JP2012533321A - ヒト胚性幹細胞の分化 - Google Patents
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Abstract
Description
本発明は、米国特許公開番号第61/226,936号(2009年7月20日出願)に対する優先権を請求する。
本発明は、多能性幹細胞からのインスリン産生細胞への分化を促進させるための方法を提供する。具体的には、本発明は、動物への移植後にインスリンを産生することのできる細胞を産生するための方法を提供する。
a.多能性幹細胞を培養する工程、
b.多能性幹細胞を、胚体内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程、及び
c.胚体内胚葉系に特徴的なマーカーを発現している細胞を、FGF7を添加した第1培地で処理し、次いで細胞を、FGF7、BMP阻害能を持つ因子、TGF−β受容体アゴニスト、レチノイン酸、及びヘッジホッグ(hedgehog)シグナル経路阻害剤を添加した第2培地で培養することにより、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程。
幹細胞は、単一の細胞レベルにて自己複製し、分化して後代細胞を生成する、それら両方の能力で定義される未分化細胞であり、後代細胞としては、自己複製前駆細胞、非再生前駆細胞、及び最終分化細胞が挙げられる。幹細胞はまた、in vitroで複数の胚葉(内胚葉、中胚葉及び外胚葉)から様々な細胞系の機能的細胞へと分化する能力によって、また移植後に複数の胚葉の組織を生じ、胚盤胞への注入後、全部ではないとしてもほとんどの組織を提供する能力によっても、特徴付けられる。
多能性幹細胞の特徴付け
多能性幹細胞は、ステージ特異的胚抗原(SSEA)3及び4、並びにTra−1−60及びTra−1−81と呼ばれる抗体によって検出可能なマーカーのうちの1つ以上を発現している(Thomsonら、Science 282:1145,1998)。in vitroで多能性幹細胞を分化させると、SSEA−4、Tra−1−60、及びTra−1−81の発現が減少し(存在する場合)、SSEA−1の発現が上昇する。未分化の多能性幹細胞は通常アルカリホスファターゼ活性を有し、これは、細胞を4%パラホルムアルデヒドで固定した後、製造業者(Vector Laboratories(Burlingame Calif.))によって記載されるようにVectorRedを基質として現像することによって検出することができる。未分化の多能性幹細胞はまた、RT−PCRにより検出されるように、一般にOCT4及びTERTも発現する。
使用が可能な多能性幹細胞の種類としては、妊娠期間中の任意の時期(必ずしもではないが、通常は妊娠約10〜12週よりも前)に採取した前胚性組織(例えば胚盤胞など)、胚性組織、胎児組織などの、妊娠後に形成される組織に由来する多能性細胞の株化細胞系が挙げられる。非限定的な例は、例えばヒト胚幹細胞株H1、H7、及びH9(WiCell)などのヒト胚幹細胞又はヒト胚生殖細胞の確立株である。それらの細胞の最初の樹立又は安定化中に本開示の組成物を使用することも想定され、その場合、供給源となる細胞は、供給源となる組織から直接採取した一次多能性細胞であろう。フィーダー細胞の不在下で既に培養された多能性幹細胞集団から採取した細胞も好適である。例えば、BG01v(BresaGen、Athens、GA)などの変異ヒト胚性幹細胞株も好適である。
一実施形態では、多能性幹細胞は、典型的にはフィーダー細胞の層上で培養され、このフィーダー細胞は、多能性幹細胞を様々な方法で支持する。あるいは、多能性幹細胞を、フィーダー細胞を本質的に含まないにも関わらず、細胞を実質的に分化させることなく多能性幹細胞の増殖を支持するような培養システム中で培養する。フィーダー細胞不含培養における多能性幹細胞の、分化を伴わない増殖は、あらかじめ他の細胞種を培養することにより馴化培地を使用して支持される。あるいはフィーダー細胞不含培養における多能性幹細胞の分化を伴わない増殖は、合成培地を使用して支持される。
一実施形態では、本発明は、多能性幹細胞から、膵臓内胚葉系に特徴的なマーカーを発現している細胞を産生するための方法を提供し、この方法は以下の工程a、b及びcを包含する:
a.多能性幹細胞を培養する工程、
b.多能性幹細胞を、胚体内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程、及び
c.胚体内胚葉系に特徴的なマーカーを発現している細胞を、膵臓内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程。
胚体内胚葉系に特徴的なマーカーを発現している細胞の形成は、以下の特定のプロトコルの前後に、マーカーの存在に関して試験することにより決定することができる。多能性幹細胞は、一般にこのようなマーカーを発現しない。したがって、多能性細胞の分化は、細胞がそれらの発現を開始した際に検出される。
一実施形態では、胚体内胚葉系に特徴的なマーカーを発現している細胞を、FGF7を添加した第1培地で培養し、次いで細胞を、FGF7、BMP阻害能を持つ因子、TGFβ受容体アゴニスト、レチノイン酸、及びヘッジホッグ(hedgehog)シグナル経路阻害剤を添加した第2培地で培養することにより、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へと分化させる。
一実施形態では、本発明の方法により産生される、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞は、膵内分泌系に特徴的なマーカーを発現している細胞へと更に分化し得る。
一態様では、本発明は、I型糖尿病に罹患しているかあるいはI型糖尿病を発症するリスクを有する患者を治療する方法を提供する。一実施形態では、本方法は、多能性幹細胞を培養することと、多能性幹細胞をin vitroでβ細胞系に分化させることと、β細胞系の細胞を患者に移植することと、を包含する。別の実施形態では、本方法は、多能性幹細胞を培養することと、多能性幹細胞を、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へとin vitroで分化させることと、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない膵臓内胚葉系細胞を患者に移植することと、を包含する。
PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へのヒト多能性幹細胞の分化
本実施例は、アクチビンAをノギン及びレチノイン酸と組み合わせて使用することで、NKX6.1発現の上方制御を促進できることを示す。簡潔に述べると、ヒト胚性幹細胞株H1細胞を、MATRIGEL(商標)(1:30希釈)をコートしたディッシュと、2%のBSA、100ng/mLのアクチビンA、20ng/mLのWNT−3a、8ng/mLのbFGFを添加したRPMI培地により1日培養した後で、2%のBSA、100ng/mLのアクチビンA、8ng/mLのbFGFを添加したRPMI培地で更に2日間処理し(ステージ1)、次いで
a.2%のBSA及び50ng/mLのFGF7を添加したDMEM/F12で3日間にわたって培養し(ステージ2)、次いで
b.1%のB27、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン、及び20ng/mLのアクチビンA若しくは50ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって培養した(ステージ3)。
ヒト胚性幹細胞株H1細胞を、MATRIGEL(1:30希釈)をコートしたプレート上で培養し、以下のプロトコルを使用して膵内分泌前駆細胞へと分化させた:
a.2%のBSA(カタログ# 152401,MP Biomedical,Ohio)、100ng/mLのアクチビンA(R&D Systems,MN)、20ng/mLのWNT−3a(カタログ# 1324−WN−002,R&D Systems,MN)及び8ng/mLのbFGF(カタログ# 100−18B,PeproTech,NJ)を添加したRPMI培地(カタログ#22400,Invitrogen,Ca)で1日培養した後に、2%のBSA、100ng/mLのアクチビンA、8ng/mLのbFGFを添加したRPMI培地で更に2日間にわたって処理し(ステージ1)、次いで
b.2%のBSA及び50ng/mLのFGF7を添加したDMEM/F12(カタログ#11330,Invitrogen,Ca)で3日間にわたって培養し(ステージ2)、次いで
c.処理1:1%のB27(Invitrogen,CA)、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)及び100ng/mLのノギンを添加したDMEM(高グルコース)で4日間にわたって処理するか(ステージ3)、又は
d.処理2:1%のB27(Invitrogen,CA)、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン及び20ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって処理するか(ステージ3)、又は
e.処理3:1%のB27(Invitrogen,CA)、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン及び1μMのALK5阻害剤IIを添加したDMEM(高グルコース)で4日間にわたって処理した(ステージ3)。
PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵内分泌前駆細胞への、膵臓内胚葉系に特徴的なマーカーを発現している細胞の分化
これまでの研究により、膵臓内胚葉系に特徴的なマーカーを発現している細胞は、更なる分化を受けた際に、インスリンを発現している細胞よりも、グルカゴンを発現している細胞を産生しやすいということが示されている。この示唆の一部は、膵臓内胚葉細胞でのNGN3発現に起因するものであり得る。本発明の方法は、NGN3を発現しない、膵臓内胚葉細胞集団を産生することから、これらの細胞集団はインスリンを発現している細胞へとより分化しやすいものと考えられる。しかしながら、NGN3発現は、膵内分泌細胞又は膵内分泌前駆細胞(例えばグルカゴン又はインスリンを発現している細胞を形成することのできる細胞)の形成に必要とされる。したがって、膵内分泌前駆細胞の最終的な運命を誘導するにあたり、NGN3の一時的な制御が重要である。
a.2%のBSA及び50ng/mLのFGF7を添加したDMEM/F12で3日間にわたって培養し(ステージ2)、次いで
b.1%のB27、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン及び20ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって処理し(ステージ3)、次いで
c.1%のB27、100ng/mLのノギン及び1μMのALK5阻害剤IIを添加したDMEM(高グルコース)で3日間にわたって培養するか(ステージ4)、又は
d.1%のB27のみを添加したDMEM(高グルコース)で3日間にわたって培養した(ステージ4)。
膵内分泌細胞への、PDX1とNKX6.1を共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞の分化
本実施例は、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞が、膵内分泌前駆細胞から、膵内分泌細胞へと更に分化するための能力について試験することを目的に設計された。
a.2%のBSA及び50ng/mLのFGF7を添加したDMEM/F12で3日間にわたって培養し(ステージ2)、次いで
b.1%のB27、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン及び20ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって処理するか(ステージ3)、又は
c.1%のB27、50ng/mLのFGF7、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン及び20ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって処理し(ステージ3)、次いで
d.1%のB27、100ng/mLのノギン及び1μMのALK5阻害剤IIを添加したDMEM(高グルコース)で3日間にわたって培養し(ステージ4)、
e.1%のB27、100ng/mLのノギン、1μMのALK5阻害剤II及び20ng/mLのベータセルリンを添加したDMEM(高グルコース)で5〜7日間にわたって培養した(ステージ5)。
STZ誘導型糖尿病の重症複合免疫不全(SCID)−beige(Bg)マウスへの、本発明の細胞の移植
ヒト胚性幹細胞株H1細胞を、MATRIGEL(登録商標)をコートしたディッシュ(1:30希釈)と、0.2%のFBS、100ng/mLのアクチビンA、20ng/mLのWNT−3aを添加したRPMI培地で1日培養し、次いで0.5%のFBS及び100ng/mLのアクチビンAを添加したRPMI培地により更に2日間にわたって処理し(ステージ1)、次いで
a.2%のFBS及び50ng/mLのFGF7を添加したDMEM/F12で3日間にわたって処理し(ステージ2)、次いで
b.1%のB27、0.25μMのシクロパミン−KAAD、2μMのレチノイン酸(RA)、100ng/mLのノギン、50ng/mLのFGF7及び20ng/mLのアクチビンAを添加したDMEM(高グルコース)で4日間にわたって処理し(ステージ3)、次いで
c.1%のB27、100ng/mLのノギン及び1μMのALK5阻害剤IIを添加したDMEM(高グルコース)で4日間にわたって処理した(ステージ4)。
Claims (1)
- PDX−1及びNKX−6.1は共発現するが、CDX−2及びNGN−3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へと、多能性幹細胞集団を分化させる方法であって、以下の工程a、b及びcを包含する方法:
a.前記多能性幹細胞を培養する工程、
b.前記多能性幹細胞を、胚体内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程、及び
c.前記胚体内胚葉系に特徴的なマーカーを発現している細胞を、FGF7を添加した第1培地で処理し、次いで前記細胞を、FGF7、BMP阻害能を持つ因子、TGF−β受容体アゴニスト、レチノイン酸、及びヘッジホッグ(hedgehog)シグナル経路阻害剤を添加した第2培地で培養することにより、PDX1及びNKX6.1は共発現するが、CDX2及びNGN3は発現しない、膵臓内胚葉系に特徴的なマーカーを発現している細胞へと分化させる工程。
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RU2012105923A (ru) | 2013-08-27 |
KR101785626B1 (ko) | 2017-10-16 |
KR101893021B1 (ko) | 2018-08-29 |
GB201202847D0 (en) | 2012-04-04 |
WO2011011302A2 (en) | 2011-01-27 |
ES2693088T3 (es) | 2018-12-07 |
HK1170262A1 (en) | 2013-02-22 |
US20110014702A1 (en) | 2011-01-20 |
US8785184B2 (en) | 2014-07-22 |
JP5819825B2 (ja) | 2015-11-24 |
SG177483A1 (en) | 2012-02-28 |
ZA201201221B (en) | 2013-07-31 |
AU2010276440A1 (en) | 2012-01-19 |
WO2011011302A3 (en) | 2011-05-05 |
EP2456858B1 (en) | 2018-08-29 |
CN102482640A (zh) | 2012-05-30 |
RU2540021C2 (ru) | 2015-01-27 |
PL2456858T3 (pl) | 2019-01-31 |
CA2768644A1 (en) | 2011-01-27 |
MX2012000899A (es) | 2012-02-13 |
KR20120034793A (ko) | 2012-04-12 |
AU2010276440B2 (en) | 2014-07-03 |
GB2485112B (en) | 2014-02-26 |
GB2485112A (en) | 2012-05-02 |
BR112012001557A2 (pt) | 2016-03-08 |
CN102482640B (zh) | 2015-03-11 |
MX340952B (es) | 2016-07-29 |
AR077767A1 (es) | 2011-09-21 |
EP2456858A2 (en) | 2012-05-30 |
KR20170117231A (ko) | 2017-10-20 |
EP2456858A4 (en) | 2015-03-18 |
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