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 border="0" cellpadding="0" cellspacing="0">
	<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 0.01M 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>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</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 colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII 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>25cm2 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

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

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

科研

发育生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

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 ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

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 ...