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>
	<li>Copley, M. R., Beer, P. A., Eaves, C. J. <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+cell+heterogeneity+takes+center+stage.">Hematopoietic stem cell heterogeneity takes center stage.</a> Cell Stem Cell. 10 (6), 690-697 (2012).</li><li>Medvinsky, A., Rybtsov, S., Taoudi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+the+adult+hematopoietic+system:+advances+and+questions.">Embryonic origin of the adult hematopoietic system: advances and questions.</a> Development. 138 (6), 1017-1031 (2011).</li><li>Kumaravelu, P., 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=Quantitative+developmental+anatomy+of+definitive+haematopoietic+stem+cells/long-term+repopulating+units+(HSC/RUs):+role+of+the+aorta-gonad-mesonephros+(AGM)+region+and+the+yolk+sac+in+colonisation+of+the+mouse+embryonic+liver.">Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.</a> Development. 129 (21), 4891-4899 (2002).</li><li>Taoudi, S., 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=Extensive+hematopoietic+stem+cell+generation+in+the+AGM+region+via+maturation+of+VE-cadherin+CD45++pre-definitive+HSCs.">Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs.</a> Cell Stem Cell. 3 (1), 99-108 (2008).</li><li>Rybtsov, S., 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=Hierarchical+organization+and+early+hematopoietic+specification+of+the+developing+HSC+lineage+in+the+AGM+region.">Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.</a> J Exp Med. 208 (6), 1305-1315 (2011).</li><li>Rybtsov, S., 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=Tracing+the+origin+of+the+HSC+hierarchy+reveals+an+SCF-dependent,+IL-3-independent+CD43(-)+embryonic+precursor.">Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.</a> Stem Cell Reports. 3 (3), 489-501 (2014).</li><li>McGrath, K. E., 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=Distinct+Sources+of+Hematopoietic+Progenitors+Emerge+before+HSCs+and+Provide+Functional+Blood+Cells+in+the+Mammalian+Embryo.">Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.</a> Cell Rep. 11 (12), 1892-1904 (2015).</li><li>Lin, Y., Yoder, M. C., Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+progenitor+emergence+in+the+murine+embryo+and+yolk+sac+precedes+stem+cell+detection.">Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection.</a> Stem Cells Dev. 23 (11), 1168-1177 (2014).</li><li>Medvinsky, A., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+hematopoiesis+is+autonomously+initiated+by+the+AGM+region.">Definitive hematopoiesis is autonomously initiated by the AGM region.</a> Cell. 86 (6), 897-906 (1996).</li><li>Sheridan, J. M., Taoudi, S., Medvinsky, A., Blackburn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+generation+of+reaggregated+organotypic+cultures+that+permits+juxtaposition+of+defined+cell+populations.">A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.</a> Genesis. 47 (5), 346-351 (2009).</li><li>Yoder, M. C., Hiatt, K., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+repopulating+hematopoietic+stem+cells+are+present+in+the+murine+yolk+sac+at+day+9.0+postcoitus.">In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.</a> Proc Natl Acad Sci U S A. 94 (13), 6776-6780 (1997).</li><li>Arora, N., 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=Effect+of+developmental+stage+of+HSC+and+recipient+on+transplant+outcomes.">Effect of developmental stage of HSC and recipient on transplant outcomes.</a> Dev Cell. 29 (5), 621-628 (2014).</li><li>Butler, J. M., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascular+niche+for+studying+stem+cell+homeostasis.">Generation of a vascular niche for studying stem cell homeostasis.</a> Methods Mol Biol. 904, 221-233 (2012).</li><li>Butler, J. M., 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=Endothelial+cells+are+essential+for+the+self-renewal+and+repopulation+of+Notch-dependent+hematopoietic+stem+cells.">Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells.</a> Cell Stem Cell. 6 (3), 251-264 (2010).</li><li>Kobayashi, H., 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=Angiocrine+factors+from+Akt-activated+endothelial+cells+balance+self-renewal+and+differentiation+of+haematopoietic+stem+cells.">Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells.</a> Nat Cell Biol. 12 (11), 1046-1056 (2010).</li><li>Hadland, B. 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=Endothelium+and+NOTCH+specify+and+amplify+aorta-gonad-mesonephros-derived+hematopoietic+stem+cells.">Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells.</a> J Clin Invest. 125 (5), 2032-2045 (2015).</li><li>Hadland, B. 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=A+Common+Origin+for+B-1a+and+B-2+Lymphocytes+in+Clonal+Pre-+Hematopoietic+Stem+Cells.">A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.</a> Stem Cell Reports. 8 (6), 1563-1572 (2017).</li><li>Wilson, N. 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. B., 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=Role+of+RUNX1+in+adult+hematopoiesis:+analysis+of+RUNX1-IRES-GFP+knock-in+mice+reveals+differential+lineage+expression.">Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression.</a> Blood. 103 (7), 2522-2529 (2004).</li><li>Holmes, R., Z&#250;&#241;iga-Pfl&#252;cker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009 (2), pdb.prot5156 (2009).</li><li>Yoshimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+wave+of+B+lymphopoiesis+develops+independently+of+stem+cells+in+the+murine+embryo.">The first wave of B lymphopoiesis develops independently of stem cells in the murine embryo.</a> Ann N Y Acad Sci. 1362, 16-22 (2015).</li><li>Kobayashi, M., 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=Functional+B-1+progenitor+cells+are+present+in+the+hematopoietic+stem+cell-deficient+embryo+and+depend+on+Cbf&#946;+for+their+development.">Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbf&#946; for their development.</a> Proc Natl Acad Sci U S A. 111 (33), 12151-12156 (2014).</li><li>Yoshimoto, M., 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=Autonomous+murine+T-cell+progenitor+production+in+the+extra-embryonic+yolk+sac+before+HSC+emergence.">Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.</a> Blood. 119 (24), 5706-5714 (2012).</li><li>Inlay, M. A., 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=Identification+of+multipotent+progenitors+that+emerge+prior+to+hematopoietic+stem+cells+in+embryonic+development.">Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.</a> Stem Cell Reports. 2 (4), 457-472 (2014).</li><li>Palis, J. <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+cell-independent+hematopoiesis:+emergence+of+erythroid,+megakaryocyte,+and+myeloid+potential+in+the+mammalian+embryo.">Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo.</a> FEBS Lett. 590 (22), 3965-3974 (2016).</li></ol>

著者が明らかに何もありません。

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>AGM-EC culture media </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for culture of endothelial cells</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&#39;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>AGM-EC culture media (500 mls)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;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>Materials for AGM index sort</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 &#181;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>Antibodies for AGM index sort</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>AGM-serum free media (10mls)</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>Materials for Staining co-cultured cells</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 colspan="3">Antibodies for analysis of Index sorted clones co-cultured with endothelial cells</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>Materials for tail vein injection for transplant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 ml insulin syringes with 29G 1/2&#34; needles</td>
			<td>BD Biosciences</td>
			<td>309306</td>
			<td>Use for tail vein injection for transplantation</td>
		</tr>
		<tr>
			<td>Antibodies for Peripheral Blood analysis following translant</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>Equipment</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

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

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

胚性メッケル軟骨でZebrabowクローン細胞分析によって軟骨細胞のインターカレーションと方向性増殖の可視化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

発生生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

失活ヒト真皮を用いて遺伝子組換え器官皮膚培養物の生成

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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, ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

競争力の移植は、造血幹細胞のフィットネスを評価します

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

転写因子との再プログラミングマウス胚線維芽細胞造血プログラムを誘導するために

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

赤血球の溶血症に対する造血幹細胞移植後の残留ドナー赤芽球系前駆細胞の検出

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

生活そのままゼブラフィッシュの単一セルの光電

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

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

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