作者没有确认。

实验过程和善待动物组织批准匹兹堡IACUC大学（协议＃1104674）。 1.制备G6PD染色混合注:确保每个试剂的最终浓度如下: 5mM葡萄糖-6-磷酸（G6P）; 2mM的NADP，5mM的MgCl 2的，0.35毫1-甲氧基-5-甲基吩甲基硫酸（MMPMS）和0.8mM的NBT。1,8,9-对于每个试剂的初始浓度推导对于40 ml的终体积。解决方案新鲜制备，每天使用。 <ol><li>加入2毫升200毫pH 7.4的磷酸盐缓冲液（PB）到35 ml的十月培养基的50ml管中，并剧烈振荡。确认用pH试纸，该溶液是pH为〜7.4。如果不丢弃溶液。 </li><li>加入61毫克G6P溶解在0.94毫升200mM的PB，61毫克NADP溶解在0.94毫升200mM的PB和41毫克的MgCl 2溶于毫升100mM的0.96 PB。再次确认该pH值为7.4〜机智^ h pH试纸。如果没有〜7.4，丢弃的解决方案。 </li><li>添加5毫克MMPMS溶解在0.25毫升200mM的PB（pH 7.4）中的。用力摇动所得的反应混合物以产生一种清晰的暗红色匀浆。如果溶液混浊，使混合物应该被丢弃。被包裹在箔含有反应混合物的管，以尽量减少暴露于光。在室温储存的反应混合物，而最后一个组件，NBT，制备。 </li><li>通过加入0.4 ml的无水二甲甲酰胺（DMF）和0.4 ml的无水乙醇，以164毫克的NBT在2 ml管的O形环盖单独准备5mM的NBT溶液。松散盖上盖子（未密封），并把该溶液到剧烈沸腾的油浴中，直到NBT是在溶液中。8 </li><li>允许溶解的NBT冷却至RT，然后加入所有溶液到OCT反应混合物并剧烈振荡，获得澄清的溶液。观察到的暗红色或purp从初始橙红色的溶液的颜色变化乐颜色随着时间的推移。这是正常的。 </li><li>在37℃的温暖的室内使用前1小时将最终G6PD染色的混合物。 </li></ol> 2.动物处理和组织收集<ol><li>治疗6周龄的C57BL / 6雄性小鼠与DNA损伤剂，例如，10mg / kg的氧化偶氮甲烷（AOM）的体积为200μl的PBS腹腔注射。治疗的对照组小鼠用200微升PBS腹腔注射。 </li><li> 90日，由二氧化碳安乐死小鼠2窒息颈椎脱位批准的IACUC协议。 </li><li>手术从刚肛门到胸骨的基部上方打开腹腔。移动小肠出体腔，但不切除它。切除肠的下部，正下方的盲肠到直肠。10 </li><li>用微型剪刀解剖沿结肠的一侧仔细切片和通过用干净无菌PBS洗涤除去从打开结肠的粪便。瑞士卷消费税ð结肠与腔侧朝外，并置于组织模具10 </li><li>完全沉浸在OCT中，地方模具结肠中含液态氮杜瓦。保持所述模具以使塑料底仅仅与液体N 2接触，直到在OCT介质被冻结固体。 </li><li>存放冷冻组织块在-80至切割冰冻切片。不使用任何固定剂，这将导致在G6PD酶的失活。 </li></ol> 3.准备冰冻切片的<ol><li>切冰冻组织切片在使用低温恒温器在-25℃的厚度为7微米。 4切连续7微米的部分，并把他们两个人在同一张幻灯片（ 图1）。准备2幻灯片，或四个连续的7微米的部分来表示结肠（图1）中的一个级别。 </li><li>对于一个新的水平&gt; 50微米来自PR的最后一节的最小距离重复evious水平。这个距离保证了评估每个级别隐窝不重复彼此。 </li><li>经常擦拭低温恒温器刀片和100％的乙醇以除去OCT的残余物在切割期间积聚。切片被恢复之前，将干叶片擦拭用抗静电干燥器片消散累积静电荷。存储幻灯片冰冻切片在-80℃。 </li></ol> 4.染色冰冻切片组织<ol><li>请在37℃温暖的室内高湿度组织化学反应。 注意:试图在孵化烤箱或幻灯片上温暖的酶促反应导致穷人和不一致的G6PD染色。 </li><li>建井使用两个1.5厘米×0.15厘米钢垫圈（内径×高），该键合在一起使用硅脂（图1）组织染色。切断封口膜，以适应井与中心的底座切出和阱地方ð在幻灯片上的组织切片。 注:在垫圈的底部的封口膜将创建幻灯片和维持对组织切片粘性G6PD染色混合物垫圈之间形成密封。该构建给出了一个很好地与一个0.4 - 0.5 ml的体积，使得在研究中的每个组织部分接收G6PD反应染色混合物相同体积。 </li><li>在温暖的房间10分钟孵育冷冻组织切片在37℃。在37℃，45-50分钟的葡萄糖-6-磷酸脱氢酶染色反应混合物添加到每个组织切片以及（每张幻灯片2段）。从温暖的房间中取出的幻灯片，并小心地将不锈钢油井关闭的幻灯片。 </li><li>在其长边组幻灯片，以使反应介质排出。在100mM的PB（pH 7.4）中为30〜60分钟的地方滑动以除去来自组织的葡萄糖-6-磷酸脱氢酶染色反应混合物，而不会干扰组织染色。淹没在蒸馏水中的幻灯片5-10分钟，以除去铅盐</li><li>密封由加载组织从滴管荷兰国际集团氟 - 凝胶到组织上并等待〜30分钟它干。避免气泡可以混淆突变隐窝的识别。 注意:如果形成气泡，该过程可以去除氟 - 凝胶后重复，通过浸泡在蒸馏水中。不要使用盖玻片，因为他们往往​​陷阱气泡。 </li></ol> 5.测定葡萄糖-6-磷酸脱氢酶突变隐窝和突变频率<ol><li>在光学显微镜下分析幻灯片G6PD突变隐窝。 注意:用于识别一个完全突变体隐窝的标准是:（i）该整个隐窝是缺乏蓝色; （ii）所述隐窝的外结构完整，（ⅲ）的突变体隐窝上观察到相邻的7微米的部分和，（ⅳ）两个观察者不得不标识相同隐窝作为突变体。上相邻的7微米的部分观察到的突变体计为一个突变。 </li><li>采用500万像素的摄像头，图像中的每个葡萄糖-6-磷酸脱氢酶突变隐窝放大40倍和目录的图像图像处理软件。 </li><li>通过选择有代表性的部分，用于每只小鼠从具有最小表面面积和图像它在10倍的放大倍率（图1C）的水平量化隐窝检查在小鼠的总数。 注:A级代表四个连续7微米的部分（ 图1D），这是由于他们的接近常表现突变隐窝上的多个部分。的水平通过&gt;50μm的分离，以可视化从结肠的不同区域隐窝。 </li><li>打开每个图像文件和视觉通过分析文件（ 图 1C中示出为框）依靠一个水平隐窝数。图像文件将在从低至20至&gt; 300隐窝在一个文件中观察到隐窝的数目而变化。 </li><li>通过筛选突变体的G6PD隐窝每个结肠级别数相乘从所选择的水平（图1C）对每只小鼠隐窝的总数。例如，总计数每个选定的级别1250隐窝乘以8级筛选突变隐窝等于10000隐窝的总计数。评价最低的万隐窝每个结肠进行统计分析。 </li><li>组合由隐窝筛选以计算对MF每个治疗的总数在所有动物的观察突变体的数量。未处理小鼠背景干细胞的MF水平是&lt;0.1×10 -4（表1），这是接近先前在C57BL / 6小鼠的报道。11 </li></ol>

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The authors have nothing to disclose.

常的化合物的基因毒性作用是通过其来修改DNA的能力来确定。这通常是通过分离组织和测量DNA加合物的全球水平上进行。对于AOM，这将涉及量化O 6 -甲基鸟嘌呤加合物在结肠。上使用的特定细胞类型，如在干细胞小生境中的损害此方法的信息，将丢失。此外，DNA加合物的是不一样的突变，因为加合物的只有一小部分被最终转化成固定的突变。12我们提供本文所述细节基于由格里菲思所述的初始方法可再现地量化干细胞突变在结肠等人 1和基于由VanNorden开发G6PD染色。4,8,9,13,14 有这种酶组织化学方法产生可量化的结果是几个关键步骤。一个是在组织的切片。显著更多的组织宗派离子必须产生，并且比将典型的做法用H＆E组织学分析来分析。我们确定，7微米的部分，得到最好的葡萄糖-6-磷酸脱氢酶染色的5，6，7，8和10微米厚的切片评估后。 其次，染色的有价值的组织切片的必要数量需要可再现的反应混合物中。我们发现，使用在原始协议中使用聚乙烯醇（PVA）的是有问题的，因为它是非常粘稠的溶液百分比（18％和更高）必要以促进同时防止G6PD酶的扩散从组织化学反应组织。试剂和pH值控制的更好溶解通过与华侨城介质，其含有10％的PVA代替PVA完成。 pH值可精确地确定用pH试纸，如果pH值不接近7.4，该溶液被丢弃，而不会浪费宝贵的组织样本和时间。这种变化导致了更好的和M矿石中的组织切片染色一致。在测量G6PD活性所需要的pH值（7.2-7.4），完整的反​​应混合物具有的特性明确深橙红色，这将慢慢改变到紫色。不管颜色的，如果反应染色溶液混浊它应该被丢弃，因为它不会得到好的染色。我们发现，有些大量十月介质的提供外的所需pH范围的混合物因此建议通过加入该PB（7.4）到OCT介质和确定pH值，以测试手。我们也用作先前建议MMPMS，8,9，而不是在原纸 1用于消除G6PD染色反应混合物的感光度5- methylphenazenium甲基硫酸盐。 最后，该方法用于导出与其他方法相比的MF是新颖以前使用。1,2-染色工件可以导致假G6PD突变体隐窝潜在鉴定。要解决Ť他的问题，我们分析了4个连续的7微米的组织切片，使对于相同的突变隐窝在一个以上的组织切片（图4D）的可视化。此验证降低染色影响MF分析工件的机会。此外，我们的方法使用计数来估计隐窝计算对MF进行分析时的总数总隐窝的实际值。 因为在小肠和大肠中发现的唯一的隐窝结构，有可能来确定干细胞的位置和细胞谱系每个隐窝。一个主要的限制的方法是，它被限制在肠因为其它组织的形态没有得到很好的定义为在结肠。因此，不可能确定在那些易患癌症大多数其他组织中的干细胞的MF。由于该测定法是基于G6PD的酶活性的损失表型分析，实际的MF中的干细胞群体将高于我们由于基因突变在摆动碱基和点突变不显著影响酶活性报告为G6PD。 而有局限性和挑战染色G6PD活性结肠组织中量化体干细胞突变，重大修改在本文中引入，以提高利用该方法的可行性。我们发现，这些变化提高了生成葡萄糖-6-磷酸脱氢酶染色用试剂和定量的MF的可重复性。此外，染色G6PD的酶活性作为标记成体干细胞突变并不需要在免疫组织化学染色的专业知识，将测试对于金属硫蛋白，15的成体干细胞突变的另一个潜在标记物的表达。 在未来，我们将分析结肠干细胞突变符合环保的化合物治疗的小鼠，如2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PHIP）和烤肉发现的其它芳族胺，其是与人结肠癌有关。

结肠癌被认为涉及暴露于环境剂和膳食成分，可以破坏DNA和产生激活体细胞突变在致癌基因（例如，β-catenin的）或失活突变的肿瘤抑制基因（例如，APC）。据推测，这些关键突变发生在结肠的干细胞。由于结肠上皮的独特隐窝结构的，有可能让动物接触与结肠癌的发生​​相关联的化学品之后测量干细胞突变在结肠。几个X连锁酶可作为指标发生在干细胞突变，如突变将存在于一个隐窝内的所有细胞。 以前，一个过程被出版表明化学品诱导结肠肿瘤小鼠也通过在X连接的葡萄糖-6-磷酸脱氢酶（G6PD）基因突变的分析生成体干细胞突变在结肠1-3该方法量化了结肠干细胞随机体细胞突变的发生没有任何选择压力。该过程涉及从处理的小鼠和对照小鼠未固定的冷冻切片结肠癌的生产和隐窝缺乏产生官能G6PD活性细胞的鉴定。这些突变隐窝，呈现白色，表明突变发生在给人们带来的后代也携带突变基因G6PD干细胞。在酶测定中，葡萄糖-6-磷酸脱氢酶缺陷突变隐窝不能氧化葡萄糖-6-磷酸，这是需要的硝基四氮唑蓝（NBT）的还原。当G6PD酶是功能性的，在NBT在染色混合物降低到不溶性甲臜和沉淀物，积累在酶和"染色"细胞蓝色的位置。细胞，葡萄糖-6-磷酸脱氢酶突变体仍然发白而相比之下，蓝染野生型隐窝。这种方法测量的是有一个"空"的表型变异。因为恩P450酶活，如葡萄糖-6-磷酸脱氢酶，可以扩散上，如果节放在水溶液未定影组织部分的细胞出来，有必要稳定酶在组织切片进行分析。4，在酶的稳定化组织不得干扰必需的G6PD酶反应试剂的小分子的扩散。 我们已经取得了一些显着的变化，以原来的程序。用于关键酶染色的介质已从聚乙烯醇到最佳切削温度化合物（OCT），这有利于控制pH值，并在测定中使用的组分的增溶。 0.5 ml的井由钢制成的垫圈，以便每个组织部分接收的葡萄糖-6-磷酸脱氢酶反应混合物相同体积的 - 使用0.4进行染色。的程序被开发来估计在成像的部分，它提供了结肠组织的大采样，而无需隐窝数手动计数&gt; 10 5隐窝每个冒号。相邻的7微米的组织切片的分析，得到的突变体隐窝可视化多个幻灯片，这减少了潜在的染色工件。这些变化使程序更加高效和可重复性。 使用这个修订协议，我们已量化结肠干细胞的突变频率之C57B1 / 6小鼠，以氧化偶氮甲烷，在啮齿类动物中公知的结肠的致癌物质的暴露后5-7

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nitroblue tetrazolium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NADP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glucose-6-phosphate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-methoxy-5-methylphenazinium methyl sulfate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl formamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer (pH 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature Compound (OCT)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>light microscope equipped with 5 megapixel camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cryostat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>warm room&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of  colonic stem cell mutations

测量干细胞突变的能力是一个强有力的工具中的一个关键的细胞群进行量化如果，以及在何种程度，化学可诱导的突变可能导致癌症。使用的酶测定法来量化干细胞突变在X连接的葡萄糖-6-磷酸脱氢酶基因已被报道 。1此方法要求与反应混合物能够产生一个在制备冰冻切片的切片组织和培养如果细胞产生功能葡萄糖-6-磷酸脱氢酶（G6PD）酶的蓝色。如果不是，该细胞出现发白。我们已修改代替聚乙烯醇使用最佳切削温度化合物（OCT）中的反应混合物中。这有利于pH测量，提高了G6PD染色成分的溶解和限制G6PD酶的扩散。为了证明突变发生在干细胞中，整个隐窝必须缺乏G6PD酶促活动。只有当一个干细胞藏着表型G6PD基因突变都会在地下室的后代缺乏G6PD酶的活性。要确定隐窝干细胞突变，连续四个相邻的冰冻切片（A级）切成7微米的厚度。使得相邻削减这种方法提供了构象地穴充分突变，因为相同的突变隐窝将相邻路段进行观察。幻灯片与组织样本均大于50μm相距已准备共&gt; 10 4每只小鼠隐窝来评估。突变频率是所观察到的突变的（白色）由野生型（蓝色）隐窝中一个治​​疗组的数量÷隐窝数。

衡量结肠干细胞基因突变的动物的能力提供了一个独特的方式来关联突变诱发癌症。通常情况下，假定在癌变的关键步骤包括激活突变在癌基因和/或失活的肿瘤抑制基因的突变。我们注射C57BL / 6小鼠用200μl的PBS或10mg / kg的急性中耳炎在200μlPBS中。 AOM是一种已知的致癌物质冒号5-7在90日进行了结肠干细胞基因突变分析。这个时间是必需的，以允许干细胞能够充分填充隐窝，只有当整个隐窝中不产生葡萄糖-6-磷酸脱氢酶是鉴定为干细胞突变一个隐窝。完全突变体隐窝例子示于图2中 。该图显示了两个不同的方位发生在冷冻切片的制备隐窝（垂直和水平）。垂直视图俯视隐窝的长轴，而水平提供了侧视图。该哭点是野生型葡萄糖-6-磷酸脱氢酶染色蓝与白一些是由于来自杯状细胞粘液生产。通过计数上滑动（图1）和突变隐窝数隐窝的总数，对MF为突变隐窝可以计算出来。没有突变隐窝任何检查对照的&gt; 100,000野生型隐窝观察（PBS处理）的小鼠。我们设置MF为对照小鼠为&lt;0.1×10 -4，这是接近之前对C57BL / 6小鼠的报道。1 <img alt="图1" src="/files/ftp_upload/53240/53240fig1.jpg" /> 图1.准备幻灯片G6PD染色试剂。 （A）两节1.5厘米×0.15厘米钢垫圈（内径×高），使用硅润滑脂密封在一起。石蜡膜，然后切成合适的井的基座与中心切出和阱放置在瑞士上轧制结肠组织切片该幻灯片。 （B）中从相邻7微米的削减两组织切片放置在相同滑动并染色的同时。比例尺1.0公分。 （C）的部分隐窝的数量通过查看组织在10倍的放大倍率确定的。字段数（示为框）与每个部分而不同。隐窝中每个字段的数目由视觉计数测定，并加入到得到隐窝该级别的总数。 （D）A级定义为四个连续7微米的组织切片。该水平&gt; 50微米分开，以避免与先前评估隐窝重叠。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53240/53240fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53240/53240fig2.jpg" /> 图2.代表性的例子在观察到AOM突变隐窝处理之C57B1 / 6不产生功能性的G6PD雄性小鼠的葡萄糖-6-磷酸脱氢酶的突变体隐窝是有严格规定，但实际上缺乏蓝染色。野生型隐窝组成大多数实地隐窝是蓝色/紫色指示G6PD的酶活性。规模:10微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53240/53240fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td>治疗</td><td>突变隐窝发病率</td><td>突变隐窝一 </td><td>总隐窝分析</td><td> MF（×10 -4） </td></tr><tr><td>溶剂</td><td> 0/6小鼠</td><td> 0 </td><td> &gt; 10 5 </td><td> &lt;0.10 </td></tr><tr><td> AOM </td><td> 10月12日的小鼠</td><td> 43 </td><td> 96821 </td><td>60; 4.44 </td></tr></tbody></table> 表1突变频率在未处理和氧化偶氮甲烷处理之C57B1 / 6小鼠。12 结肠干细胞突变在WT C57BL / 6小鼠暴露于10mg / kg的氧化偶氮甲烷（AOM）或溶剂对照后90天。

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

结肠干细胞突变定量

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

quantification-of-colonic-stem-cell-mutations

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.5K 次观看.  University of Pittsburgh. We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

视频: Quantification of  Colonic Stem Cell Mutations

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

科研

发育生物学

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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

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

转基因器官皮肤培养使用灭活人真皮代

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

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

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 ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...