JP2012223191A - 多能性幹細胞の非腫瘍形成性の増殖 - Google Patents
多能性幹細胞の非腫瘍形成性の増殖 Download PDFInfo
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Abstract
【解決手段】本発明は、フィーダーとして、ヒト臍帯由来間葉系幹細胞(HUCMSCs)を含む培地におけるヒト胚性幹(hES)細胞を増殖させる方法を提供する。前記ヒト胚性幹(hES)細胞は、前記培地中で、例えば分化万能性、未分化状態での無限増殖及び正常核型といった胚性幹細胞の特性を維持する。本発明は、さらに、奇形腫を形成しないヒト胚性幹(hES)細胞の非腫瘍形成性増殖に関する方法をも提供する。
【選択図】なし
Description
HUCMSCs又はMEFフィーダー層において培養された未分化の、又は分化したhES細胞を、RLT溶解緩衝液(Qiagen社)により処理し、機械的に除去した。SuperScript III One−Step RT−PCRキット(Invitrogen社)を用い、そのメーカーの指示に従い、最初のcDNA鎖を合成した。表1に、その配列、アニーリング温度、及び各対のプライマーの生成物サイズを示す。すべてのPCRサンプルについて、0.5μg/mlの臭化エチジウム(Sigma社)を含有する2%アガロースゲルで電気泳動分析を行った。定量性RT−PCR(qRT−PCR)分析を行うために、GAPDHを内部対照とした、ABI Step One Plusシステム(Applied Biosystems社)におけるFastStartユニバーサルSYBRグリーンマスター(ROX、Roche社、米国)遺伝子発現分析を使用した。表1に、プライマーの配列と、アニーリング温度を示す。
ハンクス平衡塩溶液(HBSS:Gibco/BRL 14185−052)を含む無菌箱中において、ヒト臍帯サンプル(長さ:20cm、重さ:20g)を収集した。ヒト臍帯の収集と使用のためのプロトコールは、慈濟大学病院の治験審査委員会(Institutional Review Board)によって承認された。同意書は陣痛前の妊婦から取得した。
フローサイトメトリック分析によりMSC特異性表面マーカーの特性を確認した。その細胞を、燐酸塩緩衝食塩水(PBS)中の2mM EDTAで剥離し、洗浄してからフルオレセインイソチオシアネート(FITC)又はフィコエリスリン(PE)をコンジュゲートした適切な抗体と共にインキュベートした。抗体としては、CD1q、CD3、CD10、CD13、CD14、CD29、CD31、CD34、CD44、CD45、CD49b、CD49d、CD56、CD73、CD90、CD105、CD117、CD166、HLA−ABC及びHLA−DR(BD,PharMingen)を使用した。次に、その細胞を、Becton Dickinson社製フローサイトメーター(Vantage SE、Becton Dickinson社、米国カリフォルニア州サンノゼ)で分析した。
骨形成性と脂質生成性の分化を誘発するために、HUCMSCsを骨形成性培地(10%CBS、0.1μmol/L デキサメタゾン、10mmol/L β−グリセロール燐酸塩、50μmol/L アスコルベイトを補充したDMEM培地)と脂質生成性培地(10%CBS、1μmol/L デキサメタゾン、5μg/mL インスリン、0.5mmol/L イソブチルメチルキサンチン及び60μmol/L インドメタシンを補充したDMEM培地)に3週間転写した。骨形成性の潜在能力は、アリザリンレッドS(Sigma社、米国)で染色することで測定するカルシウムの鉱化作用により評価した。脂質生成性分化を評価するために、顕微鏡下で細胞内の脂質の小滴を観察した。当該脂質の小滴を、オイルレッドOで染色することにより検証した。
TW1株化細胞(P22、即ち、22回継代培養で得た細胞株)を工業技術研究院生物医学技術及び装置研究実験室より取得し、提供者である食品工業発展研究所(FIRDI、台湾)の指導に従い、MEFs上で培養を開始し、MEFs(P3、即ち、3回継代培養したもの)、又はHUCMSCsのどちらかをミトマイシンCで失活した後に、フィーダー細胞としてhES細胞の培養に使用した。これらは、200,000細胞数/9.4cm2/ウェルの密度で6ウェルプレートにおいて培養した。hES細胞の培養培地は、80%(v/v)ノックアウト(KO)DMEM,20%(v/v)KO血清置換、2mM L−グルタミン、10mM非必須アミノ酸(すべてInvitrogen社製)、50μM B−メルカプトエタノール(Sigma社)及び4ng/mL bFGFより構成される。
分化が発生する前にhES細胞を収集し、bFGFを欠いたhES細胞培地に再懸濁した。その後、このhES細胞を、低接着6ウェルプレートにおいて、懸濁液中の凝集した状態で培養した。5日後に、牛胎児血清(FBS)(最終濃度5%)を添加した。通常7〜10日後に、その凝集hES細胞は胚様体(EB)を形成し、その後、成熟した(嚢胞性の)EBが20〜80%のEBから出現する。その後、生成固形物又は嚢胞性のEBを、ゼラチン処理したチュンバースライド又は35mmの培養皿にプレートし、更に分化を続けさせ、その後、直接的に接着培養で育てられた細胞で同様に処理した。分化したhES細胞について、固定後に、三つの胚性胚葉に特異的な蛍光ラベル抗体を用いて免疫細胞化学的研究を行った。当該胚葉は、外胚葉としてMAP2、NF200(Chemicon社)、中胚葉として短尾奇形突然変異体、内胚葉としてATBF1(Santa Cruz社)を使用した。
hES細胞を、ガラスキャピラリを用いた機械的スライシングにより分離し、ペレット後、マトリゲル(BD Bioscience社)とPBS(1:1の比率)中に再懸濁し、非肥満性糖尿病−重度複合免疫不全症(NOD−SCID)マウスの背部皮下組織(n=17)又は腎被膜(n=4)に注入した。細胞を血球計算板を用いて測定した後、PBSとマトリゲル(1:1)との異なる濃度の混合液に懸濁した。hES細胞は注入に先立って、最適な成育能力を有するように45分間未満氷上に保持した。奇形腫の形成を触診によって追跡し、生成した腫瘍を切り離し、固定して、パラフィン中に包埋するなど、組織学的に処理した。
細胞を溶解緩衝液(150mMの食塩、50mMのトリス−塩酸[pH7.4]、1%のNonidet P−40)にプロティナーゼ阻害因子カクテル(Roche社、米国インディアナ州インディアナポリス)を加えた混合液に溶解した。タンパク質を10%のドデシル硫酸ナトリウム−ポリアクリルアミドゲルで電気泳動(SDS−PAGE)した後、ニトロセルロース膜(Hybond−C Super;Amersham社、英国リトルチャルフォント)に転写した。当該膜を抗−c−myc(2μg/m)、又は抗−α−アクチン(1:10,000;Sigma−Aldrich社)モノクロナール抗体とインキュベートした。二次抗体としてヤギ抗マウスIgG−HRPコンジュゲート(Jackson Immuno−Research Laboratories社)を用いた。増強化学発光試薬(ECL;Amersham社)を用いて結合抗体を検出した。
Claims (20)
- ヒト多能性幹細胞と臍帯由来幹細胞とを共培養することにより構成される、ヒト多能性幹細胞を増殖させる方法。
- 前記臍帯由来幹細胞は、前記ヒト多能性幹細胞の増殖用の培地中に、フィーダー層を形成する請求項1に記載の方法。
- 前記臍帯由来幹細胞は、ヒト臍帯由来間葉系幹細胞(HUCMSCs)である請求項1に記載の方法。
- 前記ヒト臍帯由来間葉系幹細胞(HUCMSCs)は、ヒト臍帯のホウォートンゼリー由来である請求項3に記載の方法。
- 前記臍帯由来幹細胞は、前記ヒト多能性幹細胞を未分化状態に維持する請求項1に記載の方法。
- 前記臍帯由来幹細胞は、一つ又は一つ以上のCD10、CD13、CD29、CD44、CD73、CD90、CD166及びHLA−ABCに対して陽性である請求項1に記載の方法。
- 前記臍帯由来幹細胞は、一つ又は一つ以上のCD1q、CD3、CD34、CD45、CD56、CD117及びHLA−DRに対して陰性である請求項1に記載の方法。
- 前記臍帯由来幹細胞は、骨形成性と脂質生成性の分化能力を有する請求項1に記載の方法。
- 前記ヒト多能性幹細胞は、ヒト胚性幹(hES)細胞である請求項1に記載の方法。
- 前記ヒト多能性幹細胞は、一つ又は一つ以上のアルカリ性ホスファターゼ(AP)、Oct−4、SSEA−1、SSEA−4,TRA−1−60、TRA−1−81、NANOG、SOX2及び分化マーカーに対して陽性であり、前記分化マーカーは、一つ又は一つ以上のNF−200、短尾奇形突然変異体、ATBF1及びMAP2である請求項1に記載の方法。
- 前記ヒト多能性幹細胞は、一つ又は一つ以上の分化遺伝子を発現し、前記分化遺伝子は、GDF9、GATA4、HAND1又はTUJ−1遺伝子である請求項1に記載の方法。
- 前記ヒト多能性幹細胞において、MYCは下方調節される請求項1に記載の方法。
- 前記ヒト多能性幹細胞の増殖は、非腫瘍形成性増殖である請求項1に記載の方法。
- 前記ヒト多能性幹細胞は、奇形腫を形成しないものである請求項13に記載の方法。
- 前記ヒト多能性幹細胞は、胚様体を形成する請求項1に記載の方法。
- 臍帯由来幹細胞を含有して構成される、ヒト多能性幹細胞を増殖させるための培地。
- 前記臍帯由来幹細胞は、前記培地中でフィーダー層を形成する請求項16に記載の培地。
- 前記臍帯由来幹細胞は、ヒト臍帯由来間葉系幹細胞(HUCMSCs)である請求項16に記載の培地。
- 臍帯由来幹細胞を含有する培地と、その取扱説明書とにより構成されるヒト胚性幹(hES)細胞を増殖させるためのキット。
- 前記臍帯由来幹細胞は、ヒト臍帯由来間葉系幹細胞(HUCMSCs)である請求項19のキット。
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JP2022120128A (ja) * | 2016-04-27 | 2022-08-17 | ロート製薬株式会社 | Cd201、cd46、cd56、cd147及びcd165からなる群より選択される少なくとも1種の細胞表面マーカーを発現する間葉系幹細胞及びその調製方法、並びに上記間葉系幹細胞を含む医薬組成物及びその調製方法 |
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