Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1.媒体准备注:协议依赖于使用两个单独的媒体混合的:DMEM / F12补充有改性N2添加剂和的Neurobasal补充有B27添加剂，典型地以1:1的比例。 <ol><li>通过混合各组分在一个15毫升管制备改性N2添加剂。不要涡旋或过滤这一点;通过倒转该管，直到溶液澄清混合。 <ol><li>开始吹打7.2毫升的DMEM / F12的，然后加入1毫升25毫克/毫升胰岛素（由在0.01M的HCl中），通过颠倒试管混匀。这需要几分钟的时间，直到解决方案是明确的。 </li><li>加入1毫升100毫克/毫升脱辅基转铁蛋白（由水），33微升0.6毫克/毫升progesternone（由乙醇中），100微升160毫克/毫升（1μM）腐胺（由在水），10微升3毫亚硒酸钠（由在水中）和666微升7.5％的牛血清白蛋白和混合管井。分装成2.5毫升并储存在-20°下长达2个月。 </li></ol></li><li>准备媒体。 <ol><li>向250ml DMEM / F12的加入2.5毫升氮气。拌匀但不要摇晃或过滤器。 </li><li> 250ml的神经基础介质添加5毫升B27的补充。拌匀但不要摇晃或过滤器。 </li><li>混合来自步骤1.2.1和1.2.2媒体以1:1的比例。在某些情况下，以1:3的混合，可能需要（补充作为1:1混合在步骤1.2.4）。 </li><li>到混合添加0.5毫升L-谷氨酰胺（最终浓度为0.2mM）和3.5微升2-巯基乙醇（终浓度为0.1mM）拌匀不摇动。存储介质在4°C至3周，避免暴露于光。这最后的混音被称为N2B27。 </li></ol></li><li>制备明胶溶液。 <ol><li>准备在超纯水中和高压釜1％明胶溶液在121℃，15磅，15分钟。重要的是，用于该瓶是从洗涤剂或消毒剂完全干净，因此，建议一个新瓶是用于此，并且永远只漂洗在超纯水中使用之间。分装1％溶液到50ml等份，并保持在4℃下几个月。 </li><li>暖1％明胶的等分试样在37℃水浴中直至溶解，并添加至500ml温热的磷酸盐缓冲盐水（PBS）中的。拌匀，吸管，足以覆盖每个孔的底部或板。允许明胶涂布的表面为至少30分钟。 </li></ol></li></ol> 2.电镀的细胞注意:此协议同样适用于在10％血清与白血病抑制因子（LIF），血清替代品与LIF或无血清培养基用LIF和骨形态发生蛋白4（BMP4），或MEK和GSK3抑制剂（2I媒体）生长小鼠ESC有或不含LIF。然而，分化的时间和效率可以依赖于介质和细胞（见讨论）而有所不同。对于这里显示的实验中，我们使用的鼠标46C线ESC（即有绿色荧光蛋白记者撞到Ø内源性SOX1等位基因ね），生长在GMEM含10％血清和LIF。为了达到最佳效果，重要的是细胞解离和再接种在N2B27媒体;简单地从GMEM /血清/ LIF改变媒体N2B27总是导致降低效率的差异比较replating细胞。 <ol><li>生长中的细胞GMEM含10％血清，1mM丙酮酸钠，1×非必需氨基酸，0.1M 2-巯基乙醇，和100单位/ ml LIF 13。为了获取鼠标ESC，板的汇合培养1×10 6个细胞变成T25培养瓶中，这将需要2 - 3天。 </li><li>观察明场显微镜下的细胞。当细胞是亚汇合并准备通道，在PBS两次冲洗。加入1毫升细胞解​​离试剂，并返回到培养箱中2 - 5分钟。尽管胰蛋白酶和其它酶也可用于细胞分离，也可以有对细胞附着有负面影响这样的电镀密度将需要adjuSTED。 </li><li>一旦细胞开始从板抬起，挖掘船撞出成单细胞悬液。收集细胞成总体积为10ml的介质和细胞解离试剂和吸管入15ml离心管中。 </li><li>旋转细胞在300×g离心3分钟，在室温。 </li><li>小心吸出上清液，并通过上下抽吸3悬浮在10ml预热N2B27的粒料 - 5倍。当上下吹吸的悬浮液，吸移管对管的侧面，以避免产生气泡。计数细胞用自动细胞计数器或血球并记录浓度。 </li><li>准备将每孔预温N2B27镀含有量的细胞的期望数量的每单位的细胞悬浮液。每平方厘米10500细胞在6孔培养皿的密度（ 即，每6孔培养皿的孔1×10 5个细胞）是最佳的。 见表1每平方厘米2和PLA细胞的数量建议廷介质卷，适用于各种细胞培养容器。 </li><li>吸从培养容器中的明胶和板按照表1所建议的密度和体积的细胞悬浮液。不要摇动板，因为这将集中在细胞的中心。放置在潮湿的培养箱中培养，在37℃，5％的CO 2。 </li><li>用新鲜的N2B27全部更换介质2日 - 改变媒体每1。吸管轻轻用作冲洗细胞可能会影响密度，减少分化的效率在接下来的几天。其中，电镀后的初始生存能力较差，板上的细胞以1:3的混合N2和B27为辅媒体和初二后更改为N2B27。细胞将开始分化，并应表现出SOX1表达式中4（经记者在这里使用的46C细胞系绿色荧光显示） - 6天。 </li></ol> 3.免疫荧光染色 NOTE:该协议可以被应用到任何容器类型。各试剂的体积每孔中描述的6-孔板中并可以扩展到任何表面区域。 Replating不建议由于低可行性之后。 <ol><li>除去分化培养基，并通过添加500微升4％（体积/体积）的多聚甲醛固定细胞，进行15分钟离开。 </li><li>洗和透化的细胞用2ml PBS-T（磷酸盐缓冲盐水含有0.5％（体积/体积）吐温20）洗涤两次。细胞可以保持在PBS-T中长达2个月，在4℃。 </li><li>更换PBS-T用1ml的PBS-T，用10％（体积/体积）的血清（来自相同物种作为第二抗体）。孵育1小时在板摇杆在RT以阻止任何非特异性结合。 </li><li> 1小时后，用2ml的PBS-T洗细胞两次，并孵育在打击βIII微管蛋白500微升小鼠IgG（稀释1:1000于PBS-T的10％血清）。把上盘摇杆，并留下O / N在4℃。 </li><li>从这个步骤，始终保护血管从光。用PBS-T清洗细胞两次，然后加入500μl荧光标记的抗小鼠IgG抗体（稀释至2微克/毫升在PBS-T中，用10％血清）。把它放在板摇杆1小时，在室温。 </li><li>用PBS-T冲洗细胞两次，然后加入500μl的DAPI（稀释至0.5微克/毫升在PBS-T）和离开5分钟。 </li><li>再次洗涤细胞，并让他们在PBS-T。细胞准备要被拍摄，并且可以保持长达2周，在4℃。注意，GFP信号消失时间的推移所以是不适当的比较固定的和活细胞的细胞固定在不同的时间或GFP的强度。 </li></ol>

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The authors have nothing to disclose.

单层神经分化协议已经使用了十多年6。该协议是高效率的，由确定的培养基，并在单层系统，使该系统更适用于临床前完成（ 例如，药物筛选）使用。然而，也有确定分化效率一些关键的因素。本文指出的那些因素，并针对每个障碍物的溶液。 电镀在分化条件后密度的细胞可能是最关键的因素。在太高的密度铺板细胞减少分化的效率，同时在太低的密度电镀?...

胚胎干细胞是从早期胚胎与体外无限增殖的能力，同时保留分化成以下再引入适当阶段胚胎所有成年细胞类型（通过形成嵌合体的能力），注射到同基因或免疫妥协的宿主衍生的多能细胞（通过形成畸胎瘤）或体外受到适当的线索1。 在体外分化的小鼠胚胎干细胞分化为神经谱系的于1995年首次被描述和所涉及的形成多细胞悬浮聚集体（胚体，胚）在补充有形态发生维甲酸2-4含血清介质。自那时以来，各种协议已被开发以允许神经分化5。许多人仍利用聚合，更为共培养与诱导细胞类型和几个涉及使用无血清介质。所有的协议有优势第二缺点和神经的确切性质或神经细胞产生的也有所不同，根据使用的协议。 理想的协议将是健壮的，可扩展和使用完全确定的培养基和基板，是适合于非侵入性监控分化过程的，并导致在神经祖细胞的纯群体的产生能够由外部线索和分化来图案成所有的神经元和神经胶质亚型具有高效率和产率在相当短的时间。在过去的十几年，我们一直在使用，用于从小鼠ESC神经祖细胞和神经元密度低，无血清贴壁单层培养6-10的方法。这个协议来完成许多上文所列的标准:在我们手中分化的效率已经相当一致多年来和各种细胞系，它可被放大或缩小（我们成功地使用从96孔板的船只直径15厘米迪畲族）和所使用的介质是良好定义的。这个过程是适合于缩时显微术为分化的监测和各种图案形成线索可加入以诱导不同类型的神经元亚型的的产生（ 例如，Shh和FGF8为多巴胺能神经元6）。 尽管如此，还是有一些挑战这一协议的成功应用。其中一个重要方面是精心准备的媒体。我们始终准备介质内部尽管商业替代品的可用性。其中所使用的添加剂的（N2;见协议 ）有修改过的标准的商用版本。最后，这种方法的成功应用的最重要的步骤之一是在电镀的最优的细胞密度。这主要是因为，而诱导信号中的一个（FGF4 11）的自分泌性质要 ​​求足够细胞存在，以允许最佳viabil性和分化，在太高的密度分化受损（可能部分是由于自分泌生产LIF 12）。因此重要的是，这两个介质制备和细胞电镀仔细进行和一致，以确保最佳的效果。

<table><tbody><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>10270-106</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-074</td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Life Technologies</td><td>A11105-01</td><td></td></tr><tr><td>B-27 Supplement, serum free</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I6634</td><td>Reconstitute with sterile 0.01 M HCl</td></tr><tr><td>Apo-transferrin</td><td>Sigma</td><td>T1147</td><td>Reconstitute with sterile water</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td>Reconstitute with ethanol</td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td>Reconstitute with sterile water</td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>S5261</td><td>Reconstitute with sterile water</td></tr><tr><td>Bovine albumin fraction V</td><td>Life Technologies</td><td>15260-037</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030-081</td><td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td></tr><tr><td>Gelatine</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>6-well tissue culture dish</td><td>Thermo Scientific</td><td>140675</td><td></td></tr><tr><td>24-well tissue culture dish</td><td>Thermo Scientific</td><td>142475</td><td></td></tr><tr><td>96-well tissue culture dish</td><td>Thermo Scientific</td><td>167008</td><td></td></tr><tr><td>GMEM</td><td>Life Technologies</td><td>11710-035</td><td></td></tr><tr><td>MEM Non-essential amino acids solution</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Leukaemia inhibitory factor</td><td>prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td><td></td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma</td><td>D9542</td><td>Protect from light</td></tr><tr><td>Mouse anti &amp;beta;III tubulin IgG antibody</td><td>Covance</td><td>MMS-435P</td><td></td></tr><tr><td>Fluorescence-labelled anti-mouse IgG antibody</td><td>Life Technologies</td><td>A31571</td><td>Protect from light</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask</td><td>Thermo Scientific</td><td>156367</td><td></td></tr></tbody></table>

neural differentiation of mouse embryonic stem cells in serum-free monolayer culture

分化的小鼠胚胎干细胞的能力（ESC）的神经祖细胞允许控制神经规范以及产生成熟的神经细胞类型的进一步研究的机制的研究。在这个协议中，我们描述了ESC的使用无血清，单层培养神经祖细胞分化的方法。该方法具有可扩展性，高效率和结果在4内生产70％的神经祖细胞〜 - 6天。它可以从在各种条件下生长的各种菌株施加到ESC。神经祖细胞可以允许进一步分化为功能性神经元和神经胶质细胞或分析用显微镜，流式细胞仪或分子技术。分化过程是适合于延时显微镜，并且可以与使用记者行监测神经规范过程进行组合。我们提供培养基制备和细胞密度优化的详细说明，使该过程为bë施加到最ESC线和各种细胞培养容器。

在这个实验中，我们使用的46C细胞系14，小鼠胚胎干细胞与内源SOX1-GFP报告，以跟踪神经分化。通过使用该细胞系SOX1，一个标记为神经祖的，表达，可以通过绿色荧光来检测。电镀密度是实现神经元分化的关键因素。小鼠胚胎干细胞以不同的密度从10500到88500个细胞/ cm 2的变化的铺于6孔板中。 图1A示出了由不同的电镀密度在一个6孔板来实现分化效率。对分化的第6天，?...

Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

14.0K 次观看.  University of Dundee. This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

视频: Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

科研

发育生物学

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

生物学

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

神经科学

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Source: Wongpaiboonwattana, W., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52823">Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture.</a> J. Vis. Exp.(2015)
The video demonstrates a protocol that uses serum-free media to generate neural progenitors from mouse embryonic stem cells. The desired concentration of embryonic stem cells, suspended in serum-free media, is plated on gelatin-coated coverslips. At an optimal cell density, cells produce autocrine signaling factors, which, along with the neural differentiation-inducing factors in the serum-free media, trigger the differentiation of embryonic stem cells into neural progenitors.

在无血清单层培养物中从胚胎干细胞生成神经祖细胞

1. Media Preparation
NOTE: The protocol relies on using a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://appjove.unicartagenaproxy.elogim.com/v/56358">Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors.</a> J. Vis. Exp. (2017)
This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

从小鼠胚胎干细胞产生皮质中间神经元前体

1. Media Preparation
NOTE: Warm all media to 37 &#38;#176;C before use in cell culture.

	Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	
	...

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

将小鼠胚胎干细胞分化为联网神经元群

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...