这项工作是支持部分由美国国立卫生研究院：HD054347和NS063997 - 01到VLS的。通过国务院卫生合同＃C024324到VLS的纽约帝国州干细胞基金中的一部分，这项工作也得到了支持。这里所表达的意见仅为作者个人的，不一定反映帝国干细胞，纽约州卫生署，纽约州的。 VLS的是一个多丽丝公爵临床科学家发展奖得主。我们也感谢他的反O1，反物理学抗体的礼物教授蒂莫西Vartanian。

1。神经干细胞培养的清扫和维护的解决方案和材料的制备<ol><li>提前准备100毫升夹层中（KNOCKOUT的DMEM/F12，Invitogen）和冷藏。 </li><li> Prepare100毫升培养液（干临NSC可持续森林管理，Invitrogen公司），并保持在37 ° C在水浴。 </li><li>准备长期长期冷冻保存的细胞的细胞冷冻培养基（KNOCKOUT的DMEM/F12 +10％胎牛血清+ 5％DMSO）。 </li><li>如果需要，准备为4％多聚甲醛（PFA）组织固定。 </li><li>消毒，灭菌镊子和手术刀片，带手柄用于清扫。 </li><li>抛开移液枪，10毫升转移移液器，和40微米的细胞过滤器解离（屋宇署猎鹰352340）。 </li><li>预留10厘米的几个文化菜解剖光盘（BD），50 mL离心管解离（BD），组织储存和冷冻小瓶冷冻细胞（BD）的1.5 mL离心管。 </li></ol> 2。隔离人类神经干细胞从人类胎儿大脑<ol><li>收获后，立即终止对胎儿的可行的组织是手术成功的关键。选修的程序，定时获取样品可以提前安排，以尽量减少胎儿死亡后的时间。概念产品收获后2小时内的程序，但最好可以在几分钟之内的选任程序实现。胎儿组织中，往往是支离破碎。然而，在一般情况下，大脑的很大一部分仍然完整的视觉识别。胎龄（GA）的限制是由成文法，但已使用此协议，GA 18-22周之间。 </li><li>胎儿的大脑被放置在一个10毫升的Petri菜含有冰冷的基因敲除的DMEM/F12解决方案。识别解剖标志的皮质的不同部位。正面和parieto枕部皮层的边界推断中央沟和裂裂交集为导向。从中央沟前额叶皮层和沿边境的手术刀裂裂的解剖组织，一定要保持完好的心室和完好。 </li><li>从额叶皮层的分离块删除任何残留的血液和脑膜。如果样品是足够的质量，它是理想的解剖成几个较小的样本，作各种用途的块：部分（固定在4％多聚甲醛，煤灰）和蛋白质/基因检测（快于-80℃冷冻）。 </li><li>选定的脑块转移到50 mL离心管中，并加入组织的货量约3倍，冰冷的基因敲除的DMEM/F12解决方案。轻轻机械吹打分离10毫升移液管组织，直到所有的组织变得支离破碎（一般为20-30倍），然后通过一个40微米的细胞滤网（屋宇署猎鹰352340）过滤的细胞获得单或接近的单细胞停牌。 </li><li>在2000转和室温离心5分钟，细胞悬液，在10毫升的新鲜温暖的培养液（干临NSC SFM），重悬细胞沉淀，血球计数细胞数。 </li><li>添加到5毫升的温暖的培养液， 每 25平方厘米培养瓶和 2X10 6细胞转移到每个烧瓶中。文化是保持在37 ° C / 5％的CO 2培养箱中培养1周前分析。更改为进一步培养或实验一周一次中期的一半。 </li></ol> 3。神经干细胞的进一步鉴定或experimentations Manipulaton <ol><li>神经球通常建议文化与NSC直径200至400微米不等的条件下形成1至2周。在此阶段神经球，可解离为15分钟获得单细胞为0.2 g / L的EDTA钙和镁的自由汉克斯介质（汉克斯）在37 ° C。细胞悬液纺在2000转，新鲜汉克斯冲洗，在温暖的亚文化的培养基replated。 </li><li>要开始分化，游离细胞均镀上涂层的盖玻片poly-D-lysine/laminin 1 1X10 5％盖玻片细胞（24mmX24mm）的密度。少突胶质细胞分化是通过维持细胞KNOCKOUT的DMEM/F12（Invitrogen公司，主，MD）2％B27（50X，Invitrogen公司，主，医师），10 ng / ml的碱性成纤维细胞生长因子100毫微克/毫升SHH +在10ng/ml，PDGF - AA 2天没有生长因子，然后切换到同一介质中，另外5天。神经元的分化是通过维持细胞击倒7天的DMEM/F12 +2％B27（50X）。星形胶质细胞的分化是通过击倒了一个星期的DMEM/F12 1％胎牛血清培养细胞。 </li><li>从预制神经球分离细胞，神经干细胞基因转染可以做。在这里，我们表现出神经干细胞与EGFP - C1转染，电。电EGFP - C1的构造是利用小鼠神经干细胞AMAXA Nucleofector套件（VPG1004）AMAXA Nucleofector设备（龙沙AAD - 1001）后，该公司的指令。总之，1 × 10 6细胞的5微克的DNA与100μL转染介质，混合和脉冲电后，细胞悬浮在神经干细胞维持进一步的文化媒介。转染细胞的分化与神经球后，电的3-4天的分解进行了处理，下面的步骤3.2中所描述的相同程序。 </li></ol> 4。冻结的神经干细胞和亚文化<ol><li>游离细胞悬液，离心和冻结介质悬浮1X10 7细胞/瓶/毫升的浓度。慢慢冻结的细胞在-20℃，-80 ° C，然后转移存放时间过长，以液态氮。 </li><li>这些细胞快速解冻37 ° C水浴和悬浮在温暖的DMEM/F12 10％血清一洗，离心除去冷冻介质，并在温暖的培养基中悬浮。 </li></ol> 5。代表性的成果： 下面描述的方法和神经球，可以看到一个圆形，光滑的边框和相当同质的大小（图2a）一周后，从18孕周的正常胎儿的神经干细胞培养。这些神经球，可转与EGFP - C1或其他结构和荧光显微镜下（图2b）。成立神经球，然后用EDTA和镀涂盖玻片分散的细胞分离。 4％parafamaldehyde，并根据各自的协议分化的细胞固定与不同的细胞类型特异性标志物染色。 Multipotentiality观察与表达的标记指示（Fig2C D，若丹明）星形胶质细胞神经​​元（Fig2E，女，罗丹明）和少突胶质细胞（Fig2G H，若丹明）。没有经历电EGFP的细胞也分化成不同细胞类型和不同的细胞特异性标志物染色。 Multipotentiality观察与表达的标记指示（Fig3A，B fluoroscein）星形胶质细胞神经​​元（Fig3C，若丹明和Fig3D，fluoroscein）和少突胶质细胞（Fig3E，女，若丹明）。 <img src="/files/ftp_upload/2681/2681fig1.jpg" alt="图1"/> 隔离丢弃人类胎儿大脑的神经干细胞的实验过程示意图图1 。 <img src="/files/ftp_upload/2681/2681fig2.jpg" alt="图2"/> 图2，未分化和分化的人类神经细胞，在体外propogated。 （一）神经球相差显微镜下表现出光滑，圆形的边界和文化的快速增长后1个星期以上。 （二）各种质粒和构造的介绍，可以通过转。三天EGFP - C1转染，多个单元格显示fluoroscein免疫荧光显微镜下看到的绿色荧光蛋白的表达。 EGFP - C1转染神经球是分离和分化成神经元（C，D），星形胶质细胞（E，F）和少突胶质细胞（G，H）的不同条件下，罗丹明荧光下所见。同时，转染EGFP的阳性细胞fluoroscein荧光下显示。转染细胞（白色箭头头），未转染细胞（白色箭头）无异。 Hoechst33342细胞核染色。比例尺为200μm为A，B的100微米和25微米的CH。 <img src="/files/ftp_upload/2681/2681fig3.jpg" alt="图3"/> 图3。神经球未转分离和分化成神经元（A，B fluoroscein），星形胶质细胞和少突胶质细胞（C，D，若丹明，fluoroscein）（E，F，若丹明）不同条件下。 Hoechst33342细胞核染色。比例尺是25微米的AF。

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是对文化的新鲜组织和人类细胞系生产的各种方法。从历史上看，已经收获和新鲜组织培养，立即在中枢神经系统中产生的各种细胞类型。然而，这种做法显然是有限的，可这在人类样本的情况下，通常是相当小的样本数量。鉴于操纵的最小程度，新鲜培养的神经细胞提供最可靠的实验系统，通过限制延长培养潜在的工件。供应有限导致的第二种方法产生人体细胞线，已通过插入各种癌基因12...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> KNOCKOUT的DMEM/F12 </td><td> Invitrogen公司</td><td> 12660-012 </td><td>解离中等</td></tr><tr><td>干临国科会可持续森林管理</td><td> Invitrogen公司</td><td> A10509 - 01 </td><td>培养基</td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 10091-148 </td><td>冷冻介质</td></tr><tr><td> Hanks液（ 钙，镁2） </td><td> Invitrogen公司</td><td> 14175-095 </td><td>解离中等</td></tr><tr><td>二甲基亚砜</td><td> Sigma - Aldrich公司</td><td> D2650 </td><td>冷冻介质</td></tr><tr><td> EDTA </td><td> Sigma - Aldrich公司</td><td> 431788 </td><td>解离中等</td></tr><tr><td>多聚甲醛</td><td> Sigma - Aldrich公司</td><td> 158127 </td><td>固定的解决方案</td></tr><tr><td>碱性成纤维细胞生长因子</td><td>研发</td><td> 234 - FSE </td><td>分化培养基</td></tr><tr><td> SHH </td><td>研发</td><td> 1845 - SH </td><td>分化培养基</td></tr><tr><td> PDGF - AA </td><td>研发</td><td> 221 - AA </td><td>分化培养基</td></tr><tr><td> B27 </td><td> Invitrogen公司</td><td> 17504-044 </td><td>分化培养基</td></tr><tr><td>鼠抗- MAP2 </td><td> Sigma - Aldrich公司</td><td> M2320 </td><td> 1:200 </td></tr><tr><td>兔抗- DCX </td><td>细胞信号转导</td><td> 4604s </td><td> 1:200 </td></tr><tr><td>兔抗GFAP </td><td> DAKO公司</td><td> Z0334 </td><td> 1:200 </td></tr><tr><td>兔抗- S100B </td><td> DAKO公司</td><td> Z0311 </td><td> 1:200 </td></tr><tr><td>兔抗O1群</td><td>教授Timothy Vartanian礼物* </td><td></td><td> 1:50 </td></tr><tr><td>兔抗- O4 </td><td>教授Timothy Vartanian礼品* </td><td></td><td> 1:50 </td></tr><tr><td> 40微米细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr></tbody></table> *提摩太Vartanian，医学博士，哲学博士，美国威尔康乃尔医学院神经病学和神经科学，纽约，系

generation of neural stem cells from discarded human fetal cortical tissue

神经干细胞（NS​​Cs）居住在沿脑室区神经上皮的皮质板的发展。这些早期的祖细胞，最终上升到中间的祖先，后来，大脑皮层的各种神经元和神经胶质细胞亚型。发电量和扩大人类神经干细胞从废弃的正常胎儿组织（所谓的神经球）提供了一种手段，用以直接研究人体正常的国科会发展 1-5的功能方面。这种方法也可以直接向从已知的神经系统疾病的神经干细胞的生成，从而给予的机会，以确定疾病的进程，改变祖细胞的增殖，迁移和分化6-9。我们一直专注于识别人类唐氏综合征的神经干细胞可能有助于加速老年痴呆症的疾病表型10,11的病理机制。无论在体内还是在体外培养的小鼠模型，可以复制的基因位于人类21号染色体上的相同的剧目。 这里我们使用一个简单而可靠的的方法隔离综合征从流产的人类胚胎皮层神经干细胞，并在文化中成长。该方法提供有限的解剖标志，细胞分选，电镀和人类神经干细胞传代收获组织，解剖的具体方面。我们还提供一些基本的协议更有选择性的细胞亚型的人类神经干细胞诱导分化。

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

描述一个简单和可靠的方法是从丢弃的人类胚胎皮层组织的神经干细胞的分离和培养。从已知的人类神经系统疾病的病理细胞和分子过程的特性，可用于文化，以及提供一个平台，以评估药理药效。

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

generation-neural-stem-cells-from-discarded-human-fetal-cortical

Research

JoVE Journal

Neuroscience

15.4K 次观看.  Beth Israel Deaconess Medical Center. 描述一个简单和可靠的方法是从丢弃的人类胚胎皮层组织的神经干细胞的分离和培养。从已知的人类神经系统疾病的病理细胞和分子过程的特性，可用于文化，以及提供一个平台，以评估药理药效。

视频: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

科研

神经科学

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...