这项工作得到了西班牙经济与竞争部（RTC-2014-2626-1至JE）和卡洛斯三世健康研究所（PI15/01458至JE）的资助。EC得到了Atracción de Talento Investigador拨款2017-T2 / BMD-5766（马德里市和UAM）的支持。

所有小鼠饲养和实验程序必须符合当地、国家、国际立法和动物实验指南。
1.诱导毛发生长，烧伤诱导和识别尾部皮肤上皮整体中的长期BrdU LRC
注意:使用10天或7周大的C57BL / 6小鼠，最好是同窝小鼠，用于下面描述的实验设计。在所有实验程序中，动物将通过3%异氟醚吸入麻醉或通过颈椎脱位安乐死，如所示。
<ol><li>在休止期（休息）阶段（出生后约50天）诱导小鼠背部皮肤的毛发生长<ol><li>用吸入3%异氟醚麻醉小鼠。通过无踏板反射（脚趾用力捏）确认完全深度麻醉。使用理发器和脱毛膏在每只小鼠中剃除背部皮肤的两个独立的并排区域（材料表）。使用左侧进行对照，使用右侧进行治疗。 注意:检查剃须后，下颌背皮肤是否为粉红色而不是灰色/黑色，这是该小鼠品系黑色素生成和进入生长期（生长）阶段的指标。</li>
		<li>用PBS彻底清洗以去除所有乳霜残留物，并按照第2.1节所述诱导非致命ROS水平的瞬时原位产生。</li>
		<li>通过每天采集对照组和治疗后每只动物背部皮肤区域的高分辨率图像（例如，使用与5-20倍双目镜头耦合的高清摄像机）来记录毛囊生长。</li>
	</ol></li>
	<li>在休止期（静息）阶段（出生后约50天）诱导小鼠背部皮肤中的2度烧 伤病变<ol><li>麻醉小鼠。使用理发器和脱毛膏剃除每只小鼠的整个背部皮肤区域，并用PBS彻底清洗以去除所有乳霜残留物。 注意:在燃烧程序之前，在无菌盐水中原 位 皮下注射0.5%（2mg / ml）利多卡因，不超过7mg / ml。</li>
		<li>浸入沸水中，在95°C下预热黄铜棒（横截面为1cm），然后在每只小鼠背背皮肤表面的中心区域施用5秒。</li>
		<li>在烧伤产生后，腹膜内在电热毯上向动物注射1mL生理溶液（0.9%NaCl）以防止脱水。让动物恢复24小时，并按照第2.3节所述诱导非致命ROS水平的瞬时原位产生。</li>
		<li>通过每天采集对照组和治疗过的每只动物背部皮肤区域的高分辨率图像（例如，使用与5-20倍双目镜头耦合的高清摄像机）来记录烧伤创面的进展。</li>
	</ol></li>
	<li>尾部皮肤上皮中长期BrdU LRC的产生和鉴定<ol><li>腹膜内注射10/14天大的同窝伴侣（无麻醉），连续4天，将50mg / kg体重BrdU溶解在PBS中。在标记阶段之后，让小鼠在任何处理前生长50-60天。 注意:每次注射使用新针头。</li>
		<li>按照第2.3节所述进行，在制备组织整体之前的不同时间诱导尾部皮肤中瞬时原位非致命ROS的产生。</li>
		<li>为了准备整个尾巴表皮，通过颈椎脱位对小鼠实施安乐死，并用手术剪刀夹住尾巴。<ol><li>用手术刀沿着尾巴做一个直的纵向切口，并将整个皮肤从脊椎上剥成一块。将剥落的皮肤在PBS中的5mM EDTA中在5mL管中的5mM EDTA中在37°C下孵育4小时，并使用镊子小心地将完整的表皮片与真皮分开。</li>
			<li>在室温（RT）下将组织固定在PBS中的4%甲醛中至少72小时，并使用适当的抗体进行BrdU检测。</li>
			<li>使用荧光/共聚焦显微镜来识别和量化每个实验条件下的LRC，包括在制备组织整体之前不同时间的光对照和光动力处理，如前所述详细描述19,20。 注意:固定表皮片可以在含有0.02%叠氮化钠的PBS中在4°C下储存长达三个月。固定表皮片可用于按照标准组织学切片程序对所需蛋白质进行免疫定位。</li>
		</ol></li>
	</ol></li>
</ol>2.诱导小鼠皮肤中非致死性ROS水平的瞬时产生
注意:为了诱导小鼠皮肤中瞬时产生非致命ROS水平，将使用内源性光敏剂PpIX的前体进行光动力处理，在这种情况下，甲基氨基乙酰丙酸酯（mALA）和红光。
<ol><li>要开启瞬时ROS产生以诱导背部皮肤的毛发生长，请按照第1.1节中的说明准备动物。<ol><li>在右侧区域以外用乳膏（材料表）的形式涂抹~25mg的mALA，保持左侧作为内部对照，避免个体间差异。在黑暗中孵育2.5小时，用PBS彻底洗掉多余的奶油。为了确认麻醉深度，每10分钟监测一次动物，直到完全恢复。 注意:背部皮肤中PpIX的产生应在蓝光（407 nm）激发下通过其红色荧光原位测试。</li>
		<li>麻醉动物。</li>
		<li>用足够的红光源照射整个背部皮肤（材料表），总剂量为2.5-4J / cm2。将小鼠放在电热毯上，直到它们完全恢复，并按照步骤1.1.3中所述进行。 注意:辐照度应通过操纵光源和组织之间的距离来调整，并使用功率能量计（材料表）进行测量。当在每只动物的任何独立剃光区域观察到毛发生长时，该实验被认为是完成的。整个过程只涉及一次照片处理。</li>
	</ol></li>
	<li>要开启瞬时 ROS 生产以改善2 度烧 伤病变的愈合，请按照第 1.2 节中的指示准备动物。<ol><li>沿烧伤表面以外用乳膏的形式涂抹~25mg的mALA，包括约4mm的相邻组织。在黑暗中孵育2.5小时，用PBS彻底洗掉多余的奶油。为了确认麻醉深度，每10分钟监测一次动物，直到完全恢复。</li>
		<li>麻醉动物。</li>
		<li>用足够的红光源照射整个背部皮肤，总剂量为 2.5−4 J/cm2。将小鼠放在电热毯上，直到它们完全恢复，并按照步骤1.2.4中所述进行。 注意:当观察到完全烧伤愈合时，每只动物的实验程序被视为已完成。整个过程只涉及一次照片处理。</li>
	</ol></li>
	<li>为了开启尾部皮肤中的瞬时ROS产生，如第1.3节所示制备小鼠，沿背侧组织区域以局部乳膏的形式施用~25mg的mALA，并按照第2.1节中所述进行背部皮肤。在动物安乐死前 24 小时、48 小时或 72 小时进行光处理和相应的光控制，并进一步提取尾部皮肤整个坐骑。 注意:在所有实验设计中，通过使用抗氧化剂ROS清除剂测定该过程的ROS依赖性（例如，通过在PBS中腹膜内注射20mg / mL溶液，pH 7.2，每天接种100mg / kg体重N-乙酰半胱氨酸，在mALA处理前5天开始，或者，在50%乙醇中加入两剂100mg / mL抗坏血酸， 间隔30分钟，在mALA治疗和红光照射之间的时间间隔内局部施用于皮肤）。</li>
</ol>3. 皮肤中的ROS检测
<ol><li>使用氢乙啶进行光动力处理后尾部皮肤ROS产生的离体评估 注意:氢乙啶是一种非荧光分子，与ROS特异性反应产生荧光染料2-羟基乙锭（hET）。<ol><li>将如第1.3.3节所述获得的整个尾皮在37°C下在PBS溶液（对照样品）中的5mM EDTA中孵育3小时或另外含有2mM mALA（光动力处理样品）。</li>
		<li>在所有情况下，从25mg / mL储备液中的二甲基亚砜（DMSO）中加入氢乙啶至终浓度为3.2μM，并在室温下在黑暗中孵育1小时。</li>
		<li>用指尖将尾皮样品拉伸到玻璃表面上，并以 636 J/cm 2 的通量照射2 nm 红光。</li>
		<li>立即进行将表皮与真皮分离，并按照第1.3.3节所述固定表皮片。</li>
		<li>使用绿色激发光在荧光/共聚焦显微镜下评估hET红色发射，捕获高质量图像并进行进一步分析。 注意:在没有光动力处理的情况下使用组织样品的hET染色作为hET自氧化的阴性对照。</li>
	</ol></li>
	<li>体内检测诱导毛发生长和烧伤愈合后背部皮肤中ROS的产生，然后进行光动力治疗 注意:此步骤使用2′，7′-二氯二氢荧光素二乙酸酯（DHF-DA）进行，这是一种细胞通透性非荧光化合物，在细胞内酯酶切割后，与ROS特异性反应，产生2′，7′-二氯荧光素（DCF）荧光染料。<ol><li>使用为诱导毛发生长（第1.1节）或烧伤愈合（第1.2节）而准备的动物。在局部mALA乳膏治疗之前（见第2.1和2.2节），在所有靶标对照/治疗皮肤区域局部分配100μL 1mg / mL的50%乙醇DHF-DA，让皮肤充分吸收材料并继续进行局部mALA乳膏应用。</li>
		<li>将处理过的动物在黑暗中孵育4小时，用PBS彻底洗去皮肤上的局部mALA霜，并在皮肤上分配第二剂100μL的DHF-DA溶液。为了确认麻醉深度，每10分钟监测一次动物，直到完全恢复。</li>
		<li>将治疗过的动物在黑暗中孵育50分钟，使用LED灯麻醉并照射整个背部皮肤，通量范围为2.5至4 J /cm 2 的636nm红光。</li>
		<li>照射后，立即使用体内成像系统评估皮肤中产生的ROS水平（材料表）。将滤光片盒设置为445−490 nm激发和515-575 nm发射，捕获高质量图像并进行进一步分析。 注意:在没有光动力处理的情况下使用组织样品的DHF-DA染色作为DHF-DA自氧化的阴性对照。</li>
	</ol></li>
</ol>

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本作品中描述的程序的所有商业应用均受CSIC-UAM专利（EP2932967A1）的保护，该专利由EC，MIC和JE撰写，并授权给Derma Innovate SL进行商业开发。JE和JJM在Derma Innovate SL担任顾问职位。

在这里，我们提出了一种方法，该方法允许在小鼠皮肤中瞬时激活体内内源性ROS产生，并具有生理效应。该方法基于光动力程序，以诱导内源性光敏剂PpIX的受控和局部刺激（图1B）。这种实验方法是在体内实验系统中研究ROS生物学的有趣工具，与使用外部ROS源（通常是过氧化氢）的方法相比，该方法取得了重大进展，并允许在组织/样品中受控和局部产生ROS。
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活性氧（ROS）是分子氧化学还原形成水的结果，包括单线态氧、超氧阴离子、过氧化氢和羟基自由基1,2,3。由于其极强的化学反应性，ROS的寿命非常短。在需氧生物中，ROS是在线粒体中有氧呼吸（电子传递链）的主要泄漏副产物在细胞内偶然形成的。细胞中高水平的ROS的瞬时积累导致氧化应激条件，可能导致蛋白质，脂质和糖的不可逆失活，并在DNA分子中引入突变2,3,4,5。细胞，组织和整个生物体中氧化损伤的逐渐积累随着时间的推移稳步增加，并且与细胞死亡程序，几种病理和衰老过程的诱导有关2,3,4,6。
需氧生物已经稳步进化出有效的分子机制，以解决细胞和组织中过量的ROS积累。这些机制包括超氧化物歧化酶（SOD）蛋白家族的成员，其催化超氧化物自由基歧化为分子氧和过氧化氢，以及不同的过氧化氢酶和过氧化物酶，它们使用抗氧化剂库（谷胱甘肽，NADPH，过氧化物还原蛋白，硫氧还蛋白7,8）催化随后过氧化氢转化为水和分子氧。
然而，一些报告支持ROS作为调节关键细胞功能的分子回路的关键组成部分的作用，包括增殖，分化和迁移2,3,4。这一概念得到了需氧生物中专用ROS产生机制的初步鉴定和表征的进一步支持，包括脂氧合酶环氧合酶和NADPH氧化酶9,10。从这个意义上说，ROS在脊椎动物胚胎发育过程中表现出积极作用11,12,13，并且这些分子在调节特定体内生理功能中的关键作用已在不同的实验系统中报道，包括果蝇14中造血祖细胞的分化程序，斑马鱼的愈合诱导或非洲爪蟾蝌蚪的尾部再生15.在哺乳动物中，ROS参与了神经球模型中神经干细胞的自我更新/分化潜力16，以及结直肠癌起始期间肠道干细胞功能的失调17。在皮肤中，ROS信号传导与表皮分化以及皮肤干细胞生态位和毛囊生长周期的调节有关18,19。
从这个角度来看，确定ROS在正常或病理条件下在生物系统中的生理作用的一个主要实验限制是缺乏足够的实验工具来诱导细胞和组织中这些分子的受控产生，准确地类似于它们作为第二信号信使的生理产生。目前，大多数实验方法涉及外源性ROS的施用，主要以过氧化氢的形式。我们最近实施了一种实验方法，以在小鼠皮肤中开启内源性，非致命的内源性ROS的体内产生，基于内源性光敏剂原卟啉IX的前体（PpIX;例如氨基拉伏林酸或其甲基衍生物甲氨基乙酰丙酸）的施用，并用红光进一步照射样品以诱导细胞内分子氧原位形成ROS（图1).这种光动力程序可以有效地用于刺激常驻干细胞生态位，从而激活组织的再生程序19,20，并为皮肤再生医学中的新治疗方式开辟道路。在这里，我们提供了该方案的详细描述，显示了刺激干细胞生态位的代表性示例，测量为毛囊19,21的凸起区域中长期5-溴-2'-脱氧尿苷（BrdU）标记保留细胞（LRCs）数量的增加，以及随后由瞬态诱导的再生程序的激活（头发生长和烧伤愈合过程的加速）， C57Bl6小鼠品系皮肤中非致命ROS产生。

<table border="0" cellpadding="0" cellspacing="0" style="width:812px;" width="811">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">D6883-50MG</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">5&#39;-bromo-2&#39;-deoxiuridine</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td style="width:115px;">B5002-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Anti-Bromodeoxyuridine-Fluorescein</td>
			<td style="width:104px;">Roche</td>
			<td>11202693001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Depilatory cream (e.g., Veet)</td>
			<td style="width:104px;">Veet</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Dihydroethidium</td>
			<td style="width:104px;">Sigma Aldrich</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">In Vivo imaging system, e.g., IVIS Lumina 2</td>
			<td style="width:104px;">Perkin Elmer</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:432px;">mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td>
			<td style="width:104px;">Galderma</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Power energy meter (e.g., ThorLabs Model PM100D)</td>
			<td style="width:104px;">ThorLabs</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Red light source, e.g., 636 nm Aktilite LED lamp</td>
			<td style="width:104px;">Photocure ASA</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

stimulation of stem cell niches and tissue regeneration in mouse skin by switchable protoporphyrin ix-dependent photogeneration of reactive oxygen species in situ

在这里，我们描述了一种在小鼠皮肤中诱导内源性活性氧（ROS）的可切换体内光生的方案。这种原位ROS的瞬时产生有效地激活了干细胞壁龛中的细胞增殖，并刺激组织再生，这通过加速烧伤愈合和毛囊生长过程得到了强烈体现。该方案基于可调节的光动力处理，该处理用内源性光敏剂原卟啉IX的前体处理组织，并在严格控制的物理化学参数下进一步用红光照射组织。总体而言，该协议构成了分析ROS生物学的有趣实验工具。

在小鼠背部和尾部皮肤中局部施用mALA前体导致PpIX在整个组织中显着积累，并且显着地在毛囊中，如该化合物在蓝光（407nm）激发下的红粉红色荧光所证明的那样（图2A，C）。随后以2.5−4J / cm 2的通量用红光（636nm）照射处理的组织，促进组织中ROS的瞬时产生，特别是在毛囊的凸起区域（图2B，D）。
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Watch this Scientific Journal Video about Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In 

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

该协议的目的是诱导小鼠皮肤中瞬时体内产生非致死水平的活性氧（ROS），进一步促进组织中的生理反应。

通过可切换原卟啉IX依赖性原位活性氧物种光生成刺激小鼠皮肤中的干细胞壁龛和组织再生

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

stimulation-stem-cell-niches-tissue-regeneration-mouse-skin

Research

JoVE Journal

Biology

1.9K 次观看.  Ramón y Cajal University Hospital. 该协议的目的是诱导小鼠皮肤中瞬时体内产生非致死水平的活性氧（ROS），进一步促进组织中的生理反应。

视频: Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

科研

生物学

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

在蝾螈肢体再生过程中通过全皮瓣手术抑制伤口表皮的形成

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.