茉莉Kresse的熟练技术援助深表感谢。

1.人类ESC分化对定形内胚层<ol><li>培养在培养箱中的人胚胎干细胞（ESC），在37℃和5％ 的 CO 2。 </li><li>涂覆新的6孔细胞培养板用1ml基底膜基质孵育培养餐具在RT至少30分钟。有关具体细节，请谈谈各自制造商的说明。 </li><li>确认该培养的人胚胎干细胞已达到使用低倍率（ 例如，4X）在显微镜下的80％-90％汇合。吸通过吸走培养基，用无菌玻璃巴斯德吸管从空腔的介质。用磷酸盐缓冲盐水（PBS）溶液洗涤细胞一次。对于这一点，加入2毫升的PBS至每孔轻轻摇动板和吸出溶液，以去除死细胞和细胞碎片。 </li><li>加入1毫升的无酶传代解决方案，试剂的细胞团的解离温柔。在37℃的细胞D 5％的CO 2，直到细胞显示中断的迹象明显分成小簇。 注:培养时间取决于所使用的试剂。在材料部分中提到的无酶传代溶液，温育时间大约是7分钟。 </li><li>加入1ml的DMEM / F-12培养基并通过上下抽吸用1毫升枪头破坏剩余的细胞聚集成单个细胞。使用此冲洗从表面的细胞和细胞转移到一个离心管中。检索所有细胞，用1ml的DMEM / F-12培养基中洗每个孔，并在介质加入到离心管中。 </li><li>离心细胞5分钟，在300×g的。吸出上清液并重新悬浮于5毫升的ES细胞培养基含有10μM的Rho激酶（ROCK）抑制剂的细胞。 </li><li>算使用血球在显微镜下的细胞和种子150000 - 400,000个单元格每6孔或在另一块板的布局，这取决于所用的胚胎干细胞系。使用CULTURE培养基含有10μM的ROCK抑制剂，以避免细胞凋亡和培养在培养箱中的细胞在37℃和5％的CO 2。 </li><li>大约24小时后播种抽吸介质用无菌玻璃巴斯德吸管，并加2ml原条诱导培养基中。 注意:此培养基含有高级RPMI-1640培养基1％谷氨酰胺，0.2％FCS，5μMCHIR-99021和50ng / ml激活素A的最终浓度。通常，使用两毫升培养基用于培养在6孔平板上。 </li><li>接种后48小时，更换为内胚层诱导培养基的媒介。培养细胞的另一个48小时，每天更换培养基这个网上平台。 注意:此培养基含有高级RPMI-1640培养基1％谷氨酰胺，0.2％FCS和50ng / ml激活素A。正在朝着定型内胚层致力于细胞表达表面标志CXCR4。 CXCR4的染色可以用于量化的DE-致力于细胞的数目。 </li></ol><p类="jove_title"&gt; 2。 CXCR4 +定形内胚层细胞的染色流式细胞仪分析<ol><li>收获前的最后24小时，但至少1小时的细胞中添加10μMROCK抑制剂的培养基。 </li><li>涂层的细胞培养器皿，将用于重新播种与基底膜基质， 即用于免疫荧光染色定量PCR分析或室载玻片12孔板。孵育所述板或幻灯片在室温至少30分钟。 </li><li>吸与用于染色和/或分选的分化的细胞的孔中的无菌玻璃巴斯德吸管的介质。 </li><li>加入1ml无酶传代溶液试剂为细胞团的温和解离。孵育细胞在37℃和5％的CO 2，直至细胞显示中断的明显迹象成小簇。 注:培养时间取决于所使用的试剂。在马泰提到的无酶传代溶液ALS部，温育时间大约是7分钟。 </li><li>加入1ml的DMEM / F-12培养基并破坏通过上下吹打使用1毫升尖剩余细胞聚集成单个细胞。使用此冲洗从表面的细胞和细胞转移到一个离心管中。检索所有细胞，用1ml的DMEM / F-12培养基中的w / o FCS洗每个孔，并在介质加入到离心管中。 </li><li>算使用血球在显微镜下的细胞。从6孔板的三个孔细胞应后分化三天导致10-15×10 6个细胞。离心细胞5分钟，在300×g的。 注意:得到的细胞的确切数目将取决于用于分化多能干细胞系。 </li><li>吸出上清液并重新悬浮在含有10μM的ROCK抑制剂PEB缓冲器细胞。使用100微升该缓冲液中达10 7个细胞。添加10μM的ROCK抑制剂上staini的天NG。使用该缓冲液用于所有下游应用（被称为PEB缓冲液+ RI）。 注:PEB缓冲液含有0.5％BSA和2mM EDTA的PBS中。如果有更多的细胞被染色，相应地调整缓冲体积。 </li><li>每10 7个细胞加入10微升CXCR4-APC抗体对100微升，这大致表示1:10的稀释度。 注意:一个APC连接的抗体的使用不是强制的。相反，它可以根据所用的微珠被取代。如果有更多的细胞被染色相应地调节抗体量。 </li><li>通过用手指轻轻翻转管混合并在冰箱中在4℃孵育15分钟。重悬以1-2毫升PEB缓冲器细胞。离心细胞5分钟，在300×g的。 </li><li>吸用无菌玻璃巴斯德吸管将上清液和重悬细胞沉淀在每10 7个细胞80微升的PEB，并添加20微升抗APC微珠。 注意: </s仲&gt;如果更多的细胞是被染色，相应地调整微珠体积。 </li><li>通过用手指轻轻翻转管混合并在冰箱中在4℃孵育15分钟。重悬以1-2毫升PEB缓冲液+ RI细胞。离心细胞5分钟，在300×g的。吸出缓冲器。悬浮于500μlPEB缓冲区+ RI。 </li></ol> 3. CXCR4 +细胞磁选<ol><li>放置在磁场中的中等大小的磁列按照制造指令。预冲洗用500μlPEB缓冲区+ RI列。申请的全部细胞悬浮液上柱。收集通过，因为不是所有细胞中的流动将与柱结合。确保不打扰列CXCR4 +细胞的最佳检索。 </li><li>洗柱用500μlPEB缓冲+ RI三次。通过收集第一流动并与收集的细胞来自步骤3.1相结合。从磁场去除磁列，并将其放置在一个合适的收集管。加入1ml PEB缓冲+ RI在柱子上。为了洗脱细胞柱塞用力按下到列中。 </li><li>可选的:分别收集通过样品的所有流动和使用各20微升分析使用流式细胞仪的CXCR4 +细胞的数目。其数量应与每个洗涤步骤下降。 注:在至少2×10 4个活，门控细胞应该算为可靠的结果。 </li><li>重复步骤3.1-3.3从3.1步收集流过样品，并通过使用样品新列第一，由步3.2。不要重复使用前一列。通过使用柱塞空气被压入柱，其阻断它。 </li><li>算使用血球在显微镜下的细胞。根据不同的分化达6×10 6个细胞的效率可以最初排序和另一个1×10 6个细胞使用10 7个细胞的过程时使用的第二列。 </LI&gt; <li>离心细胞，在300×g离心5分钟。吸出上清液，用无菌玻璃巴斯德吸管和来自步骤1.9与附加10μM的ROCK抑制剂悬浮在1ml内胚层的诱导培养基中的细胞。 </li><li>算使用血球在显微镜下的细胞。种子的细胞以合适的密度， 即，〜4×10 5个细胞每一个12孔板的孔（表观3.6 cm 2的表面）或者〜每8孔腔室载玻片孔1.5×10 5个细胞。 </li></ol> 4.可选:纯净定形内胚层人口分析<ol><li>对于免疫荧光染色固定来自步骤3.7纯化的细胞大致与4％多聚甲醛和染色为定形内胚层（DE）和/或多能性标记蛋白接种后24小时。 注:常用的DE标志物包括FOXA2和SOX17，常用的多能性标志物包括OCT3 / 4，NANOG和SOX2 6,8。 </li><li>佛- [R RT-qPCR的分析，收获纯化细胞直接或24小时后播种，提取总-RNA和反向从提取的总-RNA样品的cDNA转录。使用10纳克的cDNA作为每RT-qPCR的反应（一式三份）模板来分析DE和多能性标记基因6,8的表达。 注:常用的标记基因在步骤4.1提及。循环条件是95℃5分钟，并在95℃15秒的40个循环，在60℃1分钟，接着通过熔化曲线分析。 </li></ol>

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The authors declare that they have no competing financial interests.

目前使用的分化协议很少导致100％的分化的细胞。对于仍然有要解决的原因，某些细胞抗蚀分化过程。取决于所用的分化方案的效率和ESC的倾向排队甚至分化成定形内胚层后，通常观察到残余的多能细胞的一定数量。这些残余细胞会削弱下游分化或进一步的分析，如转录组学，蛋白质组学和miRNA表达分析。残留的多能干细胞或其它不想要的谱系也可以表现出可与分化目标干扰旁分泌的影响。因此?...

多能干细胞，如胚胎干细胞（ESC）具有分化成几乎人体的任何细胞类型的能力。因此， 在体外分化协议可以被用于产生许多成人细胞类型，如心肌1，肝细胞2，β细胞3，肺上皮4或神经元细胞5。这使得胚胎干细胞对各种退行性疾病3的潜在治疗的有价值的工具。 胚胎干细胞的朝向肺，肝脏和胰腺的成人组织中体外分化需要一个伪原肠胚成让人想起定形内胚层（DE）6的细胞。因为向上述体细胞类型的下游分化是显著效率较低，最佳的内胚层分化被视为限速7。被朝内胚层系致力于细胞进行查在他们的基因表达谱racteristic变化。多能性主控调节基因被下调，而其他转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员和表面受体CXCR4被高度上调6，8，9。CXCR4已知由SMAD2被反式表达/ 3，交点/ TGF-β信号传导和SOX17的下游，由于在其启动子区域10特异性结合位点。因此，它是在一些报告6,8 11-13中使用的非常合适的标记。这些表达的变化反映了伪原肠事件，其中胚胎干细胞首先获取原始的条纹状的细胞群的特点，并随后提交到内胚层胚层6。 然而，分化的协议很少是100％有效，因为一些细胞可能会抵制分化过程或向其他意外谱系分化14。这些细胞可能负面influeNCE进一步分化。此外，剩余的未分化细胞海港供以后移植实验很大的风险，并可能导致畸胎瘤15-17。 除去这些不想要的细胞早期的表面标记物CXCR4的可用于该朝向DE 18致力于细胞的纯化。在这里，我们描述了从DE分化培养物的CXCR4 +细胞的阳性选择的方法。对于这一点，表面标志物CXCR4的是由再依次结合至磁微珠的抗体结合。不像FACS分选过程中的恶劣条件下，磁性标记DE样细胞然后可以很容易地在一个台式格式使用温和的纯化方法进行纯化。这个协议提供了用于抵制对DE分化过程中除去细胞群的一个简单的方法。

<table><tbody><tr><td>Hues8 human embryonic stem cell line</td><td>Harvard Department of stem cell &amp;amp; regenerative biology</td><td></td><td>Suitable cell line for endoderm generation</td></tr><tr><td>Hes3 human embryonic stem cell line</td><td>ES Cell International</td><td></td><td>Suitable and robust cell line for endoderm generation</td></tr><tr><td>mTeSR1</td><td>Stemcell Technologies</td><td>5850</td><td>ESC culture medium</td></tr><tr><td>FCS</td><td>Biowest</td><td>S1860</td><td></td></tr><tr><td>Advanced RPMI 1640</td><td>Life Technologies</td><td>12633012</td><td></td></tr><tr><td>CD184 (CXCR4)-APC, human</td><td>Miltenyi Biotec</td><td>130-098-357</td><td></td></tr><tr><td>anti-APC MicroBeads</td><td>Miltenyi Biotec</td><td>130-090-855&amp;nbsp;</td><td></td></tr><tr><td>OctoMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-109</td><td>magnetic field</td></tr><tr><td>Y-27632</td><td>Selleck Chemicals</td><td>S1049</td><td>ROCK inhibitor</td></tr><tr><td>CHIR-99021</td><td>Tocris Bioscience</td><td>4423</td><td></td></tr><tr><td>Activin A</td><td>Peprotech</td><td>120-14</td><td></td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>Stemcell Technologies</td><td>7174</td><td>Enzyme-free passaging solution, alternative: Trypsin/EDTA</td></tr><tr><td>Matrigel*</td><td>Corning</td><td>354277</td><td>basement membrane matrix&lt;br/&gt; * solve and store in aliquots at -80 &amp;deg;C as outlined in the suppliers manual. Upon use, thaw on ice, dilute in 25 ml ice-cold knockout DMEM/F-12.&lt;br/&gt; Add 1 ml to each well of a 6-well plate and incubate for 45 min at room temperature.&lt;br/&gt; Remove the matrigel and use immediately. </td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec</td><td>30-042-201</td><td></td></tr><tr><td>MACS Separator</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>Human FOXA2 FW&lt;br/&gt;gggagcggtgaagatgga</td><td>Life Technologies</td><td>NA</td><td></td></tr><tr><td>Human FOXA2 REV&lt;br/&gt;tcatgttgctcacggaggagta</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human GSC FW&lt;br/&gt;gaggagaaagtggaggtctggtt</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human GSC REV&lt;br/&gt;ctctgatgaggaccgcttctg</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>SOX17 TaqMan assay</td><td>Applied Biosystems</td><td>Hs00751752_s1</td><td></td></tr><tr><td>Human SOX7 FW&lt;br/&gt;gatgctgggaaagtcgtggaagg</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human SOX7 REV&lt;br/&gt;tgcgcggccggtacttgtag</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human POU5F1 FW&lt;br/&gt;cttgctgcagaagtgggtggagg</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human POU5F1 REV&lt;br/&gt;ctgcagtgtgggtttcgggca</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human Nanog FW&lt;br/&gt;ccgagggcagacatcatcc</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human Nanog REV&lt;br/&gt;ccatccactgccacatcttct</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human TBP FW&lt;br/&gt;caa cag cct gcc acc tta cgc tc</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human TBP REV&lt;br/&gt;agg ctg tgg ggt cag tcc agt g</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human TUBA1A FW&lt;br/&gt;ggc agt gtt tgt aga ctt gga acc c</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human TUBA1A REV&lt;br/&gt;tgt gat aag ttg ctc agg gtg gaa g</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human G6PD FW&lt;br/&gt;agg ccg tca cca aga aca ttc a</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Human G6PD REV&lt;br/&gt;cga tga tgc ggt tcc agc cta t </td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Anti-SOX2</td><td>Santa Cruz Biotechnology</td><td>sc-17320</td><td></td></tr><tr><td>Anti-FOXA2</td><td>MerckMillipore</td><td>07-633</td><td></td></tr><tr><td>Anti-SOX17</td><td>R&amp;amp;D Systems</td><td>AF1924</td><td></td></tr></tbody></table>

a quick and efficient method for the purification of endoderm cells generated from human embryonic stem cells

多能干细胞，如胚胎干细胞（ESC）的分化能力允许对细胞替代疗法的潜在的治疗应用。终末分化的细胞类型可以用于各种退行性疾病的治疗。 在体外 ，这些细胞向肺，肝脏和胰腺的组织的分化需要作为第一步定型内胚层细胞的产生。这个步骤是限速为朝向末端成熟细胞类型，如产生胰岛素的β细胞，肝细胞或其它内胚层来源的细胞类型进一步分化。即朝向内胚层谱系定型细胞高度表达的转录因子如FOXA2，SOX17，HNF1B，该GATA家族的成员，且表面受体CXCR4多种。然而，分化的协议很少是100％的效率。这里，我们描述了一种用于一CXCR4阳性细胞群的分化后的纯化入DE通过使用磁微珠。这种净化此外删除不需要的谱系的细胞。平缓的纯化方法是快速，可靠的，并且可以用于改善下游应用和分化。

在分化时 胚胎干细胞进行基因和蛋白质表达的急剧变化。 图1描绘了可用于验证成功定形内胚层分化典型的标记基因。对于基因表达分析的主要目标是GSC，FOXA2和 SOX17在一个相对的基因表达分析尤其是FOXA2和SOX17相比未分化的胚胎干细胞时，增加了&gt; 2000倍。GSC原条的形成过程中已经表示24小时内很早但它仍?...

Watch this Scientific Journal Video about A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells at JoVE.com

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

在这里，我们描述了该朝向为下游应用和进一步分化的改善的定形内胚层致力于分化的人类胚胎干细胞的纯化方法。

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

a-quick-efficient-method-for-purification-endoderm-cells-generated

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.3K 次观看.  Hannover Medical School. 在这里，我们描述了该朝向为下游应用和进一步分化的改善的定形内胚层致力于分化的人类胚胎干细胞的纯化方法。 

视频: A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

科研

癌症研究

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

生物学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

发育生物学

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

生物工程

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

从 iPSC 高效生成人肠道类器官的基质比较

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

作者聚焦：使用 iPSC 推进用于药物开发的类器官生成

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

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基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

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