JP2018166512A - 三次元不均一分化組織培養 - Google Patents
三次元不均一分化組織培養 Download PDFInfo
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Abstract
Description
プラスミド構築物及び材料。shRNAの同時エレクトロポレーション及びライブ造影に使用されるGFPプラスミドは、pCAG-GFP(Addgene plasmid 11150)であった。ヒトCDK5RAP2を標的とするshRNAsを、pSuper shRNA発現ストラテジ(OligoEngine)を使用してクローン化した。標的配列は、以下のとおりであった:shRNA1 AGGACGTGTTGCTTCAGAAAT(配列番号1)、shRNA2 AGAGTCAGCCTTCTGCTAAAG(配列番号2)、shRNA3 GTGGAAGATCTCCTAACTAAA(配列番号3)、shRNA4 ACTATGAGACTGCTCTATCAG(配列番号4)。CDK5RAP2発現構築物を、AttB部位を有するプライマーを使用してMGCヒトCDK5RAP2 cDNA(クローンID:9052276)からCDK5RAP2のPCR増幅によるゲートウェイシステム(Invitrogen)を使用して生成した:フォワード:GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGGACTTGGTGTTGGAAGA(配列番号5)、リバース:GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCTTTATTGGCTGAAAGTTCTTCTC(配列番号6)。CDK5RAP2を、ディスティネーションベクターpcDNA3.1/nV5-DEST内にクローン化した。
様々な臓器系のインビトロモデルの最近の進展が、多能性幹細胞が全組織を形成する高い自己組織化能力を実証した。ヒト脳の複雑さと不均一をモデル化するアプローチを開発する際に、我々は、この概念を当てにし、人工的に特定の脳領域を駆動する成長因子のあらゆるパターニングを省略した。代わりに、我々は、外発的に特定の脳領域の形成を駆動するより、組織の成長要求性を改善すること、及び本来備わっているきっかけが発生に影響を及ぼすのに必要な環境を提供すること焦点を当てた。
全体的な形態分析により、脳オルガノイドが不均一な脳領域を示したことが示唆されたので、次に、我々はこれらの組織の領域独自性を特徴づけしようと考えた。これらの組織のうまくいっている神経誘導を示して、我々は最初に多能性及び神経の独自性の数種類のマーカーについてRT−PCRをおこない(図8)、そして多能性マーカーOct4及びNanogがオルガノイド分化の過程で減少したが、神経の独自性マーカーSox1及びPax6が上方制御されたことがわかった。
我々はヒト背側皮質の発生と疾患のモデリングに興味を持っていたので、次に、我々は脳オルガノイド内の背側皮質領域の組織化を調べた。放射状グリア前駆体(RGs)及び新生ニューロンのマーカーに関する染色(図3a)により、脳室を連想させる大きい体液の充填した腔に隣接してRGsが層を形成する典型的な前駆帯の組織化が明らかになり、脳室帯(VZ)の形成が示唆された。Tbr1を染色することにより(図11a)、神経の独自性及び発生プレプレート(CPに対する前駆体)への放射状移動の適切な発生が明らかになった。更に、神経の前駆体及び神経特異的BAF成分を染色することにより、神経運命指定中、クロマチン再モデリング複合体における特徴スイッチが明らかになった(図16a)。更に、中間前駆体(IP)のマーカーTbr2(図3b)を染色することにより、VZに隣接するIPsの薄層が明らかになり、それが脳室下帯(SVZ)を連想させた。これにより、背側皮質組織はインビボで見られるのと酷似した典型的な前駆帯の組織化を表す。
放射状に組織化されるCPの形成は、前駆体であるプレプレートの形成で始まる。この初期の組織化を試験するために、我々は、プレプレートのマーカーTbr1並びに神経マーカー38であるMap2について30日オルガノイドについて染色した(図12a)。これにより、プレプレートを連想させる基底神経層の存在、及びIZを連想させる尖状隣接部位が明らかになった。更に、我々は基底面に沿ってリーリン陽性ニューロンを観察することができ、CPアーキテクチャの生成において重要な集団であるカハール−レツィウス細胞の存在が示唆された。
脳オルガノイドが神経発生におけるヒト固有のプロセスを試験するのに使用できるか調べるために、我々は発生的により高度な背側皮質組織において前駆帯の形態を調べた。これらの領域は、より高度な段階まで発生させた場合、一般的にはるかに厚くて、且つ非常に大きくなった(オルガノイド内のただ一つの背側皮質領域が直径最大1mmまで成長できた)。我々は、RGs及びニューロンを染色し、そして尖状表面から置き換えられるように見える多くのSox2陽性前駆体を観察した(図5a、図18a)。これらの前駆体のマーカーの独自性及び位置は、その前駆体がマウスや他の下等哺乳類と比べて、ヒト大脳皮質では代表が多すぎる最近同定された前駆体タイプである外側放射状グリア(oRGs)を表す可能性を指摘する。
小頭症は、(平均を2標準偏差より大きく下回る)小さい頭囲で示される神経発生障害であり、そしてそれは、大きく低減した脳サイズの発生が原因である。いくつかの遺伝子が原発性小頭症、並びに小頭性骨異形成性原発性小人症(MOPD)やゼッケル症候群などのいくつかの重複疾患で同定された。モデル系における徴候は、これらの疾患で同定された遺伝子の多くが中心体又はDNA修復において機能し得ることを示唆していたが、マウス変異体は同じ重症度の表現型を示さないことが多かったので、ヒト小頭症表現型はモデル化することが著しく難しかった。この疾患が発生中の脳の肥大の欠損を反映し、且つ、ヒト脳は増殖機構において重要な分岐を呈するので、我々は、ヒト脳オルガノイドの方がこの疾患の側面のより良好なモデルとなり得ると仮定した。
ヒトの脳の発生は、我々が紐解き始めるだけの多くの独特な特徴を呈している。我々がヒトの脳の発生に関して知っていることの大部分は、齧歯動物や他の下等哺乳類と共通した基本的なプロセスに限定されていた。脳発生の基本的な機構を理解するのにこれらの洞察が必要不可欠であるのに対して、これらの神経発生的な試験は、モデル系の利用可能性によって制限されてきた。
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Claims (17)
- 少なくとも2つの異なる前駆体及び神経分化層の不均一細胞集団を含むインビトロ(in vitro)培養人工三次元神経組織培養物であって、
少なくとも1つの前駆体層が、外側放射状グリア細胞を含み、前記組織が更に、外側又は余剰皮質脳室下帯(outer or extra cortical subventricular zone)と、皮質内側線維層(cortical inner fiber layer)細胞とを含み、
前記培養物が三次元マトリックスを含み、前記三次元マトリックスはヒドロゲルであるか、或いは、前記培養物が、神経分化多細胞凝集体を三次元マトリックス内で培養して得られる、組織培養物。 - 前記組織の部分は少なくとも2つの層を形成し、好ましくは少なくとも1つの層は球状組織体周囲に形成される、請求項1に記載の組織培養物。
- 前記組織は尖状(apical)組織部分及び背側(dorsal)組織部分を発生させる、請求項1又は2に記載の組織培養物。
- 前記組織は脳組織である、請求項1〜3の何れか一項に記載の組織培養物。
- 前記培養物の細胞は、前脳マーカーBF1及びSix3並びに後脳マーカーKrox20及びIls1から選択される1又は2以上の遺伝子発現マーカーを発現し、好ましくは前脳マーカーの発現量は後脳マーカーと比べて増加している、請求項1〜4の何れか一項に記載の組織培養物。
- 前記培養物の細胞は、Otx1、Otx2、FoxG1、Auts2、Tuj1、Brn2、Satb2、Ctip2、カルレチニンから選択される1又は2以上の遺伝子発現マーカーを発現する、請求項1〜5の何れか一項に記載の組織培養物。
- 前記培養物が、多能性細胞を培養することにより得られる、請求項1〜6の何れか一項に記載の組織培養物。
- 人工組織培養物を生成する方法であって、
a)多能性幹細胞の多細胞凝集体を提供し、
b)前記多細胞凝集体を神経誘導培地中で培養することにより、前記多細胞凝集体を神経組織へと分化させ、
c)前記多細胞凝集体を三次元マトリックス、好ましくはゲル中で培養し、これにより多細胞凝集体内の前記細胞を増殖させつつ、前記細胞の分化を許容し、次いで
d)前記c)の増殖した細胞の多細胞凝集体を、懸濁培養物中で培養する
ことを含む方法。 - 前記多能性細胞は人工多能性細胞、特に患者より単離された人工多能性細胞である、請求項8に記載の方法。
- 前記増殖細胞は分化単能性幹細胞へと分化する、請求項8又は9に記載の方法。
- 前記三次元マトリックスは、Engelbreth-Holm-Swarm腫瘍由来のコラーゲン又は細胞外マトリックス、或いはラミニン、コラーゲン、エンタクチン、及びヘパラン硫酸化プロテオグリカン、又はこれらの組み合わせから選択されるその成分を含む、請求項8又は9に記載の方法。
- 発生的神経組織作用を調べる方法であって、請求項8〜11の何れか一項に記載の方法の何れかの段階で、細胞内での所望の遺伝子の発現を減少又は増加させることを含む方法。
- 所望の発生的神経組織欠損の治療に適した候補治療薬を選別する方法であって、請求項12に記載の方法を実施し、前記方法の何れかの段階で、好ましくは全ての段階で、前記候補薬剤を前記細胞に投与することを含む方法。
- 候補薬物の神経作用を試験する方法であって、候補薬物を請求項1〜7の何れか一項に記載の人工培養物に投与し、前記培養物の細胞について所望の活性を決定し、当該活性を、前記候補薬物を投与せずに培養した細胞の活性と比較することを含み、活性の差分が神経作用を示す、方法。
- 分化神経細胞を得る方法であって、請求項1〜7の何れか一項に記載の人工培養物を提供し、所望の分化神経細胞を単離することを含み、或いは、請求項8〜11の何れか一項に記載の人工組織培養物生成方法を実施し、更に所望の分化神経細胞を単離することを含む方法。
- 請求項8〜11の何れか一項に記載の方法により得られうる人工組織培養物。
- 請求項1〜7の何れか一項に記載の人工三次元神経組織培養物を生成し、或いは請求項8〜15の何れか一項に記載の方法を実施するためのキットであって、
i)三次元マトリックス及び栄養素を含む培地と、
ii)レチノイン酸及び栄養素を含む培地と、
iii)a)栄養素及びb)ROCK阻害剤、インシュリン、又はヘパリンを含む培地とを含み、
更に任意により、栄養素を含むが、神経組織を特定の運命へと分化させる成長因子を欠損する培地を更に含む、キット。
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