Name: generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds
Uploaded: 2016-05-05T15:00:00
Description: Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

我们要感谢Rossant博士和碧萝博士在图1-3中描述的实验中使用的Nkx2-1 mcherry ESC。 FACS是在病童医院，UHN流式细胞仪进行融资。这项工作是由操作从加拿大卫生研究院的研究，并从创新的加拿大基金会的资助基础设施（CSCCD）资助。

动物实验按照医院的病童研究院动物护理委员会准则进行。 1.脚手架准备<ol><li>肺脱细胞<ol><li>使用二氧化碳安乐死室成年Wistar大鼠。放置动物在腔室，并以每分钟室体积10-30％的填充率开始100％的CO 2的曝光。 <ol><li>观察动物的意识障碍;在此之后大约2-3分钟会发生。如果神志不清不这段时间内发生，检查填充率和腔密封。以下失去知觉，对于褪眼睛颜色和缺乏呼吸的观察动...

<ol>
	<li>Discher, D. E., Mooney, D. J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324 (5935), 1673-1677 (2009).</li><li>Daley, W. P., Peters, S. B., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+dynamics+in+development+and+regenerative+medicine.">Extracellular matrix dynamics in development and regenerative medicine.</a> J. Cell Sci. 121 (3), 255-264 (2008).</li><li>Ghaedi, 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=Human+iPS+cell-derived+alveolar+epithelium+repopulates+lung+extracellular+matrix.">Human iPS cell-derived alveolar epithelium repopulates lung extracellular matrix.</a> J. Clin. Invest. 123 (11), 4950-4962 (2013).</li><li>Huang, S. X. 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=Efficient+generation+of+lung+and+airway+epithelial+cells+from+human+pluripotent+stem+cells.">Efficient generation of lung and airway epithelial cells from human pluripotent stem cells.</a> Nat. Biotechnol. 32 (1), 84-91 (2014).</li><li>Jensen, 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=A+rapid+lung+de-cellularization+protocol+supports+embryonic+stem+cell+differentiation+in+vitro+and+following+implantation.">A rapid lung de-cellularization protocol supports embryonic stem cell differentiation in vitro and following implantation.</a> Tissue Eng. Part C: Methods. 18 (8), 632-646 (2012).</li><li>Longmire, T. 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=Efficient+derivation+of+purified+lung+and+thyroid+progenitors+from+embryonic+stem+cells.">Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells.</a> Cell stem cell. 10 (4), 398-411 (2012).</li><li>Wong, A. 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=Directed+differentiation+of+human+pluripotent+stem+cells+into+mature+airway+epithelia+expressing+functional+CFTR+protein.">Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein.</a> Nat. Biotechnol. 30 (9), 876-882 (2012).</li><li>Gilpin, S. 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=Enhanced+Lung+Epithelial+Specification+of+Human+Induced+Pluripotent+Stem+Cells+on+Decellularized+Lung+Matrix.">Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix.</a> Annal. Thorac. Surg. 98, 1721-1729 (2014).</li><li>Cortiella, 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=Influence+of+Acellular+Natural+Lung+Matrix+on+Murine+Embryonic+Stem+Cell+Differentiation+and+Tissue+Formation.">Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation.</a> Tissue Eng. Part A. 16 (8), 2565-2580 (2010).</li><li>Princivalle, M., De Agostini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+roles+of+heparan+sulfate+proteoglycans:+a+comparative+review+in+Drosophila,+mouse+and+human.">Developmental roles of heparan sulfate proteoglycans: a comparative review in Drosophila, mouse and human.</a> Int. J. Dev. Biol. 46, 267-278 (2002).</li><li>Thompson, S. M., Jesudason, E. C., Turnbull, J. E., Fernig, 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=Heparan+sulfate+in+lung+morphogenesis:+The+elephant+in+the+room.">Heparan sulfate in lung morphogenesis: The elephant in the room.</a> Birth Defects Res. Part C, Embryo Today. 90 (1), 32-44 (2010).</li><li>Zimmermann, 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=Improved+reproducibility+in+preparing+precision-cut+liver+tissue+slices.">Improved reproducibility in preparing precision-cut liver tissue slices.</a> Cytotechnology. 61 (3), 145-152 (2009).</li><li>Ying, Q. -. 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=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Fox, 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=Three-Dimensional+Culture+and+FGF+Signaling+Drive+Differentiation+of+Murine+Pluripotent+Cells+to+Distal+Lung+Epithelial+Cells.">Three-Dimensional Culture and FGF Signaling Drive Differentiation of Murine Pluripotent Cells to Distal Lung Epithelial Cells.</a> Stem Cells Dev. 24 (1), 21-35 (2014).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Shojaie, 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=Acellular+lung+scaffolds+direct+differentiation+of+endoderm+to+functional+airway+epithelial+cells:+requirement+of+matrix-bound+HS+proteoglycans.">Acellular lung scaffolds direct differentiation of endoderm to functional airway epithelial cells: requirement of matrix-bound HS proteoglycans.</a> Stem Cell Reports. 4, 1-12 (2015).</li><li>Kubo, 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=Development+of+definitive+endoderm+from+embryonic+stem+cells+in+culture.">Development of definitive endoderm from embryonic stem cells in culture.</a> Development. 131 (7), 1651-1662 (2004).</li><li>Longmire, T. 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=Efficient+Derivation+of+Purified+Lung+and+Thyroid+Progenitors+from+Embryonic+Stem+Cells.">Efficient Derivation of Purified Lung and Thyroid Progenitors from Embryonic Stem Cells.</a> Cell stem cell. 10 (4), 398-411 (2012).</li><li>Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P., Henkelman, R. M. <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+3D+mouse+embryo+atlas+based+on+micro-CT.">A novel 3D mouse embryo atlas based on micro-CT.</a> Development. 139 (17), 3248-3256 (2012).</li></ol>

这里描述的协议产生只使用天然肺支架直接，没有其他补充了分化成熟的ESC源性上皮气道。这种文化建立的定义，无血清，价格低廉，重现性好。没有基础分化培养基中生长因子的补充是必要的。用于产生干细胞衍生的肺上皮细胞中以前发表的方法已使用2维的策略与生长因子补充，以促进谱系限制3,4,8,18,19。此处所描述的技术是在这些方法中有几个原因，包括有利：定义培养条件下，分?...

多能细胞对肺谱系定向分化是依赖于微环境1,2-精确信号事件。由于此过程的动态本质，已经挑战模仿肺器官的准确事件体外 。最近的报道已经用于与二维培养物的可溶性生长因子补充分步进行谱系限制策略，实现肺分化3-8。在步分化方案，多能细胞，是否胚胎干细胞（ESC）或诱导的多能干细胞，首先分化为定形内胚层胚层。内胚层细胞随后推到前部内胚层的命运和其后肺祖细胞，所确定包含同源结构域转录因子NKX2-1的表达。这些肺祖细胞进一步分化近端（气）或远端（肺泡）肺上皮细胞的Wi日继续生长因子的补充。这种2维策略曾在产生肺上皮细胞取得了一些成功，但也有一些限制，包括不明效率，从其它内胚层谱系可能的污染，缺少一个3维（3D）结构，并且在某些情况下使用未定义培养物血清补充。上脱细胞肺支架多能或分化的细胞的培养日益用作测定，以评估在形成肺上皮结构3,5,6,8,9接种的细胞的再生潜能。这些报告接种文化与持续生长因子或血清补充支架的细胞。 龙开发涉及的划分，迁移，基因响应环境线索的表达和个体细胞的分化。细胞外基质（ECM）是糖蛋白的一个格子，除了提供结构支撑，引导Tissu酒店通过整合和调节这些过程10,11ê形态。通过使用肺ECM支架作为天然平台内胚层培养，以更好地模仿体内肺发育环境，我们已经产生干以限定三维培养细胞来源的呼吸道上皮细胞具有高效率和再现性设置。 由脱细胞以及小鼠ESC衍生的内胚层细胞产生的大鼠肺的ECM支架生成并随后接种到这些支架。 CXCR4和C-KIT蛋白的表达双重指示定型内胚层细胞的身份和阳性细胞都SOX2和NKX2-1的表达被确定为气道（近端肺）祖细胞。定形内胚层细胞在空气液体界面（ALI）长达三周生成官能呼吸道上皮细胞在体外进行培养。 该协议促进defini肺系分化略去内胚层早7天，用NKX2-1 + / + SOX2早期肺癌近祖的出现观察。第14天，文化成熟的气道上皮细胞群的21冒出包括纤毛（TUBB4A +），俱乐部（SCGB1A1 +），和基底（TRP63 +，KRT5 +）细胞的形态和功能相似本地鼠标气道。这个协议说明了3D-基质微环境的实现健壮分化到呼吸道上皮细胞的重要性。

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Reagents</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">H0777</td>
			<td style="width:281px;">10U/mL heparin</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Perfusion solution</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">14170112</td>
			<td style="width:281px;">dissolved in Hank&#39;s balanced salt solution (HBSS-)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">CHA003</td>
			<td style="width:281px;">8mM CHAPS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">E9884</td>
			<td style="width:281px;">25mM EDTA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">BioShop</td>
			<td style="width:168px;">SOD002</td>
			<td style="width:281px;">1M NaCl</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Decellularization solution</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">dissolved in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Novagen</td>
			<td style="width:168px;">70664-3</td>
			<td style="width:281px;">90U/mL Benzonase nuclease&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Benzonase nuclease</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15140</td>
			<td style="width:281px;">200U/mL penicillin streptomycin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td align="right" style="width:168px;">15290</td>
			<td style="width:281px;">25&#956;g/mL amphotericin B&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antimicrobial solution&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">14190-144</td>
			<td style="width:281px;">diluted in PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Trypsinization&#160;</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12605-028</td>
			<td style="width:281px;">TrypLE</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">IMDM 2440-053, F12 11765-054</td>
			<td style="width:281px;">3:1 ratio of IMDM and Ham&#8217;s modified F12 medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">12587-010</td>
			<td style="width:281px;">B27 supplement (50x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">17502-048</td>
			<td style="width:281px;">N2 supplement (100x dilution)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">15260-037</td>
			<td style="width:281px;">0.05% (Fraction V) bovine serum albumin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Gibco</td>
			<td style="width:168px;">35050-061</td>
			<td style="width:281px;">200mM Glutamax&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">M6145</td>
			<td style="width:281px;">4&#956;M monothioglycerol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Serum free differentiation media (SFDM)</td>
			<td style="width:163px;">Sigma</td>
			<td style="width:168px;">A4403&#160;</td>
			<td style="width:281px;">0.05mg/mL ascobic acid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Endoderm induction</td>
			<td style="width:163px;">R&#38;D</td>
			<td style="width:168px;">338-AC/CF</td>
			<td style="width:281px;">Activin A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Antibodies</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CDH1</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">610181</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">C-KIT</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558163</td>
			<td style="width:281px;">Rat, PE-Cy7, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">CXCR4</td>
			<td style="width:163px;">BD Biosciences</td>
			<td align="right" style="width:168px;">558644</td>
			<td style="width:281px;">Rat, APC, 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">KRT5</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab24647</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">NKX2-1</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:168px;">ab76013</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Laminin</td>
			<td style="width:163px;">Novus Biologicals</td>
			<td style="width:168px;">NB300-144</td>
			<td style="width:281px;">Rabbit, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SCGB1A1</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-9772&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SOX2</td>
			<td style="width:163px;">R&#38;D Systems</td>
			<td style="width:168px;">AF2018&#160;</td>
			<td style="width:281px;">Goat, non-conjugated, 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TRP63</td>
			<td style="width:163px;">Santa Cruz</td>
			<td style="width:168px;">sc-8431</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">TUBB4A</td>
			<td style="width:163px;">BioGenex</td>
			<td style="width:168px;">MU178-UC</td>
			<td style="width:281px;">Mouse, non-conjugated, 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Goat IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-11055</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21202</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Mouse IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31571</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-21206</td>
			<td style="width:281px;">Donkey, Alexa Fluor 488, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Rabbit IgG&#160;</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:168px;">A-31573</td>
			<td style="width:281px;">Donkey, Alexa Fluor 647, 1:200</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:291px;">Other Materials</td>
			<td style="width:163px;"></td>
			<td style="width:168px;"></td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low adherent plates</td>
			<td style="width:163px;">Nunc</td>
			<td style="width:168px;">Z721050&#160;</td>
			<td style="width:281px;">Low cell binding plates,&#160; 6 wells&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air-liquid interface membranes&#160;</td>
			<td>Whatman</td>
			<td align="right">110614</td>
			<td>Hydrophobic Nucleopore membrane, 8&#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1200S&#160;</td>
			<td>Leica Vibratome</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tissue Adhesive</td>
			<td>Ted Pella</td>
			<td align="right">10033</td>
			<td>Pelco tissue adhesive</td>
		</tr>
	</tbody>
</table>

generation of esc-derived mouse airway epithelial cells using decellularized lung scaffolds

肺谱系分化需要复杂的环境线索，包括生长因子信号传导，细胞 - 细胞相互作用和细胞 - 基质相互作用的集成。由于这种复杂性， 在体外肺发育的重演，促进干细胞肺上皮细胞的分化已经挑战。在这个协议中，脱细胞的肺支架被用来模仿肺的3维环境，并产生干细胞衍生的呼吸道上皮细胞。小鼠胚胎干细胞首先分化为使用具有激活素A.内胚层细胞的胚状体（EB）培养方法的内胚层谱系，然后接种于脱细胞的支架，并在长达21天的空气 - 液体界面中培养。这种技术促进无需额外生长因子的补充接种细胞功能的气道上皮细胞（纤毛细胞，俱乐部细胞和基底细胞）分化。这种文化建立的定义，塞鲁M-免费，价格低廉，重现性好。虽然有有限污染来自非肺胚层谱系中培养，该协议仅产生呼吸道上皮人群和不引起肺泡上皮细胞。与此协议产生的气道上皮细胞可用于在肺器官发生和疾病建模或气道相关的疾病，如囊性纤维化的药物发现平台来研究细胞 - 基质相互作用。

在本协议所述，定型内胚层分化稳健成熟的气道可以用脱细胞上的脚手架肺切片种子细胞的扩展培养来实现上皮细胞。建议将脱细胞支架的特征在于，以确保（1）的宿主细胞被完全除去，和（2）的细胞外基质蛋白在使用前支架分化保留。脱细胞可以通过组织染色用苏木精和曙红（H＆E）和4&#39;，6-二脒基-2-苯基吲哚（DAPI）进行评估，如在图1A中所示。高倍率?...

Watch this Scientific Journal Video about Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds at JoVE.com

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

该协议有效地引导小鼠胚胎干细胞衍生定形内胚层成熟呼吸道上皮细胞。这种分化技术使用的3维脱细胞肺支架直接肺谱系说明书中，在一个确定的，无血清培养的设置。

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Research

JoVE Journal

Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

科研

生物工程

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

由去细胞化肺切片制备的工程肺组织

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Dissection and Isolation of Region-Specific Decellularized Lung Tissue (Video) | JoVE 

区域特异性去细胞化肺组织的解剖和分离

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...