| Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs)
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Author:
Date:
2021-02-05
[Abstract] Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for ...
[摘要] [摘要]人类诱导的多能干细胞（iPSC）及其后代具有组织特异性，为再生医学的研究铺平了道路，并在疾病的病理机制阐明和候选药物的发现等领域进行了研究。iPSC集-来源的神经元是特别有价值的，因为它是难以分析神经细胞获自人类的中枢神经系统。对于用iPSC诱导神经元，最常用的方法之一是在形成胚体（EB）之后分离和扩展神经玫瑰花结。然而，该过程费力，效率低下，并且需要进一步纯化细胞。为了克服这些限制，我们已经开发出一种高效神经诱导方法，该方法允许来自于7天内的iPSC神经干/祖细胞（NSCs / NPC的）的产生和功能的成熟神经元的。我们的方法产生一个PAX6 -阳性同质细胞群中，皮质神经干细胞/ NPC的，和t他所得的NSCs / NPC的可冷冻保存，膨胀，并分化在功能性成熟神经元。此外，我们的协议将比其他方法便宜，因为该协议在神经诱导期间需要较少的神经补充。本文还介绍了FM1 - 43成像测定法中，其是用于所述的iPSC衍生的突触前评估中有用的人类神经元。该协议为生成NSC / NPC和神经元提供了一种快速且简化的方法，使研究人员能够建立体外细胞模型来研究脑部疾病的病理学。
[背景]人类iPSC于2007年通过使用四种转录因子（Oct4，Sox2，Klf4和c-Myc）对皮肤成纤维细胞进行重编程而首次建立，并且表现出与胚胎干细胞（ESCs）相似的特征，包括其多能性和自我-更新（Takahashi等，2007； ...
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| Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
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Author:
Date:
2018-07-05
[Abstract] Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.
[摘要] 培养细胞的免疫细胞化学是用于确定细胞结构内蛋白质的组成和定位的常用且有效的技术。 然而，传统的培养细胞固定和染色方案不能有效地保存培养的细胞色素，长期专门用于形态发生转运的丝状伪足。 结果，进行了有限的机械审讯以评估其监管。 我们开发了一种用于培养细胞的固定方案，该方案保留了细胞质，允许对内源性和过表达的蛋白质进行免疫荧光分析，这些蛋白质定位于脆弱的细胞结构。
【背景】Cytonemes被分类为薄的（~200nm直径）基于肌动蛋白的丝状伪足，长度超过2μm，可以转运形态发生素（Ramírez-Weber和Kornberg，1999）。这些信号结构首先在发育中的 Drosophila 翼成像盘中进行了详细分类和描述，随后在小鼠，小鸡和斑马鱼模型生物中进行了观察（Ramírez-Weber和Kornberg，1999; Sanders et al。，2013; Stanganello et al。，2015）。在大多数情况下，只有对过表达的荧光标记蛋白进行实时成像才能进行细胞色素检测。由于传统的固定方案未能保存这些脆弱的细丝，因此对培养细胞的细胞色素的检查受到限制。这些并发症一直是决定在发育和组织稳态期间驱动细胞色素形成和功能的细胞机制以及确定这些过程是否在疾病中被破坏的限制因素。
为了克服这些限制，我们开发了一种基于修饰电子显微镜固定剂（MEM-fix）的方案，该方案可以保留培养细胞的细胞质。 ...
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| Chick Neural Tube Explant Culture
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Author:
Date:
2015-10-05
[Abstract] The neural tube explant culture technique allows in vitro culturing of small pieces of neural tissue isolated from e.g., chick or mouse embryonic tissue in a matrix of collagen for defined periods of time. This method can be used to study the effects of defined molecules on developmental processes such as neural progenitor proliferation and neuronal differentiation and/or survival. Since the explant material can also be prepared from embryonic tissue electroporated with expression vectors, this technique can be adapted to study gene function in the presence of specific environmental signals. Different regions of the neural tube can also be isolated during the dissection step, allowing specific regions of the neural tube to be studied separately. Here, we present a neural ...
[摘要] 神经管外植体培养技术允许在胶原基质中从例如小鸡或小鼠胚胎组织分离的小片神经组织的体外培养定义的时间段。 该方法可用于研究限定分子对发育过程如神经祖细胞增殖和神经元分化和/或存活的影响。 由于外植体材料也可以从用表达载体电穿孔的胚胎组织制备，该技术可以适于在特定环境信号的存在下研究基因功能。 也可以在解剖步骤期间分离神经管的不同区域，允许单独研究神经管的特定区域。 在这里，我们提出了我们已经在几个研究中使用的神经管外植体培养方法（Dias等人，2014; Lek等人，2010; Vallstedt， et al。，2005）。
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