JP2012507289A - ヒト胚性幹細胞の膵内分泌系への分化 - Google Patents
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Abstract
Description
本発明は、多能性幹細胞の分化を促進する方法を提供する。具体的には本発明は、膵内分泌系に関連付けられるマーカーの発現量を増加させる方法を提供する。
a.多能性幹細胞を培養する工程と、
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に分化させる工程と、
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に分化させる工程と、
d.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、膵内分泌系に特徴的なマーカーを発現している細胞に分化させる工程と、を含む。
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、後代細胞には、自己複製前駆細胞、非再生前駆細胞、及び最終分化細胞が含まれる。幹細胞はまた、インビトロで複数の胚葉(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に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殆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の1以上、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を発現し得る(Thomsonら、Science 282:1145,1998)。インビトロで多能性幹細胞を分化させると、SSEA−4、Tra−1−60、及びTra−1−81の発現が消失し(存在する場合)、SSEA−1の発現が増大する。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.))によって述べられるようにVector Red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。未分化多能性幹細胞はまた、RT−PCRで検出されるように、一般にOct−4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(例えば胚盤胞等)、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含まれる。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、源となる細胞は、源となる組織から直接採取した一次多能性細胞である。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、一般に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、フィーダー細胞を基本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条件づけした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ホルモン産生細胞を作製するための方法を提供し、かかる方法は、
a.多能性幹細胞を培養する工程と、
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に分化させる工程と、
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に分化させる工程と、
d.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、膵内分泌系に特徴的なマーカーを発現している細胞に分化させる工程と、を含む。
多能性幹細胞は、当該技術分野のいかなる方法、又は本発明で提案されるいかなる方法によって胚体内胚葉系に特徴的なマーカーを発現する細胞に分化させてもよい。
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、一般にかかる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。
胚体内胚葉系に特徴的なマーカーを発現している細胞は、当該技術分野の任意の方法、又は本発明で提案する任意の方法により、膵臓内胚葉系に特徴的なマーカーを発現している細胞に分化され得る。
膵臓内胚葉系に特徴的なマーカーは、当業者に周知であり、膵臓内胚葉系の特徴を示す追加のマーカーが、継続して同定されている。これらのマーカーは、本発明に従って処理された細胞が分化して膵臓内胚葉系の特徴を示す性質を獲得したことを確認するために使用され得る。膵臓内胚葉系に特異的なマーカーとしては、例えば、Hlxb9、PTF−1a、PDX−1、HNF−6、HNF−1βなどの転写因子の1つ以上のものの発現が挙げられる。
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、当該技術分野の任意の方法、又は本発明で提案する任意の方法により、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分化させることができる。
膵内分泌系に特徴的なマーカーは当業者に周知であり、膵内分泌系の特徴を示す追加のマーカーが継続して同定されている。これらのマーカーは、本発明に従って処理された細胞が分化して膵内分泌系の特徴を示す性質を獲得したことを確認するために使用され得る。膵臓内分泌系に特異的なマーカーとしては、例えば、NGN−3、ニューロD(NeuroD)、Islet−1などの転写因子の1つ以上のものの発現が挙げられる。
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。本方法は、多能性幹細胞を培養し、この多能性幹細胞をインビトロでβ細胞系へと分化させ、β細胞系の細胞を患者に埋め込むことを含む。
ウシ胎児血清の非存在下での、ヒト胚性幹細胞の細胞株H1の膵内分泌細胞への分化
継代数52のヒト胚性幹細胞株H1の細胞をMATRIGEL(商標)(1:30希釈)をコートしたディッシュ上で培養し、2% 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)を加えたRPMI培地に1日にわたって曝露した後、2% BSA、100ng/mLのアクチビンA、及び8ng/mLのbFGFを加えたRPMI培地で更に2日間にわたって処理した。次いで培養物をDMEM/F12+2% BSA+50ng/mLのFGF7+0.25μMシクロパミン−KAAD(#239804,Calbiochem,CA)で2日間にわたって処理し、次いでDMEM/F12+1% B27(Invitrogen,CA)+50ng/mLのFGF7+0.25μMシクロパミン−KAAD+2μMレチノイン酸(RA)(Sigma,MO)+100ng/mLノギン(R&D Systems,MN)中で4日間にわたってインキュベートした。
ステージ6の培養物へのアクチビンAの添加は、膵内分泌マーカーの発現量を有意に増加させた
継代数43のヒト胚性幹細胞株H1細胞をMATRIGEL(商標)(1:30希釈)コートディッシュ上で培養し、実施例1に記載の方法に従って膵内分泌細胞へと分化させた。ステージ6で、一部の培養物を10ng/mLのアクチビンA(R&D Systems,MN)で7日間にわたって処理した。7日目に、細胞を室温で5分間にわたって1Xアキュターゼ(Sigma,MO)で処理した後、続いてアキュターゼは除去してDMEM/12+1% B27を加えた。接着した細胞をセルスクレーパーを用いて取り外し、穏やかに再懸濁し、40μmのセルストレイナーで濾過した。ストレイナーに保持されたフロースルーと細胞を取り外し、サンプルをPCRとジチゾン染色のために回収した。ジチゾン(DTZ)は、島に存在することが示されてきた亜鉛へと、選択的に結合する色素である。図3のパネルa〜cは、40μmセルストレイナーを用いて分離する前、及び分離した後にDTZ染色した培養物を示す。40μmを超えるクラスターの有意な(およそ80%)部分がDTZに対して陽性に染色された。これは内分泌腺に富むクラスターを濃縮する、迅速で簡単な方法として提供される。図4のパネルa〜fは、ステージ6で+/−10ng/mLのアクチビンAで処理し、続いて40μmのフィルタを用いて濃縮した細胞の、遺伝子発現プロファイルを示す。ステージ6へのアクチビンAの添加は、全ての重要な膵内分泌マーカーの発現を、有意に増幅させた。
Claims (7)
- 膵内分泌系と関連付けられるマーカーの発現量を増加させる方法であって、該方法は、膵内分泌系に関連付けられるマーカーを発現している細胞を、十分な量のTGF−β受容体作動剤を含む培地で処理することで、膵内分泌系に関連付けられるマーカーの発現量を増加させることを含む、方法。
- 前記TGF−β受容体作動剤が、アクチビンA、アクチビンB、アクチビンC、アクチビンAのペプチド変異体、GDF−8、GDF−11及びGDF−15からなる群から選択される、請求項1に記載の方法。
- 前記膵内分泌系に関連付けられるマーカーを発現している細胞が濃縮される、請求項1に記載の方法。
- 多能性幹細胞を分化させるための方法であって、
a.多能性幹細胞を培養する工程と、
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に分化させる工程と、
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、膵臓内胚葉系に特徴的なマーカーを発現している細胞に分化させる工程と、
d.前記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、膵内分泌系に特徴的なマーカーを発現している細胞に分化させる工程と、を含む、方法。 - 前記膵内分泌系に特徴的なマーカーを発現している細胞を、十分な量のTGF−β受容体作動剤を含む培地で処理することで、前記膵内分泌系に関連付けられるマーカーの発現量を増加させることにより、前記膵内分泌系に特徴的なマーカーの発現量が増加する、請求項4に記載の方法。
- 前記TGF−β受容体作動剤が、アクチビンA、アクチビンB、アクチビンC、アクチビンAのペプチド変異体、GDF−8、GDF−11及びGDF−15からなる群から選択される、請求項4に記載の方法。
- 前記膵内分泌系に関連付けられるマーカーを発現している細胞が濃縮される、請求項4に記載の方法。
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JP2019050810A (ja) * | 2012-06-08 | 2019-04-04 | ヤンセン バイオテツク,インコーポレーテツド | 膵内分泌細胞へのヒト胚性幹細胞の分化 |
KR20200057112A (ko) * | 2012-06-08 | 2020-05-25 | 얀센 바이오테크 인코포레이티드 | 인간 배아 줄기 세포의 췌장 내분비 세포로의 분화 |
KR102285014B1 (ko) * | 2012-06-08 | 2021-08-03 | 얀센 바이오테크 인코포레이티드 | 인간 배아 줄기 세포의 췌장 내분비 세포로의 분화 |
KR20210096323A (ko) * | 2012-06-08 | 2021-08-04 | 얀센 바이오테크 인코포레이티드 | 인간 배아 줄기 세포의 췌장 내분비 세포로의 분화 |
KR102468315B1 (ko) * | 2012-06-08 | 2022-11-16 | 얀센 바이오테크 인코포레이티드 | 인간 배아 줄기 세포의 췌장 내분비 세포로의 분화 |
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CN102333862A (zh) | 2012-01-25 |
EP3517605A1 (en) | 2019-07-31 |
MX2011004563A (es) | 2011-06-01 |
RU2528861C2 (ru) | 2014-09-20 |
KR20170127576A (ko) | 2017-11-21 |
JP2015165826A (ja) | 2015-09-24 |
BRPI0919883A2 (pt) | 2015-08-11 |
US20100112692A1 (en) | 2010-05-06 |
US20150191700A1 (en) | 2015-07-09 |
KR101798474B1 (ko) | 2017-11-16 |
JP2018011610A (ja) | 2018-01-25 |
CN107435038A (zh) | 2017-12-05 |
CA2742267C (en) | 2019-06-04 |
CN107435038B (zh) | 2021-07-09 |
US9388387B2 (en) | 2016-07-12 |
CN102333862B (zh) | 2018-04-27 |
JP6806656B2 (ja) | 2021-01-06 |
US9012218B2 (en) | 2015-04-21 |
MX349178B (es) | 2017-07-17 |
CA2742267A1 (en) | 2010-05-06 |
RU2011121843A (ru) | 2012-12-10 |
KR102025158B1 (ko) | 2019-09-25 |
EP2350265B1 (en) | 2019-04-17 |
EP2350265A1 (en) | 2011-08-03 |
AU2009309044A1 (en) | 2010-05-06 |
ES2727950T3 (es) | 2019-10-21 |
WO2010051213A1 (en) | 2010-05-06 |
JP6230572B2 (ja) | 2017-11-15 |
KR20110077017A (ko) | 2011-07-06 |
AU2009309044B2 (en) | 2015-07-09 |
ZA201103983B (en) | 2018-11-28 |
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