Name: clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture
Uploaded: 2018-05-08T13:00:00.000+00:00
Duration: 9 min 32 s
Description: Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

フレッド ハッチンソン流れ Cytometry コア援助を FACS でアンドリュー ・ ベルガー、ステイシー堂園とブライアン螺鈿に感謝したいと思います。この作業は、付属の共同助成金 #HL099997 と NIDDK 付与 #RC2DK114777 国立機関の健康 NHLBI UO1 グラント #HL100395、によって支えられました。ブランドン Hadland をサポートするには、アレックスのレモネードは、財団スタンドとホイール基礎現代希望。

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Single-Cell+Functional+and+Gene+Expression+Analysis+Resolves+Heterogeneity+within+Stem+Cell+Populations.">Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.</a> Cell Stem Cell. 16 (6), 712-724 (2015).</li><li>Schulte, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Index+sorting+resolves+heterogeneous+murine+hematopoietic+stem+cell+populations.">Index sorting resolves heterogeneous murine hematopoietic stem cell populations.</a> Exp Hematol. 43 (9), 803-811 (2015).</li><li>Morgan, K., Kharas, M., Dzierzak, E., Gilliland, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+early+hematopoietic+stem+cells+from+murine+yolk+sac+and+AGM.">Isolation of early hematopoietic stem cells from murine yolk sac and AGM.</a> J Vis Exp. (16), (2008).</li><li>deBruijn, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+localize+to+the+endothelial+cell+layer+in+the+midgestation+mouse+aorta.">Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.</a> Immunity. 16 (5), 673-683 (2002).</li><li>Lorsbach, R. 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萌芽期の開発中に HSC 創世記の研究は、まだ移植成人レシピエントにおける血液造血系長期を提供する能力の欠けている hemogenic 前駆体の潜在的な HSC を検出するための手段を必要とします。このプロトコルで提案するクローン胚 hemogenic 前駆体法血管ニッチ ECs に間質の混合培養による HSC の前駆体の成熟化をサポートするには、AGM から移植アッセイで後続の機能解析と.設立許可の並べ替え?...

クローン研究は、高齢化1時に HSC サブタイプと HSC の動作の変更に新しい洞察力を提供する大人の HSCs の長期生着特性における不均一性を明らかにしました。ただし、胚性造血の研究およびその前駆物質によっては、難しくされています。初期胚の中に造血前駆細胞呼ばれる非定常過程の hemogenic 内皮細胞として知られている人口から生じる内皮細胞から造血転移2として。最初の HSC では、エアコンの成人のレシピエントに移植に続く堅牢で、長期の多能生着を提供するために彼らの能力によって定義されている超低周波3 でマウスの胚の胚日 10.5 (E10.5) 後まで検出されません。.HSC (pre-HSC) hemogenic 内皮細胞から発生する前駆物質開発の間に移植の試金4,5で効率的な生着を可能にする大人の HSC のプロパティを取得する前に成熟を受ける必要があります。,6。 まれな HSC の起源、赤芽球系、造血前駆細胞の多数の研究を隠すこと、骨髄性とリンパの可能性がすでに前 HSC7,8から HSC の出現する前に検出されました。したがって、事前 HSC を区別する他の造血前駆細胞クローン細胞を分離し、HSC では、移植の試金でその生着プロパティを検出するために十分な成熟のための信号を提供するためのメソッドが必要です。
Hsc の前のヴィヴォや生体内での成熟によって前 HSC の検出を実現するアプローチの数が記載されています。前のヴィヴォメソッドは、AGM 地域、開発9で最初の HSC を検出する場所などの萌芽期ティッシュの文化に依存しています。これらのメソッドは、プロトコルは、解離を組み込む建物並べ替え、および再集計 AGM 組織の許可している傍大動脈内 E11.5 E9.5 から開発中に HSC 前駆体を含む並べ替えられた個体群の特性評価splanchnopleura (P Sp)/AGM 地域4,5,10;ただし、これらのアプローチはクローン解析に必要な単一セルのレベルで前駆物質のハイスループット解析に従順ではありません。同様に、新生マウスへの移植による生体内で成熟、微小環境は HSC の前駆体の初期の段階のサポートに適して見なされどこにも有効な卵黄嚢と AGM から並べ替えられた個体群の研究/P Sp (P Sp は AGM に前駆体領域) pre-HSC では、これらのメソッドの特性とも単一の堅牢なプラットフォームを提供するために失敗する細胞分析11,12。
Rafiiらの研究は、その Akt 活性化血管内皮細胞 (EC) 間質は大人 HSC の自己複製の in vitro13,14,15のサポートのためニッチ基板を提供することができますを示しました。我々 は最近、AGM 地域 (AGM-EC) から派生した Akt 活性化 EC は適した体外ニッチ、分離早くも大人接が hsc の開発で E9 およびそれに続くのと同様、hemogenic の前駆体の成熟化の判断生成された HSC16の自己更新します。このシステムは、単純な 2次元の共培養を採用し、それは個々 に絶縁されて hemogenic 前駆体の HSC 可能性のクローン解析の容易に適応可能であります。
我々 は最近インデックスの AGM EC 共培養と移植後の機能分析とマウス胚からの個々 の hemogenic 前駆体の並べ替えを組み合わせることによりクローン hemogenic 前駆体の HSC の可能性を試金する方法を報告しています。17をの試金します。蛍光活性化のモードなどそれぞれの個別に並べ替えられたセルの表現型パラメーター (すなわち、前方散乱 (FSC-A)、側散布 (SSC-A)、蛍光パラメーター) すべて (インデックス) を記録する (FACS) をソートするセル インデックスの並べ替えは、これらの並べ替えを次以降の機能解析機能を遡及的に関連付けることができます。FACS ソフトウェアは、各セルに配置された 96 ウェル プレートの位置/よく表現型情報の両方を記録します。この手法は以前エレガントな大人の HSC の不均一性を識別、さらの HSC では、長期的な engrafting のサブセットを豊かにし、転写と HSC の表現型のパラメーターを関連付ける表現型のパラメーターを確認する使用されて1 つのプロパティはセル レベル18,19です。ここでは、胚の開発の初期段階中にユニークな表現型のパラメーターの同定と pre HSC の系統貢献を可能にするこの方法の詳細な方法論を提供します。

<table><tbody><tr><td>&lt;strong&gt;AGM-EC culture media &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials for culture of endothelial cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12605-028</td><td>Use to dissociate AGM-EC monolayers</td></tr><tr><td>Gelatin 0.1% in water</td><td>StemCell Technologies</td><td>7903</td><td>Use to adhere AGM-EC monolayers to plastic</td></tr><tr><td>96-well tissue culture plates</td><td>Corning</td><td>3599</td><td>Use to co-culture AGM-EC monolayers and index sorted clones</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190-144</td><td>Use for dissection and FACS staining</td></tr><tr><td>500 ml filter bottles</td><td>Fisher Scientific</td><td>9741202</td><td>Use to sterile filter Endothelial media</td></tr><tr><td>&lt;strong&gt;AGM-EC culture media (500 mls)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco</td><td>12440-053</td><td>400 mls</td></tr><tr><td>Hyclone Fetal Bovine Serum</td><td>Fisher Scientific</td><td>SH30088.03</td><td>100 mls</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>5 mls</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td>50 mg dissolved in 10 mls media</td></tr><tr><td>L-glutamine (200 mM)</td><td>StemCell Technologies</td><td>7100</td><td>5 mls</td></tr><tr><td>Endothelial Mitogen (ECGS)</td><td>Alfa Aesar</td><td>J64516 (BT-203)</td><td>Add 10 mls media to dissolve and add</td></tr><tr><td>&lt;strong&gt;Materials for AGM index sort&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase (0.25%)</td><td>StemCell Technologies</td><td>7902</td><td>Use for dissociation of embryonic tissues</td></tr><tr><td>3ml syringe</td><td>BD Biosciences</td><td>309657</td><td>Use to sterile filter antibody solutions for FACS</td></tr><tr><td>0.22 &amp;micro;M syringe-driven filter</td><td>Millipore</td><td>SLGP033RS</td><td>Use to sterile filter antibody solutions for FACS</td></tr><tr><td>5 ml polystyrene tube with cell-strainer cap</td><td>Corning</td><td>352235</td><td>Use to remove cell clumps prior to FACS</td></tr><tr><td>DAPI (prepared as 1 mg/ml stock in H2O)</td><td>Millipore</td><td>268298</td><td>Use to exclude dead cells from sort</td></tr><tr><td>anti-mouse CD16/CD32 (FcR block)</td><td>BD Biosciences</td><td>553141</td><td>Use to block non-specific staining to Fc receptors</td></tr><tr><td>&lt;strong&gt;Antibodies for AGM index sort&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse CD144 PE-Cyanine7</td><td>eBioscience</td><td>25-1441-82</td><td>Staining</td></tr><tr><td>Rat IgG1 kappa Isotype Control, PE-Cyanine7</td><td>eBioscience</td><td>25-4301-81</td><td>Isotype control for CD144 PE-Cyanine7</td></tr><tr><td>Anti-mouse CD201 (EPCR) PerCP-eFluor710</td><td>eBioscience</td><td>46-2012-80</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PerCP-eFlour710</td><td>eBioscience</td><td>46-4031-80</td><td>Isotype control for CD201 PerCP-eFluor710</td></tr><tr><td>Anti-mouse CD41 PE</td><td>BD Biosciences</td><td>558040</td><td>Staining</td></tr><tr><td>Rat IgG1 kappa Isotype Control, PE</td><td>BD Biosciences</td><td>553925</td><td>Isotype control for CD41 PE</td></tr><tr><td>Anti-mouse CD45 FITC</td><td>eBioscience</td><td>11-0451-85</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, FITC</td><td>eBioscience</td><td>11-4031-81</td><td>Isotype control for CD45 FITC</td></tr><tr><td>&lt;strong&gt;AGM-serum free media (10mls)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>X-Vivo 20</td><td>Lonza</td><td>04-448Q</td><td>10 mls</td></tr><tr><td>recombinant murine stem cell factor (SCF)</td><td>Peprotech</td><td>250-03</td><td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td></tr><tr><td>recombinant human FLT3 Ligand (FLT3L)</td><td>Peprotech</td><td>300-19</td><td>10 ml (100 mg/ml stock) Final concentration 100 ng/ml</td></tr><tr><td>recombinant human thrombopoietin (TPO)</td><td>Peprotech</td><td>300-18</td><td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td></tr><tr><td>recombinant murine interleukin-3 (IL3)</td><td>Peprotech</td><td>213-13</td><td>2 ml (100 mg/ml stock) Final concentration 20 ng/ml</td></tr><tr><td>&lt;strong&gt;Materials for Staining co-cultured cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 well plate, V-bottom</td><td>Corning</td><td>3894</td><td>Use for FACS analysis</td></tr><tr><td>&lt;strong&gt;Antibodies for analysis of Index sorted clones co-cultured with endothelial cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse CD45 PerCP-Cyanine5.5</td><td>eBioscience</td><td>45-0451-82</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PE-Cyanine5.5</td><td>eBioscience</td><td>35-4031-80</td><td>Isotype control for CD45 PerCP-Cyanine5.5</td></tr><tr><td>Anti-mouse CD201 (EPCR) PE</td><td>eBioscience</td><td>12-2012-82</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PE</td><td>eBioscience</td><td>12-4031-81</td><td>Isotype control for CD201 PE</td></tr><tr><td>Anti-mouse Ly-6A/E (Sca-1) APC</td><td>eBioscience</td><td>17-5981-83</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC</td><td>eBioscience</td><td>17-4321-81</td><td>Isotype control for Ly-6A/E APC</td></tr><tr><td>Anti-mouse F4/80 FITC</td><td>eBioscience</td><td>11-4801-81</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, FITC</td><td>eBioscience</td><td>11-4321-41</td><td>Isotype control for F4/80 FITC</td></tr><tr><td>Anti-mouse Ly-6G/C (Gr1) FITC</td><td>BD Biosciences</td><td>553127</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, FITC</td><td>BD Biosciences</td><td>556923</td><td>Isotype control for Ly-6G/C and CD3 FITC</td></tr><tr><td>&lt;strong&gt;Materials for tail vein injection for transplant&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1/2 ml insulin syringes with 29G 1/2&quot; needles</td><td>BD Biosciences</td><td>309306</td><td>Use for tail vein injection for transplantation</td></tr><tr><td>&lt;strong&gt;Antibodies for Peripheral Blood analysis following translant&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse Ly-6G/C (Gr1) PerCP</td><td>Biolegend</td><td>108426</td><td>Staining</td></tr><tr><td>Rat IgG2b kappa Isotype Control, PerCP</td><td>Biolegend</td><td>400629</td><td>Isotype control for Ly-6G/C PerCP</td></tr><tr><td>Anti-mouse F4/80 PE</td><td>eBioscience</td><td>12-4801-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, PE</td><td>eBioscience</td><td>12-4321-80</td><td>Isotype control for F4/80 PE</td></tr><tr><td>Anti-mouse CD3 FITC</td><td>BD Biosciences</td><td>555274</td><td>Staining</td></tr><tr><td>Anti-mouse CD19 APC</td><td>BD Biosciences</td><td>550992</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC</td><td>BD Biosciences</td><td>553932</td><td>Isotype control for CD19 APC</td></tr><tr><td>Anti-mouse CD45.1 (A20) PE-Cyanine7</td><td>eBioscience</td><td>25-0453-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, PE-Cyanine7</td><td>eBioscience</td><td>25-4321-81</td><td>Isotype control for CD45.1 PE-Cyanine7</td></tr><tr><td>Anti-mouse CD45.2 (104) APC-eFluor780</td><td>eBioscience</td><td>47-0454-82</td><td>Staining</td></tr><tr><td>Rat IgG2a kappa Isotype Control, APC-eFluor780</td><td>eBioscience</td><td>47-4321-80</td><td>Isotype control for CD45.2 APC-eFluor780</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD FACSAria II with DIVA software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSCanto II with plate reader</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Haemocytometer</td><td>Fisher Scientific</td><td>S17040</td><td>For counting cells</td></tr><tr><td>Multi-channel pipette</td><td>Fisher Scientific</td><td>14-559-417</td><td>For dispensing cells</td></tr><tr><td>FACS analysis software</td><td>FlowJo/BD Biosciences</td><td></td><td>https://www.flowjo.com/solutions/flowjo</td></tr></tbody></table>

clonal analysis of embryonic hematopoietic stem cell precursors using single cell index sorting combined with endothelial cell niche co-culture

萌芽期の開発中に造血幹細胞 (HSC) の起源を調査する機能は、初期胚における HSC 前駆体の希少性と長期生着能性を識別する機能の試金の不足によって制限されており個々 の推定 HSC の前駆体。ここでは、分離・単一セルのレベルで機能的に検証された HSC 前駆体の同定を可能にする方法論について述べる。まず、駆使し個別に並べ替えの各セルの正確な表現型パラメーターをカタログにインデックス並べ替え HSC の前駆物質の実験的分析のための追加のマーカーと豊かに表現型マーカーの組み合わせを使用しています。第二に、インデックスの並べ替えの各セルは非接が HSC 前駆体多能、長期生着の潜在性と機能的な HSC の成熟を支える大動脈性腺発生 (AGM) 地域から血管性ニッチ間質との共培養移植の試金。この方法論により、機能性生着に秘めたクローン hemogenic 前駆体の表現型のプロパティまたは転写プロファイル、HSC 前駆体の詳細な分析手段を提供するなど他のプロパティの相関単一セルのレベルで開発。

図 1 aは、実験的なデザインの概略を示しています。P-Sp/AGM 組織は、解剖をプールし、コラゲナーゼで解離が、一度インデックス ソートの VE カドヘリンおよび EPCR に抗体と染みが。Pre HSC は、VE カドヘリン+EPCR高(図 1 b) 細胞で濃縮されています。他の蛍光色素標識された抗体遡及的各セルのインデックス ソ...

Watch this Scientific Journal Video about Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture at JoVE.com

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセル インデックスを使用して胚性造血幹細胞前駆体のクローン解析

ここでマウスの萌芽期の開発中に造血幹細胞前駆体のクローンの解析手法を提案する.内皮細胞共培養と表現型特性と単一造血前駆細胞の生着を潜在的な特徴への移植胚大動脈性腺発生地域から単一セルの並べ替えインデックスを組み合わせています。

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

clonal-analysis-embryonic-hematopoietic-stem-cell-precursors-using

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.5K ビュー.  Fred Hutchinson Cancer Research Center. ここでマウスの萌芽期の開発中に造血幹細胞前駆体のクローンの解析手法を提案する.内皮細胞共培養と表現型特性と単一造血前駆細胞の生着を潜在的な特徴への移植胚大動脈性腺発生地域から単一セルの並べ替えインデックスを組み合わせています。

ビデオ: Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

発生生物学

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

組合せの単一セルは分子を特徴付ける方法・肺浸潤異質性ひと幹と前駆細胞集団

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

遺伝学

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

生物学

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

腫瘍特異的内皮細胞の単離および培養拡大

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

医学

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 ...

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able to generate large datasets, necessitate the destruction of the cell of interest, making it impossible to assess its functional fate. Heterogeneous biological differentiating systems like hematopoiesis are therefore difficult to describe. Building on cell division tracking dyes, we further developed a protocol to simultaneously determine kinship, division number, and differentiation status for many single hematopoietic progenitors. This protocol allows the assessment of the ex vivo differentiation potential of murine and human hematopoietic progenitors, isolated from various biological sources. Moreover, as it is based on flow cytometry and a limited number of reagents, it can quickly generate a large amount of data, at the single-cell level, in a relatively inexpensive manner. We also provide the analytical pipeline for single-cell analysis, combined with a robust statistical framework. As this protocol allows the linking of cell division and differentiation at the single-cell level, it can be used to quantitatively assess symmetric and asymmetric fate commitment, the balance between self-renewal and differentiation, and the number of divisions for a given commitment fate. Altogether, this protocol can be used in experimental designs aiming to unravel the biological differences between hematopoietic progenitors, from a single-cell perspective.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

フローサイトメトリー による 造血幹細胞と前駆細胞の血縁関係、分裂数、表現型の同時評価

Few techniques can assess phenotype and fate for the same cell simultaneously. Most of the current protocols used to characterize phenotype, although able ...

免疫・感染学

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

マウス骨髄造血幹・前駆細胞および間質ニッチ細胞のフローサイトメトリー解析

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

ヒト間葉系幹細胞の蛍光活性化シングルセルソーティング

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

著者スポットライト:間葉系幹細胞の精製集団の単離における単一細胞ソーティングの重要性 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

ヒト剥離乳歯由来の免疫表現型間葉系幹細胞の単一細胞選別

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセルインデックスを使用して胚性造血幹細胞前駆体のクローン解析

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マウス胚性造血内皮細胞の単離

組合せの単一セルは分子を特徴付ける方法・肺浸潤異質性ひと幹と前駆細胞集団

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

腫瘍特異的内皮細胞の単離および培養拡大

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

フローサイトメトリーによる造血幹細胞と前駆細胞の血縁関係、分裂数、表現型の同時評価

マウス骨髄造血幹・前駆細胞および間質ニッチ細胞のフローサイトメトリー解析

ヒト剥離乳歯由来の免疫表現型間葉系幹細胞の単一細胞選別

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセル インデックスを使用して胚性造血幹細胞前駆体のクローン解析

要約

さらに動画を探す

この動画の章

マウス胚性造血内皮細胞の単離

組合せの単一セルは分子を特徴付ける方法・肺浸潤異質性ひと幹と前駆細胞集団

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

フィーダーとゼノフリーの定義条件におけるヒト多能性幹細胞からの血原性内皮分化

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

腫瘍特異的内皮細胞の単離および培養拡大

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

フローサイトメトリー による 造血幹細胞と前駆細胞の血縁関係、分裂数、表現型の同時評価

マウス骨髄造血幹・前駆細胞および間質ニッチ細胞のフローサイトメトリー解析

ヒト剥離乳歯由来の免疫表現型間葉系幹細胞の単一細胞選別

並べ替え内皮細胞ニッチ共培養と組み合わせて 1 つのセルインデックスを使用して胚性造血幹細胞前駆体のクローン解析

フローサイトメトリーによる造血幹細胞と前駆細胞の血縁関係、分裂数、表現型の同時評価