JP7471084B2 - 幹細胞由来星状細胞、作製方法および使用方法 - Google Patents
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Description
本出願は、2017年3月21日に出願された米国仮出願第62/474,429号、および2017年3月21日に出願された米国仮出願第62/474,596号の優先権を主張し、これらの内容はそれぞれ、その全体が参照により組み込まれ、これらのそれぞれの優先権が主張される。
1.イントロダクション
星状細胞は、脳内のアミノ酸、栄養素およびイオン代謝を調節し、神経活動および脳血流をつなぎ、興奮性シナプス伝達をモジュレートするように機能するグリア細胞である。プリオン病、アルツハイマー病およびパーキンソン病を有する患者の脳では、星状細胞は封入体を有すると報告されており、死後脳において疾患の重症度と相関する。加えて、Mecp2を星状細胞からノックアウトすると、細胞は野生型ニューロンの正常な樹状形態を支持することができず、さらに、ミトコンドリアを放出し、脳卒中後にこれがニューロンに入ることが観察されている。したがって、星状細胞は、いくつかの神経疾患の病因において役割を果たし、傷害による神経損傷の重症度をモジュレートする可能性がある。このため、安定な星状細胞系を生成するインビトロ方法は、このような状態の研究に有用であろう。さらに、インビトロ分化の方法により調製された星状細胞は、神経障害を有する患者に治療的に投与され得るか、または神経損傷を患っている患者における損傷の重症度を軽減するために投与され得る。しかしながら、ヒト多能性幹細胞(hPSC)由来星状細胞は、75~200日間の培養後にインビトロで確立されているのみであるので、hPSCから星状細胞を誘導するためのロバストな方法はない。
本開示の主題は、例えばインビトロ分化による幹細胞由来の星状細胞およびグリアコンピテント細胞(例えば、星状細胞前駆体)に関する。
(a)形質転換成長因子ベータ(TGFβ)/アクチビン-ノーダルシグナリングの1つまたはそれを超えるインヒビター、
(b)BMPシグナリングの1つまたはそれを超えるインヒビター;
(c)NFIAの1つまたはそれを超えるアクチベーター;
(d)LIF、その誘導体、その類似体および/またはそのアクチベーター;ならびに
(e)1つもしくはそれを超えるグリアコンピテント細胞マーカーおよび/または1つもしくはそれを超える星状細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団への該幹細胞の分化を誘導するための指示
の1つまたはそれよりも多くを含むキットを提供する。
4.図面の簡単な説明
本明細書に記載される本開示の主題は、グリアコンピテント細胞(例えば、「星状細胞前駆体」)および/または幹細胞由来星状細胞を調製する方法、ならびにこのような細胞を産生するための方法に関する。神経変性障害を処置するためのこのような細胞の使用も提供される。
5.1 定義
5.2 幹細胞を分化させる方法
5.3 局所星状細胞への幹細胞のインビトロ分化
5.4 分化細胞集団を含む組成物
5.5 神経変性障害を予防および/または処置する方法 および
5.6 キット
5.1 定義
5.2幹細胞の分化方法
5.2.1.星状細胞への幹細胞のインビトロ3段階分化
5.2.1.1.NSCへの幹細胞のインビトロ分化
5.2.1.2.グリアコンピテント細胞へのNSCのインビトロ分化
5.2.1.2.1.NFIAシグナリングの促進
5.2.1.2.2.細胞周期のG1期の延長
5.2.1.3.星状細胞へのグリアコンピテントNSCのインビトロ分化
5.2.2 FBSを使用した星状細胞への幹細胞のインビトロ分化の方法
5.3 局所星状細胞への幹細胞のインビトロ分化
5.3.1.皮質星状細胞への幹細胞のインビトロ分化
5.3.2.脊髄星状細胞への幹細胞のインビトロ分化
5.4.分化細胞集団を含む組成物
5.5 神経変性障害を予防および/または処置する方法
5.6 キット
本開示の主題は、本開示の主題の例として、限定のためではなく提供される、以下の実施例を参照することにより、より良く理解される。
6.1 実施例1:幹細胞の集団におけるNFIA発現レベルをモジュレートすることにより、幹細胞由来星状細胞を調製する方法
6.2 実施例2:野生型星状細胞は、脊髄運動ニューロン(sMN)および眼球運動ニューロン(oMN)の細胞死を減少させる。
6.3 実施例3:NFIAは、G1細胞周期の長さをモジュレートすることにより、多能性幹細胞からの機能的ヒト星状細胞の迅速な誘導を可能にする
概要
方法および材料
細胞培養
hPSCからの前部前脳神経外胚葉の誘導
脊髄運動ニューロン(SMN)誘導プロトコール
星状細胞誘導プロトコール
免疫組織化学およびCD44のFACS分析
NFIAおよびSOX9誘導性構築物の生成およびレンチウイルス産生
遺伝子発現およびATAC-Seq分析
細胞周期分析
成体皮質へのNSC、グリア前駆体および星状細胞の移植
組織処理
カルシウムイメージング
グルタミン酸興奮毒性アッセイ
バイサルファイト変換および配列決定
結果
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本発明は、例えば以下の項目を提供する。
(項目1)
多能性幹細胞を分化させるためのインビトロ方法であって、1つまたはそれを超える神経幹細胞マーカーを発現する細胞の集団におけるNFIAシグナリングを促進して、1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目2)
幹細胞を分化させるためのインビトロ方法であって、幹細胞の集団をSMADシグナリングの有効量の1つまたはそれを超えるインヒビターに曝露すること、および該細胞におけるNFIAシグナリングを促進して、1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目3)
NFIAシグナリングの初期促進が、SMADシグナリングの前記1つまたはそれを超えるインヒビターへの前記幹細胞の該初期曝露から少なくとも約8日間である、項目2に記載の方法。
(項目4)
検出可能レベルの前記1つまたはそれを超えるグリアコンピテント細胞マーカーが、前記細胞におけるNFIAシグナリングの前記初期促進から少なくとも約5日間存在する、項目1~3のいずれか一項に記載の方法。
(項目5)
検出可能レベルの前記1つまたはそれを超えるグリアコンピテント細胞マーカーの存在後、複数の細胞において、機能的NFIA活性の発現レベルが減少する、項目1~4のいずれか一項に記載の方法。
(項目6)
機能的NFIA活性の前記発現レベルが少なくとも約90%減少する、項目1~5のいずれか一項に記載の方法。
(項目7)
前記分化細胞が、検出可能レベルの1つまたはそれを超えるニューロンマーカーを発現しない、項目1~6のいずれか一項に記載の方法。
(項目8)
前記1つまたはそれを超えるニューロンマーカーが、Tuj1、MAP2およびDCXからなる群より選択される、項目7に記載の方法。
(項目9)
NFIAシグナリングを前記促進することが、前記細胞をNFIAの1つまたはそれを超えるアクチベーターに曝露することを含む、項目1~8のいずれか一項に記載の方法。
(項目10)
NFIAの前記1つまたはそれを超えるアクチベーターが、前記幹細胞に外因的に曝露されたNFIAタンパク質を含む、項目9に記載の方法。
(項目11)
NFIAの前記1つまたはそれを超えるアクチベーターが、前記幹細胞により発現される組換えNFIAタンパク質を含む、項目9に記載の方法。
(項目12)
NFIAの前記1つまたはそれを超えるアクチベーターが、NFIAの上流アクチベーターを含む、項目9に記載の方法。
(項目13)
NFIAの前記上流アクチベーターがTGFβ1である、項目12に記載の方法。
(項目14)
NFIAシグナリングを前記促進することが、NFIAの発現を増加させることを含む、項目1~8のいずれか一項に記載の方法。
(項目15)
NFIAの発現を前記増加することが、NFIAの過剰発現を誘導するようにNSCを改変することを含む、項目14に記載の方法。
(項目16)
過剰発現した前記NFIAが、NFIA核酸により発現される組換えNFIAタンパク質である、項目13に記載の方法。
(項目17)
前記NFIA核酸が、誘導性プロモーターに作動可能に連結されている、項目17に記載の方法。
(項目18)
幹細胞を分化させるためのインビトロ方法であって、幹細胞の集団をSMADシグナリングの1つまたはそれを超えるインヒビターに曝露すること、および該細胞をウシ胎仔血清に曝露して、1つまたはそれを超える星状細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目19)
前記ウシ胎仔血清への前記幹細胞の前記初期曝露から少なくとも約30日間に、該幹細胞を前記細胞集団に分化させる、項目18に記載の方法。
(項目20)
多能性幹細胞を分化させるためのインビトロ方法であって、1つまたはそれを超える神経幹細胞マーカーを発現する細胞の集団の細胞周期のG1期を延長して、1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目21)
前記神経幹細胞マーカーが、PAX6、ネスチン、SOX1、SOX2、PLZF、ZO-1およびBRN2からなる群より選択される、項目1または20に記載の方法。
(項目22)
前記神経幹細胞マーカーが、PAX6、SOX1、PLZFおよびZO-1からなる群より選択される、項目1~21に記載の方法。
(項目23)
幹細胞を分化させるためのインビトロ方法であって、幹細胞の集団をSMADシグナリングの1つまたはそれを超えるインヒビターに曝露すること、および該細胞の細胞周期のG1期を延長して、1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目24)
前記G1期の初期延長が、SMADシグナリングの該1つまたはそれを超えるインヒビターへの前記細胞の初期曝露から少なくとも約8日間である、項目23に記載の方法。
(項目25)
検出可能レベルの前記1つまたはそれを超えるグリアコンピテント細胞マーカーが、前記G1期の前記初期延長から少なくとも約10日間存在する、項目20~24のいずれか一項に記載の方法。
(項目26)
前記G1期の前記延長が、前記細胞を1つまたはそれを超えるG1期を延長させる化合物に曝露することを含む、項目20~25のいずれか一項に記載の方法。
(項目27)
前記1つまたはそれを超えるG1を延長させる化合物が小分子化合物を含む、項目26に記載の方法。
(項目28)
前記小分子化合物がオロモウシン(Olo)を含む、項目27に記載の方法。
(項目29)
前記細胞の前記細胞周期の前記G1期を前記延長させることが、FZR1の発現を増加させることを含む、項目20~25のいずれか一項に記載の方法。
(項目30)
前記1つまたはそれを超えるグリアコンピテント細胞マーカーが、CD44、AQP4、SOX2およびネスチンからなる群より選択される、項目1~29のいずれか一項に記載の方法。
(項目31)
1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する前記細胞が、皮質グリアコンピテント細胞または脊髄グリアコンピテント細胞である、項目1~30のいずれか一項に記載の方法。
(項目32)
1つまたはそれを超えるグリアコンピテント細胞マーカーを発現する少なくとも約10%の細胞を含む前記細胞集団を、1つまたはそれを超える星状細胞マーカーを発現する細胞への該細胞の分化を促進するために適切な条件に供することをさらに含む、項目1~31のいずれか一項に記載の方法。
(項目33)
前記条件が、前記細胞集団を白血病抑制因子(LIF)、1つもしくはそれを超えるその誘導体、1つもしくはそれを超えるその類似体および/または1つもしくはそれを超えるそのアクチベーターに曝露することを含む、項目32に記載の方法。
(項目34)
LIF、1つもしくはそれを超えるその誘導体、1つもしくはそれを超えるその類似体および/または1つもしくはそれを超えるそのアクチベーターへの前記幹細胞の初期曝露が、SMADシグナリングの前記1つまたはそれを超えるインヒビターへの該幹細胞の該初期曝露から少なくとも約10日間である、項目33に記載の方法。
(項目35)
前記1つまたはそれを超える星状細胞マーカーが、GFAP、AQP4、CD44、S100b、SOX9、NFIA、GLT-1およびCSRP1からなる群より選択される、項目32~34のいずれか一項に記載の方法。
(項目36)
前記1つまたはそれを超える星状細胞マーカーがGFAPを含む、項目35に記載の方法。
(項目37)
1つまたはそれを超える星状細胞マーカーを発現する細胞が皮質星状細胞または脊髄星状細胞である、項目33~36のいずれか一項に記載の方法。
(項目38)
SMADシグナリングの前記1つまたはそれを超えるインヒビターが、形質転換成長因子ベータ(TGFβ)/アクチビン-ノーダルシグナリングの1つまたはそれを超えるインヒビターおよび骨形成タンパク質(BMP)シグナリングの1つまたはそれを超えるインヒビターを含む、項目2~16および23~37のいずれか一項に記載の方法。
(項目39)
TGFβ/アクチビン-ノーダルシグナリングの前記1つまたはそれを超えるインヒビターが、SB431542、その誘導体およびその混合物からなる群より選択される化合物を含む、項目38に記載の方法。
(項目40)
骨形成タンパク質(BMP)シグナリングの前記1つまたはそれを超えるインヒビターが、LDN193189、その誘導体およびその混合物からなる群より選択される化合物を含む、項目38または39に記載の方法。
(項目41)
幹細胞を分化させるためのインビトロ方法であって、幹細胞の集団をSMADシグナリングの有効量の1つまたはそれを超えるインヒビターに曝露すること、ならびに該細胞をレチノイン酸(RA)シグナリングの有効量の1つまたはそれを超えるアクチベーター(「RAアクチベーター」)およびソニックヘッジホッグ(SHH)シグナリングの有効量の1つまたはそれを超えるアクチベーター(「SHHアクチベーター」)に曝露して、1つまたはそれを超える脊髄前駆体マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団を得ることを含む、インビトロ方法。
(項目42)
SMADシグナリングの前記1つまたはそれを超えるインヒビターへの前記幹細胞の初期曝露から少なくとも1日間、該幹細胞の集団を1つまたはそれを超えるRAアクチベーターおよび1つまたはそれを超えるSHHアクチベーターに初期曝露する、項目41に記載の方法。
(項目43)
検出可能レベルの前記1つまたはそれを超える脊髄前駆体マーカーが、前記1つまたはそれを超えるRAアクチベーターおよび1つまたはそれを超えるSHHアクチベーターへの前記細胞の前記初期曝露から少なくとも約12日間存在する、項目42に記載の方法。
(項目44)
前記1つまたはそれを超える脊髄前駆体マーカーが、HOXB4、ISL1およびNKX6.1からなる群より選択される、項目41~43のいずれか一項に記載の方法。
(項目45)
前記幹細胞がヒト幹細胞である、項目1~44のいずれか一項に記載の方法。
(項目46)
前記幹細胞が多能性幹細胞である、項目1~45のいずれか一項に記載の方法。
(項目47)
多能性幹細胞が、胚性幹細胞、人工多能性幹細胞およびそれらの組み合わせからなる群より選択される、項目46に記載の方法。
(項目48)
前記幹細胞が、胚性幹細胞、人工多能性幹細胞、ヒト単為生殖幹細胞、始原生殖細胞様多能性幹細胞、胚盤葉上層幹細胞、Fクラス多能性幹細胞およびそれらの組み合わせからなる群より選択される、項目1~47のいずれか一項に記載の方法。
(項目49)
少なくとも約10%の1つもしくはそれを超えるグリアコンピテント細胞マーカーおよび/または1つもしくはそれを超える星状細胞マーカーを発現するインビトロ分化細胞の集団であって、項目1~40のいずれか一項に記載の方法により誘導される、集団。
(項目50)
項目49に記載の集団を含む、組成物。
(項目51)
医薬組成物である、項目50に記載の組成物。
(項目52)
被験体における神経変性障害を処置するかまたは神経傷害による損傷を軽減する方法であって、有効量の項目49に記載の集団または項目50もしくは51に記載の組成物を、それを必要とする被験体に投与することを含む、方法。
(項目53)
前記被験体が、神経変性障害と診断されているかまたはそれを有するリスクがある、項目52に記載の方法。
(項目54)
前記神経変性障害が、パーキンソン病、アルツハイマー病、筋萎縮性側索硬化症(ALS)およびレット症候群からなる群より選択される、項目52または53に記載の方法。
(項目55)
神経変性障害を処置するためのまたは神経傷害による損傷を軽減するための医薬の製造における、項目49に記載の集団または項目50もしくは51に記載の組成物の使用。
(項目56)
幹細胞の分化を誘導するためのキットであって、
(a)形質転換成長因子ベータ(TGFβ)/アクチビン-ノーダルシグナリングの1つまたはそれを超えるインヒビター、
(b)BMPシグナリングの1つまたはそれを超えるインヒビター;
(c)NFIAの1つまたはそれを超えるアクチベーター;
(d)LIF、1つもしくはそれを超えるその誘導体、1つもしくはそれを超えるその類似体および/または1つもしくはそれを超えるそのアクチベーター;ならびに
(e)1つもしくはそれを超えるグリアコンピテント細胞マーカーおよび/または1つもしくはそれを超える星状細胞マーカーを発現する少なくとも約10%の分化細胞を含む細胞集団への該幹細胞の分化を誘導するための指示
の1つまたはそれよりも多くを含む、キット。
(項目57)
インビトロ分化細胞の集団を含む組成物であって、細胞の該集団の少なくとも約50%が1つまたはそれを超えるNSCマーカーを発現し、細胞の該集団の約25%未満が1つまたはそれを超える幹細胞マーカーを発現する、組成物。
(項目58)
インビトロ分化細胞の集団を含む組成物であって、細胞の該集団の少なくとも約50%が1つまたはそれを超えるグリアコンピテントNSCマーカーまたはグリアコンピテント細胞マーカーを発現し、細胞の該集団の約25%未満が、幹細胞マーカー、NSCマーカーおよびニューロンマーカーからなる群より選択される1つまたはそれを超えるマーカーを発現する、組成物。
(項目59)
インビトロ分化細胞の集団を含む組成物であって、細胞の該集団の少なくとも約50%が1つまたはそれを超える星状細胞マーカーを発現し、細胞の該集団の約25%未満が、幹細胞マーカー、NSCマーカー、ニューロンマーカーおよびグリアコンピテント細胞マーカー/グリアコンピテントNSCマーカーからなる群より選択される1つまたはそれを超えるマーカーを発現する、組成物。
(項目60)
前記1つまたはそれを超える幹細胞マーカーが、OCT4、NANOG、SOX2、LIN28、SSEA4およびSSEA3からなる群より選択される、項目57~60のいずれか一項に記載の組成物。
(項目61)
前記1つまたはそれを超えるニューロンマーカーが、Tuj1、MAP2およびDCXからなる群より選択される、項目58~60のいずれか一項に記載の組成物。
(項目62)
前記1つまたはそれを超えるNSCマーカーが、PAX6、ネスチン、SOX1、SOX2、PLZF、ZO-1およびBRN2からなる群より選択される、項目58~61のいずれか一項に記載の組成物。
(項目63)
前記1つまたはそれを超える神経幹細胞マーカーが、PAX6、SOX1、PLZFおよびZO-1からなる群より選択される、項目58~63のいずれか一項に記載の組成物。
(項目64)
前記1つまたはそれを超えるグリアコンピテント細胞マーカーが、CD44、AQP4、SOX2およびネクチンからなる群より選択される、項目58~64のいずれか一項に記載の組成物。
(項目65)
前記1つまたはそれを超える星状細胞マーカーが、GFAP、AQP4、CD44、S100b、SOX9、NFIA、GLT-1およびCSRP1からなる群より選択される、項目59~65のいずれか一項に記載の組成物。
Claims (12)
- 少なくとも1つの神経幹細胞マーカーを発現する神経幹細胞(NSC)の集団を分化させるためのインビトロ方法であって、
工程a)少なくとも1つの神経幹細胞マーカーを発現する前記NSCの集団におけるNFIAシグナリングを促進して、少なくとも1つのグリアコンピテント細胞マーカーを発現する少なくとも10%の分化したグリアコンピテント細胞を含む細胞集団を得る工程であって、検出可能レベルの前記少なくとも1つのグリアコンピテント細胞マーカーが、前記細胞におけるNFIAシグナリングの最初の促進から少なくとも5日間存在する工程;および
工程b)工程a)における、少なくとも1つのグリアコンピテント細胞マーカーを発現する少なくとも10%の分化したグリアコンピテント細胞を含む前記細胞集団において、NFIAシグナリングの促進を減少させて、前記グリアコンピテント細胞の、少なくとも1つの星状細胞マーカーを発現する細胞への分化を促進する工程、
を包含する、インビトロ方法。 - NFIAシグナリングを前記促進することが、
i)前記細胞をNFIAの少なくとも1つのアクチベーターに曝露すること、または
ii)NFIAの発現を増加させることを含む、請求項1に記載の方法。 - NFIAの前記少なくとも1つのアクチベーターが、a)前記幹細胞に外因的に曝露されたNFIAタンパク質、b)前記幹細胞に発現された組換えNFIAタンパク質、またはc)NFIAの上流アクチベーターを含む、請求項2に記載の方法。
- NFIAの前記上流アクチベーターがTGFβ1である、請求項3に記載の方法。
- NFIAの発現を前記増加することが、NFIAの過剰発現を誘導するように前記NSCを改変することを含む、請求項2に記載の方法。
- 過剰発現した前記NFIAが、NFIA核酸により発現される組換えNFIAタンパク質である、請求項5に記載の方法。
- 前記NFIA核酸が、誘導性プロモーターに作動可能に連結されている、請求項6に記載の方法。
- 前記神経幹細胞マーカーが、PAX6、ネスチン、SOX1、SOX2、PLZF、ZO-1およびBRN2からなる群より選択される、請求項1に記載の方法。
- a)前記少なくとも1つのグリアコンピテント細胞マーカーが、CD44、およびAQP4から選択される;および/または
b)少なくと1つのグリアコンピテント細胞マーカーを発現する前記細胞が、皮質グリアコピンテント細胞または脊髄グリアコンピテント細胞である、請求項1~8のいずれか一項に記載の方法。 - 工程b)が、工程a)における少なくとも1つのグリアコンピテント細胞マーカーを発現する少なくとも10%の分化したグリアコンピテント細胞を含む前記細胞集団を、少なくとも1つの星状細胞マーカーを発現する細胞への前記グリアコンピテント細胞の分化をさらに促進するために適切な条件に供することをさらに含む、請求項1~7のいずれか一項に記載の方法。
- (a)前記条件が、前記細胞集団を白血病抑制因子(LIF)および/または少なくとも1つのそのアクチベーターに曝露することを含む;および/または(b)前記少なくとも1つの星状細胞マーカーが、GFAP、AQP4、CD44、S100b、SOX9、NFIA、GLT-1およびCSRP1から選択される;および/または(c)少なくとも1つの星状細胞マーカーを発現する細胞が皮質星状細胞または脊髄星状細胞である、請求項10に記載の方法。
- 前記NSCが
a)ヒト幹細胞;および/または
b)胚性幹細胞、人工多能性幹細胞、ヒト単為生殖幹細胞、始原生殖細胞様多能性幹細胞、胚盤葉上層幹細胞、Fクラス多能性幹細胞およびそれらの組み合わせ
から分化したものである、請求項1~7のいずれか一項に記載の方法。
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