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。

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			<td height="26" style="height:26px;width:64px;">MgCl2&#160;</td>
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			<td height="43" style="height:43px;width:64px;">Mytomycin C&#160;</td>
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			<td style="width:64px;">#M4287</td>
			<td style="width:64px;">10ug/ml</td>
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			<td height="43" style="height:43px;width:64px;">Propidium iodide&#160;</td>
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			<td style="width:64px;">50ug/ml</td>
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			<td height="43" style="height:43px;width:64px;">TRIzol</td>
			<td style="width:64px;">Ambion</td>
			<td style="width:64px;">#15596018</td>
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			<td height="85" style="height:85px;width:64px;">M-MLV reverse transcript-tase</td>
			<td style="width:64px;">Promega</td>
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			<td height="127" style="height:127px;width:64px;">Power SYBR Green PCR master mix&#160;</td>
			<td style="width:64px;">Applied Biosystems</td>
			<td style="width:64px;">#4367659</td>
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			<td height="106" style="height:106px;width:64px;">HDFs, NHDF-Ad-Der-Fibroblast&#160;</td>
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			<td style="width:64px;">Corning</td>
			<td style="width:64px;">#430513</td>
			<td style="width:64px;">0.2&#956;m</td>
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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

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

在无馈体和化学定义的培养条件下，使用人类多能干细胞的后多能感感神经元的有效分化

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...