这项工作是supportedby美国癌症协会的研究学者格兰特（RSG-12-148-01-DDC）和CIRM基础生物学奖（RB4-05779）。

根据加州大学圣迭戈分校的研究伦理委员会的指导方针进行了人体皮肤的协议。人的皮肤可以从废弃的手术样本中获得，或从皮肤银行购买（皮肤银行被列在材料/设备表 ）。其中皮肤是从主或施主的年龄衍生的位置不是本实验只要基底膜区蛋白（胶原蛋白/层粘连蛋白）在真皮临界不会降解。 1.准备失活的人真皮的<ol><li>在接收到人体皮肤时，将组织在组织培养罩解冻（〜3-5分钟）。根据皮肤的大小，放置解冻组织在一个无菌的一次性50ml锥形管或125毫升瓶装。从皮肤银行皮肤的典型大小为0.75平方英尺。 </li><li>填充容器75％满，支持4倍青霉素和链霉素（笔/链球菌）的1X PBS。剧烈摇晃5分钟和处置的液体。重复此步骤3次以上。 </li><li>在最后一次洗涤后，将组织转移到新的试管/瓶，加上新的4倍青霉素/链霉素/ PBS中以填充容器的75％。孵育在组织培养箱此混合物在37℃下进行2周，以允许从表皮真皮的分离。 </li><li>在无菌条件下，使用镊子剥离表皮从真皮。真皮是全白，而表皮会有着色根据供体（ 图1A）的比赛。根据该机构的协议处理人体组织丢弃表皮。 </li><li>将在管或瓶真皮和4倍笔/链球菌稀释于1×PBS中剧烈振荡（5分钟洗4次）清洗。在最后一次洗涤后，将真皮转移到新的试管/瓶在4倍青霉素/链霉素稀释于1×PBS中，并储存在4℃直至准备使用。真皮可以储存在4℃下长达一年。 </li></OL&gt; 2.遗传修饰小学的人角质形成细胞注：生长在完全DMEM培养基[DMEM + 10％胎牛血清（FBS）+青霉素/链霉素]，制造病毒感染的原代人角质形成细胞用嗜凤细胞。病毒包装基因编码蛋白质如的gag-pol和信封被稳定地整合到凤细胞基因组中时，用逆转录病毒载体转染其允许病毒的生产。凤凰细胞可被转染的高效率，可用于高病毒效价的生产。从这个包装线生产的病毒也可以感染多种哺乳动物细胞，包括人。 <ol><li>第1天：转染前一天，种子凤细胞在6孔板中以每孔80万细胞在完全DMEM培养基中的密度。 </li><li>第2天：转染的当天，用3微克的逆转录病毒载体的每孔。等分试样3微克逆转录病毒载体导入1.5ml试管。使用100μ组合;升的DMEM加6微升转染试剂的每3微克载体的构建。拌6微升转染试剂用在1.5ml管100微升DMEM中。 （对所使用的逆转录病毒载体中列出的设备/材料表）。 <ol><li>孵育5分钟，并添加106微升混合物到预等分管含有3微克的逆转录病毒载体的构建。混合并在室温下孵育30分钟。添加这种混合物全部滴凤凰细胞的每一个孔。 </li></ol></li><li>第3天：转染后的第二天，从凤凰细胞取出媒体和加入2毫升新鲜完整的DMEM培养基。在同一天，种子初级人角质形成细胞到6孔板中，每孔75000个细胞的密度。保持在角质形成细胞无血清培养基（KCSFM）抗生素（青霉素/链霉素）的角质形成细胞。 注：原代人角质形成细胞购买。细胞可以从多家供应商（在材料/设备表中列出）购买。 </li><li>第4天：HAR从转染的凤细胞，其现在包含病毒颗粒归属介质。通过使用注射器0.45μm的过滤器传递介质（以除去任何污染的凤细胞），并放置2 ml的到角质形成细胞（前一天接种在75000个细胞/孔：参见步骤2.3）。加入聚凝胺（5微克/毫升）向媒体帮助调解感染过程。病毒滴度是〜1×10 7 TU /毫升。 <ol><li>旋6孔板在离心机中，在200×g离心1小时，在室温。旋转后，取出含有病毒的媒体和添加KCSFM前，用1×PBS洗一次细胞。 </li></ol></li><li>第5天：感染同一批次的角质形成细胞的再按照步骤2.4。 注意：选择使用药物的角化细胞是否存在的逆转录病毒载体的选择标记和扩大用于下游分析或应用，例如器官型培养使用。 </li></ol> 3.设置器官皮肤文化<ol><li>构建从实验室中常用的材料或盒录像带器官可以通过金属加工店进行定制（ 图2A - F）。从3.5厘米组织培养板使这些磁带盒，使用烧灼来从一个3.5厘米培养皿（ 图2A-B）的盖子的中心除去1.0厘米×1.0厘米的正方形。为此无菌条件下。 <ol><li>附加方形栓用透明指甲油（ 图2C）纸盒（3.5 cm培养皿的盖子）的底部。允许5分钟的指甲油干，翻转磁带上，使其搁在方形栓（ 图2D）。放置盒插入直径6cm培养皿。 注：方形栓可以从各种手工艺品商店购买。 </li></ol></li><li>制备角质形成细胞生长培养基（KGM）由66％的DMEM，22％的Ham的F12，100单位/毫升青霉素，100微克/ ml链霉素，10％FBS，2.67微克/毫升腺嘌呤，40 N克/ ml氢化可的松，10纳克/毫升的霍乱毒素，4.94微克/毫升的胰岛素，5微克/毫升转铁蛋白，1.36纳克/毫升三碘左旋甲腺氨酸，10纳克/毫升的EGF，和10微克/毫升盐酸环丙沙​​星。用0.22微米的过滤和储存过滤介质在4°C，直到准备使用。 </li><li>取真皮出在4x青霉素/链霉素+ PBS溶液和在KGM中洗2次，然后孵育在KGM中，在37℃下在使用前两天。 </li><li>切割用手术刀真皮至1.5厘米×1.5厘米大小的块，然后放置到器官盒。放置真皮朝上真皮的顶部。真皮的顶部将是进入与角质形成细胞（ 图1B）接触的一侧。 </li><li>将预切真皮到器官盒朝上（图3A）的真皮的顶侧的方孔。 </li><li>翻转整个器官盒含有真皮过使用镊子（图3B）。冰上解冻的细胞外基质的解决方案。使用针头和注射器绘制出100微升每盒细胞外基质的解决方案。加入5滴到真皮（光面）的底部，并轻微振荡以确保整个真皮（图3B）的均匀分布。 </li><li>允许用于商业细胞外基质溶液5分钟后，完全固化（图3C）。凝固后，使用镊子翻动录像带回去。添加4毫升KGM到直径6cm培养皿。 </li><li>放置0.5-1百万遗传修饰的角质形成细胞在含有真皮（图3D）的器官型盒。 <ol><li> Trypsinize角化细胞通过将4 ml的0.05％胰蛋白酶的10个到厘米板5分钟，骤冷用4ml完全DMEM介质。 </li><li>计数使用血球的角化细胞，然后降速，在200×g离心5分钟。重悬0.5-1百万个细胞在90μ（取决于可用的细胞数）升KGM和放置所有单元格到真皮层。 注意：它放置细胞相同数量的上真皮同样的实验中，以确保出现在再生表皮的厚度的任何差异不是由于在初始细胞数的差异是很重要的。在真皮层放置少于0.5万个细胞可导致分层穷人和分化。 </li></ol></li><li>隔日变化对器官文化KGM媒体。嘉实组织对真皮的角质形成细胞放置后1-14天。表皮完整的分层和分化通常在5天后发生。 </li></ol>

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遗传操作在人皮肤器官型培养提供许多优点常用研究2D培养细胞以及小鼠模型。 2D培养缺乏完整的组织和器官中发现的三维细胞 - 细胞和细胞 - 细胞外基质相互作用。最近的研究还发现，与三维培养的细胞显示多基因表达的相似性对原代人皮肤肿瘤16的二维和三维培养的皮肤癌细胞之间的巨大差异。人体器官皮肤的文化也有过小鼠模型的几个优点。小鼠模型是耗时且昂贵的产生以及人类和?...

人类表皮是一个分层上皮，它可通过细胞外基质被称为基底膜zone.The表皮不仅作为不可渗透的屏障，以防止水分的损失，而且作为防御的第一道防线，以保护连接到下面的真皮身体从国外和有毒物质1。基底层，这是表皮的最深处层，包含表皮干细胞和祖细胞的产生的已分化后代，形成了表皮2的其余部分。表皮祖细胞分化它们迁移向上，以形成被称为棘层3分化的细胞的第一层。在棘层，细胞打开角蛋白1和10的表达，其然后提供强度来承受物理应力为表皮的分化层。作为棘层细胞进一步分化，它们向上移动在表皮为米颗粒层其特征是，形成的透明角质和片状颗粒以及其组装下面质膜结构蛋白。随着细胞继续在分化过程中，质膜下方的蛋白质交联到对方，而层状颗粒从细胞挤出 ​​，以形成富含脂质屏障称为角质层4。 疾病涉及在表皮生长和分化的影响〜人口5的20％的改变。因此，了解该方法的机制是非常重要的。因为许多这些疾病的表现是在细胞-细胞或细胞-基质接触队伍，器官型培养其中人表皮复原在3D环境中已创建6-10。这些方法通常包括使用接种在细胞外基质的主要或转化角化细胞如失活的人类的真皮层，基质胶或胶原蛋白。 要明白，在表皮生长和分化的重要的基因调控机制，角化细胞可以遗传通过逆转录病毒载体操纵以拦截或过表达的基因在二维培养，然后在三维重构。这些方法已被广泛用于表征涉及表皮干细胞和祖细胞的自我更新和分化以及进展为肿瘤11-21基因。这里，提供了深入协议，关于如何通过利用逆转录病毒的改变在表皮器官型培养的基因表达。

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

器官型培养允许它模仿功能和其在体内的组织对应的生理3D环境进行细胞-细胞接触和细胞-基质相互作用至关重要的重构。这是通过皮肤器官培养忠实地概括了表皮分化和分层方案例证。原代人表皮角质形成细胞是通过逆转录病毒基因在那里可以很容易地过度表达或撞倒的基因操纵。这些遗传修饰的角质形成细胞随后可以用来再生人表皮在器官型培养物的皮肤提供了强大的模型来研究遗传途径冲击表皮生长，分化和疾病进展。这里介绍的协议描述方法以制备灭活人真皮以及遗传操作初级人角质形成细胞，以便产生皮肤器官培养物。再生人体皮肤可downstr使用EAM的应用，如基因表达分析，免疫染色，和染色质免疫沉淀，随后通过高通量测序。因此，这些代转基因器官皮肤培养物将允许基因是保持皮肤稳态的关键的确定。

在产生器官型人皮肤的第一步是从真皮除去表皮。皮肤在37℃下在4×青霉素/链霉素/ PBS两周孵化应该允许真皮从表皮（图1A）的分离。如果分离表皮和真皮层，很难然后将组织在37℃于4笔/链球菌/ PBS一个星期，然后再次尝试剥离使用镊子。 一个关键的灭活真皮再生人表皮的是，确保了角质形成细胞被放置在真皮的正确的一面。真皮具有顶部和底部。真皮的顶部是?...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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 ...

generation-genetically-modified-organotypic-skin-cultures-using

Research

JoVE Journal

Developmental Biology

11.1K 次观看.  UCSD Stem Cell Program, University of California, San Diego. 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. 

视频: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

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

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

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 ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

易受伤害的墨西哥美国青少年的改良特里尔社会压力测试

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...