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><tbody><tr><td>2&amp;prime;,7&amp;prime;-Dichlorofluorescin diacetate</td><td>Sigma Aldrich</td><td>D6883-50MG</td><td></td></tr><tr><td>5&#039;-bromo-2&#039;-deoxiuridine</td><td>Sigma Aldrich</td><td>B5002-500MG</td><td></td></tr><tr><td>Anti-Bromodeoxyuridine-Fluorescein</td><td>Roche</td><td>11202693001</td><td></td></tr><tr><td>Depilatory cream (e.g., Veet)</td><td>Veet</td><td></td><td></td></tr><tr><td>Dihydroethidium</td><td>Sigma Aldrich</td><td>37291-25MG</td><td></td></tr><tr><td>In Vivo imaging system, e.g., IVIS Lumina 2</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>mALA in the form of topical cream, e.g.,METVIX Crema 160 mg/g</td><td>Galderma</td><td></td><td></td></tr><tr><td>Power energy meter (e.g., ThorLabs Model PM100D)</td><td>ThorLabs</td><td></td><td></td></tr><tr><td>Red light source, e.g., 636 nm Aktilite LED lamp</td><td>Photocure ASA</td><td></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

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 ...

科研

发育生物学

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 ...

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 ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

将小鼠成纤维细胞直接重编程为黑素细胞

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

生物学

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

在 vivo小鼠创面模型中活性氧的成像

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

免疫与感染

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses.

在自抗性混合奇美体模型中, 光激活询问个体自反应萌发中心

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.