Name: stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates
Uploaded: 2016-06-17T14:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates at JoVE.com

这项工作是由新加坡国立大学（R-397-000-192-133）和ETPL峡基金（R-397-000-198-592）支持的启动资助。 GS是新加坡国立大学的研究学者。作者想感谢Jiangwa兴博士为她的细胞微技术支持。

备注:本协议描述PDMS模具的制造与1000微米的图案通过激光切割和图案化的人类胚胎干细胞系，H9使用PDMS模板。 1.设计和微图案PDMS模板的制作<ol><li>设计具有所需的几何形状和尺寸（ 例如 ，1000微米圈）和使用计算机辅助设计软件10模版垫片的贯通孔的制版薄板。 </li><li>激光切割的120-150微米和2毫米厚的聚二甲基硅氧烷（PDMS）片分别使用CO 2激光切割器10的制版薄板和垫片。 </li><li>粘结的PDMS垫片用液体未固化的PDMS和烘烤的PDMS蜡纸在60℃下3-4小时，以获得用于微图案的PDMS模具。 </li><li>消毒通过高压与PDMS模版，在120℃下，在使用前的烘箱30分钟并干燥。 </li></ol> 2.人类胚胎干细胞主tenance <ol><li>培养的hESC细胞系在无饲养维持培养基上1×的hESC合格基底膜基质涂覆的细胞培养板在37℃和5％的CO 2。 </li><li>通道的时候都大致由3分钟温育70％汇合，在37℃用分散酶处理和机械解离胚胎干细胞集落入100-200微米的团块的人类胚胎干细胞。 6分光比:30-50团块每生长面积9.6 平方厘米的基底膜基质涂层组织培养聚苯乙烯在1密度板材的人类胚胎干细胞团块。 </li></ol> 3.人类胚胎干细胞的模具微图案<ol><li> 准备细胞图案前 <ol><li>通过分配在超纯水700μl的70％的分析纯的乙醇放入培养皿，并把模版上顶部密封一个PDMS模版到60毫米的培养皿。 </li><li>将生物安全柜内培养皿一夜之间令t他乙醇干燥。 </li><li>检查模板下方存在无气泡，保证了模具形成具有培养皿良好的密封。这可以防止的ECM涂覆溶液泄漏。密封在无菌条件下在培养皿和存储直到它准备好细胞接种。 </li><li>据分析证书准备的hESC合格基底膜基质的等分。确保每个等分试样产率1×的hESC合格基底膜基质溶液在25ml的Dulbecco改良Eagle培养基中稀释时:营养混合物F-12（DMEM / F12），根据制造商的产品片。 </li><li>通过加入的hESC合格基底膜基质的一种分装成16.7毫升的DMEM / F12，以使1.5×的hESC合格基底膜基质溶液制备的ECM涂覆溶液。保持冰的所有ECM涂层解决方案，以防止凝胶化。 </li><li>人类胚胎干细胞的补充维持培养基用10μMROCK抑制剂（Y27632）。 </li></ol></li><li><s仲&gt; 的钢印基板上涂ECM <ol><li>对待培养皿100 WO 2等离子90秒。以确保无菌性，只打开等离子体处理之前的等离子体室内部的培养皿盖，并迅速覆盖背面治疗结束后的培养皿。这有利于表面润湿，并防止另外的ECM涂布溶液过程中的通孔的微图案形成气泡。 </li><li>添加450微升的1.5×的hESC合格基底膜基质溶液覆盖整个模具。密封培养皿具有自密封膜，如石蜡膜，以防止该溶液干涸，并在使用前于37℃孵育5小时。 </li></ol></li><li> 人类胚胎干细胞播种到钢印基板 <ol><li>检查在6孔板的hESC集落，以确定表示典型的hESC形态损失的分化的细胞区域（ 例如，圆形的，紧密PACKE的损失ð上皮形态，高核/质比核仁明显）。除去使用真空吸气器有区别的区域。 </li><li>以每孔2mL的DMEM / F12洗涤两次。 </li><li>加入1 ml消化酶，诸如的Accutase的，每6孔板的孔中并在37℃孵育8分钟。轻轻敲击板从基材分离所有殖民地。 </li><li>与至少4毫升每毫升消化酶的DMEM / F12的冲洗每个孔，并收集细胞悬液分成15毫升锥形管中。 </li><li>离心在200×g下将细胞悬浮液在室温下3分钟。 </li><li>抽吸除去上清，加入400微升补充有ROCK抑制剂（ROCKi）hESC细胞维持培养基来重新悬浮细胞。吸取细胞悬液上下轻轻3次打破团块成单个细胞。 </li><li>混合单细胞悬液好，稀释10微升细胞样本为190微升DMEM / F12（1:20稀释）的。使用hemocytome之三，以确定在股票细胞悬浮液的细胞密度。 </li><li>计算对于给定的模版所需的细胞接种密度。 注意:例如，我们已通过实验确定的细胞接种密度，以获得单细胞的汇合单层是约4,444个/ mm 2。因此，随着为450mm 2的面积和400微升的接种体积模版将需要一个细胞悬浮液以200万个细胞/ 400微升的密度。 </li><li>稀释股票细胞悬液到所需的接种密度（见步骤3.3.8注）补充了人类胚胎干细胞ROCKi培养基。 </li><li>添加包含所需数量的细胞到每个模版细胞悬浮液的指定体积和离开的培养皿在室温下在通风橱中不受干扰5分钟以使细胞沉淀。 </li><li>转移培养皿进入培养箱，孵育1小时，以允许细胞附着。请注意在保持培养皿水平转移过程中，使细胞保持在模板单层。 </li></ol></li><li> 模板拆除和无图案基板的钝化 <ol><li>在显微镜下检查培养皿，以检查是否细胞被适当地附着在下面的衬底。 </li><li>从模具吸走细胞悬液。 </li><li>加的在DMEM / F12 0.5％细胞培养物相容的非离子表面活性剂为2ml /皿周围模版的区域。 </li><li>使用一对蒸压镊子轻轻撕下模板。漩涡非离子表面活性剂溶液周围的模版被剥离，以防止细胞干燥。目视观察微图案化细胞在培养皿。 </li><li>孵育微图案化细胞在0.5％的非离子表面活性剂的DMEM / F12溶液在37℃10分钟。 </li><li>抽吸掉非离子表面活性剂的DMEM / F12溶液。用2ml / DMEM / F12的碟洗3次。 </li><li>人类胚胎干细胞加入2毫升/皿维持培养基补充有ROCKi并在37℃下孵育过夜。 </li><li>过夜孵育后，补充有ROCKi抽吸人类胚胎干细胞维持培养基，用DMEM / F12洗涤一次，并加入2毫升/分化培养基的培养皿补充有100纳克/毫升活化素，25纳克/毫升BMP4和10ng / ml的FGF2诱导mesoendoderm分化。 </li><li>评估使用相衬成像的微图案以及如前面8中所述计算平均的Brachyury（T）的强度分布为每个图案。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+substrate+stiffness+on+early+human+embryonic+stem+cell+differentiation.">Effect of substrate stiffness on early human embryonic stem cell differentiation.</a> J Biol Eng. 7 (1), 7 (2013).</li></ol>

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微图案模板的制作 模板缩微提供生成HPSC微图案调查利基介导的分化图案的理想方法。比其他图案化技术，例如微接触印刷和光图案模版图案形成的主要优点是，它不需要表面改性，可在常规的TCPS衬底来实现。因此，对于不同的HPSC线进行了优化培养基和ECM涂料可以很容易地转移到模版图案化。 有有助于良好微图案的产生的细胞模版的...

人多能干细胞（hPSCs），包括胚胎干细胞（胚胎干细胞）和诱导多能干细胞（人iPS细胞），被广泛利用于再生医学以及正常和病变器官的实验模型，因为它们的分化潜能的进入所有的细胞谱系三种胚层1,2。 hPSCs分化命运是可以调节通过物理线索3-5介导的自分泌或旁分泌信号1以及机械传导过程当地的环境因素非常敏感。细胞微图案包括一组已发展到在空间上组织的细胞群的几何形状和位置作为平均值来控制本地细胞微环境，如细 ​​胞-细胞相互作用6和细胞-基质相互作用3的技术。在hPSCs的背景下，细胞微已被用来获得显著的认识到如何利基依赖耳鼻喉科自分泌信号调节胚胎干细胞多能性分化的第7和组织成早期胚胎分化的模式6。二维和三维微图案hPSCs已被用来控制多细胞型态，这反过来又影响分化决定成三种胚层8,9的菌落尺寸。我们采用多HPSC微图案来调制HPSC菌落内的细胞-细胞和细胞-基质相互作用的程度来探测整联E-钙粘蛋白的串扰可以如何产生细胞命运异质10。从朝hPSCs作为实验模型用于发育疾病11，要研究的组织或器官发育过程中的生长因子和激素的影响药毒性筛选的多细胞微图案的应用上述报告打开新的途径的示范，并解开的形成组织模式。 细胞micropatter有无数宁技术已被开发作为由Falconnet 等审查。人 12，但只有一小部分，如微接触印刷7,8,13，微孔文化14,15，光图案6和microstencils 16已成功地实施hPSCs。与缩微hPSCs的挑战在于他们的脆弱性和具体的细胞外基质（ECM）和生长条件的细胞附着和生存的严格的要求。对于2D HPSC模式，微接触印刷是最常用的方法，以产生对组织培养和玻璃基板13 HPSC微图案之一。该方法可用于在HPSC培养，包括层粘连蛋白和基底膜基质，如基质胶中使用图案共同的ECM。然而，它通常需要用聚-D-赖氨酸辅助的两步涂层的方法，并且需要特定的惰性大气和湿度条件下，使稳定的ECM微图案为hPSCs到6,13- ​​附加。每个图案化方法的首要考虑因素是表面修饰制度是否能够产生在所需的几何分辨率HPSC粘性ECM模式，同时尽量减少非特异性的细胞附着到周边地区。 在这里，我们报告的模板使用缩微作为一种简单的方法来产生HPSC微图案无粘结ECM模式产生之前的其他表面改性步骤hPSCs附加上。小区模版包括薄膜， 例如，聚二甲基硅氧烷（PDMS）片材，具有微米到毫米的通孔密封到一个细胞培养基材的物理上包含ECM的涂料，并随后接种hPSCs大小。作为模版图案形成工程通过物理约束，其中HPSC可以访问和连接直接到下面的ECM涂覆的衬底的位置，该方法是与能够支持HPSC培养各种基材相容。唯一requirement是该模版材料的选择可以形成与基材的可逆密封。这些底物包括常规组织培养聚苯乙烯（TCPS）17，配体缀合的基片18，以及与可调刚性的弹性体基底（ 例如 ，PDMS）19。这种方法还允许不同的ECM的涂层，例如玻连蛋白（或VTN蛋白），层粘连蛋白和基底膜基质（ 例如 ，基质胶和Geltrax），以允许适当附着和hPSCs的分化。为特定HPSC线。因此，我们可以传送优化的ECM-基板配置到模版图案化以获得最佳的细胞 - 基质粘附，存活和分化。最近，一个类似的方法也有报道直接通过使用聚甲基丙烯酸甲酯微图案的hESCs（PMMA）微模版阵列16肝分化。 细胞的模板可以由不同的材料制成，包括满足ALS 20,21，聚（对苯二甲基）聚合物22,23，聚甲基丙烯酸甲酯16和最常见的是，PDMS 24-28。硅和聚（对-亚二甲苯）聚合物模具需要具有专门的设备20-23，这限制了它们的可访问生物用户通孔的直接蚀刻。 PDMS模具可以通过根据所需的特征尺寸，其通常从3微米至2,000微米11,26-29范围不同的方法来制造。如果小的功能需要，薄板材制版可通过冲压成型PDMS在包含微图案28的浮雕精细加工硅模板预聚物生产。为特征&gt; 1000微米，CO 2激光切割器提供了一种简单和低成本的方法模版制造期间直接切上的预铸的PDMS片的图案。 PDMS模具的可回收也使得它们具有成本效益的，进行了一系列的具有足够一致性的实验。 10。

<table><tbody><tr><td>&amp;nbsp;2 mm thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.005</td><td>Used to form reservoir for stencil</td></tr><tr><td>120-150 &amp;mu;m thick PDMS sheet</td><td>Specialty Silicone Products Inc., USA</td><td>SSPM823-.040</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>60 mm Petri dish</td><td>Nunc Nunclon Delta</td><td>150326</td><td>Substrate for micropatterning</td></tr><tr><td>Accutase</td><td>Accutase, Merck Millipore, Singapore</td><td>SCR005</td><td>Enzyme to break H9 cells into single cells</td></tr><tr><td>Activin&amp;nbsp;&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-AC-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>BMP4&amp;nbsp;</td><td>R&amp;amp;D Systems, Singapore</td><td>338-BP-010</td><td>Growth factor for H9 differentiation</td></tr><tr><td>Plasma system&amp;nbsp;</td><td>Femto Science, Korea</td><td>CUTE-MP</td><td>For plasma oxidation of stencil</td></tr><tr><td>Dispase</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>7923</td><td>Enzyme used to weaken the cell-ECM adhesion during passaging</td></tr><tr><td>DMEM/F12</td><td>GIBCO, USA</td><td>11330032</td><td>Basal medium for H9 cells</td></tr><tr><td>FGF2</td><td>R&amp;amp;D Systems, Singapore</td><td>233&amp;ndash;FB&amp;ndash;025</td><td>Growth factor for H9 differentiation</td></tr><tr><td>H9 cell line</td><td>WiCell Research Institute, Inc., USA</td><td>WA09</td><td>Human embryonic stem cells</td></tr><tr><td>hESC-qualified basement membrane matrix</td><td>Matrigel, BD Biosciences, Singapore</td><td>354277</td><td>Extra-cellular matrix coating to support growth of H9 cells</td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems, Singapore</td><td>DMi1</td><td>For capturing bright-field images</td></tr><tr><td>Laser cutter</td><td>Epilog Helix 24 Laser System</td><td></td><td>Used to generate through holes in PDMS sheet</td></tr><tr><td>mTeSR&lt;sup&gt;&amp;trade;&lt;/sup&gt;1 medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5850</td><td>Maintainence medium for H9 cells</td></tr><tr><td>PDMS&amp;nbsp;</td><td>SYLGARD&amp;reg; 184, Dow Corning Co., USA</td><td>3097358-1004</td><td>Used for sticking the PDMS stencil and reservior</td></tr><tr><td>ROCKi Y27632</td><td>Calbiochem, Merck Millipore, Singapore</td><td>688000</td><td>Maintains H9 cells as single cells&amp;nbsp;</td></tr><tr><td>STEMdiff&lt;sup&gt;&amp;trade;&lt;/sup&gt; APEL&lt;sup&gt;&amp;trade;&lt;/sup&gt; medium&amp;nbsp;</td><td>StemCell&amp;trade; Technologies, Singapore</td><td>5210</td><td>Differentiation medium for H9 cells</td></tr><tr><td>Polyethylene terephthalate film</td><td>SureMark Singapore</td><td>SQ-6633</td><td>Used to form stencil&amp;nbsp;</td></tr><tr><td>Cell culture compatible non-ionic surfactant</td><td>Pluronic acid F-127, Sigma, Singapore</td><td>P2443</td><td>Passivating reagent to repel cell adhesion in non-micropatterned substrates</td></tr></tbody></table>

stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

在本文中，我们通过使用激光切割机产生1000微米的特征描述模板电池的制造。模版被由两部分组成:一薄镂花片含有通孔的微图案（大约100-200微米厚），以及将PDMS垫片以包含ECM的包衣溶液或细胞悬浮液。这里，127微米和2毫米厚的市售的PDMS片分别用作制版片和垫圈。制备可控厚度的PDMS片，例如旋涂等方法，也可用于制造的部件。两种成分通过使用液态未固化的PDMS预聚物?...

Watch this Scientific Journal Video about Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates at JoVE.com

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

人多能干细胞（hPSCs）具有分化和自我组织成不同的组织型态的内在能力;尽管这需要空间环境梯度的表现。我们提出模版图案化作为一种​​简单而可靠的方法，以产生生物化学和机械梯度用于控制HPSC分化模式。

人类多能干细胞的模板微图案用于探测分化命运的空间组织

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

stencil-micropatterning-human-pluripotent-stem-cells-for-probing

Research

JoVE Journal

Developmental Biology

8.4K 次观看.  National University of Singapore. 人多能干细胞（hPSCs）具有分化和自我组织成不同的组织型态的内在能力;尽管这需要空间环境梯度的表现。我们提出模版图案化作为一种​​简单而可靠的方法，以产生生物化学和机械梯度用于控制HPSC分化模式。 

视频: Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

科研

发育生物学

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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

使用激光切割模具实现简单的无光刻单细胞微模式

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

生物学

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

生物工程

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

<meta charset="utf8"></head><body>通过基于图像的 ML 策略监测多能干细胞分化

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

<meta charset="utf8"></head><body>作者聚焦:使用基于活细胞明场成像的 ML 模型增强 PSC 到功能性细胞的分化

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

一种基于活细胞图像的机器学习策略，用于监测多能干细胞分化

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...