我々は、この報告書の重要なレビューと編集のために博士Soshanaスベンセンに感謝します。この作品は、NIH / NINDS 1U24NS078370-01とCIRM DR2A-05320によって寄贈されました。

著者らは、開示することは何もありません。

<img alt="図6" fo:content-width="5in" src="/files/ftp_upload/51219/51219fig6highres.jpg" width="500" /> 図6。チョッピング回路図 。機械的なチョッピング法を用いて、培養中の回転楕円体幹/前駆細胞を拡大する。 重要なステップチョッピング膨張パラダイムの概要を図6に示す。HNPC球サイズがニューロスフェアを継代前に観察するための重要な基準の1つである。球体サイズの大きな分散があるが、球体の少なくとも30％が300ミクロンよりも大きい直径を有するべきである。球が通路に小さすぎると1-4日後に、単に新鮮なMM（25％-50％）でCMを交換し、再評価する。球体は直径が小さすぎるで継代したとき悪い膨張率が発生します。 HNPC膨張率も球密度及びcのメディアに依存している原因分泌される因子47にonditioning。メディアが過度に代謝され、重要な成長因子が減少している場合は、細胞は、細胞48の核型異常な集団を取得してもよい。そのため、交換する栄養素や成長因子は分泌された栄養因子とバランスをとらなければならない。 hNPCs後CHOPまたは解凍後に播種すると、メディアの1cm 3当たりの神経球の数は可変である。しかしながら、一般的なルールは、密度も大きい、高速メディアは調整され、より高い膨張率は、必要なときにメディアが交換された。メディアが完全に代謝されたときに黄色に、未代謝ときピンクの範囲であるメディア·コンディショニングカラースペクトル（ 図5D）を使用します。対数増殖のための理想的なメディアの色が赤みがかったオレンジ色、色見本の＃3です。これは、分泌されたtrophの適切な濃度を維持しながら、媒体に十分な栄養素および成長因子が含まれていることを示しICの要因。メディアは、色見本、過去＃4を条件付けるべきではありません。あるいは、細胞は（色見本の＃1）すぐにメディアを代謝していないとき、hNPCsが遅く成長し、7〜10日ごととは対照的に、すべての10-20日チョップを必要とする。それは、順番に伸張率を改善するコンディショニングを高めるために培養密度を高めることが重要である。 チョッピング手順の間に、次の重要なステップに注意してください。ブレードはチョッパアームと平行に設置されていることを確認してください。ブレードは、ペトリ皿またはシムディスクの表面上に平らになっていない場合は、多くの球体を細断しなくてもよい。ステップ4.11で説明した細胞の広がりに注意してください。球はすべての球がみじん切りされた前壁に接触しますペトリ皿またはブレードの中央に残しておく必要があります。最後に、細断処理中に長時間hNPCsを乾燥避けることが重要である。皿に球を置いた後、みじん切りにし、できるだけ早く新しい培地でそれらをあふれさせる可能。 この技術は複数の機器を必要とするように、文化の無菌性へのリスクがある場合もあります。このリスクを考えると、適切な無菌技術が手順全体で使用されている必要があります。基礎研究レベルの生産のために、70％のIPAまたは同等のすべての機器を下にふくと、15分の最小のオプションの紫外線インキュベーションと一緒に十分なものである。無菌BSCの内部のすべての手順を実行します。製造元が推奨するようにcGMP量の生産のために、チョッパーはエチレンオキサイドで滅菌されなければならない。他のすべての電源は、滅菌購入またはオートクレーブ処理することができます。 将来の応用枕元にベンチからの基礎研究療法の移行は、挑戦することができます。チョッピング手順は、この点を考慮して移行して設計されました。手順は既にウィスコンシン大学Waismanセンターバイオ製造設備においてcGMPに準拠HNPCバンクを作製するために使用されている。 T帽子HNPCバンクは、連邦医薬品局（FDA）の承認は、次の新しい前の治験薬の研究およびフェーズ1臨床試験のために使用される医薬品グレードHNPC細胞ロットの拡大のために調達した。拡張のため、この手順を使用し、他の細胞の種類があります。例えば、多能性幹細胞41から発生する神経幹細胞EZ球である。この技術は、膨張の間、一定の細胞間接触を必要とする他のタイプの組織で使用するための大きな可能性を秘めている。

A correction was made to <a href="/video/51219">A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue</a>. The corresponding author was changed from:
Brandon C. Shelley 
to:
Clive Svendsen

A correction was made to <a href="/video/51219">A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue</a>. The corresponding author was changed.

Erratum: A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

単層1-3または集約ニューロスフェア4-7のいずれかとして、培養中のげっ歯類の神経幹細胞を増殖の長い歴史があります。また、現像8-17中枢神経系の種々の領域から単離されたヒト神経前駆細胞（hNPCs）をインビトロで増殖されている。これらの細胞は、双方向性、強力、星状細胞及び神経細胞の両方に分化することができると神経の発達18,19及び疾患メカニズム20,21を研究する上で非常に有用なツールとなっている。 hNPCsまた、統合、生存および機能的効果22〜24種々のレベルの中枢神経系疾患の多くの異なる動物モデルに移植されている。 多くの場合、上皮成長因子（EGF）および/ ​​または線維芽細胞増殖因子-2（FGF-2）25-28 - - 、付着29、三両方伝統的には、齧歯類またはヒト胎児由来のNPCは、増殖因子に暴露される次元スフェロイドシステムは、典型的には単一細胞懸濁液30-34に酵素的解離を使用して継代する。研究や臨床用細胞を拡大する標準的な方法は、容易な操作のために接着単層としてある。しかし、我々は、酵素的または化学的溶液で継代単層およびニューロスフェアhNPCs、早期老化35をもたらしたことを示した。加えて、酵素的解離は、胚性幹細胞36〜38で実証されたデータに基づいて、分化および核型異常のレベルの増加をもたらし得る。継代hNPCsの標準的な方法は、現在の適正製造基準相に1臨床試験（細胞株、Neuralstem社幹細胞）行っている（cGMP）のグレードの製品を生産しているが、この方法では限界、細胞増幅のほんの数ラウンドを許可銀行の可能性。 明らかに、大規模な研究の実験と今後の臨床試験では、能力から利益を得ることができる大規模な増殖および細胞バンキングを可能にするためにバルク及び遅延された老化で細胞を伝播する。この必要性に対処するために、我々は細胞間接触を維持する小さなクラスターにそれらを「チョッピング」によって、新規かつ機械的に継代無傷の神経球の自動化された方法を開発した。この方法は、大幅に代替3Dバイオリアクター培養方法40で見られるようなそれらの寿命39および懸濁培養物は、単層培養物と比較して、インキュベータ·スペースのより効率的な使用を可能にする増加した。チョッピング設けプロトコルは、通路10よりも1つ大きい胎児試料、標準継代方法を用いて難い偉業から大規模銀行の産生を可能にする。継代hNPCsためのこの方法は、型破りであるが、人気が高まっており、最近では、ヒト胚性及び誘導多能性幹細胞由来の神経幹細胞のような他の細胞型、V INを含む様々な用途のための大規模拡大を可能にしてパブリッシュされた病気のモデリング41-46 itro。重要なことに、cGMPのグレードHNPC細胞バンクは、既に技術は、将来の臨床応用に向けて適用することができることを実証し、チ​​ョッピング方法で製造されている。

<table><tbody><tr><td>Beaker, 50 ml</td><td>Fisherbrand</td><td>FB-100-50</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Bio-Safety Cabinet, class II</td><td>Baker</td><td>SG-603A</td><td>4 ft. or 6 ft. model.&amp;nbsp; 6 ft. model recommended; multiple manufacturers/suppliers</td></tr><tr><td>Blades, Double-edge Prep</td><td>Personna</td><td>74-0002</td><td>multiple manufacturers/suppliers.&amp;nbsp; CAUTION: Sharp</td></tr><tr><td>Cell Freezing Media</td><td>Sigma-Aldrich</td><td>C6295-50ML</td><td>DMSO, serum-free</td></tr><tr><td>Centrifuge, swing-bucket with 15 ml inserts</td><td>Eppendorf</td><td>5810 R</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Conical Tubes, 15 ml</td><td>Fisherbrand</td><td>S50712</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Conical Tubes, 50 ml</td><td>BD Falcon</td><td>352074</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Controlled Rate Freezer</td><td>Planer</td><td>Kryo 750</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Cryovials, 2 ml</td><td>Corning</td><td>430488</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T12.5</td><td>BD Falcon</td><td>353107</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T25</td><td>BD Falcon</td><td>353081</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T175</td><td>BD Falcon</td><td>353045</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Culture Flask, Vented, T75&amp;nbsp;</td><td>BD Falcon</td><td>353110</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 1 L</td><td>Millipore</td><td>SCGPU11RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 150 ml</td><td>Millipore</td><td>SCGVU01RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 500 ml</td><td>Millipore</td><td>SCGPU05RE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter, 0.22 &amp;micro;m, attached cup, 50 ml</td><td>Millipore</td><td>SCGP00525</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Filter Paper, 8.5 cm circles</td><td>Whatman/GE</td><td>1001-085</td><td></td></tr><tr><td>Forceps, Standard Pattern - Serrated/Curved/18 cm</td><td>Fine Science Tools</td><td>11001-18</td><td></td></tr><tr><td>Freezing Chamber, Isopropyl Alcohol</td><td>Nalgene</td><td>5100-0001</td><td>&quot;Mr. Frosty&quot;</td></tr><tr><td>Incubator, 37 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma</td><td>370 series</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Hemacytometer, Phase</td><td>Hausser Scientific</td><td>1475</td><td>multiple manufacturers/suppliers</td></tr><tr><td>McIlwain Tissue Chopper</td><td>Lafayette Instruments</td><td>TC752-PD</td><td>&amp;nbsp;Petri dish modification required.&amp;nbsp;&amp;nbsp; CAUTION:&amp;nbsp; Moving, sharp blade.</td></tr><tr><td>Micropipettor, 1-10 &amp;mu;l</td><td>Gilson</td><td>F144562</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 100-1,000 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 2-20 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Micropipettor, 20-200 &amp;mu;l (starter kit)</td><td>Gilson</td><td>F167700</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Nutdriver, Autoclavable, 5/16&quot;</td><td>Steritool</td><td>10302</td><td></td></tr><tr><td>Pasteur Pipets, cotton-plugged</td><td>Fisherbrand</td><td>13-678-8B</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Petri Dish, Glass, Autoclavable</td><td>Corning</td><td>3160-100</td><td></td></tr><tr><td>Pipet Aid</td><td>Drummond</td><td>4-000-101</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Shim disc</td><td>McMaster-Carr</td><td>VARIABLE</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 10 &amp;mu;l</td><td>AvantGuard</td><td>AV10R-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 1,000 &amp;mu;l</td><td>AvantGuard</td><td>AV1000</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 20 &amp;mu;l</td><td>AvantGuard</td><td>AV20-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile barrier pipet tips, 200 &amp;mu;l</td><td>AvantGuard</td><td>AV200-H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 10 ml</td><td>Fisherbrand</td><td>13-676-10J</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 2 ml</td><td>Fisherbrand</td><td>13-675-3C</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 25 ml</td><td>Fisherbrand</td><td>13-676-10K</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterile Disposable pipettes, all-plastic wrap, 5 ml</td><td>Fisherbrand</td><td>13-676-10H</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Sterilization Pouches, 19 x 33 cm</td><td>Crosstex</td><td>SCL</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Strainer, 40 &amp;micro;m</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Tissue Culture Dishes, 60 mm</td><td>BD Falcon</td><td>351007</td><td></td></tr><tr><td>Tube Racks, Interlocking Four-Way</td><td>Fisherbrand</td><td>03-448-17</td><td></td></tr><tr><td>Water Bath</td><td>Fisherbrand</td><td>S52602Q</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Neural Progenitor Cell-Specific Processing Reagents</td><td></td><td></td><td></td></tr><tr><td>Neural Stem Cell Expansion Medium (Stemline)</td><td>Sigma-Aldrich</td><td>S3194-500ML</td><td>Important to use the Stemline brand</td></tr><tr><td>Recombinant Human Epidermal Growth Factor (EGF)</td><td>Millipore</td><td>GF316</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Recombinant Human Leukemia Inhibitory Factor (LIF)</td><td>Millipore</td><td>LIF1010</td><td>multiple manufacturers/suppliers</td></tr><tr><td>Trypan Blue (0.4%)</td><td>Sigma-Aldrich</td><td>T8154-100ML</td><td>multiple manufacturers/suppliers</td></tr><tr><td>TrypLE Select (1x)</td><td>Life Technologies</td><td>12563-011</td><td></td></tr></tbody></table>

a cgmp-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue

研究実験および臨床試験のための単一の試料から多数の細胞を蓄積するための細胞増殖技術が大幅に幹細胞のコミュニティに利益をもたらす。現在の多くの拡張方法は、労力を要し、高価であり、かつ完全な解離を伴うものは、いくつかの幹細胞および前駆細胞型の分化または早期老化を受ける可能性があります。これらの問題を克服するために、我々は、簡単かつ安価である「チョッピング」と呼ばれる自動化された機械的な継代方法を開発した。この手法は、単一の細胞に化学的または酵素的解離を回避し、代わりに一定の細胞/細胞接触を維持中断、スフェロイド培養の大規模な拡張が可能になります。チョッピング方式は、主に胎児脳由来の神経前駆細胞又はニューロスフィアのために使用されており、最近では、胚及び誘導多能性幹細胞由来の神経幹細胞で使用するために公開されている。プロシージャinvolvES組織培養ペトリ皿にニューロスフェアを播種し、その後効果的に手動で機械的に球を解離させる退屈なプロセスを自動化するセルを鋭い、無菌刃を通す。培養中で細胞を懸濁する良好な表面積対体積比を提供する。 50万の細胞は直径が0.5mm未満の単一神経球内で成長させることができるように。 1 T175フラスコ中に、5000万個以上の細胞でのみ1500万接着培養中に比べて懸濁培養で成長することができます。重要なのは、チョッピング手順は、臨床グレードの細胞産物の大量量産を可能とする、現在の適正製造基準（cGMP）の下で使用されています。

<img alt="図5" fo:content-width="5in" src="/files/ftp_upload/51219/51219fig5highres.jpg" width="500" /> 図5。代表的なデータ。A）は、チョッピングメソッドを介して継代したニューロスフェアに比べて酵素的解離を使用して接着単層として解凍し、拡大し、P19で凍結hNPCsの細胞数を予測した。細胞はP20で解凍したとき、0日目に表しています。B）は球の代表的な画像を事前にチョップ、球後CHOP、10X、D）色見本は、メディアのコンディショニングの範囲を記述するために使用されるのcGMPの10倍。C）代表的な画像比較可能なプロトコルをサポートします。E）1日のためにプレーティングし、ネスチン（赤）の発現について染色した解凍されたP29のhNPCsの免疫細胞。ヘキスト核対比染色（青）、F）7日間と汚れのためにプレーティングした解凍されたP29のhNPCsの免疫細胞化学GFAP（赤）とβ-IIIチューブリン（緑）のED。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51219/51219fig5highres.jpg" target="_blank">拡大画像を表示するにはここをクリックしてください。</a> 図5Aは、P20（0日）で解凍し、ラミニン被覆組織培養フラスコまたはチョッピングメソッドを介して継代し、非接着ニューロスフェアとして付着性を維持hNPCsを表しています。付着細胞を酵素的に毎週選びのTrypLE用いて解離し、培養液中の70日間（7月10日継代）の後に老化した。これとは対照的に、チョッピング技術を介して通路20で解凍し、拡張されたニューロスフェアは、老化の前に40を超える継代の成長した。 （ 図5B）をチョッピングする前に、典型的なhNPCsは主に直径≥300程度にする必要があります。一度みじん切り、球は一般的に大部分が約200ミクロン、直径（ 図5C）、可変サイズのセクションに四等分される。それはhNPCsがchoppin経由で​​拡大していることに留意することが重要であるグラムHNPC方法は、マーカーの発現を維持し、両方の星状細胞およびニューロン分化後に生産することができる。後期継代hNPCsは、解離つまたは七日の期間、ラミニン被覆カバースリップ上に播種し、続いて4％パラホルムアルデヒドで固定した。 hNPCsは1日に前駆細胞のマーカーであるネスチン（ミリポア、1:1,000）を発現hNPCs（図5E）だけでなく、分化した神経マーカーグリア線維性酸性タンパク質（GFAP、1:500）およびβ-IIIチューブリン（1メッキ：それぞれ7日間メッキhNPCs（ 図5F）、星状細胞および未成熟な神経細胞のマーカー、上2000）。

Watch this Scientific Journal Video about A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue at JoVE.

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

このプロトコルは、単一の細胞懸濁液に解離することなく、球状の神経幹細胞および前駆細胞集合体の拡大を可能にする新たな機械的なチョッピング方法が記載されている。細胞/細胞接触を維持することは、40以上の継代するための迅速かつ安定的な成長を可能にします。

ヒト神経の集合体のためのcGMP適用可能な展開法は、幹細胞および前駆細胞は、多能性幹細胞や胎児の脳組織由来

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

a-cgmp-applicable-expansion-method-for-aggregates-human-neural-stem

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.4K ビュー.  Cedars-Sinai Medical Center. このプロトコルは、単一の細胞懸濁液に解離することなく、球状の神経幹細胞および前駆細胞集合体の拡大を可能にする新たな機械的なチョッピング方法が記載されている。細胞/細胞接触を維持することは、40以上の継代するための迅速かつ安定的な成長を可能にします。 

ビデオ: A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

成体ラットおよびヒト脊髄の脳室周囲領域から神経幹/前駆細胞の単離

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

発生生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

ヒト人工多能性幹細胞由来の錐体ニューロンからの樹状突起棘の三次元定量

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

ヒト神経幹細胞を継代

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

生物学

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

廃棄されたヒト胎児脳組織から神経幹細胞の生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

神経科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

初期の神経発達障害をモデル化するためのiPSC由来のヒト脳オルガノイドの生成

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

神経毒性試験のための、ヒト誘導多能性幹細胞のニューロンとグリアの混合培養物への分化のためのプロトコール

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

血液由来神経細胞のGMフリー生成

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

3次元浮遊バイオリアクターにおけるヒトニューロゲニン2誘導性ニューロンの生産

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...