラット、ブタ、ウシ、およびヒツジを含む、これらの動物目の種から多能性細胞を得る方法については文献に記載されている。例えば、米国特許第5,453,357号;第5,523,226号;第5,589,376号;第5,340,740号;および第5,166,065号(いずれも特異的に参照により本明細書に組み入れられる);ならびにEvans, et al., Theriogenology 33(1): 125-128, 1990;Evans, et al., Theriogenology 33(1): 125-128, 1990;Notarianni, et al., J. Reprod. Fertil. 41(Suppl.):51-56, 1990;Giles, et al., Mol. Reprod. Dev. 36:130-138, 1993;Graves, et al., Mol. Reprod. Dev. 36:424-433, 1993;Sukoyan, et al., Mol. Reprod. Dev. 33:418-431, 1992;Sukoyan, et al., Mol. Reprod. Dev. 36:148-158, 1993;Iannaccone, et al., Dev. Biol. 163:288-292, 1994;Evans & Kaufman, Nature 292:154-156, 1981;Martin, Proc Natl Acad Sci USA 78:7634-7638, 1981;Doetschmanet al. Dev Biol 127:224-227, 1988);Gileset al. Mol Reprod Dev 36:130-138, 1993;Graves & Moreadith, Mol Reprod Dev 36:424-433, 1993 and Bradley, et al., Nature 309:255-256, 1984を参照されたい。
いくつかの態様では、本発明は、成体幹細胞または成体幹細胞から調製された抽出物を含む組成物を提供する。いくつかの好ましい態様では、細胞または抽出物は、以下に詳述する局所適用用に製剤化される。成体幹細胞は、分化した(特殊化した)組織中に存在する未分化の(特殊化していない)細胞であり;これは自然に再生可能であり、および特殊化して、起源を有する組織の特殊化した細胞タイプを産生する。このような前駆細胞は、組織中に分散した細胞集団としての動物、植物、原生生物、および真菌の界の、あらゆる多細胞生物の成体の分化した組織内に存在する。成体に由来する前駆細胞は、分化能力を元に3つのカテゴリーに分けられる。前駆細胞のこの3つのカテゴリーは、胚盤葉上層様の幹細胞、生殖層系統の幹細胞、および前駆細胞である。前駆細胞は、骨格筋、真皮、脂肪、心筋、肉芽組織、骨膜、軟骨膜、脳、髄膜、髄鞘、靱帯、腱、血管、骨髄、気管、肺、食道、胃、肝臓、腸、脾臓、膵臓、腎臓、膀胱、および睾丸を含むが、これらに限定されない、さまざまな組織から単離されている。前駆細胞は、体内の結合組織区画から、機械的な破壊および/または酵素による消化によって放出させることが可能であり、ならびに新生期、思春期、および老齢期のマウス、ラット、およびヒト、ならびに成体のウサギ、イヌ、ヤギ、ヒツジ、およびブタほかの動物から単離されている。
いくつかの態様では、本発明は、臍帯血細胞、または臍帯血細胞から調製された抽出物を含む組成物を提供する。いくつかの好ましい態様では、細胞または抽出物は、以下に詳述する局所適用用に製剤化される。臍帯血の移植は、血液疾患患者の処置に良好に実施されており;ドナーには、HLAが患者と完全に適合する新生子の同胞が使用されている。移植用の造血幹細胞の供給源としての臍帯血の利点は明らかである。第1に、臍帯血中の造血幹細胞の増殖能は、成体由来の骨髄または血液中の細胞の増殖能を凌ぐ。これらは速やかに増殖するので、1単位の臍帯血中の幹細胞で造血系全体を再構成することができる。第2に、臍帯血の使用は、造血幹細胞の同種間移植の成功に対する主要な障害である移植片対宿主病のリスクを低減する。移植片対宿主病は、レシピエントにおけるHLA抗原に対する移植片中のT細胞の反応に起因し;臍帯血中のリンパ球の未熟さは、この反応を低下させる。欧州における合同研究では、HLAが同一の同胞の臍帯血のレシピエントでは、急性または慢性の移植片対宿主病のリスクが、HLAが同一の同胞の骨髄のレシピエントより低いことが明らかにされている。非血縁ドナーに由来するHLAが適合しない臍帯血の移植を受けた急性白血病児の移植片対宿主病のリスクは、非血縁ドナーに由来するHLAが適合しない骨髄のレシピエントに見られるリスクより低い(Hematopoietic stem-cell transplants using umbilical-cord blood, New England Journal of Medicine, 2001, 344(24):1860-1861, editorial)。
いくつかの態様では、本発明の組成物は、上綱顎口上綱(Superclass Gnathostomata;有顎脊椎動物)、正真口類(Euteleostomi;骨を有する脊椎動物)、条鰭亜綱(Class Actinopterygii;条鰭類)、肉鰭亜綱(Class Sarcopterygii;肉鰭綱および陸生脊椎動物)、四足類(Tetrapoda;四足類)、有羊膜類(Amniota;有羊膜類)、単弓類(Synapsida;爬虫綱単弓亜綱)、哺乳類網(Class Mammalia;哺乳類)、初期獣弓目(Early Therapsida;early therapsids)、爬虫網(Class Reptilia;爬虫類)、無弓亜綱(Anapsida;カメ(tortoiseおよびturtle))、カメ目(Order Testudines;カメ(tortoiseおよびturtle))、双弓類(Diapsida;鳥類、ワニ、トカゲ、ヘビ、および近縁種)、主竜類(Archosauria;鳥類およびワニ)、ワニ目(Order Crocodilia;カイマン、ワニ、および近縁種)、鱗竜亜綱(Lepidosauria;ミミズトカゲ、トカゲ、ヘビ、およびムカシトカゲ)、喙頭目(Order Rhynchocephalia;ムカシトカゲ)、有鱗目(Order Squamata;ミミズトカゲ、トカゲ、およびヘビ)、両生網(Class Amphibia;両生類)、肺魚亜綱(Subclass Dipnoi;ハイギョ)、総鰭綱(Actinistia)、シーラカンス目(Order Coelacanthiformes;シーラカンス)、軟骨魚綱(Class Chondrichthyes;エイ、サメ、および近縁種)、板皮綱(Placodermi;甲冑魚類および板皮綱)、頭甲綱(Class Cephalaspidomorphi)、より好ましくは魚類、エビ、ウニ、または両生類の卵もしくは胚を含むが、これらに限定されない脊椎動物の細胞、卵、および胚の抽出物を使用する。いくつかの態様では、魚類、エビ、ウニ、または両生類の未受精ではあるものの活性化された卵が使用される。本発明は、任意の特定のタイプの卵の使用に限定されない。実際、ツメガエル、エビ、ウニ、サケ、マス、またはゼブラフィッシュの卵を含むが、これらに限定されない、さまざまな卵の使用が想定される。いくつかの態様では、卵は成熟した雌から採取され、水と接触すると自発的に活性化する。別の態様では、卵は、リンゲル生理食塩水で洗浄される。いくつかの態様では、卵は鳥類種に由来しない。
本発明の抽出物は、セクションA〜Dに記載された任意の供給源から調製される。いくつかの態様では、抽出物は細胞抽出物である。本発明の細胞抽出物は、幹細胞や卵などが破壊された細胞の組成物である。細胞は、機械的な剪断もしくは混合、超音波処理、または浸透圧溶解を含むが、これらに限定されない、さまざまな方法で破壊することができる。いくつかの態様では、細胞抽出物は好ましくは、さらに処理されて、細胞膜成分などの、天然の状態では細胞と結合する脂質を実質的に含まない組成物を得る。脂質を実質的に含まないという表現は、天然の状態では、細胞抽出物の作製に使用される細胞と結合する脂質を、約1%未満、好ましくは約0.5%未満、およびより好ましくは約0.1%未満の濃度でしか含まないことを意味する。いくつかの態様では、抽出物は、約1%未満、および好ましくは0.1%未満のコレステロールまたはオボアルブミンを含む。したがって、いくつかの態様では、細胞抽出物は、炭水化物、タンパク質、グリコシル化タンパク質または他の修飾タンパク質、ペプチド、アミノ酸、RNA(mRNA、sRNA、miRNA、rRNA)、DNA、水など、およびこれらの組み合わせを含む。いくつかの態様では、細胞抽出物は、天然の状態では細胞と結合する少量の脂肪、ならびに染色体、核酸、および核タンパク質などの核成分を含む場合がある。いくつかの態様では、細胞抽出物は好ましくは、核、細胞膜、および天然の状態では細胞と結合する、水に不溶性の他の材料の除去によって調製された細胞質抽出物である。いくつかの態様では、これらの成分は、破壊された細胞の遠心分離または分画によって除去される。いくつかの態様では、細胞抽出物は好ましくは、タンパク質、mRNA、および炭水化物などの水溶性の細胞成分を含む水性抽出物である。
いくつかの態様では、本発明の組成物は、エピジェネティックな阻害剤をさらに含む。好ましい態様では、1種類もしくは複数のエピジェネティックな阻害剤は、セクションA〜Eに記載される1種類もしくは複数の細胞抽出物と混合される。本発明は、任意の特定のエピジェネティックな阻害剤の使用に制限されない。実際に、合成されたエピジェネティックな阻害剤、および天然の供給源から単離されたか、または由来するエピジェネティックな阻害剤を含むが、これらに限定されない、多様なエピジェネティックな阻害剤の使用が想定される。エピジェネティックな阻害剤の例は、ヒストンデアセチラーゼ阻害剤、DNAメチルトランスフェラーゼ阻害剤、および数種類のビタミンを含むが、これらに限定されない。
いくつかの態様では、上記の抽出物(または抽出物の成分)は、局所輸送用に製剤化される。局所輸送用の一般的な製剤は、Remington's Pharmaceutical Sciences、第18版、Mack Publishing、p.1288〜1300 [1990]に記載されている。したがって、いくつかの態様では、抽出物は、水ベースのゲルもしくはペースト、軟膏、クリーム(無水もしくは含水)、ローション(無水もしくは含水)、エマルジョン、スプレー、溶液、エアロゾル、スティック(固形クリーム)、液体バンドエイド、粉末、吸入スプレー、鼻内スプレー、べーサル・ドロップ(basal drop)、チーク・ドロップ(cheek drop)、舌下薬(sublingual drop)、点眼液またはスプレー、点耳薬(ear drop)またはスプレー、および経皮パッチとして製剤化される。
いくつかの態様では、上記の抽出物(または抽出物の成分)は、さまざまな方法による輸送用に製剤化される。いくつかの態様では、上記の抽出物は、皮膚、消化器路、沈着脂肪、軟骨、骨、結合組織、筋肉、または内臓への輸送用に製剤化される。いくつかの態様では、抽出物またはこの成分は、錠剤、丸剤、糖衣錠、カプセル剤、液剤、液体、スラリー、懸濁液、および乳濁液中に、デンプン、ショ糖または乳糖などの、適切な担体を添加または添加せずに、経口投与用に製剤化される。いくつかの態様では、経口輸送用の溶媒は、腸溶コーティングを含む。他の態様では、抽出物もしくはこの成分は、カプセル、クリーム、坐薬、または液剤として直腸内投与用に製剤化される。いくつかの態様では、抽出物またはこの成分は、シリンジによって腹腔に、または内臓もしくは組織内に注入される。いくつかの態様では、抽出物またはこの成分は、浸透圧ポンプによる輸送用に製剤化される。
いくつかの態様では、局所投与用および/または内臓投与用の組成物は、抽出物と脂質および/または水および/または炭水化物および/または核酸および/またはタンパク質および/またはシグナル伝達物質の混合物である。いくつかの態様では、本明細書に記載された抽出物は、抽出物が作製されるか、または合成バージョンおよび/または天然バージョンの脂質および/または炭水化物および/または核酸および/またはタンパク質および/またはシグナル伝達物質に由来する、細胞全体、または細胞の脂質および/または炭水化物および/または核酸および/またはタンパク質および/またはシグナル伝達物質の組み合わせを含む。シグナル伝達プロファイルは、細胞の抽出物中に含まれる、細胞から放出される活性物質の組み合わせを含み、ならびに抽出物に添加された合成バージョンおよび/または天然バージョンのこれらのシグナル伝達物質を含む。想定されるシグナル伝達物質は、成長因子、エンドルフィン、ホルモン、アミノ酸伝達物質、免疫調節性のサイトカイン、および他の免疫関連因子を含むが、これらに限定されない。
いくつかの態様では、上記の抽出物またはこの成分に他の成分が混合される。いくつかの態様では、このような追加的な成分は、抽出物成分の取り込み、生物学的利用能、または浸透を高める。好ましい態様では、抽出物成分は、天然の成分または合成成分の混合物を含む場合がある。成分は、部分合成または全合成が可能である。いくつかの態様では、細胞もしくは抽出物、または抽出物中に同定される物質から作製された合成成分は、水、皮脂線および表皮の脂肪および細胞抽出物、タンパク質、ならびにこれらの成分を含む、好ましくは重量比で約10%の脂質画分を含む、重量比で約10%のタンパク質画分を含む、および重量比で約80%の揮発性画分を含む組成物と混合される
上記の局所適用用の組成物は、美容と治療の両方の目的に有用なことが想定される。治療目的の使用については、セクションJで詳述した通りである。いくつかの態様では、上記の組成物は、皮膚または身体の他の上皮もしくは表皮の表面に直接塗布されることが想定される。組成物は、処方に適切に、1日に1回、2回、または3回、塗布される場合がある。塗布される量は一般に重要ではないが、一般に約0.001 μg〜10 gの抽出物(またはこの成分)を含む組成物が、身体の任意の表面に塗布される場合がある。上述したように、組成物は、補助剤、担体、他の活性成分などの他の成分を含む場合がある。
いくつかの態様では、細胞または抽出物の組成物は、水分補給(すなわち、血管内の脱水および創傷における浮腫の処置)、防水(すなわち、創傷における循環血液量低下の補償)、感染防止(すなわち、創傷の感染からの予防)、酸化防止(すなわち、虚血性組織の炎症反応における酸素フリーラジカル産生の予防)、創傷治癒(すなわち、低酸素条件、特に熱傷を負った皮膚または嫌気性代謝における細胞における高い代謝の亢進)に有用である。いくつかの好ましい態様では、組成物は無臭であり(すなわち、揮発性の炭素または窒素を含む化合物が存在しないことの特徴)。
本発明のいくつかの態様では、損なわれていない幹細胞(胚性および成体)、または臍帯血幹細胞を含む組成物が美容もしくは治療の目的で使用される。いくつかの態様では、液状の細胞の懸濁物が皮膚に導入される。いくつかの態様では、液状の細胞の懸濁物が開放創中に導入された後に、呼吸可能な(非密封性の)創傷包帯で覆われる。いくつかの態様では、密封性の創傷包帯が使用される。いくつかの態様では、例えば、皮膚に接着する防水性の樹脂製の膜、細胞に栄養を供給することで創傷治癒を速める栄養ゲル(抗菌剤、コラーゲン調節物質、および他の物質を含む)の層;ならびに創傷に直接接触するように栄養層中に埋め込まれたか、栄養層上に配置された皮膚幹細胞の層などの1つもしくは複数の層が使用される。いくつかの態様では、細胞は実験室で当人自身の皮膚、脂肪組織、または幹細胞から培養される。いくつかの好ましい態様では、細胞は次に回収され、懸濁物中に移されて、流体として使用されるか、または密封性の創傷包帯/プラスター/バンドエイドとして皮膚に塗布される栄養ゲル層とともに樹脂膜上に配置される。
いくつかの態様では、抽出物は、患者に由来する細胞のエクスビボ療法に使用される。簡単に説明すると、細胞が患者から回収され、増殖され、透過処理され、抽出物とともにインキュベートされ、閉鎖された後に患者の処置に使用される。この過程において、テロメア(染色体上の中央のDNAを保護する染色体の末端で、細胞分裂の進行に伴って短くなるので、処理細胞を再生し、および寿命を長くする)の延長を含むが、これらに限定されない、いくつかの細胞特性は変化するか、または高められる。好ましい方法は実施例3に記載されている。
実施例1
細胞および細胞抽出物
NCCIT細胞、ジャーカット(クローンE6-1)細胞、および293T細胞(American Type Culture Collection, Bethesda, MD)は、10%ウシ胎児血清(FCS)、2 mM L-グルタミン、1 mMピルビン酸ナトリウム、および非必須アミノ酸を含むRPMI 1640(Sigma, St. Louis, MO)(完全RPMI)で培養する。NIH3T3 Swiss-Albino線維芽細胞(American Type Culture Collection)は、10% FCS、L-グルタミン、および0.1 mM β-メルカプトエタノールを含むダルベッコ改変イーグル培地(DMEM;Sigma)で培養する。マウスのESCは、株sv129胚盤胞の内部細胞塊から単離され、ゼラチンコーティングプレート上に1,000単位/ml(10 ng/ml)の組換え型白血病阻害因子(LIF;Sigma)が添加されたESC培地(DMEM、15% FCS、0.1 mM β-メルカプトエタノール、非必須アミノ酸、1%ペニシリン/ストレプトマイシン)中のマウス線維芽細胞のγ線照射済みのフィーダー層上にプレーティングする。抽出物を調製するための回収に先立ち、ESCを継代し、10 ng/mlのLIFを含むRPMI中で、無フィーダー条件で培養する。
バルジ毛包幹細胞
感覚毛の濾胞(vibrissa follicle)を単離するために、対象の感覚毛パッド(vibrissa pad)を含む上唇を切断し、その内表面を露出させた。ヒトでは、感覚毛に代えて、頭皮の毛髪または他の有毛部分の毛を使用することができる。感覚毛または毛包は、双眼顕微鏡下で切開する。感覚毛は、細いピンセットで頚部を穏やかに引いてパッドから抜く。抜かれた感覚毛は、B-27(GIBCO/BRL)および1%ペニシリン/ストレプトマイシン(GIBCO/BRL)を含むDMEM-F12(GIBCO/BRL)で洗浄した。全ての外科的手順は無菌環境下で実施した。感覚毛の濾胞バルジ領域は、ネスチン発現細胞を含む。細胞を、蛍光顕微鏡下で蛍光抗ネスチン抗体に曝露して単離した。単離された細胞を、1%メチルセルロース(Sigma-Aldrich)を含むB-27、およびおよび20 ng/mlの塩基性FGF(bFGF)(Chemicon)を含む1 mlのDMEM-F12に懸濁した。細胞を24ウェルの組織培養ディッシュ(Corning)で、37℃、5% CO2/95%大気の条件の組織培養用インキュベーター内で培養した。4週間後に、バルジ領域の細胞がコロニーを形成する。
エクスビボ療法
エクスビボにおける再プログラム対象の細胞を冷PBSで、ならびにCa2+-およびMg2+を含まない冷ハンクス平衡塩溶液(HBSS; Invitrogen, Gaithersburg, MD)で洗浄する。細胞を100,000個の細胞/100 μl HBSS、またはこの倍数のアリコートに再懸濁し、1.5 mlチューブ中に移し、120 g、5分間、4℃の条件で、スイングアウトローターで遠心分離する。沈降した細胞を97.7 mlの冷HBSSに懸濁し、チューブを37℃の水浴に移して2分間維持し、2.3 mlのSLO(Sigma;冷HBSSで1:10に希釈した100 mg/mlのストック)を、SLOの濃度が最終的に230 ng/mlとなるように添加する。試料を37℃の水浴中で50分間、ときおり攪拌しながら水平方向でインキュベート後に氷上に移す。試料を200 mlの冷HBSSで希釈し、細胞を120 g、5分間、4℃の条件で沈降させる。透過化を、細胞の再閉鎖および再プレーティングの24時間後に、別の試料中の70,000 Mrのテキサスレッド-結合デキストラン(Molecular Probes, Eugene, OR; 50 μg/ml)の取り込みをモニタリングすることで評価する。このような条件における透過化効率は約80%である。
細胞抽出物とともに使用するクリーム基剤
水-78%
タンパク質-10%
例えば、ケラチン、フィラグリン、および/または微量の成長因子(μM〜mM量のEGF、IGF、IGFII、インスリン、サブスタンスP、デフェンシン、NGF)
脂質-10%
スクアレン(9%)、脂肪族ロウ(12%)、ステロールエステル(33%)、ジオールエステル(7%)、トリグリセリド(26%)、遊離ステロール(9%)、他の脂質(4%)。
細胞抽出物もしくは卵の抽出物、または抽出物の成分-2%
魚の卵抽出物の調製
生殖期(晩秋)の雌から回収された、新鮮で未受精のサケ(Salmo salar)の卵を氷上で維持し、抽出物を好ましくは直ちに作製する。卵を凍結防止剤(例えば、1.5 Mの1,2-プロパンジオールおよび0.2 Mのショ糖)で、卵膜を破壊することなく凍結乾燥することが可能である。凍結は段階的に(-1℃/分)、-80℃まで行うべきである。卵を解凍し、抽出物の調製手順の間、氷上で維持することができる。
抽出物の毒性の検討
低pHで、特定の物質を含む抽出物は、細胞に毒性作用を及ぼす場合がある。個々のバッチの毒性は、再プログラムされる個々の細胞タイプを対象に検討すべきである。細胞を回収して、HBSSで2回洗浄する。約100,000個の細胞のペレットを得て、100 μlの抽出物に再懸濁し、37℃の水浴で1時間インキュベートする。抽出物の希釈率を検討し、さまざまなタンパク質濃度、pH、および浸透圧の抽出物中における細胞の生存率を評価することができる。好ましくは、タンパク質濃度は25 mg/ml以上であるべきであり、pHは7.2付近であるべきであり、および浸透圧は280 mOsmに近い値であるべきである。細胞および抽出物を、正常培地(選択される細胞のタイプに合わせたもの)を含むウェルで24時間インキュベートし、細胞の形態を顕微鏡で調べる。細胞を回収し、染色し、生細胞をカウントする。培養後に50%を超える細胞が生きていなければ、抽出物には毒性があると見なされる。
抽出物の遺伝子発現アッセイ法
再プログラム細胞における抽出物による研究対象遺伝子の発現を検証するために、抽出物から単離されたRNAを対象にRT-PCRを実施することができる。RNAは抽出物から選択法で、例えばQiagen RNeasy Plus Kit (Qiagen)を使用して単離することができる。RNAを分光光度計で定量し、-80℃で保存する。1 μgのRNAをcDNAの合成に使用する。cDNAの合成は例えば、iScript cDNA Synthesis Kit(Bio-Rad)の使用と、これに続く、選択プライマーによるPCRによって実施することができる。検討対象の各プライマーセットには正の対照を含める。PCR産物を臭化エチジウムを含む1%アガロースゲルに流し、バンドをUVランプで可視化する。
魚卵または胚の抽出物による細胞の再プログラミング
細胞タイプの選択(例えば、ヒト293T細胞や脂肪組織幹細胞(ASC tested))を回収し、氷上で維持し、および氷冷HBSSで2回洗浄する。100,000〜500,000個の細胞を遠心分離して(300 g、4℃で10分間)ペレットを得る。細胞を、再プログラミング前に37℃の水浴で50分間インキュベートすることでストレプトリシン-O(SLO)で透過化することができるが、これは魚卵または胚の抽出物の再プログラミング作用には必要ない。SLOとのインキュベーション後に、細胞を氷冷HBSSで洗浄し、遠心分離し、ペレットから過剰な液体を除去する。細胞を100,000個あたり100 μlの抽出物に再懸濁し、37℃の水浴で1時間インキュベートする。約100,000個の細胞を、選択される完全培地を含むウェルに添加する。仮にSLOによる透過化を実施したら、細胞を、再プログラミング後に、2 mM CaCl2が添加された培地で2時間培養して細胞膜を閉じる。培地は、再プログラミングの2〜12時間後に交換すべきである。SLOによる透過化の規模を評価するために、50 μg/mlのAlexia red結合デキストラン(10,000 Mrまたは70,000 Mrのデキストラン)を含む0 ng/mlまたは100 ng/mlのSLO中で50分間インキュベートされた細胞を対象に、落射蛍光顕微鏡を使用して、細胞の透過化および再閉鎖を検証する。
1.方法に記載された手順で再プログラミングを実施後に、正常細胞を培養;
2.記載通りに再プログラミング後に、0.4%のサケ卵抽出物が添加された培地(再プログラミング用と同じもの)で培養;ならびに
3.再プログラムされなかった正常細胞を、0.4%のサケ卵抽出物が添加された培地(再プログラミング用と同じもの)で培養。
抽出物で処理された細胞の形態学的変化
サケ卵の抽出物、またはゼブラフィッシュ胚の抽出物によって再プログラムされた細胞の形態は、約3日後に変化する。293T細胞は球形に近くなり、および細胞集団の一部は、割球様の球に成長を開始する。このような変化は持続的であり、および21日目(実験終了)まで観察可能であるが、条件によっては、変化は2週間後に正常293Tの形態に逆転するようである。正常293T細胞を、正常培地(10%FCSおよび0.2%抽出物を含むRPMI-1640)に添加された抽出物とともに培養すると、正常培地で培養されて再プログラムされた細胞に見られるのと類似の形態変化が観察される。加えて、特にサケ卵抽出物とともに培養された細胞は、正常細胞と比較して速い成長速度を有する。飢餓状態の細胞(0.5%のFCSを含むRPMI-1640)では、非抽出物処理細胞より成長速度は有意に低下し(示していない)、細胞形態はわずかにしか変化しない。抽出物(飢餓培地中に0.2%の抽出物)とともに成長させた飢餓細胞では変化は顕著である。この場合、細胞集団の大半は割球様の球に成長し、およびこの球は、培養容器から剥がれ、培養液中を浮遊した状態で成長を続ける。
抽出物とともにインキュベートされた細胞の成長パターンの変化
500,000個の293T細胞を中型の円形培養ディッシュに添加し、正常培地で、または抽出物が添加された培地で、または飢餓培地でインキュベートした。24時間後、41時間後、および68時間後に細胞を回収し、カウントし、ならびに成長速度を計算した。結果を表5および表6に示す。
サケ卵抽出物による創傷治癒の促進
試験の目的:
マウスの皮膚に見られる創傷治癒に対する、添加されたサケ卵抽出物の効果を調べる。
概要:
2タイプの創傷をマウスの背の皮膚に誘導した。直径1 cmの切除創(excision wound)を各マウス(n=12で計3回)の背中の左側に誘導し、および長さ2 cmの切開創(incision wound)を背中の右側に、脊椎に平行方向に誘導した。マウスの半数(無作為に選択)を、創傷誘導後に30 μlのサケ卵抽出物で3日ごとに12日間にわたって処置した。対照群には処置を行わなかった。全マウス(処置群および対照群)の創傷に1日目に、3回の試行のうち1回について液体バンドエイドを噴霧したが、これは全3回の実験(バンドエイドの噴霧を行った場合と行わない場合)において観察された対照マウスと処置マウス間に見られる差に影響しなかった。
マウス:A/JまたはNMRIのアルビノの雄。
創傷誘導前における皮膚の滅菌用のエタノール。
小型の手術用具(surgical scissor)および超小型の手術用具(micro scissor)、メスおよびピンセット。
上述の手順で調製されたサケ卵抽出物(バッチLE4)。
イソフルランガス:FORENE Isofluran Vnr 506949、ロット22397VA、exp 2009-10(Abbott, Solna, Sweden)
気化器:Datex-Ohmeda Isotec 5
生検試料用の液体窒素。
生検試料用の4% PFA(溶媒PBS)。
治癒期間中における創傷および皮膚の写真を撮影用のデジタルカメラ。
動物の創傷サイズのマーキング用の直径1 cmの円形の型。
動物
体重が25 g〜35 gの健康な近交系の雄のNMRIまたはA/Jマウス(別の研究)を、Institute of National Public Health(ノルウェー・オスロ)の動物部門から入手した。このマウスを実験開始前の1週間にわたって馴化させ、ポリプロピレン製ケージ内で、通常の餌および水を自由に与えながら飼育し、耳に標識を施し(各ケージに1〜4匹)、1週間後に実験を開始した。動物の体重を実験の前後に定期的に測定した。マウスを麻酔した後に実験的創傷を加えた。外科的介入は無菌条件で、イソフルランガス(酸素とイソフルランを気化器中で混合)を使用して実施した。感染症の徴候がないか個体を詳しく観察し;感染症の徴候が見られた個体は別にして試験から除外した。本明細書に記載された手順で、抽出物に関する急性毒性試験を実施した。本試験はノルウェーの倫理委員会によって承認された。
切除創および切開創の創傷モデルを使用して、サケ卵抽出物の創傷治癒活性の評価を行った。各個体の背中の右側に切除創を加え、背中の左側に切開創を加えた。創傷は1日目に加え、試験は12日目に終了した。
個々のマウスに1か所の切除創(Morton JJP, Malone MH. Evaluation of vulnerary activity by an open wound procedure in rats. Arch Int Pharmacodyn. 1972; 196:117-126)および1か所の切開創を加えた(Ehrlich HP, Hunt TK. Effect of cortisone and vitamin A on wound healing. Ann Surg. 1968; 167:324-328)。創傷を加える前に、マウスをイソフルランガス(マスクを使用、系については後述)で麻酔した。背中の毛を電気バリカンで剃り、作られる切除創の領域を、背中の左側に防水性の油性ペンで印をつけた。全厚(約1 mm)の幅1 cm(円の面積=0.785 cm2)の切除創を印に沿って、歯付きピンセット、メス、および先端のとがった器具(pointed scissors)を使用して作製し、創傷全体を開いた状態にした。脊椎の右側には、長さ2 cmの縦方向の傍脊椎切開を、頚部の皮膚および皮膚組織を通して行った。実験群については、水溶液状のサケ卵抽出物(30 μl)で処理し、3日ごとに創傷に局所的に塗布した。対照群の創傷には処理は行わなかった。
1日目に、切除創の作製時に切断された皮膚を正常皮膚生検として維持することで、後の生検解析における各個体のそれ自身の対照とした。生検の半分を4% PFAで固定し、残る半分を液体窒素で瞬間凍結した。切除創および切開創の創傷モデルでは、創傷上に生じた肉芽組織を、術後12日目に頸椎脱臼またはCO2ガスで動物を殺した後に切除した。切除創および切開創を、最初の創傷誘導の印に沿って外科的に除去した。生検の半数を4% PFAで固定し、残る半数については、後の解析用に液体窒素で瞬間凍結した。
切除創および切開創の創傷モデルで、全個体から12日目に得られた治癒組織の半数をパラホルムアルデヒド(リン酸ナトリウム緩衝液中に4%)で室温で2時間かけて固定し、4℃で保存し、遠心分離し、クリオスタットで皮膚表面に対して逆平行に切片を作製した。切片をH&Eで染色し、肉芽組織の厚みを含む瘢痕のパラメータを顕微鏡下で測定した。
代表的な抽出物処理個体および非処理対照個体の、1日目および12日目に採取された切除創生検の切片を、文献(Boulland et al., Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters. J Comp Neurol. 2004 Dec 13; 480(3):264-80)に記載された手順で、NANOG(ウサギポリクローナル、Abcam)およびカルビンジン(マウス、Abcam)、またはOCT3/4(ウサギポリクローナル、Santa Cruz)およびカルレチニン(ヤギ、Chemicon)のいずれかに対する抗体によって二重免疫染色して、治癒創傷中の幹細胞における発現の上昇(NANOGおよびOCT3/4の存在によって示される)について観察を行った。
サケ卵抽出物で処理された創傷の治癒率を非処理対照と比較した。
結果から、抽出物処理個体(画像は示さず)では、より速く創傷が治癒することがわかる(9日目および12日目に有意差)。また創傷サイズは、抽出物処理個体では、より速やかに縮小し、5日目および9日目に有意差が認められた。図4を参照されたい。
1日目に除去されて切除創を作るために皮膚から得られた生検、および12日目に6匹から採取された同じ部位の同等の生検をクリオスタットで切片化し、H&Eで染色し、顕微鏡画像を撮影して解析した。形態学的には、1日目の正常皮膚生検の切片は、対照個体と処理個体群で似ており、皮膚パラメータの尺度は同等であった。12日目に、瘢痕組織、特にコラーゲンの組織化は、対照動物では、抽出物処理動物と比較して、より乱れた状態であった。
代表的な抽出物処理個体および非処理対照個体の1日目および12日目に得られた切除創生検の切片を、文献(Boulland et al.)に記載された手順で、NANOG(ウサギポリクローナル、Abcam)およびカルビンジン(マウス、Abcam)、またはOCT3/4(ウサギポリクローナル、Santa Cruz)およびカルレチニン(ヤギ、Chemicon)のいずれかに対する抗体によって二重免疫標識して、治癒創傷における幹細胞の発現の上昇(NANOGおよびOCT3/4の存在によって示される)を観察した。
ヒト皮膚線維芽細胞およびHEK細胞の再プログラミング
hsF細胞(ヒト皮膚線維芽細胞)の継代培養
hsF用の完全培地
500 mlのDMEM F-12(+Glutamax)
50 ml(10%)のFCS(ウシ胎仔血清-加熱不活性化済み)
5 ml(1%)のPenStrep
hsF用の飢餓培地
500 mlのDMEM F-12(+Glutamax)
5 ml(1%)のPenStrep
1.細胞層を10〜15 mlのPBSで2回洗浄して、微量の血清を全て除去する。
2.2 mlのトリプシン-EDTA溶液を、細胞層が分散するまで(5〜7分間)添加する。
3.4 mlの培地を添加し、細胞を穏やかなピペット操作で吸引する。
4.継代培養比は1:2〜1:4とし;2〜3 mlの細胞懸濁物をフラスコに添加し、新鮮な培地を加えて計25 mlとする。細胞1:2〜1:4で2〜3日毎に(例えば1:4で週に2回)、継代培養する。
1.新鮮な凍結用培地を作製する:
a.20% FCSおよび10% DMSOを含む正常培地
2.上記のプロトコル(継代培養)を3まで行い;その後、
3.細胞を50 mlのNuncチューブに移し、300 g(1500 rpm)で10分間、4℃で遠心分離する。
4.細胞を1 mlの凍結用培地に100万個となるように再懸濁し、1 mlのアリコートをNunc cryoチューブに移す。
5.Mr. Frostyボックス中で細胞をイソプロパノールで、-80℃に一晩かけて(-1℃/分)凍結させる。
6.窒素タンクへ移す。
継代培養プロトコルを3まで行い;その後、
1.細胞を50 mlのNuncチューブに移し、300 g(1500 rpm)で10分間、4℃で遠心分離する。
2.細胞を百万個あたり1 mlの氷冷PBSで洗浄し、遠心分離する(300 g、10分間、4℃)。
3.ペレットを同量のPBSに再懸濁し、RNAペレット用に1 mlをエッペンドルフチューブに移す。
4.300x g、10分、4℃の条件で遠心分離する。
5.PBSを吸引する。
6.ペレットを氷上に静置し、液体N2で瞬間凍結する。
7.-80℃の冷凍庫に移す。
目的:
細胞を無核抽出物で再プログラムし、遺伝子発現、形態、および成長の因子を変化させること、ならびに分化の状態の変化を調べること。
カバースリップ上で24ウェルプレートで成長させたhsF細胞(1ウェルあたり約100,000個の細胞を約5日前に飢餓培地中に添加し、1ウェルあたり約50,000個の細胞を約3日前に正常培地に添加);抽出物(サケ卵抽出物);対照用培地によるインキュベーション;1xPBS;4℃のCa2+を含まないハンクス平衡塩溶液(HBSS);フラスコからhsF細胞を剥がすためのTE;HBSSで1:100に希釈した100 μg/mlのSLOストック;ATP(水を溶媒とする200 mMのストック);GTP(水を溶媒とする10 mMのストック);ホスホクレアチン(水を溶媒とする2 Mのストック);クレアチンキナーゼ(水を溶媒とする5 mg/mlストック);蒸気滅菌済みのMQ水;37℃の水浴;CaCl2(2 mM)濃縮培地:100 mMのCaCl2ストックを1.67 gのCaCl2を15 mlの蒸留水と混合して調製して濾過滅菌したもの。2 mMの濃度のCaCl2を、例えば50 μlの100 mM CaCl2と2450 μlの再プログラミング用培地を混合することで作製する。
細胞を氷冷1xPBS(1 ml)で2回洗浄する。細胞を冷HBSS(1 ml)で2回洗浄する。試料を37℃のインキュベーターで2〜3分間あらかじめ加温し、HBSSを除去する。110 μlのHBSSおよび90 μlのSLOを(最終SLO濃度が450 ng/mlとなるように)添加して混合する。200 μlのHBSSを、SLOなしに対照ウェルに添加する。インキュベーター内で30分間インキュベートする(プレートを10分毎に傾ける)。SLOを除去する(SLOがウェル中に残るように一方を平行に保つ)。再プログラミング用の抽出物を調製する:1回の再プログラミング反応物は、250 μlの抽出物(〜50〜100 Kの細胞)を含む。ATP生成系を調製して氷上で維持し:ATP、GTP、クレアチンキナーゼ、ホスホクレアチンを1:1:1:1の比で混合し、氷上で維持する。1回の反応あたり12.5 μlのATP生成系を抽出物に添加する。250 μlのサケ卵抽出物を(ATP生成系とともに)添加する。抽出物がカバースリップ上で細胞を覆っていることを確認する。プレートを傾けて混合する。インキュベーター内で60分間インキュベートする(プレートを10分毎に傾ける)。抽出物(200 μl)を吸引し、高Ca培地(約1500 μl)を各ウェルに添加する。2時間インキュベートする。細胞がカバースリップ上に接着しているか否かを顕微鏡でチェックする。接着していたら、Ca含有培地を除去して完全培地(約500 μl)を添加する。37℃、5% CO2の条件でインキュベートする。細胞を培養開始から24時間以内に評価する。位相差顕微鏡による観察を行う。コンフルエンスに至ったら細胞を分ける。再プログラミングの1日後にカバースリップを新しいウェルに動かし、数枚のカバースリップをトリプシン処理して小さなボトルに移した。細胞は剥がれなかったので、カバースリップ全体をボトルに移した。
RPE(飢餓培地)およびRPF(正常培地)による再プログラミング実験
遺伝子発現の変化:
(表8)qPCRによる評価による、GAPDHに対して、発生段階で調節されるOCT4遺伝子およびNANOG遺伝子のアップレギュレーションの倍率
再プログラミング後に、細胞培養を位相差顕微鏡で評価して正常細胞と比較した。
細胞を、再プログラミング後の7日目にカバースリップ上に固定した。免疫蛍光標識は基本的に、組織切片に関して報告された手順(Boulland et al., 2004)で実施した。簡単に説明すると、カバースリップ上で成長させた細胞を、4% PFA(室温で30分間)で固定し、PBSで洗浄し、1 Mエタノールアミンでブロックし、3xPBSで洗浄し、ブロック溶液(室温で1時間)でプレインキュベートし、インキュベーション用溶液を溶媒とするOCT 3/4(Santa Cruz)(1:200)に対する一次抗体とインキュベートし(室温で3時間)、3xPBSで洗浄し、蛍光結合二次抗体Alexa 488(1:2000)(Molecular Probes)とインキュベートし(室温で1時間)、最後に3xPBSで洗浄した。核を染色するために、DAPI(1:1000)を添加後に洗浄を行った。カバースリップをProLong Gold Antifade試薬(Molecular Probes)でマウントし、画像を蛍光顕微鏡(Olympus)または共焦点顕微鏡(Zeiss)で得た。
Cascade Biologicsのケラチノサイト培養システム
EXTENDED-LIFESPANシステム
Basal Medium EpiLife(登録商標)培地
成長用サプリメント HKGS(S-001-5)
継代培養試薬 トリプシン/EDTA(R-001-100)
継代培養試薬 トリプシン中和液(R-002-100)
抗生物質(再プログラミング後) ゲンタマイシン/アンホテリシンB(R-015-10)
HEKa(C-005-5C)の推定寿命 35〜45回の集団の倍加
HEKa用の完全培地
500 mlのEpiLife培地
5 mlのHKGS(ヒトケラチノサイト成長サプリメント)
再プログラミング後:1 mlのゲンタマイシン/アンホテリシン(GA)
細胞を75 cm2の培養フラスコで培養(コンフルエンス時に約10 mlの細胞)。
1.細胞を3 mlのトリプシン/EDTAで速やかに洗浄する。
2.1 mlの新鮮なトリプシン/EDTAを添加し、細胞が分散するまで(8〜10分間)インキュベートする。
3.3 mlのトリプシン/中和液を添加し、細胞を滅菌済みの15 mlチューブに移す。さらに3 mlの追加のトリプシン/中和液で繰り返す。
4.180x gで7分間、遠心分離する。
5.細胞ペレットを再懸濁し、新しい培養容器に1 cm2あたり2.5x103個の細胞となるように添加する。
6.48時間後に、細胞の培地を交換する。
7.培地を、培養物が約50%のコンフルエンシーになるまで1日おきに交換する。
8.培地を、培養物が約80%のコンフルエンシーになるまで1日おきに交換する。
7.新鮮な凍結用培地を作製する:
a.10% FCSおよび10% DMSOを添加した正常培地
8.上記プロトコル(継代培養)を4まで行う。
9.細胞をPBSで洗浄する(180x g、7分間)。
10.細胞ペレットを、1 mlの凍結用培地あたり百万個となるように再懸濁し、1 mlをcryoチューブに移す。
11.Mr. Frostyボックス中で細胞をイソプロパノールで、-80℃に一晩かけて(-1℃/分)凍結する。
12.窒素タンクへ移す。
継代培養プロトコルを4まで行い;その後、
8.細胞を1 mlのPBSに百万個となるように再懸濁し、RNAペレット用に1 mlをエッペンドルフチューブに移す。
9.300x g、10分、4℃の条件で遠心分離する。
10.PBSを吸引し、ペレットを氷上で維持し、液体N2で瞬間凍結する。
11.-80℃の冷凍庫に移す。
目的:
細胞を無核抽出物で再プログラムし、遺伝子発現、形態、および成長の因子を変化させて分化状態の変化を調べる。
1フラスコのHEK細胞;1フラスコの293T;抽出物(サケ卵抽出物);対照用培地におけるインキュベーション;RPMI培地(293T);EpiLife培地(HEK);1xPBS;4℃のCa2+を含まないハンクス平衡塩溶液(HBSS);HEK細胞をフラスコから剥がすためのTE;TN(トリプシン中和)溶液;ATP(水を溶媒とする200 mMのストック);GTP(水を溶媒とする10 mMのストック);ホスホクレアチン(水を溶媒とする2 Mのストック);クレアチンキナーゼ(水を溶媒とする5 mg/mlのストック);NTP(25 mMのストック);蒸気滅菌済みのmq水;75 cm2のフラスコ;15 ml、1.5 mlのチューブ。4℃に冷却遠心分離し;1.5 mlのチューブ用および15 mlチューブ用のスイングアウトバケツローター;37℃の水浴。
1.HEK細胞を回収する(1 mlのTEで洗浄し、吸引し、3 mlのTEと5〜10分間インキュベートする)。細胞を15 mlチューブに移し、200x g、10分間、4℃の条件で遠心分離する(293T細胞を回収する(PBSで洗浄し、10 mlのRPMI培地を添加し、細胞を剥がし、50 mlチューブに移す)。
a.30 mlの氷冷PBSで1回、および10 mlの氷冷HBSSで1回洗浄する。
b.1 mlのHBSSあたり500,000個となるように細胞を再懸濁する。
2.500,000個の細胞を個々の再プログラミング用チューブに添加する。
a.1200 rpm、5分間、4℃の条件で、SWローターで遠心分離する。
b.HBSSを除去する。
3.再プログラミング用の抽出物を調製する。
a.ATP生成系を調製して氷上で維持する:ATP、GTP、クレアチンキナーゼ、ホスホクレアチンを1:1:1:1の比で混合して氷上で維持する。1回の反応あたり+0.5 mMのNTPを添加する。
b.1回の反応あたり30 μlのATP生成系を添加する。
4.抽出物(ATP生成系を含む)を、チューブ1本あたり500 μl〜500,000個の細胞となるように添加する。
a.チューブをパラフィルムで覆い、37℃の水浴で60分間インキュベートする。インキュベーション中に細胞を2回、軽く叩く。
5.培地を含む1個のフラスコあたり1本の再プログラミング用チューブを添加する。
6.37℃で5% CO2の条件でインキュベートする。
HEKaの再プログラミング
再プログラミング実験RPH。
再プログラミング後に、細胞培養物を位相差顕微鏡で評価し、正常細胞と比較した。
再プログラミングを開始してから9日目に細胞をカバースリップ上に固定した。免疫蛍光標識を、hSF細胞について記載された手順で実施した。
Claims (31)
- i)分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、またはこれらの組み合わせからなる群より選択される細胞成分、ならびにii)該細胞もしくは該卵とは異なる供給源に由来する脂質成分を含む組成物。
- 精製核酸配列もしくは合成核酸配列、ポリペプチド、炭水化物、脂質、シグナル伝達物質、または天然物、またはこれらの組み合わせをさらに含む、請求項1記載の組成物。
- 分化可能細胞が、胚性幹細胞、胚性生殖細胞、および成体幹細胞からなる群より選択される、請求項1または2記載の組成物。
- エピジェネティックな阻害剤をさらに含む、請求項1〜3のいずれか一項記載の組成物。
- クリーム、ゲル、エマルジョン、軟膏、スプレー、粉末、またはローションとして提供される、請求項1〜4のいずれか一項記載の組成物。
- 分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、ならびにこれらの組み合わせからなる群より選択される細胞成分を、局所投与に適した、クリーム、ゲル、スプレー、エマルジョン、固体、樹脂もしくはマトリックス、軟膏、粉末、またはローションとして含む組成物。
- 精製核酸配列もしくは合成核酸配列、ポリペプチド、または天然物、またはこれらの組み合わせをさらに含む、請求項6記載の組成物。
- エピジェネティックな阻害剤をさらに含む、請求項6または7記載の組成物。
- 分化可能細胞以外の供給源に由来する脂質またはタンパク質の画分をさらに含む、請求項6〜8のいずれか一項記載の組成物。
- 脂質画分が、スクアレン、脂肪族ロウ、ステロールエステル、ジオールエステル、トリグリセリド、もしくはステロール、またはこれらの組み合わせを含む、請求項9記載の組成物。
- 脂質または該タンパク質の画分が、魚類、エビ、ウニ、カエルの卵もしくは魚卵(fish roe)またはこれらの両方に由来する、請求項9記載の組成物。
- ケラチンまたはフィラグリン(filaggrin)をさらに含む、請求項6〜11のいずれか一項記載の組成物。
- グルタミン、抗感染剤、抗炎症剤、抗酸化剤、および/またはニコチンアミドをさらに含む、請求項6〜12のいずれか一項記載の組成物。
- 抗酸化助剤が、ビタミンA、C、D、もしくはE、またはこれらの組み合わせである、請求項13記載の組成物。
- ゲルが、ヒアルロン酸およびキトサンからなる群より選択される化合物を含む、請求項6〜14のいずれか一項記載の組成物。
- スプレーがエアロゾルである、請求項6〜15のいずれか一項記載の組成物。
- スプレーが皮膚上で乾燥する、請求項6〜16のいずれか一項記載の組成物。
- スプレー組成物がゲル形成成分を含む、請求項6〜17のいずれか一項記載の組成物。
- 請求項1〜18のいずれか一項記載の組成物を対象の皮膚に塗布する段階
を含む、対象の皮膚を処置する方法。 - 皮膚組織を、塗布段階に先立って、化学物質、レーザー、または物理的な力によって除去する段階をさらに含む、請求項19記載の方法。
- 皮膚の外観が改善される条件下で組成物が塗布され、
皮膚の外観を改善する段階が、瘢痕の外観を改善する段階、シワまたは日焼けによる損傷(sun damage)のある皮膚の外観を改善する段階、および皮膚疾患によって損傷した皮膚の外観を改善する段階からなる群より選択される、請求項19または20記載の方法。 - 対象が、起伏のある皮膚または損傷した皮膚を有し、組成物が、該起伏のある皮膚または該損傷した皮膚に、このような皮膚が若返る条件下で塗布される、請求項19〜21のいずれか一項記載の方法。
- 以下の段階を含む、分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、ならびにこれらの組み合わせからなる群より選択される細胞成分を含む組成物の局所投与法:
a)分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、およびこれらの組み合わせからなる群より選択される細胞成分を含む組成物と、皮膚を有する対象とを提供する段階;ならびに
b)該組成物を該皮膚に塗布する段階。 - 分化可能細胞の細胞抽出物および卵の細胞抽出物、またはこれらの組み合わせからなる群より選択される細胞抽出物が、皮膚の細胞に対する栄養として有効である、請求項23記載の方法。
- 組成物が、皮膚の創傷に塗布される、請求項23または24記載の方法。
- 以下の段階を含む、分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、ならびにこれらの組み合わせからなる群より選択される細胞成分を含む組成物の局所投与法:
i)a)分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、ならびにこれらの組み合わせからなる群より選択される細胞成分を含む組成物、
b)皮膚に創傷を有する対象、ならびに
c)創傷包帯
を提供する段階;
ii)該分化可能細胞、該細胞抽出物、該卵抽出物、該細胞抽出物の成分、該卵抽出物の成分を該創傷に塗布する段階;ならびに
iii)該創傷を該創傷包帯で覆う段階。 - 創傷包帯が以下を含む、請求項25記載の方法:
i)防水層;
ii)分化可能細胞、分化可能細胞の細胞抽出物、および卵の細胞抽出物、ならびにこれらの組み合わせからなる群より選択される細胞成分を含む栄養ゲル層。 - 胚性幹細胞、前駆細胞、生殖細胞、魚類、エビ、ウニの卵および/またはカエルの卵の細胞画分を有効量含む、細胞の成長を促す組成物。
- 皮膚の処置のため、シワの除去のため、皮膚の若返りのため、創傷治癒のため、皮膚の外観の改善のため、皮膚の損傷を予防するため、皮膚の劣化を予防するため、または皮膚に栄養を提供するための、請求項1〜18のいずれか一項記載の組成物の使用。
- 以下の段階を含む、皮膚への局所塗布用の組成物の調製法:
分化可能細胞を提供する段階、または分化可能細胞もしくは卵の抽出物もしくは画分を調製する段階;および
局所投与に適したクリーム、ゲル、スプレー、エマルジョン、固体、樹脂もしくはマトリックス、軟膏、粉末、またはローションを提供するために、該分化可能細胞または該抽出物を、皮膚への局所投与用の薬剤で製剤化する段階。 - 請求項30記載の方法によって作製される組成物。
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DK2656832T3 (en) | 2019-04-08 |
US11033587B2 (en) | 2021-06-15 |
JP6177750B2 (ja) | 2017-08-09 |
EP2101724B1 (en) | 2020-12-02 |
CA2651856A1 (en) | 2008-02-21 |
EP2656832A3 (en) | 2014-08-06 |
US20090175927A1 (en) | 2009-07-09 |
RU2008148849A (ru) | 2010-06-20 |
RU2453301C1 (ru) | 2012-06-20 |
US8075920B2 (en) | 2011-12-13 |
JP2015063528A (ja) | 2015-04-09 |
US20120027705A1 (en) | 2012-02-02 |
CA2651856C (en) | 2015-09-29 |
RU2421208C2 (ru) | 2011-06-20 |
JP5641734B2 (ja) | 2014-12-17 |
US20150290252A1 (en) | 2015-10-15 |
US20130261063A1 (en) | 2013-10-03 |
US8460713B2 (en) | 2013-06-11 |
EP2101724A2 (en) | 2009-09-23 |
EP2656832A2 (en) | 2013-10-30 |
AU2007285483B2 (en) | 2011-12-22 |
WO2008020329A2 (en) | 2008-02-21 |
AU2007285483A1 (en) | 2008-02-21 |
WO2008020329A3 (en) | 2009-10-22 |
US9066883B2 (en) | 2015-06-30 |
EP2656832B1 (en) | 2018-12-19 |
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