这项工作得到了国家卫生研究机构（05A1-CSPP16-014，HJL）和科技部（MOST 103-2320-B-400-015-MY3，HJL）的资助。

所有涉及动物的研究均符合动物保护机构委员会批准的方案。 细胞外囊泡/外来体分离和鉴定<ol><li>培养乳腺上皮基底细胞NAMEC4，用500mL MCDB 170，pH 7.4 + 500mL DMEM / F12与碳酸氢钠（0.2438％）制备的新鲜无血清培养基; EGF（5 ng / mL）;氢可替康（0.5μg/ mL）;胰岛素（5μg/ mL）;牛垂体提取物（BPE;35μg/ mL）;和GW627368X（1μg/ mL）。 </li><li>用血细胞计数器计数细胞后，在第0天将1.2×10 6个细胞在每15cm培养皿中的12mL培养基中种植4天4 。 </li><li>培养4天后，使用台式离心机将培养基以300×g离心5分钟。将上清液转移到锥形管（ 图1 ）。 </li><li>离心上清液在台式离心机中以2,000 x g进行20分钟。将上清液转移到超速离心管中，留下死细胞和细胞碎片（ 图1 ）。 </li><li>在4℃下以10,000xg离心上清30分钟。将上清液转移到新的超速离心管（ 图1 ）。 </li><li>在4℃下将上清液以110,000xg离心60分钟。去除上清液并将EV /外来物质沉淀重悬于PBS中（ 图1 ）。 </li><li>在4℃下将上清液以110,000xg离心60分钟。去除上清液。重悬于PBS中的EV /外来体颗粒（ 图1 ）。将从240-480 mL NAMEC条件培养基中分离的沉淀物重悬于100μL。 </li><li>用BCA蛋白测定法测定EV悬浮液的蛋白质浓度。确保浓度在20-40μg/ 100μL左右。储存于-20°C进一步分析。 </li><li>通过纳米粒子跟踪分析（NTA）测量EVs /外来体的浓度和大小，如Gardiner 等人先前所述。 11 。用PBS稀释EVs /外来体（20μg/100μL）至NTA分析10,000倍。 注意:NTA分析的结果反映了分析的囊泡的数量和大小。 </li><li>用透射电子显微镜（TEM）对EV /外来体进行成像，如Lin 等人 4所述。 </li></ol> 2.使用密度梯度的外来体纯化<ol><li>将来自步骤1.7的110,000g沉淀物重悬于40％（w / v）碘克沙醇的PBS（2mL）中。按照在PBS（每次2mL）中的30％，20％，10％和5％（w / v）碘克沙醇的等分试样依次覆盖混合物，以在超速离心管中形成密度梯度。 </li><li>将混合物在4℃下以200,000xg离心8小时。 </li><li>收集每个梯度分数（10分）附件; 1 mL /分），从管顶部移液。 </li><li>通过SDS-PAGE 12和Western印迹分析每个级分中exosome标记物（ 如 CD81，CD9，CD63和Tsg101）的存在。将每个级分的50μL悬浮液加到含有0.1％（w / v）SDS的10％凝胶上，并用凝胶电泳分离各部分的蛋白质。 </li><li>将蛋白质从凝胶转移到PVDF膜，并在4℃下将膜与抗外来体标记物（ 例如 CD81，CD9，CD63和Tsg101）和内脏蛋白GAPDH孵育过夜（ 表材料 ）。 注意:结果确定含有外来体的部分。 </li></ol>细胞外囊泡/外来体标签<ol><li>将20μl胞外蛋白/100μL的步骤1.7获得的EVs /外来体悬浮于10μM羧基荧光素琥珀酰亚胺基二乙酸酯（CFSE）中。准备一个平行样本公司仅以相同的方式处理CFSE和PBS，作为后续EV /外来物吸收测定的阴性对照。将混合物在37℃下放置30分钟。 </li><li>将EVs /外来体悬挂在50倍体积的PBS中，并在4℃下以110,000xg离心悬浮液60分钟。去除上清液并将EV /外来物质沉淀重悬于PBS中。重复步骤3.2一次。 </li><li>将EVs /外来体悬浮在PBS中，浓度为20μg的外来蛋白/100μL，然后在将EV /外来体添加到细胞之前通过0.22μm膜过滤EV /外来体。 </li></ol>细胞外囊泡/外来体摄取测定<ol><li>为了制备培养基，将500mL的MCDB 170，pH 7.4 + 500mL的DMEM / F12与碳酸氢钠（0.2438％）混合; EGF（5 ng / mL）;氢可替康（0.5μg/ mL）;胰岛素（5μg/ mL）;和BPE（35μg/ mL） 4 。将人乳腺上皮HMLE细胞置于6孔培养皿中（1×10 6个/ sup&gt;细胞/孔），EV / exosome处理前一天。将2μg/ mL CFSE荧光标记的EVs / exosome（在步骤3.3中获得）添加到HMLE细胞的培养基中2-6小时。用平行制剂处理阴性对照组的HMLE细胞，如步骤3.1所述。 </li><li>孵育2至6小时后，在室温下用4mL PBS洗涤细胞两次。 </li><li>用0.25％胰蛋白酶分离细胞10分钟，并将细胞重新悬浮在含有0.2％FBS的PBS中。使用荧光细胞分析仪测量细胞中荧光强度的EV /外来体摄取。使用共聚焦显微镜对用EVs /外来体处理的细胞或阴性对照进行成像。 注意:细胞中的绿色荧光是由EV /外来体吸收引起的。有关显微镜设置， 请参见图6的图例。 </li></ol> 5.分离原代小鼠乳腺上皮细胞<ol><li&gt;通过向10cm的培养皿中加入12mL的0.1％明胶溶液来制备明胶包衣的菜肴。将板放在37℃的培养箱中30分钟。取出明胶溶液，并将盖子从层流罩中离开盘子4小时直到明胶涂层干燥。 </li><li>使用剪刀从12周龄的女性C57BL / 6小鼠解剖数目2,3,4和5个乳腺（ 图2 ），并使用剃须刀将腺切成小块（2mm）。 </li><li>进一步解离乳腺浆液（10只小鼠）在37℃，60分钟搅拌120毫升DMEM / F12含0.2％胶原酶IV，0.2％胰蛋白酶，5％FBS，5微克/毫升庆大霉素和1x pen-strep。 </li><li>通过在350×g离心10分钟从混合物中沉淀上皮组织。 </li><li>将上清组织悬浮于4mL含有0.1mg / mL DNA酶I的DMEM / F12中，室温5分钟。加入6mL DMEM / F12至最终体积10 mL。 </li><li>将悬浮液在室温下以400xg离心10分钟，弃去上清液。 </li><li>将沉淀重悬于10mL DMEM / F12中。离心机悬挂，当速度达到400 x g时，刹车。丢弃上清液。 注意:通过击打制动器，上皮组织被迅速沉淀下来，而成纤维细胞作为单细胞仍然停留在上清液中。 </li><li>重复5.6和5.7 5-7次。将一滴悬浮液添加到血细胞计数器中，然后在每次离心后在显微镜下检查来自有机体混合物的成纤维细胞的清除（ 图3 ）。 </li><li>将含有1x ITS，5％FBS，50μg/ mL庆大霉素，10ng / mL EGF和1×pen-strep的DMEM / F12，在步骤5.1中获得的明胶包被的培养皿中的组织样品放置48小时。 </li><li> 48小时后，用不含FBS的新鲜培养基替换培养基，去除培养物中的浮游细胞（含有ITS，50μg/ mL庆大霉素，10ng / mL EGF和1x pen-strep的DMEM / F12）。 </li><li>确认单层乳腺上皮细胞在3天内迁移并从附着的上皮组织中生长出来。 </li></ol>原代小鼠基底/乳腺上皮细胞分离<ol><li> 3天培养后，用具有蛋白水解和胶原酶活性的天然酶混合物分离小鼠原代乳腺细胞20分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。将细胞沉淀重悬于5mg / mL分散液中20分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。 </li><li>用浓度为10 7个细胞/ mL的稀释的0.2％FBS将细胞重新悬浮于100μLPBS中抗CD49f和抗EpCAM抗体在黑暗中1小时。 </li><li>用PBS洗涤细胞，然后在黑暗中将细胞与荧光团缀合的第二抗体在冰上孵育30分钟。 </li><li>用0.2％FBS洗涤并重新悬浮在PBS中的细胞。 </li><li>将细胞分选器4上的EpCAM hi / CD49f细胞乳腺上皮细胞分选。 </li></ol>细胞外囊泡/外来体治疗<ol><li>将来自步骤6.7的明胶包被的6孔培养皿（2×10 5个细胞/孔）上的分选的EpCAM hi / CD49f细胞乳腺上皮细胞种子，然后用PBS或2μg/ mL NAMEC衍生的EVs /外来体在步骤1.7中获得。 </li><li>为了确保EVs /外来体在长期培养处理中的生物学功效，每隔两天用含有PBS或2μg/ mL NAMEC衍生的EVs /外来体的新鲜培养基代替细胞培养基。不要分开鼠标prima在10天的治疗期间的腔细胞。 </li></ol> 8.脂肪垫注射乳腺上皮细胞<ol><li>麻醉3周龄雌性C57BL / 6小鼠与异氟烷2-3％吸入。 </li><li>把麻醉的老鼠放在背上。用剃刀/发乳去除中腹部的毛皮，并用70％酒精和聚维酮碘的三个交替周期清洁手术部位。 </li><li>用剪刀沿腹侧胸腹部区域穿过皮肤进行1.5厘米的垂直切口，然后交替暴露右侧和左侧第 4 个乳房脂肪垫。 </li><li>用剪刀去除腺体实质，清除每个脂肪垫。找到脂肪垫中的淋巴结，然后取出淋巴结下方的整个腺体实质。 注意:三分之二的脂肪垫应该保持在原位。 </li><li>用天然酶混合物（参见材料表 ）与蛋白水解a分离从步骤7.2获得的细胞nd胶原酶活性10分钟。用PBS中的5％FBS中和酶活性。 </li><li>在室温下将悬浮液以450×g离心10分钟，弃去上清液。用血细胞计数器计数细胞，并将细胞悬浮在PBS中，浓度使得15μL含有所需的细胞剂量（10 4 -10 2细胞/垫）。 </li><li>使用连接到27G针的100μL玻璃注射器将15μL乳腺上皮细胞悬液注入脂肪垫。 </li><li>对另一侧的脂肪垫重复步骤8.4-8.7。 </li><li>用伤口夹闭合皮肤。 </li></ol>乳腺全身<ol><li>细胞注射后8周用CO 2加颈椎脱位来安乐死小鼠（步骤8.7）。 </li><li>使用剪刀从胸部区域到腹股沟区域的皮肤层进行垂直切口，然后暴露右侧和左侧4 号乳房脂肪垫去除第4 个乳腺（ 图2 ）。 </li><li>在玻璃显微镜载玻片上涂抹脂肪垫，并在室温下用Kahle固定剂（4％甲醛，30％EtOH和2％冰乙酸）固定脂肪垫。 注意:Kahle的固定剂是刺激性的。在化学品罩中执行此步骤。 </li><li>在250mL 70％EtOH中洗涤脂肪垫15分钟，然后在250mL的dH 2 O中洗涤5分钟。在室温下用胭脂红明矾（1g胭脂红和2.5g硫酸铝钾）在500mL dH 2 O中染上脂肪垫。 </li><li>用250mL 70％EtOH洗涤脂肪垫15分钟，250mL 95％EtOH洗涤15分钟，加入250mL 100％EtOH 15分钟。 </li><li>清洁二甲苯中的脂肪垫数天，并在脂肪垫变透明时停止二甲苯培养。 注意:二甲苯是一种刺激性。在化学品罩中执行此步骤。 </li><li>装载幻灯片wi使用数字幻灯片扫描仪拍摄图像（2,400 dpi）的脂肪垫。 </li></ol>

<ol>
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作者没有什么可以披露的。

外来体通常携带释放它们的细胞的特征，并且释放的外来体的量可以由刺激诱导4 。可以收集细胞的培养基并进行差异超速离心用于EV /外来物质收集（ 图1 ）。目前对于分离EV /外来体的理想方法目前尚无普遍的共识。这里使用的最佳方法已经由下游应用14确定 。超速离心是分离EV /外来体的相对快速的方法，可以保护EV /外来体的生物活性?...

外来体可以通过在细胞之间转移膜和细胞质蛋白，脂质和RNA来介导细胞交流1 。已经证明外来体介导的通信涉及许多生理和病理过程（ 即抗原呈递，耐受性发展2和肿瘤进展3 ）。外来体通常具有与释放它们的源细胞相似的内容。因此，外来体可以从源细胞携带特定的细胞特性，并将这些性质转移到摄取它们的细胞中。 外来体是50至150纳米的双层膜囊泡，并呈现特异性标记（ 例如 CD9，CD81，CD63，HSP70，Alix和TSG101）。因此，外来体必须通过不同方面的各种方法来表征。透射电子显微镜可用于观察膜囊泡如外来体4,5 。纳米粒子跟踪分析（NTA）和动态光散射分析（DLS）用于测量纯化外来体的大小和数量4 。可以通过密度梯度验证外来体的脂质膜含量。染色体标志物，例如CD9，CD81，CD63，HSP70，Alix和TSG101,6,7可以通过Western印迹法测定。 乳腺基底细胞在植入脂肪垫时具有产生乳腺的能力，而腔细胞不能8,9,10 。因此，乳腺基底细胞也称为乳腺再生单位。通过使用乳腺基底细胞和腔细胞模型，可以检测EVs /外来体在不同细胞群体之间转运细胞特征的能力。这个工作证明了通过使用源自乳腺基底上皮细胞的EVs /外来体将腺体形成能力从乳腺基底上皮细胞转移到乳腺腔上皮细胞的方法。乳腺上皮细胞摄取基底细胞分泌的EV /外来体后获得基础细胞特性，然后形成乳腺。

<table>
	<tbody>
		<tr>
			<td>MCDB 170&#160;</td>
			<td>USBiological</td>
			<td>M2162</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo</td>
			<td>1250062</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100K ultracentrifuge</td>
			<td>Beckman</td>
			<td>393253</td>
			<td></td>
		</tr>
		<tr>
			<td>SW28 Ti Rotor</td>
			<td>Beckman</td>
			<td>342204</td>
			<td></td>
		</tr>
		<tr>
			<td>SW41 Rotor</td>
			<td>Beckman</td>
			<td>331306</td>
			<td></td>
		</tr>
		<tr>
			<td>NANOSIGHT LM10</td>
			<td>Malvern</td>
			<td>NANOSIGHT LM10</td>
			<td>for nanoparticle tracking analysis (NTA)</td>
		</tr>
		<tr>
			<td>Optiprep&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>60% (w/v) solution of iodixanol in water (sterile).</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>GeneTex</td>
			<td>GTX101766</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>GeneTex</td>
			<td>GTX100912</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>Abcam</td>
			<td>Ab59479</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>TSG101 antibody</td>
			<td>GeneTex</td>
			<td>GTX118736</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>GeneTex</td>
			<td>GTX100118</td>
			<td>1:6000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td>
			<td>Thermo</td>
			<td>V12883</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>fluorescence cell analyzer</td>
		</tr>
		<tr>
			<td>collagenase Type IV&#160;</td>
			<td>Thermo</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>Thermo</td>
			<td>27250018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;ITS</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td>
		</tr>
		<tr>
			<td>accutase</td>
			<td>ebioscience</td>
			<td>00-4555-56</td>
			<td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td>
		</tr>
		<tr>
			<td>dispase&#160;</td>
			<td>STEMCELL</td>
			<td>7913</td>
			<td>5 mg/ml = 5 U/ml</td>
		</tr>
		<tr>
			<td>anti-CD49f antibody</td>
			<td>Biolegend</td>
			<td>313611</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>anti-EpCAM antibody</td>
			<td>Biolegend</td>
			<td>118213</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>FACSAria</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>cell sorter</td>
		</tr>
		<tr>
			<td>carmine alum</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
		</tr>
		<tr>
			<td>human mammary epithelial cells (HMLE cells, NAMECs)</td>
			<td></td>
			<td></td>
			<td>gifts from Dr. Robert Weinberg</td>
		</tr>
		<tr>
			<td>permount</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Zymeset&#160;</td>
			<td>BSB101</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocoritisone</td>
			<td>Sigma-Aldrich</td>
			<td>SI-H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SI-I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE (bovine pituitary extract)</td>
			<td>Hammod Cell Tech&#160;</td>
			<td>1078-NZ</td>
			<td></td>
		</tr>
		<tr>
			<td>GW627368X&#160;</td>
			<td>Cayman</td>
			<td>10009162</td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>table-top centrifuge</td>
			<td>Eppendrof&#160;</td>
			<td>Centrifuge 3415R</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)&#160;</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>23228</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin&#160;</td>
			<td>STEMCELL</td>
			<td>7903</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture dish</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>female C57BL/6 mice</td>
			<td>NLAC (National Laboratory Animal Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>BioWest&#160;</td>
			<td>S01520</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15710072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>5PRIMER</td>
			<td>2500120</td>
			<td></td>
		</tr>
		<tr>
			<td>isofluorane&#160;</td>
			<td>Halocarbon</td>
			<td>NPC12164-002-25</td>
			<td></td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>MACRON</td>
			<td>H121-08</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH (Ethanol)</td>
			<td>J.T. Baker</td>
			<td>800605</td>
			<td></td>
		</tr>
		<tr>
			<td>glacial acetic acid</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminum potassium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>12625</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Leica</td>
			<td>3803665</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m membranes</td>
			<td>Merck Millipore</td>
			<td>Millex-GP</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm</td>
			<td>BD Biosciences</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clip Applier</td>
			<td>BD Biosciences</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask&#8482; Deep Red</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>C10046</td>
			<td>plasma membrane stain</td>
		</tr>
	</tbody>
</table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

细胞可以通过外来体，含有蛋白质，脂质和核酸的〜100-nm胞外囊泡（EVs）进行通信。非贴壁/间充质乳腺上皮细胞（NAMEC）衍生的细胞外囊泡可以通过差异超速离心从NAMEC培养基中分离。基于它们的密度，EV可以通过110,000×g的超速离心纯化。可以使用连续密度梯度进一步分离来自超速离心的EV制剂，以防止可溶性蛋白质的污染。然后可以使用纳米颗粒跟踪分析进一步评估纯化的EV，其测量制剂中囊泡的大小和数量。尺寸范围为50至150nm的细胞外囊泡是外来体。 NAMEC衍生的EVs /外来体可以通过乳腺上皮细胞摄取，可以通过流式细胞术和共焦显微镜检测。一些乳腺干细胞性质（ 如乳腺形成能力）可以通过来自NAMEC的EVs /外来体从茎状NAMEC转移到乳腺上皮细胞。分离的原代EpCAM hi / CD49f细胞乳腺上皮细胞在移植到小鼠脂肪垫后不能形成乳腺，而EpCAM lo / CD49f基底乳腺上皮细胞在移植后形成乳腺。通过EpCAM hi / CD49f细胞乳腺上皮细胞摄取NAMEC衍生的EVs /外来体允许它们在移植到脂肪垫中之后产生乳腺。衍生自干样乳腺上皮细胞的EVs /外来体转移到EpCAM hi / CD49f细胞乳腺上皮细胞的乳腺形成能力。

由于已经显示阻断PGE 2 / EP 4信号传导从乳腺基底样干细胞4引发EV /外来体释放，这项工作提出了从乳腺上皮基底细胞（NAMEC）培养物中分离诱导的EVs /外来体的方法。由于NAMECs在无血清培养基中培养，所以不存在来自血清13的预先存在的EV /外来体。对于在含有血清的培养基中培养的细胞，培养基中预先存在的外来体必须通...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

该方案描述了从非贴壁/间充质乳腺上皮细胞中纯化，定量和表征细胞外囊泡（EVs）/外来体的方法，并使用它们将乳腺形成能力转移到乳腺上皮细胞。衍生自茎状乳腺上皮细胞的EVs /外来体可以将该细胞特性转移到摄入EV /外来体的细胞中。

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

transfer-mammary-gland-forming-ability-between-mammary-basal

Research

JoVE Journal

Developmental Biology

11.6K 次观看.  National Health Research Institutes. 该方案描述了从非贴壁/间充质乳腺上皮细胞中纯化，定量和表征细胞外囊泡（EVs）/外来体的方法，并使用它们将乳腺形成能力转移到乳腺上皮细胞。衍生自茎状乳腺上皮细胞的EVs /外来体可以将该细胞特性转移到摄入EV /外来体的细胞中。 

视频: Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

从培养儿童原发性鼻腔上皮细胞和重新编程为诱导多能干细胞

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

科研

发育生物学

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

在肉瘤细胞间质上皮转变的诱导

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

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.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

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

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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.

活完好斑马鱼的单细胞 Photoconversion

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 ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

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