Name: in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas
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我们感谢布朗露西和亚历山大Spalla从流式细胞仪分析的核心希望之城协助分拣。这项工作部分由健康（NIH）全国学院授予R01DK081587和R01DK099734到HTK，并U01DK089533以ADR，并通过国家科学基金会资助NSF-DMR-1206121和加州再生医学研究所授予RB5-07398到DAT支持受支持从约瑟夫·雅各布斯J.研究所分子工程医学在加州理工学院DAT，和来自奥克斯纳德基金会和埃拉·菲茨杰拉德基金会HTK也表示感谢。 资金来源：这项工作部分由健康（NIH）全国学院授予的支持和R01DK081587向R01DK099734 HTK，并U01DK089533以ADR，并通过国家科学基金会资助NSF-DMR-1206121和加州再生医学研究所授予RB5-07398到DAT由约瑟夫·雅各布斯杂志支持研究所分子工程医学在加州理工学院DAT，和来自奥克斯纳德基金会和埃拉·菲茨杰拉德基金会HTK也表示感谢。研究本出版物中报道包含在流式细胞仪分析的核心和光学显微镜数码影像下奖号P30CA33572卫生的国家机构的美国国家癌症研究所支持的核心完成的工作。 研究赞助商：主办单位并未参与研究设计，收集，分析或数据的解释。

伦理声明：我们坚持开展研究，以确保质量和结果的完整性广泛接受的道德标准。动物实验是根据在市灵批准的机构动物护理和使用委员会的协议进行的。 1.成年鼠胰腺准备单细胞悬液注意：在现有的出版物7,11，CD-1或B6背景的小鼠使用;这两个背景产生了相似的结果。转基因小鼠线（指定的Sox9 / EGFP）与增强型绿色荧光蛋白通过Sox9的基因位点19,20驱动的，是在CD-1后台创建...

<ol>
	<li>Gu, G., Brown, J. R., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+tracing+reveals+the+ontogeny+of+pancreatic+cell+fates+during+mouse+embryogenesis.">Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis.</a> Mech Dev. 120, 35-43 (2003).</li><li>Kopp, J. L., 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=Sox9++ductal+cells+are+multipotent+progenitors+throughout+development+but+do+not+produce+new+endocrine+cells+in+the+normal+or+injured+adult+pancreas.">Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.</a> Development. 138, 653-665 (2011).</li><li>DiGiusto, D. L., 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=RNA-based+gene+therapy+for+HIV+with+lentiviral+vector-modified+CD34(+)+cells+in+patients+undergoing+transplantation+for+AIDS-related+lymphoma.">RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma.</a> Science translational medicine. 2, 36-43 (2010).</li><li>Inada, 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=Carbonic+anhydrase+II-positive+pancreatic+cells+are+progenitors+for+both+endocrine+and+exocrine+pancreas+after+birth.">Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.</a> Proc Natl Acad Sci U S A. 105, 19915-19919 (2008).</li><li>Solar, 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=Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth.">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17, 849-860 (2009).</li><li>Ku, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+pancreatic+progenitor+cells--recent+studies.">Minireview: pancreatic progenitor cells--recent studies.</a> Endocrinology. 149, 4312-4316 (2008).</li><li>Jin, L., 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=Colony-forming+cells+in+the+adult+mouse+pancreas+are+expandable+in+Matrigel+and+form+endocrine/acinar+colonies+in+laminin+hydrogel.">Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel.</a> Proc Natl Acad Sci U S A. 110, 3907-3912 (2013).</li><li>Fu, X., 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=MicroRNA-26a+targets+ten+eleven+translocation+enzymes+and+is+regulated+during+pancreatic+cell+differentiation.">MicroRNA-26a targets ten eleven translocation enzymes and is regulated during pancreatic cell differentiation.</a> Proc Natl Acad Sci U S A. 110, 17892-17897 (2013).</li><li>Ghazalli, 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=Postnatal+Pancreas+of+Mice+Contains+Tripotent+Progenitors+Capable+of+Giving+Rise+to+Duct,+Acinar,+and+Endocrine+Cells+In+Vitro.">Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.</a> Stem Cells Dev. , (2015).</li><li>Jin, L., 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=Colony-forming+progenitor+cells+in+the+postnatal+mouse+liver+and+pancreas+give+rise+to+morphologically+distinct+insulin-expressing+colonies+in+3D+cultures.">Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.</a> Rev Diabet Stud. 11, 35-50 (2014).</li><li>Jin, L., 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=In+Vitro+Multilineage+Differentiation+and+Self-Renewal+of+Single+Pancreatic+Colony-Forming+Cells+from+Adult+C57Bl/6+Mice.">In Vitro Multilineage Differentiation and Self-Renewal of Single Pancreatic Colony-Forming Cells from Adult C57Bl/6 Mice.</a> Stem Cells Dev. , (2014).</li><li>Huch, 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=Unlimited+in+vitro+expansion+of+adult+bi-potent+pancreas+progenitors+through+the+Lgr5/R-spondin+axis.">Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.</a> Embo J. 32, 2708-2721 (2013).</li><li>Greggio, C., 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=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Lee, J., 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=Expansion+and+conversion+of+human+pancreatic+ductal+cells+into+insulin-secreting+endocrine+cells.">Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.</a> Elife. 2, e00940 (2013).</li><li>Dorrell, C., 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=The+organoid-initiating+cells+in+mouse+pancreas+and+liver+are+phenotypically+and+functionally+similar.">The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.</a> Stem Cell Res. 13, 275-283 (2014).</li><li>Ku, H., Yonemura, Y., Kaushansky, K., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombopoietin,+the+ligand+for+the+Mpl+receptor,+synergizes+with+steel+factor+and+other+early+acting+cytokines+in+supporting+proliferation+of+primitive+hematopoietic+progenitors+of+mice.">Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.</a> Blood. 87, 4544-4551 (1996).</li><li>Ku, H. T., 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=Insulin-expressing+colonies+developed+from+murine+embryonic+stem+cell-derived+progenitors.">Insulin-expressing colonies developed from murine embryonic stem cell-derived progenitors.</a> Diabetes. 56, 921-929 (2007).</li><li>Winkler, 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=A+quantitative+assay+for+insulin-expressing+colony-forming+progenitors.">A quantitative assay for insulin-expressing colony-forming progenitors.</a> J Vis Exp. , e3148 (2011).</li><li>Gong, 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=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425, 917-925 (2003).</li><li>Formeister, E. J., 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+SOX9+levels+differentially+mark+stem/progenitor+populations+and+enteroendocrine+cells+of+the+small+intestine+epithelium.">Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium.</a> Am J Physiol Gastrointest Liver Physiol. 296, G1108-G1118 (2009).</li><li>Seymour, P. 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=A+dosage-dependent+requirement+for+Sox9+in+pancreatic+endocrine+cell+formation.">A dosage-dependent requirement for Sox9 in pancreatic endocrine cell formation.</a> Dev Biol. 323, 19-30 (2008).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81, 2844-2853 (1993).</li><li>Suzuki, A., Nakauchi, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+multipotent+pancreatic+progenitors+using+flow-cytometric+cell+sorting.">Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.</a> Diabetes. 53, 2143-2152 (2004).</li><li>Seaberg, R. 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=Clonal+identification+of+multipotent+precursors+from+adult+mouse+pancreas+that+generate+neural+and+pancreatic+lineages.">Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.</a> Nat Biotechnol. 22, 1115-1124 (2004).</li><li>Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J., Melton, D. A. <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+reprogramming+of+adult+pancreatic+exocrine+cells+to+beta-cells.">In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.</a> Nature. 455, 627-632 (2008).</li><li>Baeyens, L., 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=Transient+cytokine+treatment+induces+acinar+cell+reprogramming+and+regenerates+functional+beta+cell+mass+in+diabetic+mice.">Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.</a> Nat Biotechnol. 32, 76-83 (2014).</li><li>Thorel, 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=Conversion+of+adult+pancreatic+alpha-cells+to+beta-cells+after+extreme+beta-cell+loss.">Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss.</a> Nature. 464, 1149-1154 (2010).</li></ol>

胰腺菌落测定和单个菌落分析这里描述通过在解密造血祖细胞的生物学在过去几十年23都发挥主要作用的含有甲基纤维素造血集落测定法启发。在这些测定法（ 图5），解离胰腺细胞铺到与支持环，密集或内分泌/腺泡菌落7的形成适当的生长因子和ECM蛋白半固体培养基含有甲基纤维素。那就是能产生细胞的殖民地单祖细胞被称为PCFU。通过表征采用实时定量微-PCR和全挂?...

胰腺是由三个主要的细胞谱系;腺泡细胞分泌消化酶，导管分泌粘蛋白，抵御病原体和运输消化酶肠道，内分泌细胞分泌的激素，包括胰岛素和胰高血糖素，即保持血糖平衡。在胰腺的胚胎发育，早期导管细胞能够在成年动物1,2的胰腺引起三个谱系三效祖细胞的来源。因为成人干细胞和祖细胞，如骨髓干细胞，已成功地用于治疗各种疾病3，有在成人胰腺找到干细胞和祖细胞的强烈的兴趣。如果隔离和成人胰腺干细胞和祖细胞的操纵是可能的，这些细胞可用于治疗疾病，例如1型糖尿病，其中所述胰岛素分泌细胞是由自身免疫破坏。 使用体内 CRE-LOX谱系追踪技术，稻田和同事表明，标记有标记物，碳酸酐酶II成年鼠导管细胞，会引起所有三个胰腺谱系4。然而，使用其他导管标记物，如HNF1B 5和Sox9的2，可以得出结论，导管细胞不是在成年小鼠的β细胞的主要来源。 几年前，提出的是，上述讨论的原因，可能是由于缺乏，在字段6,7，的可以用于测量的自我更新和多谱系分化两个标准需要适当的分析工具定义的干细胞。提到体内 CRE-LOX谱系追踪技术以上可为在群体水平的祖后代关系的证据。然而，这一谱系追踪技术在其权力仅限于辨别是否单系祖细胞可以自我更新和分化成多种谱系。因为如果几个单效祖细胞，每一个不同的分化潜能，一起分析，它们可共同出现有多谱系分化能力的单细胞分析是重要的。此外，干细胞是通常成人器官的次要人口。一个次要的细胞群的活动可以由主要的人口被屏蔽。因此，从人群研究的否定结​​果并不一定表示不存在的干细胞。最后，CRE-LOX谱系追踪目前不允许自我更新的测量。 开始解决在胰腺祖细胞生物学，细胞集落领域的技术间隙7-11或类器官12-15 </suP&gt;使用三维培养系统测定法设计。胰腺祖细胞的两个集落测定法在我们实验室开发了：一个含有鼠细胞外基质蛋白（ECM）（见方法和设备表 ）的市售制剂，以及另一个包含层粘连蛋白的水凝胶，限定人造的ECM蛋白7-11。祖细胞在含有半固体培养基的甲基纤维素进行混合。甲基纤维素是从木纤维制得的生物惰性和粘性材料，并已在造血细胞集落测定法16被经常使用。半固体培养基中含有甲基纤维素，限制了单一祖细胞的运动，使得它们不能重新聚集。然而，该介质是足够软以允许祖细胞生长和分化成在三维空间中的细胞的集落。继血液学家的传统，这是能够引起细胞集落胰腺祖细胞为：NAmed的胰腺细胞集落形成单位（PCFU）。 PCFUs，在小鼠含ECM-集落培养生长时，引起了被命名为“环”殖民地7囊性殖民地。在添加的Wnt激动剂，R-spondin1，成鼠含ECM-文化，一些环殖民地变成“密”殖民地7。在本文中，这两种类型的小鼠的ECM培养中生长的菌落被统称为“环/密”菌落。当戒指/高密度菌落分离成单细胞悬液，重新接种到含有层粘连蛋白凝胶文化，“内分泌/腺泡”菌落形成7。 使用单个菌落分析，发现大多数环/密集和内分泌/腺泡殖民地，无论是从成人（2-4个月大）7,11或青年（1周龄）9鼠胰腺，表达所有三个系标记。这表明大多数始发PCFUs的是三效。在小鼠含ECM-集落培养，成年鼠PCFUs有力地自我更新和扩大了11周约50万次文化7。鼠的ECM优先支持导管细胞过度内分泌和腺泡谱系的分化，而 ​​在层粘连蛋白的水凝胶的情况下，被鼓励鼠PCFUs优先分化成内分泌和腺泡细胞和少这样的导管谱系7,9,11。重要的是，胰岛素+胰高血糖素-单-激素细胞在层粘连蛋白凝胶培养产生并且响应分泌胰岛素对葡萄糖刺激体外 7,9，提示功能成熟。三谱系分化潜能7,9和个人PCFUs的自我更新11由单细胞显微操作， 即证实，培养每孔一个细胞集落形成。总之，这些结果提供的证据表明有自我更新，三正POTENT，祖细胞样细胞在出生后小鼠胰腺，显示在三维培养的活动。 本文中描述的鼠PCFU测定是从现有集落检测设计用于从小鼠胚胎干细胞（mESCs）17分化的祖细胞。该协议是在另一个朱庇特出版物18记载的细节。培养组件和执行成人PCFUs鼠含ECM集落测定法所需的技术是一样的MESC衍生的祖细胞17,18。因此，测定的这些方面将不在这里重复;代替下列程序将得到解决：1）成人胰腺分离和分拣CD133 + Sox9的/ EGFP +导管细胞，从成年小鼠7,2）的分选的细胞的单细胞操纵，3）单菌落丰富PCFUs分析使用微流体的qRT-PCR和整体卡口免疫染色，和4）科勒尼的离解s转换单细胞悬浮液，重新电镀成鼠细胞外基质或层粘连蛋白的水凝胶菌落测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Murine ECM proteins (Matrigel)</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>354230</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Laminin Hydrogel</td>
			<td>Provided by David Tirrell (Pasadena, CA USA)</td>
			<td></td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Shinetsu Chemical (Tokyo, Japan)</td>
			<td></td>
			<td>1500 centipoise (dynamic viscosity unit equal to 15g/cm/s) (high viscosity)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline</td>
			<td>Mediatech (Manassas, VA, USA)</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Flacon Conical vial</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100mmx20mm Suspension culture dish</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>A8412</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase1</td>
			<td>Calbiochem (Darmstadt, Germany)</td>
			<td>260913</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Roche (CH-4070, Basel, Schweiz, Switzerland)</td>
			<td>11088831001</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/32</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>101310</td>
			<td>low endotoxin, azide free</td>
		</tr>
		<tr>
			<td>PE-Cy7 Rat IgG2a &#954; Isotype Control</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>400522</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 &#954; Isotype Control</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-4301-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD133-Biotin</td>
			<td>eBioscience (San Diego, CA, USA)</td>
			<td>13-1331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD71-PE-Cy7</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>113812</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Allophycocyanin</td>
			<td>Biolegend (San Diego, CA, USA)</td>
			<td>405207</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>3571</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Anti-mucin 1</td>
			<td>Thermo Fisher Scientific (Waltham, MA USA)</td>
			<td>HM-1630-P1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dylight 649 Goat anti-Armenian Hamster</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>127-495-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Mediatech(Manassas, VA, USA)</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Tissure Culture Biologicals (Long Beach, CA, USA)</td>
			<td>101</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Tris Ethylenediaminetetraacetic acid</td>
			<td>TEKnova (Hollister, CA, USA)</td>
			<td>T0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy Micro Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTec Reverse Transcription Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>205310</td>
			<td></td>
		</tr>
		<tr>
			<td>CellsDirect One-Step qRT-PCR Kit</td>
			<td>Ambion/Invitrogen(Grand Island, NY, USA)</td>
			<td>11753-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Santa Cruz Bio (Santa Cruz, CA, USA)</td>
			<td>SC-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>005-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Jackson Immuno (West Grove, PA, USA)</td>
			<td>0017-000-121</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>T-4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Invitrogen (Waltham, MA,&#160; USA)</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies (Waltham, MA, USA)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Gibco (Grand Island, NY, USA)</td>
			<td>15230-147</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Pasteur Pipette</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>13-678-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>40um Filter Mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um filter mesh</td>
			<td>Fisher Scientific (Pittsburgh, PA , USA)</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TC Plate 96 Well Suspension</td>
			<td>Sarstedt</td>
			<td>83.3924 (Previously 83.1835)</td>
			<td></td>
		</tr>
		<tr>
			<td>1cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10cc Syringe</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr>
			<td>48.48 Dyanmic Array Chip</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>BMK-M-48.48</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidigm GE 48.48 Dynamic Array Sample &#38; Assay Loading Reagent Kit</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td>85000800</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems (Grand Island, NY, USA)</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol sorbitan monolaurate</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dish</td>
			<td>MatTek (Ashland, MA, USA)</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm petri dish with glass bottom</td>
		</tr>
		<tr>
			<td>Mouth Piece/ Rubber Tubing</td>
			<td>Renova Life Inc. (College Park, MD, USA)</td>
			<td>MP-SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>N0636</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>293-VE</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Activin B</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>659-AB</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Extendin 4</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td>E7144</td>
			<td>Stock kept at -20oC</td>
		</tr>
		<tr>
			<td>Rspondin-1</td>
			<td>R&#38;D Systems (Minneapolis, MN, USA)</td>
			<td>3474-RS</td>
			<td>Stock kept at -80oC</td>
		</tr>
		<tr>
			<td>Falcon 5mL Polystyrene Round-Bottom Tube</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 18Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 16Gx1 1/2</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td>305198</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Ultra-Low Attachment Surface 24 well flat bottom plate&#160;</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 96 Black Well Plate</td>
			<td>Corning Inc. (Corning, NY, USA)</td>
			<td>3603</td>
			<td>Flat, clear bottom with lid.&#160; Black polystyrene TC-treated microplates</td>
		</tr>
		<tr>
			<td>Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope</td>
			<td>Carl Zeiss AG (Oberkochen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biomark HD&#160;</td>
			<td>Fluidigm (San Francisco, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aria Special Order Research Product Cell Sorter</td>
			<td>Becton Dickson (Franklin Lakes, NJ, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas

茎和从成人胰腺祖细胞可以是治疗性的β样细胞为1型糖尿病治疗的患者的潜在来源。然而，它仍然是未知在成人胰腺中是否存在干细胞和祖细胞。使用CRE-LOX在成年小鼠世系追踪研究策略已经初见成效，无论是支持或反驳，β细胞可以从管道，那里的成人胰腺祖细胞可能存在于假定的位置而产生的想法。 这些体内 CRE-LOX谱系追踪方法，但是，不能回答的自我更新和多谱系分化两个必要定义一个干细胞的标准的问题。要开始解决这个技术差距，我们发明了胰腺祖细胞的三维殖民地化验。我们最初公布后不久，其他实验室自主研发的类似，但不完全相同，方法调用的类器官检测。相比于类器官测定中，我们的方法是采用甲基纤维素，其形成粘性溶液，允许在低浓度包含细胞外基质蛋白。含甲基纤维素的测定法允许容易检测和祖细胞的单细胞水平，当祖细胞构成的小亚群这是至关重要的分析，因为对于许多成人器官的干细胞的情况下。一起，从几个实验室结果表明在小鼠胰腺祖细胞样细胞的体外的自我更新和多谱系分化。目前的协议描述了两个基于甲基纤维素集落实验表征小鼠胰腺祖细胞;一个含有鼠细胞外基质蛋白的市售制剂和其它人工细胞外基质蛋白已知为层粘连蛋白水凝胶。这里示出的技术是：1）的胰腺解离和CD133 +的Sox9 / EGFP的排序从成年小鼠+导管细胞，2）在S的单细胞操作orted细胞，3）单菌落分析使用微流体的qRT-PCR和整体卡口免疫染色，和4）主菌落解离成单细胞悬液，并重新电镀到次级菌落测定以评估自我更新或分化。

成人胰腺祖细胞可以通过荧光激活细胞分选（ 图1）来富集。这里使用的Sox9的/ EGFP转基因小鼠线作为GENSAT脑图谱项目19的结果被首先产生，并且EGFP报告是含有细菌人工染色体的控制下〜75 kb的上游和〜150 kb的Sox9的20的下游序列。在这些小鼠中，EGFP高效和特异性22标签胰管。胰腺采购和离解成单细胞悬浮液，并与生物素缀合的抗CD133抗体?...

Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

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

体外集落测定法来检测自我更新和从成年鼠胰腺分离的祖细胞的分化被设计的。在这些测定中，胰祖细胞产生细胞集落在3维空间中含有甲基纤维素半固体培养基。用于处理单个细胞和单独的菌落的特征的协议进行了描述。

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

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

Research

JoVE Journal

Developmental Biology

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

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Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

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Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

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Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

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In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...