This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006; SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry. We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work.

1.神经文化注意：在多能干细胞成纤维细胞重编程，承诺向背侧端脑谱系，推导，放大，和银行的晚期皮质祖细胞（LCP）在Boissart 等人 4进行了描述。还根据Boissart 等人 4在稍微修改执行LCP样细胞的神经元分化。其他程序已经开发了用于成纤维细胞的成诱导多能干细胞，接着通过分化成神经元的直接重编程。该协议被保留下来，因为它允许选择性生产锥体谷氨酸能神经元的。 <ol><li>治疗6孔培养板用玻璃盖玻片用聚鸟氨酸（在DPBS中稀释至1/6，原料浓度0.01％）O / N，随后洗涤三次在DPBS中。然后在至少10小时的流罩下添加层粘连蛋白（储备浓度为1mg / ml，稀释500倍的DPBS）<html> 。 </li><li>板并调度NSC以低密度N2添加剂的（50,000细胞/ cm 2）在6孔培养板用在3毫升由DMEM / F12的培养基盖玻片（500毫升），2瓶（5 ml的每种）， 2瓶B27补充剂（每次10毫升），10毫升笔，链霉素（青霉素= 10,000单位/毫升，链霉素= 10000单位/毫升），1毫升2-巯基乙醇（原液：50毫米）和层粘连蛋白（1 / 500），无生长因子。关键的一步：认真执行这一步骤，以减少细胞集群增加细胞的缓慢旋转的动作。 </li><li>取出培养基。添加含有新鲜的层粘连蛋白溶液2微克/毫升，以保持附着在玻璃盖玻片的神经元，并避免结块新鲜N2B27网上平台。改变介质每3天。为了防止电池从干燥加入新鲜培养基之前保持一些剩余的培养基（200微升）的。可替代地，进行快速改变总体积（3毫升）中。 </li></ol>e_title“&gt; 2。慢病毒转导注意：GFP的慢病毒载体承蒙以下乌韦Maskos实验室博士在巴斯德研究所（巴黎）提供及根据公布的协议5制备。 GFP表达由鼠标磷酸甘油酸激酶（PGK）启动子驱动。在这项研究中，病毒滴度为400纳克/μL（原液在PBS 1X）的。 <ol><li>加入1微升含有40纳克每培养的GFP的慢病毒载体以及（6孔板）的原液转导在成熟通过病毒颗粒的任何步骤人类源自iPSC的神经元，并培育48小时在新鲜培养基中。注意：对于这个协议，孵化时间允许脊柱结构的一个很好的标志。 </li></ol> 3.免疫注意：为了提高整个棘形态的标签，使用抗GFP抗体在透化条件下进行免疫荧光标记。 </p&gt; <ol><li>盖玻片上在4％多聚甲醛除去培养基并固定转导的细胞在室温10分钟，然后清洗3次的1×PBS（每次10分钟）。 </li><li>在PBS浸入盖玻片补充有0.05％的Triton（100×）和10％马血清的1小时在室温下，然后洗3次在1×PBS中。 </li><li>加入100微升的1×PBS的补充与针对GFP和由1000上每个盖玻片的因子稀释4％马血清和初级抗体（1/1000）。在孵育暗箱O / N在4°C，然后洗3次，1×PBS。 </li><li>稀释的Alexa Fluor 488缀合的抗体（1/200）的PBS补充有吐温20的0.5％，并孵育1小时，在室温。然后洗3次，1×PBS中并安装在载玻片盖玻片安装介质荧光显微镜。 </li></ol> 4.树突棘成像<ol><li>通过激光共聚焦扫描显微镜进行共聚焦成像。 </li><li>选择健康的神经元与锥体形态的次全树突分支，并从独立的实验量化每种条件至少10的神经元。量化60 - 每枝晶100微米。 </li><li>获得使用40X油NA = 1.3的物镜和488nm的激光线的GFP激励图像，具有典型的峰值功率在大约20微瓦样本水平。大约80纳米组像素大小适当样树突棘。 注：在随后的分析要求的图像，其噪声不影响树突棘和的正确分割。与上述空间采样和功率设置，我们观察到3.15微秒的像素停留时间是足以将收集足够的光子来构建这样的图像。为暗淡的样品，图像质量可以通过平均2至4个扫描得到改善。可能需要购置若干XY切片以覆盖感兴趣的区域，然后处理前一起必须缝合。 </li><li>品尝整个神经元的体积，获得一个Z堆栈，用为Z间距从150纳米到300纳米，得20至30ž切片。 注意：实现了与这些设置的横向空间分辨率为234 nm和轴向分辨率为591纳米。这里所选择的采样是足够的，但作为轴向分辨率比要被成像的棘的最小尺寸大，则分析有利于该横向延伸从树突棘。 </li></ol>树突棘5.三维定量注意：以下部分具体介绍了使用了Imaris软件进行分析的。备选实现存在，包括NeuronStudio 15或的Metamorph 8，它可以提供类似的结果。 使用下列键设置： <ol><li>作为一个预处理级，通过由软件提供的图像处理设备利用高斯滤波。通过图像处理&gt; Smoo执行高斯滤波事&gt;高斯滤波器。设置过滤器宽度为等于在XY像素大小。 </li><li>执行树突的半自动跟踪，使用该软件的灯丝示踪模块。 <ol><li>首先，估计枝晶直径，通过使用该软件的切片标签的距离的工具。 </li><li>在超越选项卡中，单击长丝工具。为了更好的稳健性，该协议依赖于半自动化跟踪;点击跳过自动创建 。该模块接口现在显示绘图选项卡。这里，选择AutoPath为方法， 枝蔓作为一种类型，并输入所估计的枝晶的直径。 </li><li>使用指针的选择模式 ，把光标移动到一个盒。对树突起点按住Shift键并单击鼠标右键。注：该软件进行初始计算。 </li><li>将沿枝晶的指针。从起点（代表编作为蓝色球体），黄线表示最可能的树突路径​​被示出。对树突端点按住Shift键并单击鼠标左键。 </li></ol></li><li>执行自动化的刺分割。注意：在跟踪枝晶的棘将由模块接口自动找到。 <ol><li>在模块界面中，单击创建选项卡上。 在重建下拉列表中，挑选重建枝蔓径及检查保持数据框中 。 然后单击重建。 </li><li>设置阈值 ，使得所分割的体积对应于实际树枝状结晶体积。作为一个算法 ， 然后从距离图最短距离。单击下一步按钮。 </li><li>确定最小棘头直径和最大长度，再通过使用软件的片标签的距离的工具，然后再回来了超越选项卡并输入参数。 F或者该协议，约200个值 - 300纳米的最小直径4微米的最大长度是很好的出发点。不要选中允许分局棘框。 单击下一步按钮。 </li><li>调整种子点阈值 ，使代表刺蓝色点定位到实际的脊椎头。 单击下一步按钮。注：现在的核心计算完成，可能会很长。 </li></ol></li><li>通过将模块接口的工具选项卡分类刺。点击分类刺。在提示用户界面时，请确保有四大类，由它们的形态定义如下：末节：长度&lt;1微米;蘑菇：长度（脊椎）&gt; 3和最大宽度（头）&gt;平均宽度（颈）×2;细细长长的：平均宽度（头）≥平均宽度（颈部）;丝状伪足样：长度≤4微米（无头）。 注：莫独乐界面生成包含分类的结果四个新的灯丝的对象。 </li><li>出口统计数据：在所有这四个对象接口的，去统计选项卡。 点击导出所有统计到文件按钮 。 注：其他统计值可导出为树突（如，长度，面积，平均直径，分支深度，分枝角度，体积等 ），以及用于棘（ 例如，直线度，的附着，长度和不同的卷区域脊骨零件，脊柱直径，密度等 ）。 </li></ol>

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The authors have nothing to disclose.

的锥体神经元的形态学特征的量化依靠软件。灯丝示踪接口用于神经元和刺的分割，以及XT模块用于自己的分析。 分析我们技术的准确性，我们首先比较所测量的形态学参数（长度，面积和总体积脊柱适用时），与那些使用大鼠成熟锥体神经元在培养6,7和人脑组织3出版。密度是相当的所有情况。没有量 ​​的数据是在大鼠神经元描述了使用自己的协议为容积...

皮质锥体神经元的树突棘是它们沿着这些神经元亚型的啮齿动物，灵长类动物和人类的大脑基底和顶树突分布小而薄的突起。他们最兴奋性突触的位点，并显示在学习和认知过程的关键功能。人树突棘的详细结构已经从技术上研究了电子显微镜2。然而，这种方法是费时和表示繁重的工作量。最近，使用组合的大型人工脊椎分析3特定软件的三维（3D）重建树突棘的形态的已报道在人类大脑皮质。 绿色荧光蛋白（GFP）的技术联接到免疫表示由荧光显微镜的精确工具脊柱识别和形状测定。这种方法可以容易地应用于培养的神经元。豪版本，没有数据已被报道脊柱成熟和形态上从诱导的多能干细胞（IPSC）衍生的人神经元分析。 本研究的目的是描述一种协议，它允许从体外培养的人神经元树突棘成像。绿色荧光蛋白标记，激光共聚焦显微镜和3D分析了Imaris软件的灯丝示踪模块进行了本协议中使用。培养的步骤所必需的获得层的皮层谷氨酸能神经元二至四从神经干细胞（NS​​C）也在这里简单说明。整个协议为人类NSC生产已经在别处4出版。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PD-PBS (1X), sans Calcium, Magnesium et Phenol Red</td>
			<td>Gibco/ Life Technologies</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine Solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin</td>
			<td>Dutscher Dominique</td>
			<td>354232</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco/ Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement w/o vit A (50X)</td>
			<td>Gibco/ Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/NUT.MIX F-12 W/GLUT-I</td>
			<td>Gibco/ Life Technologies</td>
			<td>31331028</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Med SFM</td>
			<td>Gibco/ Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco/ Life Technologies</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Steptomycin</td>
			<td>Gibco/ Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Rabbit Serum Polyclonal Antibody</td>
			<td>Gibco/ Life Technologies</td>
			<td>A-6455</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco/ Life Technologies</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Rabbit&#160;</td>
			<td>Gibco/ Life Technologies</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Anti-betaIII tubulin antibody</td>
			<td>Millipore</td>
			<td>AB9354</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass 13 mm</td>
			<td>VWR</td>
			<td>631-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent avec DAPI</td>
			<td>Gibco/ Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween(R) 20 Bioextra, Viscous Liquid</td>
			<td>Sigma Aldrich Chimie</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich Chimie</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Fibroblasts</td>
			<td>Coriell Cell Line Biorepository</td>
			<td>GM 4603 and GM 1869</td>
			<td>Coriell Institute for Medical Research, Camden, NJ, USA</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss (Germany)</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris Software</td>
			<td>Bitplane AG, Zurich</td>
			<td>6.4.0 version</td>
			<td>Filament Tracer and Imaris XT modules are necessary</td>
		</tr>
		<tr>
			<td>Huygens Software</td>
			<td>Huygens software, SVI, Netherlands</td>
			<td>Pro version</td>
			<td>Optional (for deconvolution testing)</td>
		</tr>
	</tbody>
</table>

three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells

树突棘是对应于兴奋性突触的中枢神经系统的突触后隔室的小突起。它们沿树突分布。它们的形态，在很大程度上取决于神经元活动，它们是动态的。树突棘在其表面表达并在突触后密度的水平谷氨酸受体（AMPA和NMDA受体）。每个脊椎使神经元能够独立控制其国家和地方的活动。脊柱形态已被广泛研究，在大脑皮质的谷氨酸锥体细胞，同时使用在体内的方法和从啮齿动物的组织得到的神经元培养物。神经病理症状可以关联到改变的脊柱感应和成熟，如图啮齿动物培养的神经元和一维定量分析1。本研究描述了一个协议，用于脊椎形态的三维定量分析使用人类cortic人神经元的神经干细胞（后期皮质祖细胞）获得。这些细胞最初从诱导的多能干细胞获得的。该协议允许脊柱形态的不同培养时间的分析，并与来自对照个体与那些从患者的精神疾病得到得到诱导多能干细胞之间的可能的比较。

<html>本研究描述了一种标准化协议，用于从iPSC的衍生锥体神经元的树突培养脊柱量化。该协议允许对人类神经元脊柱成熟及其与在标准啮齿动物神经元培养棘的成熟以及体内动物模型可以比较的分析。 图1A表示的不同步骤培养的，其允许生产皮层锥体神经元的方案。这样示意表示，以便更好地了解生产赋予不同类别刺锥体神经元的全球时间缩放提供。 Repr...

Watch this Scientific Journal Video about Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells at JoVE.com

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

锥体神经元的树突棘是哺乳动物大脑皮质最兴奋性突触的部位。此方法描述在从诱导的多能干细胞衍生的人皮质锥体谷氨酸能神经元脊柱形态的三维定量分析。

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

three-dimensional-quantification-dendritic-spines-from-pyramidal

Research

JoVE Journal

Developmental Biology

11.3K 次观看.  Institut Pasteur. 锥体神经元的树突棘是哺乳动物大脑皮质最兴奋性突触的部位。此方法描述在从诱导的多能干细胞衍生的人皮质锥体谷氨酸能神经元脊柱形态的三维定量分析。 

视频: Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

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 ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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 ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...