JP6786145B2 - 新規な筋骨格系幹細胞 - Google Patents
新規な筋骨格系幹細胞 Download PDFInfo
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- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
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Description
a)外胚葉マーカーであるネスチン(Nestin,NES)に対して陽性;
b)筋原性衛星マーカー(myogenic satellite marker)であるPax7に対して陽性;
c)中胚葉マーカーであるα−SMAに対して陽性;
d)全分化能マーカーであるLIN28に対して陰性;及び
f)中間葉幹細胞マーカーであるCD90に対して陰性。
中間葉幹細胞マーカーであるCD271に対して陰性。
全分化能マーカーであるDPPA4に対して陽性。
中胚葉マーカーであるT及びノーダル(Nodal)に対して陰性。
神経外胚葉(neuroectoderm)マーカーであるPax6に対して陽性。
腸幹細胞(intestinal stem cell)マーカーであるLGR5に対して陽性。
軟骨細胞(chondrocyte)マーカーであるSOX9に対して陰性。
筋原細胞(myoblast)マーカーであるMyoDに対して陰性。
a)外胚葉マーカーであるネスチン(Nestin,NES)に対して陽性;
b)筋原性衛星マーカー(myogenic satellite marker)であるPax7に対して陽性;
c)中胚葉マーカーであるα−SMAに対して陽性;
d)全分化能マーカーであるLIN28に対して陰性;及び
f)中間葉幹細胞マーカーであるCD90に対して陰性。
中間葉幹細胞マーカーであるCD271に対して陰性。
全分化能マーカーであるDPPA4に対して陽性。
中胚葉マーカーであるT及びノーダル(Nodal)に対して陰性。
神経外胚葉(neuroectoderm)マーカーであるPax6に対して陽性。
腸幹細胞(intestinal stem cell)マーカーであるLGR5に対して陽性。
軟骨細胞(chondrocyte)マーカーであるSOX9に対して陰性。
筋原細胞マーカーであるMyoDに対して陰性。
(i)本発明は、ESC又はiPSCから由来した筋骨格系幹細胞を提供する。
[実験材料及び実験方法]
〔実施例1.実験動物〕
Balb/c−nudeバックグラウンドの7〜10週齢のマウス(20〜24g)を全てOrient bio(seongnam,Korea)から購買した。動物関連実験の全てを全北大学校動物管理及び使用委員会のガイドラインにしたがって実施した。動物は、調整された温度(21〜24℃)及び12:12時間の明暗サイクル環境で保持され、水と食べ物に自由に接近するようにした。
H9hESC(human embryonic stem cells)をWiCell(Madison,MI,USA)から購入した。hESCは、マイトマイシンC処理により細胞分裂が停止したCF1マウス胚性線維芽細胞(mouse embryonic fibroblast,MEF)の栄養供給細胞上で培養した。hESC培養培地は、20% KnockOut Serum Replacement(以下、KSRと表記、Invitrogen,USA)、1mMグルタミン(Invitrogen,USA)、1%非必須アミノ酸(Invitrogen,USA)、0.1mM β−メルカプトエタノール(Invitrogen,USA)、及び0.1%ペニシリン/ストレプトマイシン(Invitrogen,USA)、及び15ng/ml bFGF(R&D Systems,USA)が添加されたDMEM/F12(Invitrogen,USA)で製造した。
1)250ng/mlヒトノギン(KOMA Biotech,Korea)、
2)20ng/mlヒトLIF(KOMA Biotech,Korea)、
3)15ng/ml線維芽細胞増殖因子(basic Fibroblast growth factor,FGF)(R&D Systems,USA)(FGF2信号伝達活性化剤)、
4)3μM CHIR99021(Cayman,USA)(Wnt信号活性化剤)、
5)1μM PD0325901(Cayman,USA)(ERK(extracellular signal−regulated kinase)信号抑制剤)、
6)10μM SB431542(Tocris,United Kingdom)(TGF−β/アクチビン/ノーダル(TGF−β/activin/nodal)信号抑制剤)、
7)その他:10%KSR(Invitrogen,USA)、1%N2サプリメント(supplement)(Gibco,USA)、2%B27サプリメント(Gibco,USA)、1%非必須アミノ酸(Gibco,USA)、43%DMEM/F12(Gibco,USA)、43%Neurobasal(Gibco,USA)、1mMグルタミン、0.1mM β−メルカプトエタノール、0.1%ペニシリン−ストレプトマイシン、及び5mg/mlウシ血清アルブミン(Gibco,USA)などで構成。
hiPSC(human induced pluripotent stem cells)は、Hasegawa等が開発した方式によって、sendaiウイルスを媒介にしたOCT4、KLF4、SOX2及びcMYC遺伝子をBJ線維芽細胞(fibroblast)(ATCC(登録商標)CRL2522(登録商標))に導入させて生成した(Fusaki et al.,2009,PNAS85,348−362)。hiPSCはマイトマイシンC処理で細胞分裂が停止したCF1マウス胚性線維芽細胞(MEF)の栄養供給細胞上で培養した。hiPSC培養培地は、20%KSR(Invitrogen,USA)、1mMグルタミン(Invitrogen,USA)、1%非必須アミノ酸(Invitrogen,USA)、0.1mM β−メルカプトエタノール(Invitrogen,USA)、0.1%ペニシリン/ストレプトマイシン(Invitrogen,USA)、及び15ng/ml bFGF(R&D Systems,USA)が添加されたDMEM/F12(Invitrogen,USA)で製造した。
1)250ng/mlヒトノギン(KOMA Biotech,Korea)、
2)20ng/mlヒトLIF(KOMA Biotech,Korea)、
3)15ng/ml FGF(R&D Systems,USA)(FGF2信号伝達活性化剤)、
4)3μM CHIR99021(Cayman,USA)(Wnt信号活性化剤)、
5)1μM PD0325901(Cayman,USA)(ERK(extracellular signal−regulated kinase)信号抑制剤)、
6)10μM SB431542(Tocris,United Kingdom)(TGF−β/アクチビン/ノーダル(TGF−β/activin/nodal)信号抑制剤)、
7)その他:10%KSR(Invitrogen,USA)、1%N2サプリメント(Gibco,USA)、2%B27サプリメント(Gibco,USA)、1%非必須アミノ酸(Gibco,USA)、43%DMEM/F12(Gibco,USA)、43%Neurobasal(Gibco,USA)、1mMグルタミン、0.1mM β−メルカプトエタノール、0.1%ペニシリン−ストレプトマイシン及び5mg/mlウシ血清アルブミン(Gibco,USA)などで構成。
実施例2.1で分化されたhMSSCを、実施例10.1及び実施例10.2のように、Balb/c−nudeに皮下及び腎臓内注入して分化させた試料を、2%パラホルムアルデヒド(PFA)(Wako,Japan)に4℃で一晩固定した。骨への分化の有無を調べるための試料は、4℃で2週間、PBS(pH7.2)中の0.4M EDTAで石灰を除去した。その後、試料を、エタノールとキシレンを順次に用いて脱水させた後、パラフィンに埋め込み、5μm厚に切断した。切断面をH&E及びModified Movat’s pentachrom(Cosmobio,Japan)で染色した。
Trizol試薬(Invitrogen,USA)を用いて、H9hESC、ヒト中間葉細胞(Human Mesenchymal Stem Cells;hMSCs,Lonza,Switzerland)及び実施例2.1のhMSSCなどからRNAを抽出した。RNA品質(RNA quality)は、Agilent 2100 bioanalyser及びRNA 6000 Nano Chip(Agilent Technologies,USA)で評価し、定量は、ND−2000 spectrophotometer(Thermo Inc.,USA)で行った。RNAシーケンシングのためのRNAライブラリー構築は、SENSE 3’mRNA−Seq Library Prep Kit(Lexogen Inc.,Australia)を用いて実施した。RNAシーケンシングは、NextSeq 500(Illumina Inc.,USA)で実施した。SENSE3’ mRNA−Seq readsをBowtie2 version 2.1.0でアラインメントを実施した。遺伝子発現の差異は、R version 3.2.2.内のEdgeRを用いてBIOCONDUCTOR versionで定めた。Read countsデータは、Genowiz version 4.0.5.6(Ocium Biosolutions,USA)で処理した。
本明細書で説明している「細胞免疫蛍光染色」は、次のような方法によって行った。
実施例2.1及び実施例2.2のhMSSCにトリプシン/EDTAを処理し、単一細胞懸濁液で分離させた後、PBS中の2%BSAによって非特異的結合を遮断した後、緩衝溶液[1XPBS、1%BSA、及び0.01%アジ化ナトリウム]内で、Sca、CD2、CD3、CD4、CD7、CD8、CD10、CD11b、CD14、CD19、CD20、CD31、CD34、CD44、CD45、CD51、CD56、CD73、CD90、CD105、CD146、CD166、CD235a、CD271に対するモノクローナル抗体(BD Biosciences,USA)と反応させ、洗浄した後、細胞をAlexa Fluor 488 secondary mouse−IgGs(Invitrogen、米国)で反応させ、洗浄した後、流細胞分析器(FACStar Plus Flowcytometer,BD Biosciences,USA)を用いて分析した。正常マウスIgGs(BD Biosciences,USA)を陰性対照群とした。
実施例2.1及び実施例2.2のhMSSCを骨芽細胞に分化させるために、細胞を骨形成分化培地(StemPro Osteogenesis Differentiation Kit,Life technology,USA)内37℃、5% CO2の条件で14日間培養した。骨生成の観察のためにアルカリホスファターゼ染色(alkaline phosphatase staining)(Roche,Switzerland)及びアリザリンレッドS(alizarin red S)(Sigma,USA)染色を行った。hMSC(Lonza,Switzerland)も上記の方式で同様に骨芽細胞に分化させ、比較した。
実施例2.1及び実施例2.2のhMSSCを脂肪細胞に分化させるために、細胞を脂肪形成分化培地(StemPro adipogenesis Differentiation Kit,Life technology,USA)内37℃、5% CO2の条件で14日間培養した。脂肪生成の観察のためにオイルレッドO(oil red O)(Sigma,USA)染色をした。hMSC(Lonza,Switzerland)も上記の方式で同様に脂肪細胞に分化させ、比較した。
実施例2.1及び実施例2.2のhMSSCを軟骨細胞に分化させるために、細胞を軟骨形成培地(StemPro chondrogenesis Differentiation Kit,Life technology,USA)で再懸濁した後、さらに遠心分離した。マイクロマス(micromass)の形成のために、ペレットを分化培地に1×105生菌/μlで再懸濁し、非付着された96−ウェルプレートの中央に細胞溶液5μlを点滴して接種した。高湿の条件下で2時間マイクロマスを培養した後、培養容器に、暖まった軟骨形成培地を添加し、5% CO2、37℃条件の培養器内で培養した。培養物は3〜4日ごとに再供給(re−feeded)された。培養して14日後、軟骨生成ペレットをアルシアンブルー(Alcian blue)で染色した。hMSC(Lonza,Switzerland)も上記の方式で同様に軟骨細胞に分化させ、比較した。
実施例2.1のhMSSCが内皮細胞(endothelial cells,ECs)に分化することを確認した。hMSSCをEC分化培地(endothelial growth medium(EGM)−2(Lonza,Walkersville,MD,USA)内の50ng/ml VEGF(vascular endothelial growth factor:ProSpec,Rehovot,Israel)及び10ng/ml bFGF(basic fibroblast growth factor;ProSpec,Rehovot,Israel)で6日間培養して分化させた。細胞免疫蛍光染色を実施して分化の有無を確認した。
実施例2.1及び実施例2.2のhMSSCが骨格筋細胞(skeletal muscle cells)に分化するかどうか確認した。hMSSCをマトリゲルでコートされたカバースリップ上で、骨格筋分化培地(2% B27が含まれたDMEM)で2週間培養することによって分化した。細胞免疫蛍光染色を実施して分化の有無を確認した。
神経細胞に分化させるために、実施例2.1のhMSSCをポリオルニチン及びラミニン−コートされた培養皿にプレートした。2日後、培地を神経分化培地(2% B27、2mM GlutaMAX及び抗生剤を含むNeurobasal medium)に交換した。分化7日目に、0.5mMジブチルcAMP(Sigma,USA)を3日間毎日添加した。対照群として用いるために、H9hESCから分化したヒト神経幹細胞(Gibco,USA)を、上記と同じ方式によって神経細胞に分化させた。細胞免疫蛍光染色を施して分化の有無を確認した。
実施例2.1のhMSSCのマウス腎臓内分化能を測定するために、hMSSCをMSCGM−CD(Lonza,Switzerland)培地で2〜5継代培養して単一細胞として収集した後、hMSSC(2x105cells)をアガロースゲルウェルにおいてDMEM+20%FBSで2日間培養して細胞凝集体を作り、Balb/cヌードマウスの腎臓内(kidney capsule)に移植した。移植4週後に組織化学染色と組織免疫蛍光染色を行った。
実施例2.1のhMSSCのマウス皮下における分化能を測定するために、hMSSCをMSCGM−CD(Lonza,Switzerland)培地で2〜5継代培養して単一細胞として収集した後、hMSSC(2x105cells)を1μg/mlのヒアルロン酸(Sigma,USA)の添加されたフィブリングルー(greenplast、緑十字、韓国)に搭載してBalb/cヌードマウスの皮下に移植した。移植4週後に組織化学染色及び組織免疫蛍光染色を行った。
大腿骨骨折モデルにおいてhMSCの骨形成を分析するために、hMSC(Lonza,Switzerland)をMSCGM−CD(Lonza,Switzerland)培地で7継代培養して単一細胞として収集した後、1mm×1mmで切断したコラーゲンメンブレイン(SK bioland,Korea)に細胞を吸収させた。6週齢のBalb/c−ヌードマウスにおいて片方の脛骨を1mm程度ドリル(Bosch professional,Germany)で穿孔した後、コラーゲンメンブレインが含有しているhMSCをマウスの骨折部位に挿入した。毎2週ごとにマウスを麻酔した後、骨折部位に対してMicro−CT(Skyscan1076,Antwerp,Belgium)を用いてイメージを得た。6週後に組織化学染色と組織免疫蛍光染色を行った。
大腿骨骨折モデルにおいてhMSSCの骨形成を分析するために、実施例2.1のhMSSCをMSCGM−CD(Lonza,Switzerland)培地で2〜5継代培養して単一細胞として収集した後、1mm×1mmで切断したコラーゲンメンブレイン(SK bioland,Korea)に細胞を吸収させた。6週齢のBalb/c−ヌードマウスにおいて片方の脛骨を1mm程度ドリル(Bosch professional,Germany)で穿孔した後、コラーゲンメンブレインが含有しているhMSSCをマウスの骨折部位に挿入した。毎2週ごとにマウスを麻酔した後、骨折部位に対してMicro−CT(Skyscan1076,Antwerp,Belgium)を用いてイメージを得た。6週後に組織化学染色と組織免疫蛍光染色を行った。
Micro−CT(Skyscan1076,Antwerp,Belgium)を用いて、実施例10.1で実施したhMSSCの腎臓内移植部位に生成された骨部位をスキャンすることによって、3次元の再構造化されたCT(computed tomography)イメージを得た。その後、フレームグラバを用いてデータをデジタル化し、その結果として得たイメージを、Comprehensive TeX Archive Network(CTAN)topographic reconstruction softwareを用いてコンピュータに移した。
実施例2.1のhMSSCの腎臓内移植物を500μL Trizol(Life Technologies,USA)を用いて、製造者のプロトコルにしたがってRNAを抽出した。hMSSCの腎臓内移植物にDNAse(RQ1DNase,Promega,USA)を処理した後、500ng RNAをSuperscript III RT(Life Technologies,USA)第1鎖cDNA合成プロトコルにしたがってオリゴ−d(T)及びランダムヘキサを用いてcDNAに逆転写した。qRT−PCRは、StepOne Plus PCRサイクラー(Applied Biosystems)上でSybrグリーン(Applied Biosystems,Foster City,CA)を用いて行った。mRNA発現データは△△CT方法を用いて分析し、遺伝子検出のためにグリセルアルデヒド−3−フォスフェート脱水素酵素(GAPDH)で正規化した。qRT−PCRに必要な検証されたプライマーはQuagen(USA)から購入した。対照群として用いるために、hMSSCも、同じ方法でRNAを抽出し、qRT−PCRを実施した。
〔試験例1.hESCから由来したhMSSCの分化誘導の確認〕
老化マーカー
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前記実施例2に示したように、hESCからhMSSCの分化を誘導し、誘導されたhMSSCの形態的変化を観察した結果を、図1Aに示した。図1Aに示すように、単一細胞化した未分化されたH9hESCが7継代以内に線維芽細胞の形状に単一の集団に分化したことを確認した。7継代から17継代までの10継代以上を類似の形状で成長するが、19継代以降には、老化マーカーβ−ガラクトシダーゼを染色した結果、陽性反応を示し、老化が進行されたことを観察した。
hESCから誘導されてから7継代以上が過ぎたhMSSCにおいて、全分化能マーカーの発現を免疫蛍光法で観察し、観察の結果を図1Bに示した。比較可能なように、H9hESCの全分化能マーカーの発現を免疫蛍光法で確認した結果を共に示した。
hESC、hMSC及び7継代、17継代のhMSSCにおいて全分化能、外胚葉、中胚葉及び内胚葉マーカーの発現をRNA−シーケンシングを用いて確認した結果を、図1Cに示した。H9hESC(hESC−1、hESC−2)では、TDGF、NANOG、POU5F1、SOX2、DPPA4、LEFTY1及びGDF3などの全分化能マーカーのmRNA発現が確認された。一方、H9hESCから誘導されたhMSSCでは全分化能マーカーであるDPPA4発現は観察されたが、全分化能マーカーTDGF、NANOG、POU5F1、LEFTY1及びGDF3の発現は観察されなかった。DPPA4発現は、H9hESCと類似のレベルであることを確認した。
図1Dに示すように、hMSSCの表面抗原発現を測定した。中間葉幹細胞特異細胞表面抗原の発現を調べた結果、中間葉幹細胞マーカーのうち、CD44、CD51、CD73、CD105、CD146、CD166はhMSSCで発現するが、中間葉幹細胞マーカーのうち、CD90及びCD271はhMSSCでは発現しないことを確認した。また、血液系統細胞表面標識子のCD2、CD3、CD7、CD8、CD11b、CD14、CD19、CD20、CD31、CD34、CD56は発現しなかったが、pre−B細胞マーカーであるCD10が発現していた。
図1Fに示すように、hMSSCの特性を調べるために、様々な系統の組織特異マーカー発現を分析した結果、中胚葉マーカーであるa−SMA(alpha smooth muscle actin)、神経外胚葉マーカーであるPax6、筋原性衛星マーカーであるPax7、及び腸幹細胞マーカーであるLGR5などが発現しており、軟骨細胞マーカーであるSOX9及び筋芽細胞マーカーであるMyoDなどは発現していなかった。これは、hMSSCが軟骨細胞及び筋肉細胞まで分化される前の前駆細胞であることを意味する。
hMSCと実施例2.1のhMSSCに対して試験管内骨形成、軟骨形成、脂肪形成を実験し(実施例7)、その結果を図2Aに示した。図2Aでは、hMSCが試験管内で骨、軟骨、脂肪に分化されることを確認できる。しかし、hMSCに適用した同一条件の試験管内誘導環境でhMSSCは骨、軟骨には分化されるが、脂肪にはほとんど分化されていないことを確認した。すなわち、中間葉幹細胞とは機能的に差異が存在することを確認した。
前記試験例1のhMSSCが骨格筋に分化し得る能力があるかどうかを評価した。
前記試験例1のhMSSCが内皮細胞に分化し得る能力があるかどうか評価した。
hMSSCを神経分化培地(2% B27、2mM GlutaMAX及び抗生剤を含むNeurobasal medium)で7日間インキュベーションした後、0.5mMジブチルcAMP(Sigma)を3日間毎日添加しながら培養した。その後、神経細胞への分化マーカーであるMAP2に対して細胞免疫蛍光法を行い、その結果を図2Fに示した。NSC(neuronal stem cells)を神経細胞分化の陽性対照群として用いた。図2Fに示すように、NSCは細胞形状が神経細胞形状に変わっており、神経細胞特異マーカーであるMAP2が発現したことから、神経細胞に分化することを確認し、一方、hMSSCの場合、細胞形状に変化がなく、MAP2も発現しないことから、神経細胞に分化する潜在力がないことが分かった。
実施例2と同一にして誘導されたhMSSCの分化可能性をin vivoで測定するために、hMSSCを免疫欠乏マウスの腎臓内(実施例10.1)及び皮下(実施例10.2)に移植した。マウス腎臓内にhMSSCを移植して3〜4週後に、組織をH&Eで染色し、また骨、筋肉、脂肪、腱の特異マーカーに対する免疫蛍光染色結果及びTO−PRO3で核を対照染色した結果を、図3A及び図3Bに示した。
実施例2と同一に誘導されたhMSSCの骨折回復に対する効果を確認するために、前記実施例11と共に骨折研究を行い、その結果を図4A及び図4Bに示した。
hiPSC(human induced pluripotent stem cells)は、Hasegawa等(Fusaki et al.,2009)によって開発されたプロトコルにしたがってsendaiウイルス−媒介されたOCT4、KLF4、SOX2、及びMYCの過発現によってIMR90胎児線維芽細胞をリプログラミングすることによって製造した。
腎臓内移植
マウス腎臓内に前記試験例6のhMSSCを移植して3〜4週後、組織をH&E染色時に、腎臓内で典型的な筋肉、脂肪、腱が形成されることが確認できた。移植部位の免疫組織化学分析を行うと、筋肉マーカーであるphospho−myosin light chain(pMLC)、脂肪マーカーであるPPARgamma(PPAr)、腱マーカーであるsleraxis(Scx)などが陽性であり、これらはいずれもヒト細胞マーカーであるhLA(human leukocyte antigen)に対しても陽性であることが確認できた。また、骨マーカーであるOsx(osterix)、Runx2、DMP1、OCN(osteocalin)などが陽性であることが確認できる。上記の方法により、iPSCから誘導されたhMSSCが筋肉、脂肪、腱及び骨に分化され得ることが確認できた
皮下移植
前記試験例6のhMSSCをヒアルロン酸の添加されたフィブリングルーに搭載してマウスの皮下に移植した後、H&E染色及びトルイジンブルー染色によって、前記hMSSCが軟骨に分化され得ることが確認できた。
前記実施例2のMSSC培地の7つの構成要素のうち構成要素1)のヒトノギン(Life technology)に代えて馴化培地(完全培地においてDMEM/F12をKnockout DMEMに置換した培地(20% Knockout Serum Replacement(Invitrogen,USA)、1mMグルタミン、1%非必須アミノ酸(Invitrogen,USA)、0.1mM β−メルカプトエタノール、0.1%ペニシリン−ストレプトマイシン、5mg/mlウシ血清アルブミンで補充されたKnockout DMEM)を用いてCF1細胞を24時間培養して得た培養上清液)を追加した培地(残りの構成要素2)〜7)は同様)(以下、「CM培地」という。)を用いて、MSSC培地とCM培地の分化能を比較した。
今回は前記実施例2のMSSC培地の構成要素のうち、1)〜6)の成分のいずれか1のずつを含まない場合の分化能を、全部を含む場合と比較した。実験の結果、前記1)〜6)の成分のいずれか一つの構成要素を欠乏する場合、軟骨(アルシアンブルー)や骨(ALP及びアリザリンレッドS)へと正しく分化されないことを確認した(図7、表3)。
寄託機関名:韓国細胞株研究財団
受託番号:KCLRFBP00460
受託日:20181010
(部署名)情報通信科学技術部
(研究管理専門機関)韓国研究財団
(研究事業名)幹細胞研究事業
(研究課題名)筋骨格幹細胞を用いた筋骨格系疾患治療技術開発
(寄与率)1/1
(主管機関)ジョンブク大学校
(研究期間)2017.06.30〜2019.01.29
Claims (3)
- 筋骨格系幹細胞(MSSC,musculoskeletal stem cell)への分化誘導用培地組成物であって、
ノギン(noggin)、LIF(leukemia inhibitory factor)、bFGF(basic Fibroblast growth factor)、Wnt信号活性化剤であるCHIR99021(9−ブロモ−7,12−ジヒドロ−ピリド[3’,2’:2,3]アゼピノ[4,5−b]インドール−6(5H)−オン)、ERK(extracellular signal−regulated kinase)信号抑制剤であるPD0325901(N−[(2R)−2,3−ジヒドロキシプロポキシ]−3,4−ジフルオロ−2−[(2−フルオロ−4−ヨードフェニル)アミノ]−ベンズアミド)、及びTGF−β/アクチビン/ノーダル(TGF−β/activin/nodal)信号伝達抑制剤であるSB431542(4−[4−(1,3−ベンゾジオキソール−5−イル)−5−(2−ピリジニル)−1H−イミダゾール−2−イル]ベンズアミド)を含み、
前記筋骨格系幹細胞は、骨、軟骨、腱、靭帯、筋肉および脂肪に分化し得ることを特徴とする、分化誘導用培地組成物。 - 請求項1の筋骨格系幹細胞への分化誘導用培地組成物においてESC(embryonic stem cell)又はiPS(induced pluripotent stem cell)を培養する段階を含む、筋骨格系幹細胞の製造方法。
- 前記培養は、培地組成の変化無しで5継代以上培養することを特徴とする、請求項2に記載の筋骨格系幹細胞の製造方法。
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