This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

这项研究已批准Regierungspräsidium卡尔斯鲁厄的伦理委员会。所有动物饲养无特定病原体条件下在德国癌症研究中心（DKFZ）下。对于所有的培养实验小鼠幼仔范围从1岁到7天被使用。 从胸腺1.隔离mTECs的注：如无菌条件下先前1中记载有一些修改如下进行以下的消化步骤。 <ol><li>斩首的乳鼠，取出胸腺。放置在冰上thymi在含有RPMI 1640培养基（含5％FCS）培养皿。 </li><li>切thymi成细，小块并置于圆底管〜5-30毫升RPMI培养基和轻轻搅拌用磁铁在室温10分钟。 </li><li>此后，滗主要含有胸腺细胞的上清液并与一个圆collagena的消化组织顺序的剩余本身IV型（0.2毫克/毫升和57 U / ml的最终concentartion）15分钟每个在37℃，随后通过胶原酶/分散酶（0.2毫克/毫升和1.2 U / ml的终浓度）为每25分钟，在37°下在水浴磁力搅拌下，直到thymi被完全消化。使用1ml的酶每两到三个thymi。 </li><li>搅动组织每隔7-10分钟用巴氏吸管。池胶原酶/分散酶组分，并通过一个70微米的纱布过滤。 </li><li>通过磁性细胞分选（MACS）充实mTECs。通过磁性细胞如先前11描述的，和在图1所示的排序执行mTECs的纯化。 注：对于mTECs的磁分选，我们使用了下列抗体：抗CD80-PE（16-10A1，使用以1：100稀释）和抗EpCAM-生物（G8.8，使用以1：100稀释）12。使用MACS未成熟和成熟mTECs被定义为：CD45 - EpCAM的+ CD80 -和CD45 - CD80 + respectiv伊利。 </li><li> MACS纯化（未成熟mTECs的纯度= 83.1±6.3％和成熟mTECs = 79.23±3.42％）后，种子mTECs到器官文化，如下所述（第2.3节）。 </li><li>可替换地，排序mTECs通过FACS（使用100微米的喷嘴）的CD45磁珠耗尽后使用下列抗体：抗CD45-PerCP（30-F11，使用以1：100稀释），抗Ly51-FITC（6C3，使用在1 ：100稀释），抗EpCAM-Alexa647（G8.8，使用以1：500稀释），并在1抗CD80-PE（16-10A1，用途：100稀释度）。排除使用碘化丙啶（1：5000）死细胞（4天）。使用流式细胞仪未成熟和成熟mTECs被定义为：PI - CD45 - Ly51 - EpCAM的+ CD80 -和PI - CD45 - Ly51 - EpCAM的+ CD80 +，分别为。 </li></ol> 2. 3D器官联合培养（OTCS） 注：3D-真皮构造的器官培养keratinocyt的西文被如先前9,13所述制备。在所有步骤的细胞在37℃和5％的CO 2。使用mTECs的OTCS制备稍作修改如下。 <ol><li>人成纤维细胞的制备 注意：人皮肤成纤维细胞从脱epidermised真皮外植体培养物如先前9中所述获得。 <ol><li>简言之，切开人体皮肤（〜5cm长）的带和治疗与嗜热菌蛋白酶（0.5mg / ml的盐水中，用10毫摩尔的Hepes pH7.4）中O / N在4℃。 </li><li>其后，从利用镊子真皮分离表皮。 </li><li>细切真皮成小块，放置在10cm的培养皿，并允许下一个无菌气流中干燥1-2小时。用DMEM含20％FBS定期补充外植体。 </li><li>分裂出生长的成纤维细胞时汇合（通常大约3周）用0.1％胰蛋​​白酶确保植继续坚持该板用于进一步CON组secutive轮生长的。 </li><li>扩大从同一外植体成纤维细胞为最多3次在含10％FBS和低温保存它们14。使用相同批次的成纤维细胞的每个实验系列。 注：成纤维细胞不照射为集合式真皮等同物。 </li></ol></li><li>脚手架的制备<ol><li>削减0.4-0.6毫米厚的粘胶，非织造纤维材料（在支持Excel表单产品的详细信息）以及成圈划定，用锋利的直径11mm的金属冲床恰好适合12良好的滤芯。然后将其放入12井滤芯（聚酯毛细管孔膜，3微米孔径）作为支架。放置的完整的过滤器安装到无菌的12孔板中。 </li><li>准备使用纤维蛋白胶，试剂盒进行手术，包括纤维蛋白原和凝血酶的结合的血纤维蛋白凝胶。预稀释fibrinogen-以及试剂盒的凝血酶组分至8毫克/毫升和10单位/毫升。对于一个12孔板的单个孔的步骤如下。 <ol><li>稀释100μl的纤维蛋白原（8毫克/毫升）用100μl磷酸盐缓冲盐水（PBS）不含Ca 2+和Mg 2+，pH值7.0。稀释100μl的凝血酶（10单位/ ml）与含有270000成纤维细胞加入100μlFCS中。 </li><li>分配200μl的含有成纤维细胞的混合到该支架的凝血酶，向其中加入200微升血纤维蛋白原（1：1的混合物），产生的2毫克/毫升的2.5单位/ ml凝血酶和纤维蛋白原的终浓度。拌匀，均匀地分布在支架轻柔吹打（第1天）的整个区域。 注：30分钟，在37℃后凝块包封的成纤维细胞会形成，尽显无遗支架的内部空间，并形成一个光滑的上表面。 </li></ol></li></ol></li><li>共培养mTECs <ol><li>用于预培养，浸没在器官培养含10％FBS，50μg/ ml的L-抗坏血酸和1纳克/毫升的培养基改变TGF-β1的隔日4-5天。 </li><li>上MTEC播种当天，由RFAD介质更换介质（1：1的DMEM + DMEM / F12）有10％FBS，10 -10 M霍乱毒素，0.4毫克/ ml氢化可的松，50μg/ ml的L-抗坏血酸， RANK配体（0.1微克/毫升）和500单位/ ml抑肽酶，从而防止早熟纤溶由成纤维细胞分泌的丝氨酸蛋白酶。 </li><li> 7-8小时后，建立共培养播种250000 mTECs（无论是完整的mTECs，或CD80 Io和 CD80 喜子集），在每孔100μl的成纤维细胞支架的顶端的体积。算使用Neubauer室（4天）的细胞。 注：在任何时候胸腺器官3D文化是浸没在媒体不像皮肤OTCS这是空气解除。 </li><li>经过24小时培养，提供如上述（步骤2.3.2）与介质的培养物（总培养基交换）现在containiNG减少抑肽酶，250单位/毫升（5日）的金额。 </li><li>为了评估mTECs的增殖活性，终止培养物之前的EdU（6.7μM/ ml时， 即，10μM/孔）添加到OTCS 4小时。在每一种文化标本或流两节这些EDU成像套件结合共染色角蛋白14的描述确定mTECs的增殖指数，方法是计数K14 + +的EdU细胞进行的OTC冷冻切片染色术（EDU流式细胞术，第代替Ly51-FITC 1.7，但是使用的CDR1-PB 15在1：100稀释和2.3.6.4）。 </li><li>下列4-7天共培养的，终止OTCS和方法RNA分离，冷冻切片或FACS分析（8-11天）。 <ol><li>使用镊子，分离的支架/真皮当量从井刀片的过滤终止培养。 </li><li>对于冷冻切片，嵌入整个OTC华侨城化合物，冻结李冷冻切片之前，英镑氮蒸气。准备使用低温恒温器和存储5-7微米厚的非处方药部分在-20℃下直到使用。对于免疫组化的冷冻切片OTCS使用抗角蛋白14（AF64，使用以1：1000稀释）和抗波形蛋白（GP53，使用以1：100稀释）的抗体。执行使用各自的二级抗体的间接免疫组织化学染色。 </li><li>对于RNA分离，加入1 ml变性溶液（含有苯酚和异硫氰酸胍）在2 ml无RNase管的螺旋盖。切断整个场外成用手术刀片，并添加到含有变性溶液的管中。机械地以6.0的速度切丝与FastPrep仪OTCS两次30秒，放在冰上间为2分钟（样品可以储存在-80℃这点之后）。如果在-80℃下冻结，解冻在冰上。离心管在11,500-13,000转速在4℃下10分钟。将上清液转移到新的管中，孵育5分钟，在RT和f使用酸性硫氰酸胍 - 酚 - 氯仿提取如制造商所述ollow RNA分离协议。 </li><li>用于FACS分析，除去该脚手架和分离的膜与纤维蛋白/纤维细胞/ MTEC凝胶。切细的凝胶用手术刀和消化它在在FACS管中〜20分钟，或直至完全在水浴磁力搅拌下用2ml胶原酶/分散酶的消化在37℃。搅拌用巴斯德吸管每5分钟的酶溶液。完全消化后，通过过滤70微米的过滤器细胞悬浮液;染色使用如1.7所描述并通过流式细胞术分析抗体的单细胞悬浮液。 </li></ol></li></ol></li></ol>

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沿着RTOCs，三维OTCS已经于TEC分化和PGE维护/感应（ 表1）相对于其它（ⅰ）“简化3D培养&#39;使用的术语是由远优于-的成纤维细胞单独无脚手架; （ⅱ）二维系统使用-成纤维细胞/饲养细胞共培养的TECs 10，（ⅲ）3T3-J2细胞，其中的TEC克隆发展，但PGE丢失，（ⅳ）基质胶或（v）ECM组分（未发表数据）。 PGE下保持7天，在3D OTCS 4天为最佳时间点之后，PGE开始下降。其他形态的TEC特征?...

显影胸腺细胞约占98％，胸腺，而其余的2％由多种细胞，它们共同构成了胸腺基质（ 即，上皮细胞，树突细胞，巨噬细胞，B细胞，成纤维细胞，内皮细胞）。外皮质上皮细胞（cTECs）促亲T细胞的移民从骨髓，T细胞谱系的诱导多能前T细胞和阳性选择自我的MHC限制性不成熟的胸腺细胞。所述内髓胸腺上皮细胞（mTECs）参与耐受诱导那些胸腺细胞具有高亲和性TCR为自身肽/ MHC复合物由两种诱导阴性选择或它们的偏差成T调节细胞谱系。在中央耐受诱导的上下文中，mTECs是因为它们表达了广泛的组织限制性自身抗原（的TRAs）因而镜像的周自谱是唯一的。这种现象被称为混杂基因表达（PGE）1,2。 关于这个迷人的细胞类型的最新研究依赖于体外分离的细胞，如各种短期二维培养系统总是导致PGE的损失和关键调节分子如MHC II类，FoxN1和艾尔内的第2天3-6 。它仍然不清楚然而，这尤其是元器件和胸腺的完整3D小梁的特点在2D模式不翼而飞。的再聚集胸腺器官培养（RTOC）一直迄今唯一的三维系统，使T细胞发育的研究中，一方面，与基质细胞生物学，另一方面，在一个完整的胸腺微环境7。然而，RTOCs有一定的局限性， 即，它们已经含有细胞的复杂混合物，需要胚胎基质细胞的输入和忍受的5到10天的最大培养期。 还原的体外培养体系的缺乏阻碍的研究T细胞发育和胸腺器官几个方面PGE的不仅仅是分子调控及其对mTECs的发育生物学关系。 由于紧密关联的皮肤和胸腺上皮细胞的结构化组织，我们选择了已被开发的最初仿效体外角质细胞的分化，从而创造一个真皮当量的三维器官培养（OTC）系统。场外交易系统由覆盖与被困在纤维蛋白胶，其上的角质形成细胞接种8,9真皮成纤维细胞惰性支架矩阵。在这里，我们代替角质形成细胞纯化mTECs。在保持这一模式的基本特征，我们优化的某些参数。 在通过场外交易模式mTECs增殖，进行终末分化和维护MTEC身份和铂族元素， 在体内 mTECs的发展密切相关，从而模仿<sup&gt; 10。本技术说明提供了一个详细的协议，允许逐步建立胸腺OTCS的。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

帧内胸腺T细胞的发展需要一个复杂的三维网状组织的各种基质细胞， 即非T细胞组成。胸腺遍历这个支架在一个高度协调的时间和空间秩序，同时顺序通过强制性的检查点， 即 T细胞谱系的承诺，其次是T细胞受体库的生成和选择之前，其出口到周边。形成该支架的两个主要居民小区类型是皮质（cTECs）和髓胸腺上皮细胞（mTECs）。 mTECs的一个关键特征是众多组织限制性抗原所谓混杂的表达。这些组织限制性抗原呈现，以直接或间接的不成熟的胸腺细胞通过mTECs或胸腺树突状细胞，分别导致自身耐受。 合适的体外模型模拟cTECs和mTECs的发展途径和功能在当前LAC国王。这种缺乏足够的实验模型已例如阻碍混杂基因的表达，这仍然​​知之甚少在细胞和分子水平上的分析。我们适应了3D器官联合培养模式，培养体外分离mTECs。最初设计该模型培养的角质形成细胞在这样的方式来产生皮肤等效体外 。 3D模型保存MTEC生物学重点功能特点：（I）的增殖和CD80 LO，艾尔阴性终末分化成CD80 喜 ，艾尔阳性mTECs，（二）反映RANKL，并FoxN1（三）持续表达， AIRE和CD80 喜 mTECs组织限制性基因。

我们采用了3D器官联合培养模型（3D OTC）已被最初开发的角质9 体外长期培养。 MACS富集mTECs（见MACS富集方案图1）接种到含有纤维蛋白胶包裹和成纤维细胞的支架。成纤维细胞提供必要的细胞外基质（ECM） 体外支承mTECs。 MTECs培养在OTCS为4-14天在RANKL的存在下，在浸没培养不像角化细胞，这是空气暴露模仿它们在体内环境（参见场外的建立方案图2）。<...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K 次观看.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

视频: 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

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.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

简化的3D 小脑鉴别协议, 可选2D 修改

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

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

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

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

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

小鼠髓质前体 32-脑脊液-R 细胞的增殖和分化

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

用于测试 Vivo 中巨噬菌体招募的异位化学表达模型

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...