JP2008512122A - 胚幹細胞の培地及び培養 - Google Patents
胚幹細胞の培地及び培養 Download PDFInfo
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- JP2008512122A JP2008512122A JP2007531300A JP2007531300A JP2008512122A JP 2008512122 A JP2008512122 A JP 2008512122A JP 2007531300 A JP2007531300 A JP 2007531300A JP 2007531300 A JP2007531300 A JP 2007531300A JP 2008512122 A JP2008512122 A JP 2008512122A
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Abstract
Description
本出願は、2004年9月8日に提出された米国仮特許出願Ser. No. 60/608,040からの優先権を主張する。
本発明は、以下の機関:NIH RR17721によって授与された米国政府援助によってなされた。米国は本発明における一定の権利を有する。
幹細胞は、多くの他の分化細胞タイプに分化することのできる細胞として定義される。胚幹細胞は、成熟体の、全てではないが、大部分の分化細胞タイプに分化することができる胚からの幹細胞である。幹細胞は多分化能と呼ばれ、これは多くの細胞タイプに分化するこの能力を表現している。研究団体が高い関心のある多分化能幹細胞の部類は、ヒト胚幹細胞、ここでは略してヒトES細胞であり、ヒト胚源由来の胚幹細胞である。ヒト胚幹細胞が非常に科学的関心が高いのは、それらが培養液中で無制限に増殖でき、及びそれによって、少なくとも原理的には、不備又は欠陥のあるヒト組織の代わりとなる細胞及び組織を供給することができるからである。ヒト胚幹細胞が培養液中に存在することは、ヒトの健康を補助する種々の治療プロトコルに使用するためのヒト細胞及び組織の無制限量の潜在能力を提供する。将来的には、ヒト胚幹細胞は増殖され、及び特定の系統に分化する方向に向かい、治療目的のために人体に移植することのできる分化細胞又は組織を開発することが想定される。ヒト胚幹細胞及びそれらから生じ得る分化細胞は、ヒト細胞及び発生系の研究のための強力な科学的ツールにもなる。
本発明は、支持細胞又はならし培地を必要としないヒト胚幹細胞を培養するための、塩、ビタミン、アミノ酸、グルコース、線維芽細胞成長因子、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを、全てが複数の培養継代中に該幹細胞を未分化状態に保持するのに十分な量で含む培地でヒト胚幹細胞を培養する工程を含む方法として要約される。
本発明は、幹細胞を線維芽細胞支持細胞又はならし培地を必要とせずに無制限に未分化状態で培養することのできる、高濃度の線維芽細胞成長因子、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを含む培地で培養されるヒト胚幹細胞の生体内細胞における培養も意図している。
本発明の目的は、支持細胞からの又は幹細胞が培養されているならし培地のための、動物細胞及び動物性蛋白質の使用又はそれらへの露出を回避するヒト胚幹細胞のための長期培養条件を定義することである。
本発明の別の目的は、培養液中の最大割合の細胞をできる限り未分化状態でできる限り保持する上記のヒト胚幹細胞のための培養条件を定義することである。
本発明の他の目的、特徴及び利点は、以下の明細書から明らかとなる。
未分化状態且つさらに支持細胞及びならし培地の不存在下でのヒト胚幹細胞の無限培養及び活気のある増殖を可能にする複数の培養条件及び培地を確認した。これらの培地及び培養条件の開発が、任意の種類の動物細胞に直接的又は間接的に露出されることなく、定義され且つ制御された条件でのヒトES細胞系の誘導及び保持を可能にする。これらの培地は、未分化ES細胞の増殖を、これらの培地がそのような培養を無制限に支持することが確実に明らかである少なくとも5回の複数の継代中に支持することが証明された。
特に明記しない限り、ここに記載される全ての培養液に用いたTeSR1培地の成分を、以下の表1に述べる。我々の予備実験は、未分化ヒトES細胞増殖が7.2のpH、350mOsMolのモル浸透圧濃度、及び10%CO2/5%O2の気圧で最適であることを示した。これらの条件を、ここで記載される全てのその後の培養液に用いた。
無機塩 mM
塩化カルシウム(無水物) 0.8232
HEPES 11.76
塩化マグネシウム(無水物) 0.2352
硫酸マグネシウム(MgSO4) 0.319088
塩化カリウム(KCl) 3.26144
重炭酸ナトリウム(NaHCO3) 11.2112
塩化ナトリウム(NaCl) 94.55824
リン酸ナトリウム、二塩基(dibas)(無水物) 0.392
リン酸ナトリウム、モノ(NaH2PO4-H2O) 0.355152
硝酸鉄(Fe(NO3)3-9H2O) 0.00009408
硫酸鉄(FeSO4-7H2O) 0.001176
硫酸銅(CuSO4-5H2O) 4.0768E-06
硫酸亜鉛(ZnSO4-7H2O) 0.001176
メタバナジン酸アンモニウムNH4VO3 0.000056
硫酸マンガンMnSO4 H2O 1.00592E-05
モリブデン酸アンモニウム 1.00404E-05
NiSO4 6H2O 4.94861E-06
メタケイ酸ナトリウムNa2SiO3 9H2O 0.004926108
SnCl2 5.32544E-05
CdCl2 6.21931E-05
CrCl3 9.41176E-06
AgNO3 5.00293E-06
AlCl3 6H2O 2.4855E-05
Ba(C2H3O2)2 4.99217E-05
CoCl2 6H2O 5.0021E-05
GeO2 2.5337E-05
KBr 5.04202E-06
KI 5.12048E-06
NaF 0.000500119
L-アラニン 0.1392
L-アルギニンヒドロクロライド 0.5488
L-アスパラギン-H2O 0.1392
L-アスパラギン酸 0.1392
L-システイン-HCl-H2O 0.0784
L-シスチン 2HCl 0.0784
L-グルタミン酸 0.1392
L-グルタミン 2.96
グリシン 0.296
L-ヒスチジン-HCl-H2O 0.1176
L-イソロイシン 0.326144
L-ロイシン 0.353584
L-リシンヒドロクロライド 0.391216
L-メチオニン 0.090944
L-フェニルアラニン 0.16856
L-プロリン 0.2176
L-セリン 0.296
L-トレオニン 0.352016
L-トリプトファン 0.0346528
L-チロシン 2Na 2H2O 0.167776
L-バリン 0.354368
アスコルビン酸 0.375
ビオチン 1.12112E-05
塩化コリン 0.0502544
D-カルシウムパントテナート 0.0036064
葉酸 0.004704
i-イノシトール 0.05488
ナイアシンアミド 0.012936
ピリドキシンヒドロクロライド 0.0076048
RbCl 5.00414E-05
ZrOCl2 8H2O 9.03834E-05
リボフラビン 0.0004704
チアミンヒドロクロライド 0.02460217
ビタミンB12 0.000392
GABA 0.979
ピペコリン酸 0.000984
bFGF 5.80E-06
LiCl 0.979
TGFベータ1 2.35E-08
リノール酸 0.0070976
リポ酸 0.00039984
アラキドン酸 0.001312
コレステロール 0.0113798
DL-アルファトコフェロール-アセテート 0.02962
リノレン酸 0.007184
ミリスチン酸 0.008758
オレイン酸 0.00708
パルミトレイン酸 0.007862
ステアリン酸 0.00703
D-グルコース 13.72784
ピルビン酸ナトリウム 0.392
ヒトインスリン 0.0034438
ヒトホロトランスフェリン 0.14
ヒト血清アルブミン 199.7
グルタチオン(還元型) 0.00592996
ヒポキサンチンNa 0.01176
フェノールレッド 0.0159936
プトレッシン-2HCl 0.000394352
チミジン 0.001176
2-メルカプトエタノール 0.1
セレン 0.000177304
Pluronic F-68 0.238
Tween 80 0.3358
Claims (16)
- 支持細胞又はならし培地を必要としない基質上でヒト幹細胞を未分化状態で培養するための、塩、ビタミン、アミノ酸、グルコース、線維芽細胞成長因子、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを、複数回の連続する培養継代中に該ヒト幹細胞を未分化状態で保持するのに十分な量で含む培地において、該ヒト幹細胞を培養する工程を含む、方法。
- 培地が、少なくとも40ng/mLの濃度で線維芽細胞成長因子を含む、請求項1記載の方法。
- 培地が、トランスフェリン及びインスリンも含む、請求項1記載の方法。
- ヒト幹細胞が胚幹細胞である、請求項1記載の方法。
- 基質上でヒト胚幹細胞を培養する方法において、及び塩、ビタミン、アミノ酸、及び線維芽細胞成長因子を含む培地において、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを、複数回の連続する培養継代中に該細胞を未分化状態で保持するのに十分な量で該培地に加えることを含む、方法。
- ヒト幹細胞;
幹細胞が成長することのできる基質;及び
塩、ビタミン、アミノ酸、グルコース、線維芽細胞成長因子、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを、複数回の培養継代中に該ヒト幹細胞を未分化状態で保持するのに十分な量で含む培養培地であって、支持細胞を含まず、且つ支持細胞に決して露出されない培地:
を培養容器内に含む、生体外細胞培養。 - 培地が、少なくとも40ng/mLの濃度で線維芽細胞成長因子を含む、請求項6記載の細胞培養。
- 培地が、少なくとも100ng/mLの濃度で線維芽細胞成長因子を含む、請求項6記載の細胞培養。
- 培地が、アルブミン、インスリン及びトランスフェリンからなる群から選択される蛋白質をさらに含む、請求項6記載の細胞培養。
- 蛋白質がヒト型である、請求項6記載の細胞培養。
- インスリン及びトランスフェリンが組み換え型蛋白質である、請求項10記載の細胞培養。
- 幹細胞がヒト胚幹細胞である、請求項6記載の細胞培養。
- 未分化ヒト幹細胞、基質及び培養培地を含むヒト胚幹細胞の培養であって、該培養は支持細胞又は支持細胞に露出された培地を含まず、該培地は非ヒト型動物からの生成物も含まず、該ヒト胚幹細胞は未分化状態で増殖し、且つ長期培養中に転写因子Oct4に対して少なくとも90%陽性である、培養。
- 細胞がシアル酸残渣Neu5Gcを示さない、請求項13記載のヒト胚幹細胞の培養。
- 幹細胞を培養するための培地であって、塩、ビタミン、アミノ酸、グルコース、線維芽細胞成長因子、ガンマアミノ酪酸、ピペコリン酸、リチウム及び形質転換成長因子ベータを、複数回の培養継代中に該培地において未分化状態で成長した幹細胞を保持するのに十分な量で含む、培地。
- 培地が、アルブミン、インスリン及びトランスフェリンからなる群から選択される蛋白質をさらに含む、請求項15記載の培地。
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