| Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
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Author:
Date:
2017-09-05
[Abstract] Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).
[摘要] 心脏神经嵴细胞（CNCC）起源于神经管的背部边缘，位于第3个体节的耳穴和尾缘之间，并迁移到咽弓和心脏。 我们已经表明，纤连蛋白（Fn1）通过调节CNCCs到咽弓动脉周围的血管平滑肌细胞的分化，在CNCC的发展中起重要作用（Wang and Astrof，2016）。 该方案描述了CNCC与神经管和尾尾弓的分离，以及神经嵴衍生细胞分化成平滑肌细胞。 该方案从（Newgreen和Murphy，2000; Pfaltzgraff等人，2012）改编。
【背景】以前发表的方案描述了从神经管分离神经嵴细胞。然而，在耳孔和第三体细胞之间的神经管区域中的神经嵴细胞包括有助于许多不同细胞类型的神经嵴细胞群体;例如，迷走神经嵴细胞也来自该区域。在该方案中，我们修改了用于分离心脏神经嵴细胞的常规方法。而不是使用神经管，我们在胚胎期（E）9.5（22-25个体节期）使用尾部咽部弓形区。这是在将心脏神经嵴细胞分化为血管平滑肌细胞之前。神经嵴培养物通常含有污染性间充质细胞，通常表达平滑肌基因。为了鉴定神经嵴衍生细胞，我们从以下交叉产生的胚胎中分离出神经嵴细胞：Fn1flox / flox; ROSAmTmG / mTmG雌性小鼠×Fn1 +/-;Tfap2αIRESCre/ ...
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| Retinal Differentiation of Mouse Embryonic Stem Cells
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Author:
Date:
2016-07-05
[Abstract] Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and ...
[摘要] 来自Yoshiki Sasai博士的实验室的开创性研究最近已经引入了将小鼠和人胚胎干细胞（mESC和hESC）区分为器官样3D结构的新方法，其旨在重现发育器官发生程序（Eiraku等人， ，2011; Eiraku和Sasai，2012; Nakano等人，2012; Kamiya等人，2011）。 我们利用这种方法优化3D协议以有效地生成视网膜祖细胞和随后视网膜神经元体外。 这种文化系统提供了一个宝贵的平台，既研究早期发展过程，并获得视网膜神经元的移植方法。 这里描述的协议已成功应用于几个鼠标ESC（包括R1，WD44和G4细胞系）和小鼠诱导多能干细胞（iPSCs）线。
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| EML Erythroid and Neutrophil Differentiation Protocols
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Author:
Date:
2014-06-20
[Abstract] Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al. ...
[摘要] 红细胞 - 髓样淋巴细胞（EML）是能够在SCF（干细胞因子）存在下在体外长期维持的小鼠骨髓来源的多潜能造血细胞系et al。，1994）。 EML细胞系的自我更新能力通过最初通过逆转录病毒转导递送的显性 - 负性视黄酸受体（RAR）的存在（Tsai等人，1994）来赋予，其捕获细胞在阻止从正常进展成骨髓分化的早期祖细胞阶段。 RAR转基因的存在不干扰红细胞分化，并且可能在维持培养中捕获低百分比的早期红细胞而不是骨髓，定型细胞（Pina等人， 2012; Ye等人，2005）。通过使用高剂量的视黄酸（RA），细胞可以被驱动进入粒细胞/嗜中性粒细胞分化，其克服了分化阻断。应当注意，RA的这些药理学剂量与红细胞分化不相容，因此在相同测定中获得稳定的红细胞和骨髓分化是不可行的。实际上，在CFC测定中作为混合谱系评分的集落是未分化和类红细胞的混合物（Tsai et al。，1994）。然而，在如下所述的确定的细胞因子条件下的液体培养中可以获得稳定的单谱系红细胞和嗜中性粒细胞分化。
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