Name: isolation and enrichment of liver progenitor subsets identified by a novel surface marker combination
Uploaded: 2017-02-18T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination at JoVE.com

この作品は、VLKにアレクサンダー・フォン・フンボルト財団Sofja Kovalevskaja賞によってサポートされていました。

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肝臓の炎症と異なる起源の損傷は、前駆細胞の増殖および活性化2、3を伴う肝臓での再生プロセスをトリガーします。これらの肝前駆細胞は、細胞性幹有し、おそらく、様々な肝疾患の病理学的機序において重要な役割を果たす。 肝臓前駆細胞の不均一性は、長い間示唆されています。異なる表面マーカーを運ぶサブセットを識別し、肝損傷12の間の炎症関連遺伝子の固有のセットを表現できるCD133およびGP38の新たな表面マーカーの組み合わせを使用して、肝前駆細胞サブセットの再評価。ここで説明する方法は、稀な細胞の直接のフローサイトメトリー分析および種々の肝障害モデルでの直接比較を可能にします。種々の傷害は、複数の前駆体の活性化をトリガーするように、これは、特に適切です小管反応3、4、11、12で観察された細胞の不均一性を反映することができubsets。注目すべきことに、マウスの肝臓組織（0.3 g）を小部分に記載した方法を用いて前駆細胞のフローサイトメトリー分析のために十分な細胞を単離するのに十分です。 サブセットの高純度の集団を提供する仕分けフローサイトメトリーは、独自の遺伝子発現プロファイルを探求する必要があります。以前の報告は、フローサイトメトリーベースの前駆細胞の単離15、16について説明しました 。それにもかかわらず、ここで紹介するプロトコルは、高純度、これらの細胞の生存性を確実に最適化された方法を提供します。なお、前述のインビトロ培養および拡張技術がうまくここ15に提示プロトコルと組み合わせることができ<s16、&gt;アップ。 ソーターフローサイトメトリーの多くの種類は、様々な機関で利用可能であり、それらの特定のインストルメンテーションは若干異なる場合があります。それにもかかわらず、細胞選別のための一般的な原理は、記載されたプロトコルに提示されています。一般的に、より低い圧力とより大きなノズルサイズは、マシンの種類と仕様の独立した絶対に必要です。また、適切な選別培地の利用が著しく生存率および特に遺伝子発現研究のために、重要な因子で選別された細胞のRNAの品質を高めることができます。前駆細胞の選別は、しかしながら、非常に細胞生存率が減少し、選別後の細胞の収率は、（7-10,000イベント/亜集団の高純度の並べ替えのために、4-5健康な肝臓をプールする必要がある）比較的低いです。これはさらに、in vitroでの分析のための制限要因である可能性があります。我々は、 中のため、磁気マイクロビーズベースの分離を示唆していますインビトロで、そのより高い収率および細胞生存性のアッセイ、および複数の指定されたサブセットを分析し、単離が必要とされる場合は特に、遺伝子発現解析のためにソーティングフローサイトメトリー。 細胞の生存率が大幅にソーティングフローサイトメトリーの後に減少するという事実に基づいて、前駆細胞の自動化された磁気ビーズベースの分離が開発され、ここで提示されています。これは、高純度（複数肝臓を組み合わせる）で前駆細胞のより多くの単離を可能にします。さらに、細胞の生存率は、分離プロセスのために必要な時間が大幅に削減されるため、維持されます。前駆細胞の下の数字は、in vitro 1、2、15、16 に拡張することができますが、これらのインビトロ操作は、幹/ PROGENの細胞の特徴を変化させることができる方法を検討することをお勧めしますコンデンサ細胞は、他の間葉系間質細胞集団17、18について記載しました。 提示された磁気マイクロビーズベースの分離の主な制限は、CD133 +およびGP38 +細胞集団は、異種の細胞を表していることです。追加の表面マーカーは、より小さな細胞集団を同定することが必要になる場合があります。 肝幹細胞や前駆細胞が互いにどのように関係するかを理解することは、ローラ・リード、その他1、8、19による優れた研究にもかかわらず、完成には程遠いです。したがって、このようなここで提案1として、高い収率および生存率が得られた技術を慎重に考慮すべきことは、前駆細胞サブセットの分析とそれらの相互接続された関係の理解を拡張するために助けることができます。 全体として、我々は、Tが記載されています彼は最近、12の区別された肝前駆細胞サブセットの単離および分析を詳述しました。また、新規な酵素の組み合わせを使用することにより、稀な前駆体は、直接フローサイトメトリー測定により分析することができました。さらに、プロトコルは、細胞選別または自動化されたマイクロビーズベースの技術のいずれかを使用して、高純度の前駆細胞を濃縮する方法を示しています。 トラブルシューティング： 細胞片のパーセンテージおよび死細胞が高いです消化（ステップ2）の間に、肝臓試料のピペットがあまりに過酷である場合、単一細胞懸濁液が増加中の細胞死滅のパーセンテージ。肝臓が示唆さよりも大きな片に切断されている場合は、消化が少なく効率的であり、準備中に複数の細胞死をもたらします。 ステップ5の手順を完了した後、サンプルの純度が不十分です percen場合ステップ2で調製した単一細胞懸濁液中の細胞破片のtages及び瀕死細胞は、マイクロビーズを非特異的に結合し、したがって、分離の純度が大幅に低減され、高すぎます。 マイクロビーズベース精製後の低細胞数記載されたプロトコルの成功のためには、プロトコルに示唆されるようにマイクロビーズへの細胞の割合が、最適であることが重要です。より多くのマイクロビーズを利用する純度を低減し、単離された細胞の収量と生存率を低下させます。プロトコルで提示されたもの以外の表面マーカーは、前駆体サブセットの単離のために使用されている場合は、マイクロビーズに対する細胞の最適な比率を決定しなければなりません。 GP38 + CD133の磁気マイクロビーズベースの分離-個体群ステップ5.3ではセクション5の手順に従い説明するように、その後のステップ5、6.2及び6.3に従って、さらに10μL抗CD133 +マイクロビーズを追加D。 絶対細胞数を計算します肝臓重量は、特定のサブセットの多くの細胞は、グラム肝臓組織あたりに存在するかを計算するために使用されます。 （フローサイトメトリーに基づいて、生細胞中の亜集団の％（）/ 100）は、肝臓片= Zから単離された全生細胞数をxは （肝臓片の1 /重量）Z =細胞数/ g肝臓組織にxはこの方法で単離することができる他の細胞集団 CD45 +細胞（または造血細胞の亜集団、 例えばクッパー細胞、樹状細胞、T細胞、NK細胞など ）とのLSEC：この消化プロトコルを使用して、以下の肝細胞を単離することが可能です。消化プロトコルは、肝細胞または肝星状細胞の単離には適していません。これらの細胞は比較的低い細胞数で存在し、それらは、パーソナルプラグインと比較して減少し生存率を示し利用可能な方法rを。

肝臓再生は、主に肝細胞の自己再生能と関連しています。それにもかかわらず、慢性肝損傷は、肝細胞及び胆管1、2、3、4に分化する能力と関連している前駆細胞の活性化および拡大で起こります。慢性傷害の間、肝細胞増殖が有効でない場合、これは特に重要です。前駆細胞を標的とする多数の遺伝的追跡研究にもかかわらず、肝臓再生におけるそれらの役割は、5、6、7、8議論の余地。また、前駆細胞の活性化は、負傷9の間にそれらの正確な役割について疑問を提起肝臓で増加した線維化反応、にリンクされています、10。 前駆細胞コンパートメントの不均一な性質は、長い顕微解剖または細胞選別に基づく方法1、11を使用して単一の表面マーカーを発現する前駆細胞を単離し、遺伝子発現研究によって示唆されています。実際、最近、GP38（ポドプラニン）を使用して、新規な表面マーカーの組み合わせは明確種々のサブセット12に前駆細胞の前の単一のマーカーを結合されました。重要なことは、これらのサブセットは、それらの表面マーカー発現が異なるだけでなく、怪我12の間に機能的変化を示しただけではなく。 多数の動物モデルは、前駆細胞の活性化及び肝再生を研究するために利用されています。様々な損傷の種類は、前駆細胞12の異なるサブセットの活性化を促進すると思われます。これは、pHを説明するかもしれません人間4で観察された小管反応のenotypic発散。このように、前駆細胞の複雑な表現型および機能解析は、怪我や肝疾患における小管反応の真の意義における役割を理解することが極めて重要です。 表面マーカーの組み合わせの他に、細胞単離プロトコールにおける重要な違いは、さらに、以前の研究2に基づいて結論を複雑にします。研究のかなりの量が大きく、それらの単離プロトコルが異なる前駆細胞（ 例えば、肝臓解離（酵素の組み合わせと処理の持続時間）、密度媒体、及び遠心分離スピード）の役割に対処2。希少な細胞集団とサブセットの組成物の反射のためのより良い生存可能性を提供する最適化された分離技術は、開発され、最近12公開されています。この記事の目的は、より多くのDETを提供することです技術の適切な再現を可能にするために、この肝細胞の単離手順およびサブセット解析のプロトコルをailed。さらに、プロトコルは、新しいプロトコルと比較して違いを示すために、以前の分離法との比較を含んでいます。

<table><tbody><tr><td>RPMI</td><td>Life Technologies</td><td>21875-034</td><td></td></tr><tr><td>phenol red free DMEM</td><td>Life Technologies</td><td>31053-028</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10270-106</td><td></td></tr><tr><td>Collagenase P</td><td>Sigma-Aldrich</td><td>11249002001</td><td></td></tr><tr><td>DNAse-I</td><td>Sigma-Aldrich</td><td>10104159001</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105041</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Life Technologies</td><td>A10492-01</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14025-050</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Prepare 1% stock solution</td></tr><tr><td>10% BSA</td><td>Miltenyi Biotec</td><td>130-091-376</td><td></td></tr><tr><td>autoMACS Rinsing Solution</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>add 0.5% (v/v) BSA and store on ice</td></tr><tr><td>Phenol-red free DMEM</td><td>Sigma-Aldrich</td><td>D1145</td><td></td></tr><tr><td>counting Beads Count Bright</td><td>Life Technologies</td><td>C36950</td><td></td></tr><tr><td>PI</td><td>Miltenyi Biotec</td><td>130-093-233</td><td></td></tr><tr><td>FcR Blocking Reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>anti-CD31 MicroBeads</td><td>Miltenyi Biotec</td><td>130-097-418</td><td></td></tr><tr><td>anti-CD45 MicroBeads</td><td>Miltenyi Biotec</td><td>130-052-301</td><td></td></tr><tr><td>Dead Cell Removal Kit</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></td></tr><tr><td>anti-Biotin MicroBeads</td><td>Miltenyi Biotec</td><td>130-090-485</td><td></td></tr><tr><td>CD64 Purified</td><td>BioLegend</td><td>139302</td><td>Dilution: 1:100</td></tr><tr><td>CD16/32 Purified</td><td>BioLegend</td><td>101302</td><td>Dilution: 1:100</td></tr><tr><td>CD45 APC/Cy7</td><td>BioLegend</td><td>103116</td><td>Dilution: 1:200, marks hematopoetic cells</td></tr><tr><td>CD31 Biotin</td><td>BioLegend</td><td>102504</td><td>Dilution: 1:200, marks endothelial cells</td></tr><tr><td>ASGPR1 Purified</td><td>Bio-Techne</td><td>AF2755-SP</td><td>Dilution: 1:100, marks hepatocytes</td></tr><tr><td>Podoplanin APC</td><td>BioLegend</td><td>127410</td><td>Dilution: 1:1,400, marks progenior cells</td></tr><tr><td>Podoplanin Biotin</td><td>BioLegend</td><td>127404</td><td>Dilution: 1:1,400</td></tr><tr><td>CD133 PE</td><td>Miltenyi Biotec</td><td>130-102-210</td><td>use 3 &amp;micro;L, marks progenitor cells</td></tr><tr><td>CD133 Biotin</td><td>BioLegend</td><td>141206</td><td>Dilution: 1:100</td></tr><tr><td>CD34 Biotin</td><td>eBioScience</td><td>13-0341-81</td><td>Dilution: 1:100</td></tr><tr><td>CD90.2 Pacific Blue</td><td>BioLegend</td><td>140306</td><td>Dilution: 1:800</td></tr><tr><td>CD157 PE</td><td>BioLegend</td><td>140203</td><td>Dilution: 1:600</td></tr><tr><td>EpCAM Brilliant Violet 421</td><td>BioLegend</td><td>118225</td><td>Dilution: 1:100</td></tr><tr><td>Sca-1 Biotin</td><td>Miltenyi Biotec</td><td>130-101-885</td><td>use 10 &amp;micro;L</td></tr><tr><td>Mouse IgG2b, &amp;kappa; PE</td><td>BioLegend</td><td>400311</td><td></td></tr><tr><td>Rat IgG1 PE</td><td>BioLegend</td><td>400407</td><td></td></tr><tr><td>Rat IgG2b, &amp;kappa; APC/Cy7</td><td>BioLegend</td><td>400624</td><td></td></tr><tr><td>Rat IgG2a, &amp;kappa; Biotin</td><td>BioLegend</td><td>400504</td><td></td></tr><tr><td>Rat IgG2a, &amp;kappa; Brilliant Violet 421</td><td>BioLegend</td><td>400535</td><td></td></tr><tr><td>Syrian Hamster IgG APC</td><td>BioLegend</td><td>402012</td><td></td></tr><tr><td>Syrian Hamster IgG Biotin</td><td>BioLegend</td><td>402004</td><td></td></tr><tr><td>Normal Goat IgG Control Purified</td><td>Bio-Techne</td><td>AB-108-C</td><td></td></tr><tr><td>Donkey anti-Goat IgG Alexa Fluor 488</td><td>Life Technologies</td><td>A11055</td><td>Dilution: 1:800</td></tr><tr><td>Streptavidin Alexa Fluor 405</td><td>Life Technologies</td><td>S32351</td><td>Dilution: 1:400</td></tr><tr><td>100 &amp;micro;m Filter mesh</td><td>A. Hartenstein</td><td>PAS3</td><td></td></tr><tr><td>LS Column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>QuadroMACS separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec</td><td>130-096-343</td><td></td></tr><tr><td>AutoMACS Pro Separator</td><td>Miltenyi Biotec</td><td>130-092-545</td><td></td></tr><tr><td>FACS AriaTMIII</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FACSDiva sofware</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Polypropylene Round bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Rneasy plus mini kit</td><td>Qiagen</td><td>74134</td><td>RLT lysis buffer is included</td></tr></tbody></table>

isolation and enrichment of liver progenitor subsets identified by a novel surface marker combination

慢性肝臓傷害の間、前駆細胞は、炎症性細胞浸潤および上皮細胞の活性化の出現を伴う小管反応と呼ばれるプロセスで展開します。そのような炎症反応中に前駆細胞集団は、大部分の組織学的分析によって、またはフローサイトメトリーベースの技術のいずれかによって、単一の表面マーカーを使用して研究されています。しかしながら、新規な表面マーカーは、細胞コンパートメント幹/肝前駆細胞内の種々の機能的に異なるサブセットを同定しました。ここで紹介する方法は、新規の表面マーカーの組み合わせを使用して、前駆細胞サブセットの分析サイトメトリー分離と詳細な流れを説明しています。また、種々の前駆細胞のサブセットは、自動磁気及びFACSソーティングに基づく方法を用いて高純度で単離することができる方法を示します。重要なことは、小説や肝臓の単純化された酵素的解離は高い生存率これらの希少な細胞集団の単離を可能にしますそれは、他の既存の方法に比べて優れています。これは、さらに、インビトロで前駆細胞を研究するため、または遺伝子発現プロファイルを分析するために、高品質のRNAを単離するために特に適切です。

実質及び非実質肝細胞（ 図1および図2A）を含む単一細胞懸濁液中の酵素の結果の新規な混合物を使用して、肝臓の消化のためにここに提示手順。赤血球のACK-溶解後、単一細胞懸濁液の分析サイトメトリー直接流れが（ 図1及び2）が可能です。ゲーティング戦略はダブレットと死細胞（ 図2a）の排除を伴います。 CD45、CD31、およびASGPR1のために陰性である細胞がゲートされます。この集団は、肝臓前駆細胞を含み、それらのGP38及びCD133発現（ 図2A）に基づいて分類することができます。 GP38 + CD133 +およびGP38 - CD133 +細胞はCD45の中で最も豊富な細胞集団表す- CD31 - ASGPR1 -健常成人マウスLIVで細胞をER（ 図2A、B）。これらのサブセットは、以前にこのようなEPCAM、SCA-1、およびCD34（ 図2c）のような前駆細胞に関連する付加的な表面マーカーの発現において異なります。 前駆細胞は、肝細胞、肝臓類洞内皮細胞（のLSEC）（ 図1及び図 3）、および前駆細胞は、磁気マイクロビーズベースの分離とさらに精製することができた（ 図3a、b）またはのように、造血および実質細胞から枯渇させることができましたソーティング高純度（ 図1及び4）。 90％を超える純度のCD133 +またはGP38 +細胞の結果の磁気マイクロビーズベースの分離（ 図3a、b）のいずれかの汚染CD45、CD31、またはASGPR1 +細胞について、細胞は非常に生存したままで（ 図3a、b）に 。 2の細胞組成および生存率が異なることができます。これは、組織解離のために利用する酵素混合物の選択のために特に関連しています。ここで、コラゲナーゼPの代わりに、コラゲナーゼDは、肝臓1、 図2、 図8に使用しました。重要なことには、前駆細胞のCD133の発現レベルおよび単離された細胞集団の収率が大幅コラゲナーゼDの存在下で減少した（ 図5A、B）。これに加えて、我々の混合物は、種々の細胞型13の穏やかな分解および継代培養のために非常に適している、ディスパーゼ含まれます。ディスパーゼが表すと共にコラゲナーゼP前駆細胞分析のために肝臓の細胞解離用の理想的な組み合わせ。この組み合わせはまた、肝臓（データは示していない）のトリプシン消化またはプロナーゼ系よりも優れていました。それは、このようなパーコールベースの濃縮2、14のようなその密度遠心分離を、注意することも同様に重要である、このプロトコルで提示された前駆分析のために必要ではなかったです。 <img alt="図1" src="/files/ftp_upload/55284/55284fig1.jpg" /> 図1：記載されているプロトコルのワークフロー。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55284/55284fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/55284/55284fig2.jpg" /> 図2：肝前駆細胞サブセットの新規分類。単細胞懸濁液を、健康な肝臓から調製し、続いて表面マーカーのパネルを用いて染色した：CD45、CD31、CD133およびGP38（A）。死細胞を排除するために、ヨウ化プロピジウム（PI）を用いました。ゲーティング戦略を持つ代表的なドットプロットが示されています。細胞数の決意をフローサイトメトリーのために使用されるゲートはまた、印が付けられています。 （B）の割合と、野生型の未処理動物におけるCD45陰性細胞の中で様々な間質細胞サブセットの肝臓組織1g当たりに存在する細胞の絶対数を示します。 （C）のヒストグラムは、健康な肝臓にストローマ細胞サブセットの区別マーカー特性を表示します。 CD34はB. SCA-1は、Bに存在する亜集団に特異的である一方で、高CD90.2の発現およびCD157の存在は、Aに特異的である、C、D及びEPCAMは人口CとDの平均値±SEMで唯一の存在です。データ（AC）を、2-3の独立した実験を表すのn = 1実験あたり3-4。データWEReは対になっていない、両側t検定* P &lt;0.05、** P &lt;0.005、*** P &lt;0.0001を用いて比較しました。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55284/55284fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/55284/55284fig3.jpg" /> 図3：磁気マイクロビーズベースの濃縮および自動化された細胞精製。 （A）CD45の磁気マイクロビーズベースの濃縮- 、CD31 -及びASGPR1 -細胞を濃縮する前と後に描かれています。自動化されたマイクロビーズベースの細胞の単離の（B）代表ドットプロットは左に、CD133 +およびGP38 +細胞のために描かれています。右側には、濃縮前と後の細胞集団の生存率は、の割合として示されていますヨウ化プロピジウム（PI）陰性細胞。平均±SEM;データ（AB）は、1回の実験当たりのn = 3と3つの独立した実験を表します。データは対になっていない、両側t検定* P &lt;0.05、** P &lt;0.005、*** P &lt;0.0001を用いて比較しました。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55284/55284fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/55284/55284fig4.jpg" /> 図4：高純度の前駆細胞サブセットの並べ替えフローサイトメトリー。ドットプロットは、ソートされたサンプル（ポストソート）で細胞集団の純度を示します。データは3回の独立した実験を表します。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55284/55284fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> = &quot;1&quot;&gt; <img alt="図5" src="/files/ftp_upload/55284/55284fig5.jpg" /> 図5：消化酵素の比較。単一細胞懸濁液を、健康な肝臓から、コラゲナーゼP（ステップ2に記載されるように）またはコラゲナーゼD（1mg / mlの+ DNアーゼIを0.1mg / ml）を用いて調製し、続いて表面マーカーのパネルを用いて染色した、CD45 CD31、CD133、およびGP38（A）。野生型の未処理動物におけるCD45陰性細胞の中で様々な間質細胞サブセットの肝臓組織のグラム当たりに存在する細胞の割合が示されています。 （B）CD133のメジアン蛍光強度はCD133 +細胞集団のために示されています。平均±SEM;データ（AB）は、1回の実験当たりのn = 3と2つの独立した実験を表します。データは対になっていない、両側t検定* P &lt;0.05、** P &lt;0.005、*** P &lt;0.0001を用いて比較しました。ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

Watch this Scientific Journal Video about Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination at JoVE.com

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

肝臓損傷は、不均一な細胞集団を表し、前駆細胞の増殖を伴います。この細胞区画の新規分類は、複数のサブセットの区別を可能にします。ここで説明する方法は、分析し、さらなるアッセイのために使用することができる種々のサブセットの高純度分離、フローサイトメトリーを示します。

新規の表面マーカーの組み合わせで識別離および肝前駆サブセットの濃縮

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

isolation-enrichment-liver-progenitor-subsets-identified-novel

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.0K ビュー.  Saarland University Medical Center. 肝臓損傷は、不均一な細胞集団を表し、前駆細胞の増殖を伴います。この細胞区画の新規分類は、複数のサブセットの区別を可能にします。ここで説明する方法は、分析し、さらなるアッセイのために使用することができる種々のサブセットの高純度分離、フローサイトメトリーを示します。 

ビデオ: Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下で心臓前駆細胞の内皮への分化の誘導

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

発生生物学

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

ヒト多能性幹細胞の肝細胞への効率的な分化

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

クローン性増殖のためのCD133 +肝幹細胞の単離

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

生物学

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

肝疾患研究のための末梢血からのヒト肝スフェロイド

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

生物工学

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

マウスにおける肝部分切除後の再生肝細胞の単離

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

医学

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 ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

オルガノイド培養のためのヒト肺上皮前駆細胞の単離と濃縮

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...