Name: efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors
Uploaded: 2017-03-07T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

この原稿は、トロント一般と西洋財団とバンティング＆ベスト糖尿病センター、大学健康ネットワーク大学院賞からの資金によってサポートされていました。

ソリューションとメディアの作製注：無菌環境での細胞培養のためのすべてのメディアを準備します。媒体を作製し、直ちに使用しなければなりません。試薬の詳細は材料表で提供されます。 <ol><li> 分化培地 <ol><li> 1％グルタミンを含むRPMI中、2μMCHIR 99021、100ng / mlのアクチビンA、10 4 M MTG：0日分化メディアを準備します。 </li><li> 1％グルタミン、100ng / mlのアクチビンA、10 4 M MTG、5 ngの/ mlのbFGF、50μg/ mlのアスコルビン酸を含むRPMI培地：日1-2分化のメディアを準備します。 </li><li> 1％グルタミン、1％のB27、10 4 M MTG、0.75μMドルソモルフィン、50 ngの/ mlのFGF10を含むRPMI培地：日3-5分化のメディアを準備します。 </li><li>日6-7分化メディアを準備しますDMEMを1％グルタミン、1％のB27、50μg/ mlのアスコルビン酸、50 ngの/ mlのFGF10、0.25μMのSANT-1、2μMレチノイン酸、50 ngの/ mlのノギンで。 注：レチノイン酸は、最後の培地に添加されるべきであり、メディアが酸化分解15を防止するために、光から保護されるべきです。 </li><li>日8-12分化メディアを準備しますDMEMを1％グルタミン、1％のB27、50μg/ mlのアスコルビン酸、50 ngの/ mlのノギン、100ng / mlののhEGF、10 mMのニコチンアミドと。 </li></ol></li><li> PBS中10％FBS、マイナスカルシウムとマグネシウム：FACS緩衝液を準備します。 </li></ol> 2. HESC分化注：HESC、解凍継代およびbFGF 16を補充しKOSRベースのメディアの存在下で照射したマウス胚性線維芽細胞上で展開されます。細胞は、80から95までパーセントの集密度に達したときのhESCは、分化の準備ができています。この時点でコロニーが定義された境界線と&#39;domelike」構造（ 図1A）との大きさであるべきです。すべての細胞培養は、平底、0.1％ゼラチンでコーティングした組織培養処理プレート中で行われます。典型的に、細胞は6で増殖させますかそれぞれ2ミリリットルまたは1ミリリットルのメディアボリュームで12ウェルプレート、。 <ol><li> 0日目に、0日目の分化メディアでKOSRベースのメディアを交換することによって分化を始めます。 </li><li> 1日目および2日目に、静かに吸引する前に、単層から任意の死んだ細胞を除去するためにプレートを振ります。日1-2分化のメディアと交換してください。 </li><li> 3日目に、フローサイトメトリーのための収穫細胞。細胞が90％以上CXCR4 + / CD117 +を発現した場合は、2.4に進みます。 </li><li> 3日目および5日目に、静かに前吸引に単分子層から任意の死んだ細胞を除去するためにプレートを振ります。日3-5分化のメディアと交換してください。 </li><li>日6および7に、日6-7分化のメディアと交換してください。 </li><li> 8日、10と12日、日8-12分化のメディアと交換してください。 </li><li> 13日目に、フローサイトメトリーのための収穫細胞は、PDX1とNKX6-1式を決定します。 </li></ol>フローサイトメトリー分析のため3.収穫細胞<ol><li>で細胞を解離1X商業トリプシン溶液を製造業者のプロトコールに従って、3分間37℃でインキュベートします。 </li><li> 30μlのDNアーゼIと千μlのFACS緩衝液中で単一細胞を商業トリプシン溶液を除去し、再懸濁</li><li>マイクロチューブに35μmのナイロンメッシュセルストレーナーと転送を使用してフィルタ細胞。 </li><li> 5分間、455×gで遠心分離した細胞。ライブまたは固定のいずれかの染色のための細胞を準備します。 </li></ol>フローサイトメトリー用4.染色<ol><li> 3日目のライブ染色のための準備を流れます <ol><li>千μlのFACSバッファーで細胞を再懸濁し。 （未染色および染色された両方のサンプル用）96ウェルプレートの2ウェルに200μlの（約1-3×10 5細胞）を転送。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li> APC及びCD117：PE（Mは表をaterials参照）3用の一次結合抗体、CXCR4とステイン光から保護し、室温で0分。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li> 100μlのFACS緩衝液中に再懸濁サンプル。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li> 1ミリリットルマイクロ試験管または5ミリリットル丸底チューブに300〜500μlのFACSバッファーの全体積で各サンプル、転送を再懸濁します。 </li><li> 17フローサイトメーター上で実行したサンプル。サンプルはすぐに実行することができない場合は、4°Cで保存光から保護します。 </li></ol></li><li> 13日目に固定された染色のための準備を流れます <ol><li> 4℃で24時間、商業固定/透過処理溶液中の細胞ペレットを再懸濁します。 </li><li> 5分間、455×gでサンプルを遠心。 400μlのパーマ/洗浄溶液中に再懸濁します。 </li><li> TWにサンプル（約0.5〜1×10 6細胞）の200μLを移します（IgGコントロールとPDX1 / NKX6-1染色用）を、96ウェルプレートのウェルO。 2分間931×gで遠心分離します。 </li><li>抗PDX1および抗NKX6-1プライマリまたはアイソタイプ対照抗体を含有する100μlのパーマ/洗浄溶液中で細胞ペレットを再懸濁（ 材料表を参照してください）。 4℃で一晩インキュベートします。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 100μlのパーマ/洗浄溶液中に再懸濁サンプル。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li>光から保護し、室温で1時間、二次抗体を含有する100μlのパーマ/洗浄でサンプルを再懸濁します。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li> 100μlのパーマ/洗浄溶液中に再懸濁サンプル。 </li><li> 2分間931×gでプレートを遠心。プレートを反転させ、上清を除去します。 </li><li>代表ステップ4.2.9と4.2.10を食べます。 </li><li> 1ミリリットルマイクロ試験管または5ミリリットル丸底チューブに300〜500μlのFACSバッファーの全体積で各サンプル、転送を再懸濁します。 </li><li> 17フローサイトメーター上で実行したサンプル。サンプルはすぐに実行することができない場合は、4°Cで保存光から保護します。 </li></ol></li></ol>

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成功し、in vitroで hPSCsからNKX6-1 +膵臓前駆細胞を生成する膵臓の開発中にキーの発達段階を管理する特定のシグナル伝達経路の正確なレギュレーションを伴うhPSCsの高品質の文化と向かう分化の使用に依存しています。このプロトコルは、以下の考慮がなされるべきである効率的なNKX6-1生成を確実にするために、先に示したように3 HPSC線の多様にわたってNKX6-1?...

糖尿病の有病率は増加しており、カナダ糖尿病協会によると、1型糖尿病（T1D）1を持つこれらの個体5〜10％の、カナダで1100万人が糖尿病または前糖尿病であると推定されています。 T1Dは、ランゲルハンス島の中に配置されているインスリン産生β細胞の破壊により引き起こされる自己免疫疾患です。現在、T1Dとの生活の個人は、インスリン2の外因性の源を必要とします。インスリン療法の進歩にもかかわらず、T1D患者は、血糖値を調節する困難な時期を持っており、ハイポや高血糖の両方を苦しみ続けるために続けています。 T1Dにおいて正常血糖を回復させる治療の有望な形は、in vivoおよびin vitro 3 の両方で β細胞のインスリン産生の無制限の供給を生成するために使用することができるヒト胚性幹細胞（hESC）の使用であります<sクラス= &quot;外部参照&quot;&gt; 4、5、6、7アップ。 β様する細胞は、それが可能なin vitroで糖尿病を研究することを可能にする2型糖尿病のための新たな治療標的の同定を可能にし、T1D患者への移植用の細胞を提供することができるヒトES細胞を分化。 in vitroでのヒトES細胞からのインスリン産生細胞の生成で最も成功した試みは、膵臓の開発4、5中に発生する胚のイベントを再現することです。これは、正確に開発膵臓の主要な段階をモデル化するための明確なシグナル伝達経路の操作を必要とします。膵臓の開発は、CXCR4およびCD117（C-KIT）8、9の発現によって特徴付けられる胚体内胚葉の誘導で始まります。 definitの正確なレギュレーションアイブ内胚葉組織は、その後、前方対後方と腹側背パターニングを受け、腸管の形成のために必要とされます。背側と腹側膵臓芽は、膵臓の開発10のために必要である膵臓および十二指腸ホメオボックス遺伝子（ のPdx1）を発現する後部前腸の領域から出てきます。背側と腹側芽ヒューズはその後、大規模な上皮再構築および展開11を受ける膵臓を形成します。内分泌と外分泌系統へのコミットメントを表明多能性前駆細胞（MPCは）の生成を伴っている、とりわけ、転写ははPdx1、NKX6.1およびPtf1a 12、13因子 。内分泌および導管細胞になるだろうMPCがPtf1a発現を減少させながらNkx6-1を発現し続けます。これに反して、外分泌系統細胞はexpressioを失うことになりますNkx6-1のnおよびPtf1a式 12を維持します 。 転写因子Nkx6-1、特にβ細胞する内分泌前駆細胞の分化の間に、膵臓の発達において重要な役割を有しています。前述のように、Nkx6-1の欠失は、膵臓の発達14中β細胞の障害の形成をもたらします。したがって、 インビトロおよびインビボの両方において 、インスリン産生β細胞を生成するNkx6-1の効率的な誘導を必要とします。 我々は最近、効率的にhPSCsからPDX1 + / NKX6-1 +膵臓前駆細胞を生成するためのプロトコルを開発しました。これらのHPSC由来の膵臓前駆細胞は、免疫不全マウス3への移植の際に成熟β細胞を生成します。 1）胚体内胚葉誘導：分化プロトコルは、特徴的な4段階に分けることができます、2）後部前腸パターニング、3）膵臓仕様および4）NKX6-1誘導。ここでは、有向分化の各ステップの詳細な説明を提供します。

<table><tbody><tr><td>&lt;strong&gt;Media and cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Thioglycerol (MTG)</td><td>Sigma</td><td>M6145</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D</td><td>338-AC/CF&amp;nbsp;</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma</td><td>A4544</td><td></td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Buffer</td><td>BD Bioscience</td><td>554722</td><td></td></tr><tr><td>BD Perm/Wash buffer, 1x</td><td>BD Bioscience</td><td>554723</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>CHIR990210</td><td>Tocris</td><td>4423a</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11995</td><td></td></tr><tr><td>DNase I</td><td>VWR</td><td>80510-412&amp;nbsp;</td><td></td></tr><tr><td>Dorsomorphin</td><td>Sigma</td><td>P5499</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D</td><td>236-EG</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>88150</td><td></td></tr><tr><td>FGF10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>Glutamine</td><td>Life Technologies</td><td>25030</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>NO636</td><td></td></tr><tr><td>NOGGIN</td><td>R&amp;amp;D</td><td>3344-NG</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875</td><td></td></tr><tr><td>SANT-1</td><td>Tocris</td><td>1974</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x), phenol red</td><td>Life Technologies</td><td>12605-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies for flow cytometry (working dilutions)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD117 PE (1:100)</td><td>Life Technologies</td><td>CD11705</td><td></td></tr><tr><td>CXCR4 APC (1:50)</td><td>BD&amp;nbsp; Bioscience</td><td>551966</td><td></td></tr><tr><td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td><td>Life Technologies</td><td>A-31571</td><td></td></tr><tr><td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.</td><td>705-546-147</td><td></td></tr><tr><td>Isotype Control Mouse IgG&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories Inc.&amp;nbsp;</td><td>015-000-003</td><td></td></tr><tr><td>Isotype Control Goat IgG</td><td>R&amp;amp;D&amp;nbsp;&amp;nbsp;</td><td>AB-108-C</td><td></td></tr><tr><td>NKX6-1 (1:2,000)</td><td>DSHB</td><td>F55A10</td><td></td></tr><tr><td>PDX1 (1:100)</td><td>R&amp;amp;D</td><td>AF2419</td><td></td></tr></tbody></table>

efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors

多能性幹細胞は、自己に更新し、それらの研究および糖尿病の将来の治療のために使用することができる膵臓前駆細胞の生成のための魅力的なソース作り、複数の系統に分化する能力を有します。この記事では、ヒト胚性幹細胞（ヒトES細胞）から膵臓前駆細胞を生成するように設計された4段階の分化プロトコルの概要を説明します。このプロトコルは、ヒト多能性幹細胞（HPSC）行数に適用することができます。膵臓前駆細胞を生成するために取られたアプローチは、正確に膵臓発生の主要な段階をモデル化するためにヒトES細胞を区別することです。これは、アクチビンA、塩基性線維芽細胞増殖因子（bFGF）およびCHIR990210の存在下で細胞を培養することによって達成される胚体内胚葉の誘導で始まります。線維芽細胞増殖因子10（FGF10）とドルソモルフィンとのさらなる分化とパターニングは、後部前腸に似た細胞を生成します。レチンの追加OIC酸、ノギンは、SANT-1およびFGF10は膵臓内胚葉に特徴的な細胞内に後部前腸細胞を区別します。最後に、上皮成長因子（EGF）の組合せは、ニコチンアミドとノギンは、PDX1 + / NKX6-1 +細胞の効率的な生成につながります。フローサイトメトリーは、膵臓の発達の重要な段階に特異的なマーカーの発現を確認するために行われます。ステージ4の終わりにPDX1 + / NKX6-1 +膵臓前駆細胞は、免疫不全マウスへの移植時に、成熟β細胞を生成することができ、さらに、インビトロでのインスリン産生細胞を生成するように分化させることができます。それは、in vitroでヒト膵臓の開発を研究するためのプラットフォームを提供し、に分化する能力を有する細胞の供給源を提供するようしたがって、このプロトコルで示されているようにPDX1 + / NKX6-1 +膵臓前駆細胞の効率的な生成は、非常に重要ですEVができβ細胞entually糖尿病の治療のために使用されます。

図1（a）の模式的に示したように、膵臓前駆細胞を効率的に生成するには、分化プロトコル中に特定のシグナル伝達分子の正確加え、続いて未分化細胞の適切な成長および維持に依存しています。 0日目に、未分化細胞は、80から95パーセントのコンフルエントであるべきであり、コロニーは、定義されたエッジ（ 図1A）を持っている必要があ...

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

ここでは、in vitroで NKX6-1 +膵臓前駆細胞にヒト胚性幹細胞を区別するために4段階のプロトコルを記述します。このプロトコルは、ヒト多能性幹細胞株の多様に適用することができます。

NKX6-1への多能性幹細胞の効率的分化<sup&gt; +</sup&gt;膵臓前駆細胞

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

efficient-differentiation-pluripotent-stem-cells-to-nkx6-1-pancreatic

Research

JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

7.9K ビュー.  University Health Network. ここでは、in vitroで NKX6-1 +膵臓前駆細胞にヒト胚性幹細胞を区別するために4段階のプロトコルを記述します。このプロトコルは、ヒト多能性幹細胞株の多様に適用することができます。 

ビデオ: Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

ヒト膵臓の開発にリネージュの決定要因を問い合わせるためのゲノム編集とhPSCsの指向性分化

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

遺伝学

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

発生生物学

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

インビトロコロニーアッセイでトライ強力な前駆細胞を特徴付けるための大人のマウスの膵臓から分離されました

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

効率的な世代の膵臓・十二指腸ホメオ蛋白質 1+後部の前腸・膵前駆細胞接着培養における hPSCs から

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

2D培養系におけるヒト多能性幹細胞の膵臓ベータ細胞前駆体への分化

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

ヒト多能性幹細胞のインスリン産生膵島クラスターへの分化

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

生物学

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

監督分化アプローチで細胞を産生する膵臓インスリンにヒト胚性幹細胞の内皮細胞の共培養を仲介する成熟

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

インビトロ 膵臓系統に向けたヒト歯髄幹細胞の誘導

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

生物工学

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

NKX6-1への多能性幹細胞の効率的分化

要約

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この動画の章

ヒト膵臓の開発にリネージュの決定要因を問い合わせるためのゲノム編集とhPSCsの指向性分化

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

インビトロコロニーアッセイでトライ強力な前駆細胞を特徴付けるための大人のマウスの膵臓から分離されました

効率的な世代の膵臓・十二指腸ホメオ蛋白質 1⁺後部の前腸・膵前駆細胞接着培養における hPSCs から

2D培養系におけるヒト多能性幹細胞の膵臓ベータ細胞前駆体への分化

ヒト多能性幹細胞のインスリン産生膵島クラスターへの分化

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

監督分化アプローチで細胞を産生する膵臓インスリンにヒト胚性幹細胞の内皮細胞の共培養を仲介する成熟

インビトロ膵臓系統に向けたヒト歯髄幹細胞の誘導

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

NKX6-1への多能性幹細胞の効率的分化

要約

さらに動画を探す

この動画の章

ヒト膵臓の開発にリネージュの決定要因を問い合わせるためのゲノム編集とhPSCsの指向性分化

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

インビトロコロニーアッセイでトライ強力な前駆細胞を特徴付けるための大人のマウスの膵臓から分離されました

効率的な世代の膵臓・十二指腸ホメオ蛋白質 1+後部の前腸・膵前駆細胞接着培養における hPSCs から

2D培養系におけるヒト多能性幹細胞の膵臓ベータ細胞前駆体への分化

ヒト多能性幹細胞のインスリン産生膵島クラスターへの分化

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

監督分化アプローチで細胞を産生する膵臓インスリンにヒト胚性幹細胞の内皮細胞の共培養を仲介する成熟

インビトロ 膵臓系統に向けたヒト歯髄幹細胞の誘導

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

効率的な世代の膵臓・十二指腸ホメオ蛋白質 1⁺後部の前腸・膵前駆細胞接着培養における hPSCs から

インビトロ膵臓系統に向けたヒト歯髄幹細胞の誘導