JP6923134B2 - 間葉系幹細胞由来エキソソーム - Google Patents
間葉系幹細胞由来エキソソーム Download PDFInfo
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- JP6923134B2 JP6923134B2 JP2017533112A JP2017533112A JP6923134B2 JP 6923134 B2 JP6923134 B2 JP 6923134B2 JP 2017533112 A JP2017533112 A JP 2017533112A JP 2017533112 A JP2017533112 A JP 2017533112A JP 6923134 B2 JP6923134 B2 JP 6923134B2
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Description
本発明における間葉系幹細胞としては、例えば、骨髄、脂肪、筋肉、神経、皮膚、羊膜、胎盤、絨毛膜、脱落膜又は臍帯由来の間葉系幹細胞が挙げられ、好ましくは骨髄、脂肪、胎盤由来の間葉系幹細胞であり、更に好ましくは脂肪由来の間葉系幹細胞である。
本発明における間葉系幹細胞は、例えば以下の方法によって培養することができる。即ち、組織由来の間葉系幹細胞、株化された間葉系幹細胞等の間葉系幹細胞を馴化培地中で間葉系幹細胞を培養し、続いて非馴化培地中で間葉系幹細胞を培養することができる。
本発明の間葉系幹細胞由来微粒子は、間葉系幹細胞から得られる微粒子である。間葉系幹細胞由来微粒子は、間葉系幹細胞により産生される微粒子である。典型的には、微粒子は間葉系幹細胞から分泌される。
本発明における間葉系幹細胞由来微粒子は、間葉系幹細胞を馴化培地で拡大培養を行ってサブコンフルエント(あるいはコンフルエント)にし、新しい馴化培地へと交換してから、更に通常1〜5日(例えば、1日、2〜3日、3〜4日)間培養を行って、その培養上清より回収することができる。
角膜上皮幹細胞は、増殖能(未分化性又はコロニー形成能)を有し、そして角膜上皮幹細胞特異的マーカーを発現する。角膜上皮幹細胞は、角膜上皮細胞に分化することができる。角膜と結膜の間の輪部には、角膜上皮細胞及び/又は角膜上皮幹細胞が存在する。
本発明の間葉系幹細胞由来微粒子が、角膜上皮幹細胞及び/又は角膜上皮細胞の増殖促進活性、角膜上皮幹細胞の未分化維持活性又は角膜上皮幹細胞のコロニー形成促進活性、或いは角膜上皮保護作用を有することについては、当業者に公知の手法により評価することができる。
角膜上皮幹細胞の増殖能は、例えば、本発明の間葉系幹細胞由来微粒子の存在下で、角膜上皮幹細胞を培養系へと播種し継代培養を行い、角膜上皮幹細胞が増殖することを確認することによって、評価することができるが、これに限定されない。角膜上皮幹細胞が増殖することは、継代培養でコロニーを形成することにより確認することができる。好ましくは、コロニーを形成することを確認するための継代培養は、フィーダー細胞(例えば、NIH/3T3細胞など)との共培養系で行なうこと、または、上皮系細胞や上皮幹細胞用の培地で行なうことができるが、これに限定されるものではない。前記増殖能は、自己複製能であることが好ましい。細胞が自己増殖能を有することの評価については、これに限定されるものではないが、例えば、細胞が増殖能を有することに加えて、増殖後の細胞が有する特質が継代培養によって変化しないことを指標とすることができる。
増殖能を有する角膜上皮幹細胞は、後述の角膜上皮幹細胞マーカーによって確認することができるが、これに限定されるものではない。
角膜上皮幹細胞マーカーを発現していることの具体的な検出方法は公知であるが、例えば、レポーター遺伝子の発現を検出するか、または免疫細胞化学による検出することができる。分化誘導を行なう多能性幹細胞に上記角膜上皮幹細胞マーカーのレポーター遺伝子(例えば、角膜上皮幹細胞マーカーをコードする遺伝子のプロモーター領域と、緑色蛍光タンパク質(GFP)等の蛍光タンパク質とを連結したレポーター遺伝子)を導入し、分化誘導後にレポーター遺伝子の発現を検出してもよい。抗体染色法を用いた顕微鏡観察による検出、セルソーター(フローサイトメーター)による細胞表面マーカーの検出などが例示される。
本発明は、間葉系幹細胞由来微粒子の存在下で培養することを特徴とする、角膜上皮幹細胞の培養方法にも関する。
(1)間葉系幹細胞由来微粒子の存在下で、角膜の輪部から採取した角膜上皮系細胞を培養する工程、および
(2)前記培養した細胞から、角膜上皮幹細胞を選択する工程。
本発明の角膜上皮細胞シートは、本発明の培養方法により得られる角膜上皮幹細胞および/または角膜上皮前駆細胞に由来する、角膜上皮細胞シートである。前記角膜上皮細胞シートは、角膜上皮細胞シートの重層化シートであってもよい。
本発明は、間葉系幹細胞由来微粒子を有効成分として含有する、角膜上皮疾患の予防又は治療剤にも関する。
脂肪由来間葉系幹細胞(AD−MSC)は、Poietics Adipose−derived stem cells( PT−5006、Lonza)を使用した。
ヒト角膜上皮系細胞(HCEC)は、以下のようにして調製した。ヒト輸入角膜(Sight Life、WA)の輪部からヒト角膜上皮系細胞を採取し、10cmdish(353003、Falcon)においてCnT−Prime、Epithelial Culture Medium(CnT−PR、CELLnTEC)を用いて37℃、5%CO2の環境下のインキュベーターにて培養し、70−90%の密度まで培養した。PBS(14190−144、Gibco)10mlで1回washした後、TrypLE Express(12605−010、Gibco)2mlで5〜10分間、37℃、5%CO2のインキュベーター内にて酵素反応後、PBS8mlを添加して、遠心機(LC−230、TOMY)1300rpm、5分間遠心操作を行い、上清を除去した。ペレットをSTEM−CELLBANKER(CB046、ZENOAQ)を用いて1×106cells/mlとなるように懸濁し、使用時まで-150℃の冷凍庫に保管した。
AD−MSC培養およびエキソソーム回収のための培地は、MSCGM−CD BulletKit(00190632、Lonza)を使用した。
1.エキソソーム抽出用培養上清の回収
AD−MSC(PT−5006、Lonza)(Passage4〜6)をT−75フラスコ(353136、Falcon)にて5000cells/cm2の密度にて13mlのMSCGM−CDを用いて37℃、5%CO2の環境下のインキュベーターで培養を行った。70−90%の密度に達したら新しい13mlMSCGM−CDへと交換し、それから3日間同様の環境下で培養を行い、その培養上清をエキソソーム単離用に回収した。エキソソームの単離に使用するまでは15mlチューブ(352096、Falcon)に分注して−80℃にて保管した。
以下の報告に従って超遠心を行った。参考にした方法を図1に示す。
(Thery, C., Amigorena, S., Raposo, G., & Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino [et Al.], Chapter 3, Unit 3.22. doi:10.1002/0471143030.cb0322s30)
1.エキソソームの効果検討
フィーダーとして、24well plate(353047、Falcon)にマイトマイシンC(マイトマイシン注用 2mg、協和発酵キリン株式会社) 8μg/mlで2時間処理したNIH/3T3細胞を1.0×104cells/cm2として10%FBS DMEMを用いて播種した。サブコンフルエントになるまで10cmdishで培養したHCEC(passage=2)の培養上清を除き、PBS10mlでwash後、TrypLE Express(12605−010、Gibco)2mlを添加し37℃、5%CO2の環境下のインキュベーターにて酵素反応後細胞をはがし、PBS8 mlを添加して15 mlチューブへと移し、遠心機(LC−230、TOMY)にて1300rpmで室温で5分間遠心を行いHCECのペレットを調製し、それを5%FBS KCMにて懸濁後、200cells/wellとなるよう、NIH/3T3フィーダーの上へと播種した。培地量は合計300μl/wellとなるようにした。培地は2-3日に1度、合計3度交換し、エキソソームはそのたびに新しいものへと交換し、10日間同じ環境下で培養を続けた。エキソソームはBCAアッセイにより測定したタンパク量換算で20あるいは30μg/mlの濃度となるように5%FBS KCM培地で調整し処理を行った。
培養液を除去し、PBS500μlで1回洗い、10%ホルムアルデヒド緩衝液300μlを入れ2時間以上RTで固定した。その後、超純水500μlでwellを1回洗い、2%RhodamineB溶液300μlを加え、室温で30分間以上反応させた後、0.2M HClで1〜3回ウェルを洗い、室温にて乾燥後、ウェルをスキャナー(GT−F740、EPSON)でスキャン、あるいはEVOS FL AUTO(Thermo Fisher Scientific)で撮影して、画像を得た(図2)。それを基にコロニー数を計測した。Colony−Forming Efficincy(CFE)(%)は「100×コロニー数/播種した細胞の数」で算出した。図3に示すようにコントールの細胞のCFEは、5.6%であり、エキソソーム処理した細胞のCFEは、9.1%であり、エキソソーム処理によって有意にコロニー形成が増加した。以上の結果から、AD−MSC由来のエキソソームはHCECのコロニー形成促進作用を有することが確認された。
スキャナーで取り込んだウェルの画像をImage J 1.45Sを用いて解析して、それぞれのコロニーの面積値を得た。結果を図4に示す。エキソソームによって、一つあたりのコロニーを大きくする効果があることが明らかとなった。このことからエキソソームがHCECのコロニーに対して細胞増殖促進活性を有していることが示された。
<統計解析>
統計解析はR version 3.1.1、EZR version 1.25を用いて行った。
1.K12の遺伝子発現解析
培養上清を除去後、PBS 500μlにて1度洗浄し、得られたHCECのコロニーをウェルごとにQIAzol Lysis Reagent(79306、QIAGEN)1ml添加して、TotalRNAを回収し、プロトコールにしたがって、total RNAを精製後、SuperScript III First−Strand Synthesis SuperMix for qRT−PCR (18080400、Thermo Fisher Scientific)を用いて逆転写反応を行い、cDNAを得た。GAPDH、およびK12のTaqMan(R)Gene Expression assay(それぞれ、Hs99999905_m1、Hs00165015_m1)を用いてApplied Biosystems 7500 Fastを使用し、リアルタイムPCR法を実施した。結果は比較Ct法を用いて、GAPDHの発現量に対する相対的量として示した。結果を図5に示す。
形成したコロニーをPBS500μlで1度洗い、−30℃で冷却したMeOH300μlで30分間、室温で固定した。その後5%NST(5%Normal Donkey Serum、0.3%Triton/TBS)300μlで1時間、室温でブロッキングを行い、1%NST(1%Normal donkey serum、0.3%Triton/TBS)で100倍に希釈したCytokeratin 12 Antibody(N−16)(SC−17098、Santa Cruz Biotechnology)300μlを加えて、3時間、室温で処理した。3回PBS300μlで洗った後、200倍に希釈したDonkey anti−Goat IgG(H+L) Secondary Antibody,Alexa Fluor 647 conjugate(A21447、Thermo Fisher Scientific)300μlを加えて、で1時間、室温で処理し、最後の10分間Hoechstで処理をし、核を染色した。3回PBS μlで洗った後、PBS 300 ulを加え、蛍光倒立顕微鏡AxioObserver D1(Carl Zeiss)で647の波長でサンプルを撮影した。結果を図6に示す。以上の結果から、HCECの分化マーカーに対するAD−MSC由来エキソソームの抑制作用が示された。
K3、K14、K15、p63(TP63)、N−cadherin(CDH2)、PAX6は、K12の遺伝子発現解析方法にしたがって行った。Taqman Gene Expression Assayは以下のものを用いた。K3(Hs00365080_m1)、K12(00165015_m1)、K14(Hs00559328_m1)、K15(Hs00267035_m1)、p63(Hs00978339_m1)、N−Cadherin(Hs00983056_m1)、PAX6(Hs00240871_m1)これらの結果を図5に示す。角膜上皮細胞の分化マーカーであるK3や分化の際に重要な役割を果たすPAX6などの発現を抑制する一方、角膜上皮幹細胞の幹細胞マーカーである、K14、K15、p63、N−Cadherinの発現は増加させることがわかった。以上のことからAD−MSC由来エキソソームは角膜上皮幹細胞の分化を抑制し、未分化な状態を維持することでコロニー形成の促進作用を示すことが示唆された。
・エキソソーム中タンパクのショットガン解析
前記の方法に従ってエキソソームペレットを得た。エキソソームペレットは10μlの100mMTris−HCl pH8.0に2%Sodium Deoxycholate(190−08313、Wako)、および1XProtease Inhibitor CocktailセットI(165−26021、Wako)を添加したものを加え、ボルテックス後4℃、O/Nで静置して懸濁した。。BCAアッセイキット(Pierse、23227)によるタンパク定量を行い、1μg/μlに希釈したもの10μlをショットガン解析に使用した。解析はQTRAP 5500(AB SCIEX)を使用した。
前記のエクソソームは、CD9、CD63、CD81、GAPDH,PKM、Enolase−1、40S ribosomal protein S2、S5、SA、S13、S23、S4、S16、S9、60S acidic ribosomal protein P0、P1、60S ribosomal protein L9、HSPB1、HSP7C、14−3−3 protein zeta/delta、epsilon、theta、beta/alpha、gamma、eta、Syntenin、TSG101、Actin Cytoplasmic−1、Cofilin−1、Annexin A1、A2、A5、A6、A7、A11、Rab−1B、7a、8B、11A、13、35、ICAM−1,Integrin alphaV、alpha−2、alpha−4、alpha−5、beta−1、beta−5を含むことが確認された。
骨髄由来間葉系幹細胞(BM)は、D10132(タカラ)、臍帯由来間葉系幹細胞(UC)はKW−4009(KURABO)ヒト正常皮膚繊維芽細胞(NHDF)はKF−4009(KURABO)を使用して、前記の方法によってAD-MSCを含むそれぞれの培養上清からエキソソームを単離した。コントロール群(CTL)では細胞を培養していないMSCGM−CDに対して同様の処理を行ったものを使用した。エキソソームペレットをProtease Inhibitor Cocktail Set I(Wako、165−26021)を添加したRIPA Lysis and buffer(89900、Thermo Fisher Scientific)10 ulを使用して懸濁した。微量超音波ホモジナイザーQSONICA Q125を用いて20%、5秒で処理してエキソソームを破砕し、4℃、15分間14,000Gで遠心して上清を回収した。BCAキット(Pierce、23227)によって上清のタンパク定量後、サンプルに4 x NuPAGE LDS Sample Buffer(Bio−Rad)4分の1量添加して70℃、10分間処理したヒートブロックで処理したサンプルをタンパク量換算で3 μgずつ4−12%のNuPAGE Novex Bis−Trisゲル(invitrogen)へとアプライし、SDS−PAGEを行った。iBlotシステム(Invitrogen)を用いてPVDFメンブレンに転写した後,5% skim milk/PBS中で室温にて1時間ブロッキングした。0.05% Tween20含有TBS(TBS−T)で5分間3回洗浄したメンブレンを4℃、オーバーナイトで一次抗体に反応させ,TBS−Tで5分間3回洗浄した後,二次抗体に室温で1時間反応させた。一次抗体には,抗CD63抗体(10628D、Thermo Fisher Scientific、1:1000希釈(TBS))を使用し、二次抗体には,HRP標識抗マウスIgG抗体(1:10,000倍希釈(TBS))を使用した.発光にはECL prime(GE healthcare Bio−Sciences)を使用し,ChemiDoc XRS(Bio−Rad)にて検出した。AD−MSC由来のエキソソームは、CD63を発現し、UC−MSC(臍帯由来の間葉系幹細胞)及びBM−MSC(骨髄由来の間葉系幹細胞)由来のエキソームも、AD−MSC由来のエキソソームより少ないものCD63を発現していた。これらのことから、他の細胞と比較し、AD−MSCがより多くのエキソソームを分泌している可能性が示唆された。
Claims (8)
- 間葉系幹細胞由来エキソソームを有効成分として、タンパク量換算で20μg/mL以上含有し、上記間葉系幹細胞が脂肪由来、胎盤由来又は骨髄由来である、角膜上皮幹細胞及び/又は角膜上皮細胞の増殖促進剤、角膜上皮幹細胞の未分化維持剤又は角膜上皮幹細胞のコロニー形成促進剤、或いは角膜上皮保護剤。
- 間葉系幹細胞由来エキソソームを有効成分として、タンパク量換算で20μg/mL以上含有し、上記間葉系幹細胞が脂肪由来、胎盤由来又は骨髄由来である角膜上皮疾患の予防又は治療剤。
- 角膜上皮疾患が、熱腐蝕、アルカリ腐蝕、酸腐蝕、薬剤毒性、Stevens−Johnson症候群、眼類天疱瘡、(再発)翼状片、遷延性角膜上皮欠損、角膜穿孔、角膜周辺部潰瘍、角膜潰瘍、エキシマレーザー術後の上皮剥離、放射線角膜症、無虹彩症、トラコーマ後角膜混濁、Salzmann角膜変性、角膜びらん、瞼球癒着、原因不明の角膜上皮の幹細胞の消失した疾患、輪部腫瘍、宿主対移植片疾患(GVHD)、角膜炎、点状表層角膜症、ドライアイ、乾性角結膜炎、角膜上皮幹細胞疲弊症、角膜ジストロフィー、糖尿病角膜症、及び角膜上皮障害からなる群より選択される、請求項2記載の角膜上皮疾患の予防又は治療剤。
- 角膜上皮疾患が、角膜上皮障害、点状表層角膜症、角膜びらん、角膜周辺部潰瘍、遷延性角膜上皮欠損、ドライアイ、エキシマレーザー術後の上皮剥離、熱腐蝕、アルカリ腐食、酸腐蝕、薬剤毒性、糖尿病角膜症、角膜上皮幹細胞疲弊症、Stevens−Johnson症候群、眼類天疱瘡、無虹彩症、原因不明の角膜上皮の幹細胞の消失した疾患、及び移植片対宿主病(GVHD)からなる群より選択される、請求項2記載の角膜上皮疾患の予防又は治療剤。
- 角膜上皮疾患が、角膜上皮障害、点状表層角膜症、角膜びらん、角膜周辺部潰瘍、遷延性角膜上皮欠損、ドライアイ、エキシマレーザー術後の上皮剥離、熱腐蝕、アルカリ腐食、酸腐蝕、薬剤毒性、糖尿病角膜症である、請求項2記載の角膜上皮疾患の予防又は治療剤。
- 角膜上皮幹細胞疲弊症が、外的要因、内的要因、先天的な欠損、又は腫瘍性疾患により引き起こされる、請求項4記載の角膜上皮疾患の予防又は治療剤。
- 間葉系幹細胞由来エキソソームがタンパク量換算で20μg/mL以上存在する条件下で培養することを特徴とし、上記間葉系幹細胞が脂肪由来、胎盤由来又は骨髄由来である角膜上皮幹細胞の培養方法。
- 請求項7記載の培養方法によって得られる、角膜上皮幹細胞及び/又は角膜上皮細胞。
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