这项研究是由基础科学研究发展计划（2013R1A1A2060930）和医学研究中心计划（2015R1A5A2009124）通过韩国国家研究基金会（NRF），通过科学，信息和通信技术部提供资金，以及未来规划的支持。这项研究也从病童医院（HK宋）初创工作资助。我们要感谢劳拉·巴维尔和萨拉金JS他们在编辑这个手稿和安德拉斯·纳吉博士提供G4卓制线出色的帮助。

注:在无血清和无饲养-CM集合协议的示意图示于图1。 1.材料（MEF中的制备，中，印版和解决方案） <ol><li>制成500毫升之培养基来培养的MEF。有10％FBS（ESC质量），50单位/ ml青霉素和50mg / ml链霉素补充的Dulbecco氏改良的Eagle氏培养基（DMEM）。 </li><li>从胚胎以下既定程序协议13 MEF中隔离和维护他们MEF培养基。 </li><li>制成500毫升之培养基培养的mESCs。的DMEM补充有15％FBS和2mM的L-谷氨酰胺，100μM的非必需氨基酸（NEAA），100μM的β巯基乙醇，100单位/ ml白血病抑制因子（LIF），50单位/ ml青霉素，和50毫克/毫升链霉素。 </li><li>通过用5毫升0.1％的明胶溶液涂层10 1cm池培养皿制备糊化板（5糊化板/ 1 MESC板）。孵育至少10分钟<html>在室温下。 </li><li>制成500毫升之低血清培养基用于mESCs的无血清状态。补充低血清介质使用1.2克碳酸氢钠（pH 7.0）中。通过0.2μm瓶顶过滤器进行过滤。 </li><li>制备衰老相关β半乳糖苷酶（SAβ半乳糖苷酶）用于检测衰老细胞的染色溶液:1毫克/毫升X-gal的（溶解在二甲基甲酰胺，DMF）中，40 mM柠檬酸/磷酸钠缓冲液（pH 6.0） ，5mM的铁氰化钾，5mM的亚铁氰化钾，150毫摩尔NaCl和2mM MgCl 2的14。 注意:（危险）DMF是一种有毒和腐蚀性的解决方案。处理解决方案时穿戴个人防护服（ 例如，腈或乳胶手套，实验室外套，和护目镜）。使用通风橱。 </li><li>制成500毫升之培养基培养的人皮肤成纤维细胞（HDFS，NHDF-AD-DER-成纤维细胞）。补充的DMEM含10％FBS和100单位/ ml青霉素和100mg / ml链霉素。 </li></ol>jove_title"&gt; 2。小鼠胚胎干细胞培养（图1A和2A） 注:执行在细胞培养生物安全罩的所有步骤。 <ol><li>治疗的MEF用20ml含有在15厘米的细胞培养皿10微克/毫升丝裂霉素C的MEF培养基的。孵育在37℃下2小时和5％ 的 CO 2。 </li><li>吸由MEF培养基。洗细胞用PBS洗3次。加入3 ml胰蛋白酶- EDTA（TE，1×）的孵育在37℃下3分钟和5％ 的 CO 2。 3分钟后，中和在TE用6ml MEF培养基和离心机在300×g下3分钟。 </li><li>悬浮在5毫升MEF培养基。使用台盼蓝染色和血细胞计数器确定在所得的细胞悬浮液中的细胞数。板灭活的MEF（馈线）在MEF培养基每10厘米细胞培养皿2×10 6个细胞的密度。孵育在37℃下24小时和5％ 的 CO 2。 </li><li>电镀的进纸器（上后，更换与卓制介质MEF培养基24小时次日）。 </li><li>板以2×10 6个细胞的与卓制介质供给器的密度在mESCs（G4 F1杂种ES细胞）。孵育在37℃下48小时和5％ 的 CO 2。 注:卓制-CM的抗老化作用是当低传代次数mESCs使用15-17可能更强。我们Lunenfeld-的Tanenbaum研究所，西奈山医院，25奥德街，多伦多，M5T 3H7，加拿大获得了G4卓制线由安德拉斯·纳吉博士。 </li><li>使细胞保持在在70-80％的子铺满状态相对高的密度和通道。每天更换新鲜卓制培养基中。 </li></ol> 3.血清的收集和无饲养条件培养基（图1B和2B） 注:执行在细胞培养生物安全罩的所有步骤。 <ol><li>用5毫升的PBS冲洗卓制板。加入1ml的TE（2.5倍）的孵育在37℃下3分钟和5％ 的 CO 2。 3分钟后，中和在TE用2ml卓制mediu的米和离心机在300×g下3分钟。 </li><li>悬浮在5ml MESC介质和板1毫升到卓制介质每明胶涂层培养皿（5糊化板/ 1 MESC板）。达到培养在37℃和5％的CO 2，直至80-85％汇合。 </li><li>洗mESCs具有足够的PBS覆盖于每次洗涤10分钟，将细胞（8毫升，每10cm平板），总共洗涤三次。孵育在低血清培养基在37℃，24小时，5％的CO 2。 注:洗涤步骤是很重要的，以防止FBS的污染。它遵循孵育时间18,19是重要的。 </li><li>收集MESC-CM到50ml锥形管中并离心以2500×g的20分钟。收集离心后上清液溶液（CM）。通过0.2μm瓶顶过滤器进行过滤。 </li></ol> 4.小鼠胚胎的影响干细胞条件培养基（MESC-CM） 注:卓制-CM的效应通过几种方法，诸如验证SAβ半乳糖苷酶测定，细胞周期分析，和定量RT-PCR。 <ol><li> SAβ半乳糖苷酶测定（图3A） <ol><li>种子的HDF在HDF介质每孔2×10 4个细胞在6孔板的密度。在37℃孵育过夜，5％ 的 CO 2。 </li><li>继隔夜培养后，HDF介质丢弃一半，并添加MESC-CM和控制网上平台。孵育在37℃下72小时和5％ 的 CO 2。控制介质从无血清培养基中在没有mESCs的明胶包被培养皿（低血清媒体）的。 </li><li>清洗细胞具有足够的PBS覆盖于每次洗涤30秒将细胞（2毫升，每6孔板），总计两次洗涤的。添加3.7％多聚甲醛（PFA）进行固定。孵育在室温下5分钟。 注意:（危险）多聚甲醛是一种有毒和腐蚀性的解决方案。处理解决方案时穿戴个人防护服（ 例如，腈或乳胶手套，实验室外套，和护目镜）。使用通风橱。 </li><li>吸出固定的解决方案。洗涤固定的细胞用PBS洗涤两次，如在步骤4.1.3说明。 </li><li>添加该SAβ-gal染色溶液（1-2毫升，每孔在6孔板）。在37℃下孵育17.5小时。 注意:它不以在CO 2培养箱中培养。 </li><li>吸出的SAβ-gal染色溶液和洗涤用PBS细胞两次，如在步骤4.1.3说明。 </li><li>添加反染色曙红的解决方案。孵育在室温下5分钟。用PBS洗细胞两次，如在步骤4.1.3说明。 </li><li>利用使用连接数码相机进行后续分析光学显微镜拍摄图像的图像细胞放大100倍。 注:细胞总数可以以盲方式进行计数并计算SAβ半乳糖苷酶阳性蓝色的细胞的百分比。 </li></ol></li><li> 细胞周期分析（图3B） <ol><li>在密度种子的HDF8×10 4个细胞每孔中的HDF介质将6cm细胞培养皿。在37℃孵育过夜，5％ 的 CO 2。 </li><li>继隔夜培养后，HDF介质丢弃一半，并添加MESC-CM和控制网上平台。孵育在37℃下24小时和5％ 的 CO 2。 </li><li> Trypsinize细胞，如在3.1中描述的步骤，和离心机在300×g下5分钟。用冷PBS溶液洗涤细胞（PBS用0.5mM的CaCl 2和2％FBS的1毫升每1.5mL管中）两次并离心在2500×g下3分钟。重悬在100μl冷PBS溶液。 </li><li>通过滴加冷乙醇200微升，同时涡旋固定细胞。储存在4℃下至少1小时。 </li><li>洗用冷PBS溶液中的细胞两次，如在步骤4.2.3说明。 </li><li>悬浮细胞中的柠檬酸钠缓冲液含有50微克/毫升RNA酶250微升（1.12％，pH值8.5）。在37℃下孵育30分钟。 </li><li>加入250微升柠檬酸钠含缓冲器50微克/毫升的碘化丙啶。孵育在室温下20分钟。 </li><li>测量10,000个细胞使用流式细胞仪20的每个样本。 </li></ol></li><li> 定量RT-PCR（图3C） <ol><li>种子的HDF并添加MESC-CM，如步骤4.1.1和4.1.2所述。 </li><li>隔离根据制造商的方案使用RNA提取试剂盒的HDF总RNA。量化使用分光光度计21所提取的总RNA。 </li><li>合成cDNA通过添加总RNA的1微克到20μl包含寡聚（dT）引物和M-MLV逆转录酶的反应混合物，根据生产商的方案20。 </li><li>测量cDNA的扩增用实时PCR机，采用绿色PCR主混合物和基因特异性引物（ 补编 表1）。与正常化GAPDH表达的数据。使用以下PCR协议:初始德复性在95℃10分钟; 45个循环15秒，在95℃，20秒55℃，和72℃35秒;并在95℃下，在60℃下1分钟，95℃30秒，并在60℃下15 5秒15秒的熔融曲线的阶段。 </li></ol></li></ol>

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The authors have nothing to disclose.

对于无血清和MESC-CM无饲养成功集合，下面的建议，应考虑。最关键的因素是使用早期通道mESCs为MESC-CM的集合。以前，已经显示，相比于晚期通道mESCs早期传代MESC-CM具有更好的抗衰老作用。 mESCs的通道数目已经报道影响其发育潜力16和多能性17。 而分析MESC分泌蛋白质，从而诱发抗衰老效应的具体因素，需要更多的研究，我们目前可以断定MESC-CM足以在细胞水平降...

这个协议的目标是从无血清和无饲养培养条件收集小鼠胚胎干细胞（MESC）-conditioned培养基（MESC-CM）和表征其生物学功能。 在一般情况下，胚胎干细胞（ESC）由于它们的多能性和产能自我更新1-3具有用于再生医学，细胞疗法的巨大潜力。然而，干细胞直接移植有几个限制，如免疫排斥和肿瘤形成4,5。因此，无细胞的方法可提供用于再生医学和老化干预6,7-替代的治疗策略。 衰老被视为蜂窝对口组织器官的老化，其特点是生长停滞，改变细胞的生理和行为的永久状态。老化为流行的疾病，包括癌症，心血管疾病，叔的主要危险因素YPE 2型糖尿病，和神经变性8。一个老龄化的明显特征是组织的再生潜能，这是由干细胞衰老和疲惫9造成的下降。许多显著研究显示药理分子，如雷帕霉素9，白藜芦醇10，和二甲双胍11，和血源性系统性的因素，即GDF11 12中，具有一致延缓衰老和延长寿命的能力。 在本研究中，卓制-CM已经收获无血清（胎牛血清，FBS）和馈线（小鼠胚胎成纤维细胞，MEF中）的层以排除血清因子和由MEF分泌因子的污染。这些条件允许的无血清和无饲养的CM那因此启用的MESC特异性分泌因子的准确识别。 这项拟议的协议效率高，相对成本效益，易于进行操作。该技术提供了见解的MESC衍生的可溶性因子表征可以介导的抗衰老作用，其可用于向干预一个安全的和潜在有利无细胞的治疗方法的开发对于衰老相关疾病和其它再生治疗。

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>#11960-044</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>#30044333</td><td>20%, ES cell quality</td></tr><tr><td>Penicillin and streptomycin</td><td>Invitrogen</td><td>#15140</td><td>50 units/ml penicillin and 50&amp;nbsp;mg/ml streptomycin</td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>#25030</td><td>2&amp;nbsp;mM</td></tr><tr><td>Nonessential amino acids (NEAA)</td><td>Invitrogen</td><td>#11140</td><td>100&amp;nbsp;&amp;micro;M</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>#M3148</td><td>100&amp;nbsp;&amp;micro;M</td></tr><tr><td>Leukemia inhibitory factor&amp;nbsp;</td><td>Millipore</td><td>#ESG1107</td><td>100 units/ml</td></tr><tr><td>OPTI-MEM</td><td>Invitrogen</td><td>#22600</td><td></td></tr><tr><td>X-gal&amp;nbsp;</td><td>Sigma</td><td>#B4252</td><td>1 mg/ml</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td>3.70%</td></tr><tr><td>Dimethylformamide (DMF)</td><td>Sigma</td><td>#D4551</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>Aldrich</td><td>#455946</td><td>5 mM</td></tr><tr><td>potassium ferrocyanide</td><td>Aldrich</td><td>#455989</td><td>5 mM</td></tr><tr><td>NaCl</td><td>Sigma</td><td>#S7653</td><td>150 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>#M2393</td><td>2 mM</td></tr><tr><td>Mitomycin C</td><td>Sigma</td><td>#M4287</td><td>10&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>#P4170</td><td>50&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>TRIzol</td><td>Ambion</td><td>#15596018</td><td></td></tr><tr><td>M-MLV reverse transcript-tase</td><td>Promega</td><td>#M170B</td><td></td></tr><tr><td>Power SYBR Green PCR master mix&amp;nbsp;</td><td>Applied Biosystems</td><td>#4367659</td><td></td></tr><tr><td>HDFs, NHDF-Ad-Der-Fibroblast&amp;nbsp;</td><td>LONZA</td><td>#CC-2511</td><td></td></tr><tr><td>Bottle top filter</td><td>Corning</td><td>#430513</td><td>0.2 &amp;mu;m</td></tr></tbody></table>

collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

本来，mESCs保持在与FBS等补品（ 图1A和2A）卓制中的MEF饲养。的CM是由在低血清媒体mESCs收集无饲养层，FBS，或其它补充剂（ 图1B和2B）。这种文化条件允许我们从给料机，FBS，或其他补充的因素来收集MESC特定条件培养基没有潜在的污染。对照培养基在相同的培养条件下收集，无需mESCs。 <p class="jove_content" fo:keep-together.wit...

Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

这个协议提供了小鼠胚胎干细胞的血清衍生的集合（MESC）-conditioned培养基（MESC-CM）的方法（胎牛血清，FBS） - 和馈线（小鼠胚胎成纤维细胞，MEF中）用于小区-free条件 - 免费的做法。它可以是适用于老化和老化有关的疾病的治疗。

和无血清收集无饲养小鼠胚胎干细胞条件培养基的无细胞方法

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

collection-serum-feeder-free-mouse-embryonic-stem-cell-conditioned

Research

JoVE Journal

Developmental Biology

10.2K 次观看.  Yeungnam University. 这个协议提供了小鼠胚胎干细胞的血清衍生的集合（MESC）-conditioned培养基（MESC-CM）的方法（胎牛血清，FBS） - 和馈线（小鼠胚胎成纤维细胞，MEF中）用于小区-free条件 - 免费的做法。它可以是适用于老化和老化有关的疾病的治疗。 

视频: Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

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

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

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 ...

科研

发育生物学

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

免疫与感染

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

生物学

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

神经科学

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://app.jove.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)
	
	...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

一种从小鼠胚胎干细胞生成神经祖细胞的技术

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...