JP2016512031A - 定義された条件下におけるヒト多能性幹細胞の造血内皮分化のための方法及び材料 - Google Patents
定義された条件下におけるヒト多能性幹細胞の造血内皮分化のための方法及び材料 Download PDFInfo
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Abstract
Description
約20から約50ng/mlのVEGFを添加したIF9Sを含むヒト多能性幹細胞を分化するための異種非含有培地である。いくつかの実施態様において、前記培地は、FGF2及びVEGFを含み、さらに、造血サイトカインを含む。いくつかの実施態様において、前記造血サイトカインは、約50から約100ng/mlのSCF、約50から約100ng/mlのTPO、約50から約100ng/mlのIL−6、及び約5から約15ng/mlのIL−3を含む。いくつかの実施態様では、前述の培地のいずれかは実質的にIF9S培地、及び添加された成分から構成される。いくつかの実施態様では、前述の培地のいずれかは、濃縮された形態で提供される。
組成物
定義された細胞培養培地と濃縮培地添加物
キット
hPSCsの造血内皮分化のために定義された細胞培養系
IMDM/F12ベース培地がhPSCsの造血内皮系統への分化効率を有意に向上させる
方法
(実施例2)
造血前駆細胞の分化と維持を促進する細胞外マトリックスとしてのテネイシンCで特定される、造血を支持する間質細胞のユニークな分子サインの分析
(実施例3)
FGF2、BMP4、アクチビンA、塩化リチウム、及びVEGFの時間及び用量依存的の処置は、中胚葉、内皮、及び造血の段階の分化を誘発する
(実施例4)
テネイシンCは独特にhPSCsからのTリンパ球前駆細胞の特定を支持している
(実施例5)
TGF−βの阻害は、化学的に定義された条件で造血内皮の特定を促進する
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Claims (40)
- 以下の工程を含む、ヒト多能性幹細胞を分化させる方法。
(a)ヒト多能性幹細胞を提供する工程、及び
(b)幹細胞を、間葉系血管芽細胞(mesenchymoangioblast)可能性を有するEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞の集団を形成するために、約2日間の期間低酸素条件下で、FGF2、BMP4、アクチビンA、及び塩化リチウムを構成要素として含む混合物に暴露する工程。 - さらに以下の工程を含む、請求項1に記載の方法。
(c)細胞を、工程(b)の原始中胚葉段階において、血球血管共通前駆細胞(HB−CFC)可能性とOP9細胞上で培養する際に造血内皮クラスターを形成する可能性を有する細胞に富む造血血管(hematovascular)中胚葉細胞(EMHlin−KDRhiAPLNR+PDGFRalphalo/−)と共にEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉を含む集団を得るために、約1〜2日間の期間低酸素条件下で、FGF2、及びVEGFを構成要素として含む混合物に暴露する工程。 - さらに以下の工程を含む、請求項2に記載の方法。
(d)工程(c)の造血血管(hematovascular)中胚葉段階で、CD144+CD73+CD235a/CD43−非造血内皮前駆細胞(非HEP)、CD144+CD73−CD235a/CD43−造血内皮前駆細胞(HEPs)、CD144+CD73−CD235a/CD43+41a−血管新生造血前駆細胞(AHP)、及びCD43+CD41a+造血前駆細胞の形成を達成するために、前記細胞を、構成要素FGF2、VEGF、IL6、SCF、TPO、及びIL3を含む混合物に、約1日間暴露する工程。 - さらに以下の工程を含む、請求項3に記載の方法。
(e)造血拡大(hematopoietic expansion)の結果としてCD43+CD235a+CD41a+エリスロメガカリオサイト前駆細胞及びlin−CD34+CD43+CD45+/−多能性造血前駆細胞から構成されるCD43+造血前駆細胞の集団を得るために、構成要素FGF2、VEGF、IL6、SCF、TPO、IL−3を含む混合物に常酸素下で約3日間、HEPsを曝露し続け、造血前駆細胞を出現させる工程。 - 前記混合物が、実質的に前記構成要素から構成される請求項1に記載の方法。
- 前記構成要素が、異種非含有である請求項5に記載の方法。
- 前記細胞は、テネイシンCで処理された基質上に播種された細胞である、請求項1に記載の方法。
- 約50から約250ng/mlのBMP4、
約10から約15ng/mlのアクチビンA、
約10から約50ng/mlのFGF2、及び
約1mMからの約2mMの塩化リチウム
を添加したIF9Sを含むヒト多能性幹細胞を分化するための異種非含有培地。 - 約10から約50ng/mlのFGF2、及び
約20から約50ng/mlのVEGF
を添加したIF9Sを含むヒト多能性幹細胞を分化するための異種非含有培地。 - さらに、造血サイトカインを含む請求項9に記載の培地。
- 前記造血サイトカインが、
約50から約100ng/mlのSCF、
約50から約100ng/mlのTPO、
約50から約100ng/mlのIL−6、及び
約5から約15ng/mlのIL−3
を含む請求項10に記載の培地。 - 請求項9に記載の培地の濃縮された形態。
- 少なくとも約0.25μg/cm2から約1μg/cm2の濃度で、テネイシンCの層を含む基質上の単細胞懸濁液として播種したヒト多能性幹細胞、及び
約50から約250ng/mlのBMP4、
約10から約15ng/mlのアクチビンA、
約10から約50ng/mlのFGF2、及び
約1mMからの約2mMの塩化リチウムを添加したIF9Sを含む異種非含有培地
を含む、ヒト多能性幹細胞を中胚葉、内皮細胞、及び含む造血前駆細胞へ分化するための異種非含有細胞培養系。 - 以下の工程を含む、ヒト多能性幹細胞を分化させる方法。
(a)ヒト多能性幹細胞を提供する工程、
(b)テネイシンCで処理された基質上に単細胞懸濁液として細胞を播種する工程、及び
(c)間葉系血管芽細胞(mesenchymoangioblast)潜在性を有する約30%EMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞を得るために、前記播種した細胞を、低酸素下で約2日間、BMP4、アクチビンA、FGF2、及び塩化リチウムを添加したIF9S培地中で培養する工程。 - さらに、血液血管共通前駆細胞(HB−CFC)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉、及びOP9細胞上での培養時に造血内皮クラスターを形成する潜在性を有するEMHlin−KDRhiAPLNR+PDGFRalphalo/−造血血管(hematovascular)中胚葉前駆細胞を得るために、前記細胞を、EMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉段階において、低酸素下で約1〜2日間、FGF2及びVEGFを添加したIF9S培地中で培養する工程を含む請求項14に記載の方法。
- 以下の工程を含む、間葉系血管芽細胞(mesenchymoangioblasts)の製造方法。
(a)ヒト多能性幹細胞を提供する工程、
(b)有効量のコラーゲンで処理された基質上に前記細胞を播種する工程、及び
(c)間葉系血管芽細胞(mesenchymoangioblast)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞の集団を形成するために、前記幹細胞を、低酸素下で約2日間、FGF2、BMP4、アクチビンA、及び塩化リチウムを含む混合物中に暴露する工程。 - 前記コラーゲンは、コラーゲンIVを含む請求項16に記載の方法。
- 64mg/LのL−アスコルビン酸、2−リン酸Mg2+塩、40μl/Lのモノチオグリセロール、8.4μg/Lの追加的な亜セレン酸ナトリウム、10mg/Lのポリビニルアルコール、1×のグルタマックス、1×の非必須アミノ酸、0.1×の化学的に定義された脂質濃縮物、10.6mg/Lのホロートランスフェリン、及び20mg/Lのインスリンを含むヒト多能性幹細胞の分化のための細胞培養培地。
- (a)ヒト多能性幹細胞を提供する工程、及び
(b)間葉系血管芽細胞(mesenchymoangioblast)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞の細胞集団を形成するためにヒト多能性幹細胞を、低酸素下で約2日間、FGF2、BMP4、アクチビンA、及び塩化リチウムを含む細胞培養培地中で培養する工程を含むヒト多能性幹細胞を分化するための方法。 - 前記ヒト多能性幹細胞が、テネイシンC上で培養される請求項19に記載の方法。
- 細胞培養培地中の濃度が、BMP4は約50ng/mlから約250mg/ml、アクチビンAは約10ng/mlから約15ng/ml、FGF2は約10ng/mlから約50ng/ml、かつ塩化リチウムは約1mMから約2mMである請求項19に記載の方法。
- さらに、血液血管共通前駆細胞(HB−CFC)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉、及びOP9細胞上での培養時に造血内皮クラスターを形成する潜在性を有する細胞に富む造血血管(hematovascular)中胚葉細胞(EMHlin−KDRhiAPLNR+PDGFRalphalo/−)を含む細胞集団を得るために、工程(b)で得られた細胞集団を、低酸素下で約1〜2日間、FGF2及びVEGFを含む細胞培養培地中で培養する工程を含む請求項19に記載の方法。
- 細胞培養培地中の濃度が、FGF2は約10ng/mlから約50ng/ml、かつVEGFは約20ng/mlから約50ng/mlである請求項22に記載の方法。
- さらに、(d)CD144+CD73+CD235a/CD43−非造血内皮前駆細胞(非HEP)、CD144+CD73−CD235a/CD43−造血内皮前駆細胞(HEPs)、CD144+CD73−CD235a/CD43+41a−血管新生造血前駆細胞(AHP)、及びCD43+CD41a+造血前駆細胞を含む細胞集団を得るために、工程(c)の造血血管(hematovascular)中胚葉細胞を、低酸下で約1日間、FGF2、VEGF、IL6、SCF、TPO、及びIL3を含む細胞培養培地中で培養する工程を含む請求項21に記載の方法。
- 細胞培養培地中の濃度が、FGF2は約10ng/mlから約50ng/ml、VEGFは約20ng/mlから約50ng/ml、SCFは約50ng/mlから約100ng/ml、TPOは約50ng/mlから約100ng/ml、IL−6は、約50ng/mlから約100ng/ml、かつIL−3は約5ng/mlから約15ng/mlである請求項24に記載の方法。
- さらに、(d)CD43+CD235a/CD41a+エリスロメガカリオサイト前駆細胞及びlin−CD34+CD43+CD45+/−多能性造血前駆細胞を含むCD43+造血前駆細胞の増殖した(expanded)集団を得るために、造血内皮前駆細胞(HEPs)、及び造血前駆細胞を、常酸素下で約3日間、FGF2、VEGF、IL6、SCF、TPO、IL3を含む培養培地中で培養する工程を含む請求項24に記載の方法。
- さらに、CD4+CD8+二重陽性T細胞を含む細胞集団を得るために、CD34+CD43+造血前駆細胞を、約3週間、DLL4を過発現するOP9細胞上で共培養する工程を含み、工程(b)のヒト多能性幹細胞を含む細胞集団が、テネイシンC基質上で培養される請求項26に記載の方法。
- 前記細胞培養培地はIF9S細胞培養培地を含む請求項19に記載の方法。
- (i)間葉系血管芽細胞(mesenchymoangioblast)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞の細胞集団を形成するために、ヒト多能性幹細胞を、低酸素下において約2日間、FGF2、BMP4、アクチビンA、及び塩化リチウムを含む細胞培養培地で培養する工程、
(ii)血球血管共通前駆細胞(HB−CFC)潜在性を有するEMHlin−KDR+APLNR+PDGFRalpha+、及びOP9細胞上で培養した場合造血内皮クラスター形成潜在性を持つ細胞に富む造血血管(hematovascular)中胚葉細胞(EMHlin−KDRhiAPLNR+PDGFRalphalo/−)を含む細胞集団を得るために、間葉系血管芽細胞(mesenchymoangioblast)潜在性を持つEMHlin−KDR+APLNR+PDGFRalpha+原始中胚葉細胞を、低酸素下において約1〜2日間、FGF2、及びVGEFを含む細胞培養培地で培養する工程、
(iii)CD144+CD73+CD235a/CD43−非造血内皮前駆細胞(非HEP)CD144+CD73−CD235a/CD43−造血内皮前駆細胞(HEPs)、CD144+CD73−CD235a/CD43+41a−血管新生造血前駆細胞(AHP)、及びCD43+CD41a+造血前駆細胞形成を達成するために、培養造血血管(hematovascular)中胚葉細胞(EMHlin−KDRhiAPLNR+PDGFRalphalo/−)を、低酸素下において約2日間、FGF2、VEGF、IL6、SCF、TPO、及びIL−3を含む細胞培養培地で培養する工程、
(iv)CD43+CD235a/CD41a+エリスロメガカリオサイト前駆細胞及びlin−CD34+CD43+CD45+/−多能性造血前駆細胞を含むCD43+造血前駆細胞の増殖した(expanded)集団を得るために、造血内皮前駆細胞(HEPs)、及びCD43+CD41a+造血前駆細胞を、常酸素下で約3日間、FGF2、VEGF、IL6、SCF、TPO、IL3を含む培養培地中で培養する工程、及び
(v)CD4+CD8+T細胞を含む細胞集団を得るために、CD34+CD43+造血前駆細胞を、約3週間、DLL4を過発現するOP9細胞上で共培養する工程、
の少なくとも一つを含むヒト多能性幹細胞を分化する方法。 - 基本培地、L−アスコルビン酸2−リン酸エステルのMg2+塩、モノチオグリセロール、亜セレン酸ナトリウム、ポリビニルアルコール、グルタマックスTM、非必須アミノ酸(NEAA)、定義された脂質濃縮物、ホロートランスフェリン、及びインスリンを含むヒト多能性幹細胞の造血内皮分化に適した細胞培養培地。
- さらにBMP4、アクチビンA、FGF2、及び塩化リチウムを含む請求項30に記載の細胞培養培地。
- さらにFGF2、及びVEGFを含む請求項30に記載の細胞培養培地。
- さらにFGF2、VEGF、SCF、TPO、IL−6、及びIL−3を含む請求項30に記載の細胞培養培地。
- さらにIF9S培地を含む請求項30に記載の細胞培養培地。
- 前記IF9S細胞培養培地が、表2のIF9S細胞培養培地処方を含む請求項34に記載の細胞培養培地。
- IMDM/F12基本培地中の9S濃縮培地添加物の希釈物が、IF9S細胞培養培地を生成する9S濃縮培地添加物。
- 請求項36に記載の9S濃縮培養添加物、及びBMP4、アクチビンA、FGF2、塩化リチウム、SCF、TPO、IL−6、IL−3、及びテネイシンCのうちの一つ以上を含むキット。
- ヒト多能性幹細胞の付着、成長または造血内皮系統に沿った分化した子孫のための、IF9S細胞培養培地とテネイシンCの基質を含む、ヒト多能性幹細胞の造血内皮分化に対する定義された細胞培養系。
- 前記IF9S細胞培養培地が、低酸素条件下で維持される、請求項38に記載の定義された細胞培養系。
- テネイシンC基質上に成長させたヒト多能性幹細胞を含む請求項39に記載の定義された細胞培養系。
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CN105209609B (zh) | 2020-08-21 |
KR102151210B1 (ko) | 2020-09-02 |
WO2014165131A1 (en) | 2014-10-09 |
BR112015022770A2 (pt) | 2017-07-18 |
CN105209609A (zh) | 2015-12-30 |
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CA2905786A1 (en) | 2014-10-09 |
CA3141731A1 (en) | 2014-10-09 |
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HK1218927A1 (zh) | 2017-03-17 |
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CA2905786C (en) | 2022-01-25 |
KR20150126943A (ko) | 2015-11-13 |
PL2970912T3 (pl) | 2019-08-30 |
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