这项工作是按顺序一个NCCR前沿遗传学试验奖，青少年糖尿病研究基金会的资助41-2009-775和格兰特12-126875从侦探的frieForskningsråd/ Sundhed OG Sygdom资助。作者感谢SPAGNOLI实验室主办的视频拍摄。

该协议旨在扩大从鼠E10.5派生类器官胰腺上皮分离胰腺细胞。 该协议要求的伦理批准用于动物实验。 背胰芽1。解剖从E10.5小鼠胚胎<ol><li>牺牲定时妊娠小鼠胚胎一天（五）10.5，开有一对剪刀的腹部，取出两个子宫角，并放置在10厘米的培养皿中充满了冷磷酸盐缓冲液（PBS）或贝科改良基本培养基（DMEM）中保持在冰上。总的实验从祭祀细胞种植在60-90分钟做是为了防止细胞损伤。 </li><li>子宫分成用小剪刀个别胚胎段。转移一个胚胎为35 mm培养皿用冷PBS和可视化它在解剖显微镜从上面照射下。用解剖镊子取出周围的肌肉，蜕膜和蛋黄SAc和暴露胚胎。将胚胎放回冷DMEM培养基。胚胎可以很容易地使用3毫升塑料移液管/滴管转移。在继续之前隔离每个人胚胎以类似的方式。 </li><li>将胚胎放入干净35毫米培养皿中的PBS。 <ol><li>使用薄钳（0.05毫米宽），除去前肢。轻轻插入钳子的开口分离从脊髓区域的消化道（ 图1）。可选:为了更方便地看到肚子，取出胚胎的上身，下到下面的蛋黄秆心脏和尾部区域。 </li><li>找到胃，肝和肠。使用镊子，隔离从胃到肠的消化道，并将其放置在冷的DMEM在冰上（ 图1）。背胰芽附着背侧，后到胃（ 图1）。 </li><li>隔离在西米每个人胃肠道在继续之前拉尔方式。如果胚胎需要进行基因分型，收集的尾部在夹层的时间，并保持每一个个体胃肠道在不同的井在24孔板上用冰冷的DMEM中。 </li></ol></li><li>将一个胃肠道在一个干净的35毫米培养皿中冷PBS。现在使用的照明从下面的明视场的解剖显微镜下观察并剖析背胰芽。这些条件将优化卷的可视化。使用电解削尖钨针或20 G注射器针头，分离出胰芽用尽可能少的间充质尽可能周围上皮细胞（ 图1）。 <ol><li>传输的分离的芽到含有冷分散酶溶液（1.25毫克/毫升）为2-3分钟的培养皿中。从这点来说，传输可以最方便地与连接到一个口控制的柔性管火焰拉50微升玻璃毛细管进行。备选地，但更多的里失去了芽的SK，使用10微升自动移液器（的Pipetman）用适当的塑料头。 </li><li>在微观控制执行胰芽愿望和弹射。把胰芽早在PBS中。从使用的玻璃毛细管（ 图1）针和温柔的吸间质进一步清理隔离胰芽。 </li><li>当整个间质被删除，在冲洗冷PBS胰芽;在单井每个芽转移到冷的DMEM（60孔迷你托盘装满10微升冷的DMEM）。它不删除的间质中分散酶是重要的，这使得组织很粘。 </li></ol></li></ol> 2，电镀分散细胞与文化<ol><li>来自胚胎的火焰拉玻璃毛细管进60孔迷你托盘装满10微升PBS冲洗锥形井转让解剖上皮细胞。 <ol><li>转移芽成10微升胰蛋白酶0.05％的ND让它在37℃孵育4分钟。通过传送芽成井与10微升DMEM +10％胎牛血清（FCS）的灭活胰蛋白酶。 </li><li>通过皮薄毛细拉带吸管拉马吸离解细胞悬液。避免气泡在这个阶段，而上下吹打以解离的细胞是重要的。胰腺组织体最佳地小群体的5-15细胞开始，因此部分离解，建议（ 图1）。 </li></ol></li><li>普尔从几个胚胎细胞到Eppendorf管中，以尽量减少由于个体差异的处理。稀释细胞悬液冷冻基底膜以1:3的比例。等分的该混合物到96孔板中，8微升/孔，或在用于成像的最佳化的板（见下文）。 </li><li>将培养板在37℃下进行5分钟，使基底膜变厚。填补了井70微升的选择培养基（组织体或球体，看标签莱1和2），并在含5％CO 2和95％空气中于37℃的潮湿环境中留下</li><li>每一个第 4天更换培养基。每日监控的日益胰腺殖民地和成像记录下这一过程。 </li><li>感兴趣的小分子或蛋白质可以被添加到在该阶段的培养基为干扰实验，如先前报道28。 </li></ol> 表1:类器官培养基。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>材料名称</td><td>股票集中度</td><td>在决赛中浓度</td><td>的蓄积量</td></tr><tr><td>青霉素，链霉素</td><td> 100％ </td><td> 1％ </td><td> 50微升</td></tr><tr><td>淘汰赛血清替代（补充） </td><td> 100％ </td><td> 10％ </td><td> 500微升</td></tr><tr><td> 2 - 巯基乙醇</td><td> 14.3 M </TD&gt; <td> 0.1毫米</td><td> 1微升</td></tr><tr><td>佛波酯（PMA） </td><td> 16微米</td><td> 16纳米</td><td> 5微升</td></tr><tr><td> Y-27632（ROCK抑制剂） </td><td> 50毫米</td><td> 10微米</td><td> 1微升</td></tr><tr><td>表皮生长因子</td><td> 50微克/毫升</td><td> 25纳克/毫升</td><td> 2.5微升</td></tr><tr><td>重组人R-脊椎1 </td><td> 250微克/毫升</td><td> 500毫微克/毫升</td><td> 10微升</td></tr><tr><td> - 或 - </td><td></td><td></td><td></td></tr><tr><td>重组小鼠的R-脊椎蛋白1 </td><td> 250微克/毫升</td><td> 500毫微克/毫升</td><td> 10微升</td></tr><tr><td>重组人FGF1（的aFGF） </td><td> 100微克/毫升</td><td> 25微克/毫升</td><td> 1.25微升</td></tr><tr><td>肝素（Liquemin） </td><td> 2500 U / ml的</td><td> 2.5单位/毫升</td><td> 2微升</td></tr><tr><td>推荐binant人类FGF10 </td><td> 100微克/毫升</td><td> 100毫微克/毫升</td><td> 5微升</td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25微升</td></tr><tr><td>总</td><td></td><td></td><td> 5,000微升</td></tr></tbody></table> 表2:球体介质。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>材料名称</td><td>股票集中度</td><td>在决赛中浓度</td><td>的蓄积量</td></tr><tr><td>青霉素，链霉素</td><td> 100％ </td><td> 1％ </td><td> 50微升</td></tr><tr><td> B27×50（增刊） </td><td> 100％ </td><td> 10％ </td><td> 100微升</td></tr><tr><td>重组人FGF2（bFGF）的</td><td> 100微克/毫升</td><td> 64纳克/毫升</td><td> 3.2微升</td></tr><tr><td> Y-27632（ROCK抑制剂） </td><td> 50毫米</td><td> 10微米</td><td> 1微升</td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845.8微升</td></tr><tr><td>总</td><td></td><td></td><td> 5000微升</td></tr></tbody></table>类器官发展的进展3。成像<ol><li>图片类器官每天或通过时间推移显微镜用荧光延时显微镜。对于时间的推移成像，使用一个XY（Z）自动倒置荧光显微镜。 </li><li>存款小水滴的3μl，以4孔板或玻璃底板装有2-5毫升培养基。图像与10倍的远距离目标。注意:表达荧光示踪剂的转基因小鼠可以使用。电影1所示，例如组织体从的Pdx1，Ngn3的-ER TM-IRES-nGFP +小鼠4初始扩张。核GFP使用户能够跟踪的细胞作为单独的对象，但类似的原理可以应用到带膜荧光追踪细胞如作为MT / MG小鼠29。 </li><li>接种细胞，避免焦点漂移和设置软件控制自动化取1张/小时，3天或以上的在手动定义的位置后，开始时间推移成像3小时。对于每一个位置，获得一个微分干涉对比（DIC）的形象，以及对GFP信号，报告荧光标记物的表达。 </li></ol>组织体4。恢复的组织学<ol><li>放置在96孔板上的冰和除去培养基，用冰冷的PBS代替。这部分解聚基底膜。 </li><li>轻轻地吸出每个单独的组织体，为了不破坏整体架构使用了1000微升末端除去周围的基质胶。将每个类器官，以良好的用冰冷的PBS。置于冰上板。直接固定在基质胶也是可能的。 </li><li>修复类器官15分钟在4％多聚甲醛（PFA），冷冻保存在蔗糖和嵌入在明胶。处理每个类器官为cryosectioning和组织学如前所述（Johansson 等人 ，2007）4。 </li></ol> 5，恢复组织体进行PCR和生物化学<ol><li>放置在96孔板上的冰和除去介质。加入60微升每孔的RNAlater，以稳定和保护细胞的RNA。 </li><li>通过部分地在冰上解聚破坏它在每个孔的机械凝胶。无论是为了不破坏整体架构或者通过用200μl的前端机械地破坏凝胶恢复整个井（用基质胶）用1,000微升尖恢复单个组织体。使用1,000μl吸头的良好的内容转移到一个无RNA酶，无粘性的Eppendorf管放在冰上。 </li><li>用60微升的RNAlater洗井，其余内容添加到相同的离心管中。 </li><li>旋转的管子5分钟，在500〜1000×g离心在4℃下</li><li>去除上清，只留下20-30微升的RNAlater中的管连同粒料。对于生物化学，储存样本尽可能干燥。 </li><li>不要立即冻结在RNAlater样品;在4°CO / N店（允许RNAlater中彻底穿透组织）。该组织可以被储存在-20℃下长期贮存和以后可以对组织破坏和提取少量的RNA的处理。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. 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The authors have nothing to disclose.

大规模生产的功能性β细胞在体外仍然无效1。在这个充满挑战的背景下，发育生物学的研究可能有助于破译所必需的功能性β细胞分化的确切信号。此协议允许在体外胚胎胰腺祖细胞的维持，扩展和分化。这包括形成产生胰岛素的β细胞的不共表达其他内分泌激素，有高水平的Pdx1的，表达了亲convertases的成熟胰岛素和有胰岛素处理28。系统内的重要的关键因素是FGF的活性（通过加入FGF通过肝素增效外源性刺激的）和Notch（内源）信号传导途径，以及ROCK-抑制由Y-27632:在不存在这些因素，没有或很生成组织体和球体的数量有限28。对于FGF和不要求CH活动是很容易理解的基础上的， 在体内28胰腺发育的重要性。 ROCK抑制剂可通过II型肌球蛋白被取代，从而揭示微丝动力学的过度活化分解后导致既增加了细胞死亡，祖转录因子的Pdx1强烈的抑制和缺乏扩展。有趣的是，类器官介质的许多其他组件被证明是不必要的个别，但他们的结合缺乏导致上皮分支28的损失。除了在培养基中的某些基本元件，重要的是要控制的祖细胞的离解程度。事实上，祖细胞增殖和Pdx1的维修超过4细胞组显著提升。压实可以在最初的12小时，故障的故障维护的Pdx1和扩大小巧结果中可以观察到。 ROCK抑制剂是这一重要过程。 此刻的组织体不形成FACS分选，但该系统的效率有可能通过重新聚集祖细胞的受控数得到改善后。该培养体系的另一个关键组件是三维矩阵。细胞放于基质胶或在基质胶太靠近板扩散的底部和失去的Pdx1表达。基质胶最有可能提供生化成分，特别是层粘连蛋白以及机械线索28。的确，基质的硬度起着关键性的作用。僵硬的水凝胶是不宽容的胰腺祖细胞的维护和扩展28及摊薄基底膜不是非此即彼。当基底膜1:10稀释胰腺祖无法培养。 的组织体系统可用于测试在存活，增殖，分化，偏振计的小分子和重组蛋白对胰祖细胞的影响和支28，它也可以被用来测试不同细胞类型的胰腺发育28中的合作。我们有信心，胰腺祖细胞的辅助功能也将使遗传操作，如病毒性目标，如在其他类器官系统17,27所示。这可以被用于筛选17与一个系统，使形态对比前面描述以及这里16,17,28的球体。我们还制定了培养条件呈现为无血清的优势，无饲养和缺乏间质和血管，从而减少了细胞和生物化学的复杂性。然而，在能力通道中的组织体的限制，从而获得大量祖细胞。这可以规避在未来的协议，以适应以后的发展，其中祖细胞较为丰富的阶段，胰腺祖细胞从胎产源集成电路干（ES）细胞或诱导多能干细胞（iPS细胞）。这铺平了道路，为人类胰腺发育的3D模型。 这个系统也可以潜在地在治疗的未来透视用于生产胰腺细胞。在这种情况下，所生产的功能性β细胞移植将可能在糖尿病的治疗提供帮助。该系统的人ES或iPS细胞的改编将用于这一目的的重要。目前还不清楚该组织体或球体条件是否应该被使用。该组织体条件允许用于生产的细胞，具有成熟的β细胞的几大特点，但它们的功能还有待考验的。然而，这些细胞是目前并不多，并在其他细胞混合。它也可能是异质性的组织体系统提前出现导致细胞之间的信号传导不受控制的，因此是不利于生产。 Ŧ他球系统，它维护的祖细胞原则上是优选的受控制的膨胀祖细胞和传代，但他们随后的分化仍然被控制。其他最近生产的，可以有效区分pancreatospheres。这将是重要的，以比较在当前协议缺乏馈线和血清与其他协议16,17得到的球体中得到的球的性质。从一个治疗点的复杂性和基质胶的生物来源可能构成的再现性，健康和可扩展性的问题。初步结果显示，官能化的层粘连蛋白柔软的水凝胶是容许体外胰腺祖扩张。进一步优化需要为这些凝胶还没有高效的基底膜28。 β细胞在它们的自然背景下的产量也可能是有用的试验药物，提高公测细胞活性或增加他们的生存或增殖，但为此目的首先测试的成熟的β细胞的程度，增加其分化的效率，并测试是否这里介绍的培养条件下更好地保持胰岛比当前这将是重要的悬浮培养。生产胰腺外分泌也可能是有用的，开发药物瞄准胰腺癌和胰腺炎。在这里，需要产生的外分泌细胞的成熟程度进行彻底调查。

器官培养提供了桥梁在体内研究和复杂，但具有很强的针对性之间的差距细胞系模型的方便，但近似模拟一个有用的模型。在胰腺的情况下，不存在细胞系完全等价于胰腺祖细胞虽然有转化细胞系模拟内分泌和外分泌细胞。成人全胰腺不能培养;分离的内分泌胰岛可维持数周而不细胞增殖和组织切片可以保持在体外对几个小时5。胚胎胰腺培养已被广泛使用，不仅研究其发展，还探讨上皮-间充质相互作用4,6,7，对图像进行处理8或化学上与它们9干扰。两个器官培养方法主要应用于:第一种是在纤连蛋白包被的板2，它是型转换器中培养胰芽便于您进行成像的目的;第二个选项是文化在气-液界面3,4的最佳形态保留在过滤器上的器官。虽然非常有用，这些方法会导致一定程度的平坦化的;相比于正常发育和起始群体是复杂的，包括所有类型的胰腺细胞和间充质细胞的前体细胞的扩张是非常有限的。 的能力，培养和扩大分散的初级细胞是有价值的研究谱系关系，并发现分离的细胞类型10的固有性质。杉山等 11能保持胰腺祖细胞和内分泌祖细胞是保留了一些功能特点3-5天培养的饲养层。 Pancreatospheres，类似于神经球12和13微球体，已经扩大，从成人胰岛细胞和导管细胞虽然祖细胞的性质/干细胞产生这些球体是不明确的。另外，在与生理发育相反，pancreatospheres含有一些神经元14,15。领域也从最近胚胎胰腺祖细胞16,17和再生胰腺18具有良好的祖扩张和随后的分化产生，但未能概括形态。 从分散，通常被定义细胞自组织成微型器官3D模型最近已蓬勃发展，并模拟多个器官如肠道19,20，胃21，肝脏22，前列腺23和气管的开发或成年营业额24。在某些情况下，发育和形态发生分化已扼要重述在三维由ES细胞，因为是光学杯25，小肠26或脑27的情况。 在这里，我们DEScribe一个扩大分离的多能胰腺祖细胞在三维基质胶支架在那里他们可以分化和自我组织的方法。

<table><tbody><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>KnockOut Serum replacement (supplement)</td><td>Gibco</td><td>10828-028</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma Aldrich</td><td>3148-25ML</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Phorbol Myristate Acetate (PMA)</td><td>Calbiotech</td><td>524400-1MG</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Y-27632 (ROCK inhibitor)</td><td>Sigma Aldrich</td><td>ab120129</td><td>Stock kept at -20 &amp;deg;C. Attention! Stability/source is a frequent source of problems.</td></tr><tr><td>EGF</td><td>Sigma Aldrich</td><td>E9644-2MG</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human R-spondin 1</td><td>R&amp;amp;D</td><td>4645-RS-025/CF</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Mouse R-spondin 1</td><td>R&amp;amp;D</td><td>3474-RS-050</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF1 (aFGF)</td><td>R&amp;amp;D</td><td>232-FA-025</td><td>Stock kept at -80 &amp;deg;C - do not include to increase beta cell production</td></tr><tr><td>Heparin (Liquemin)</td><td>Drossapharm</td><td></td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF10</td><td>R&amp;amp;D</td><td>345-FG-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>DMEM/F-12</td><td>Gibco</td><td>21331-020</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15070-063</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>B27 x50 (supplement)</td><td>Gibco</td><td>17504-044</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Recombinant Human FGF2 (bFGF)</td><td>R&amp;amp;D</td><td>233-FB-025</td><td>Stock kept at -80 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td>Stock kept at 4 &amp;deg;C</td></tr><tr><td>RNAlater - RNA stabilizing reagent</td><td>Qiagen</td><td>76104</td><td>Store at RT</td></tr><tr><td>Dispase&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4818-2MG</td><td>Working concentration: 1.25 mg/ml. Stock kept at -20 &amp;deg;C</td></tr><tr><td>BSA for reconstitution</td><td>Milipore</td><td>&lt;a href=&quot;http://www.millipore.com/catalogue/item/81-068-3&quot;&gt;81-068&lt;/a&gt;</td><td>For reconstituition of cytokines&amp;nbsp; - stock kept at -20 &amp;deg;C</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Gibco</td><td>16141079</td><td>Stock kept at -20 &amp;deg;C</td></tr><tr><td>60-well MicroWell trays</td><td>Sigma Aldrich</td><td>M0815-100EA</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>95-well plates F bottom</td><td>Greiner Bio</td><td>6555180</td><td></td></tr><tr><td>Glas bottom plates</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Disposal glass micropipettes</td><td>Blaubrand</td><td>708745</td><td></td></tr><tr><td>Microscope</td><td></td><td></td><td>Cell&amp;#174; imaging station (motorized inverted Olympus IX81 stand) equipped with a Hamamatsu ORCA ER B7W camera and the Ludin Cube and Box.&lt;br /&gt;
Leica DMI6000 B stand surrounded with a Ludin Cube and Box, equipped with a Leica DFC365 FX camera and the AF6000 Expert/Matrix software command interface.</td></tr><tr><td>Objective</td><td></td><td></td><td>Olympus UPLAN FL NA 0.30 air 9.50 mm 10X long distance;&lt;br /&gt;
Leica HC PL FLUOTAR NA 0.30 air 11.0 mm 10X long distance</td></tr></tbody></table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

分离并接种于三维基质胶E10.5胰背祖概括胰腺发育。祖细胞可以最容易遵循的荧光记者。在我们的例子中，我们使用了转基因小鼠，表达不激活Neurog3 4（ 图2），在没有他莫昔芬，从而通过的Pdx1启动子（ 的Pdx1，Ngn3的-ER TM-nGFP）（电影1）控制的核GFP蛋白。 与组织体介质中，小簇细胞的初始压实发生在第一个小时。膨胀然后可以由簇在第4天的扩大（ 图2A）来检测。从第5天，树枝形成在20％最大类器官。单细胞不扩大而失去的Pdx1表达，而在大型集群保留的Pdx1表达28。 在这些条件下，祖细胞经历一个规范tacular形态与支上皮结构的出现。这个过程发生时，才祖细胞接种于≥4细胞簇，提示社区信号的强烈需求。组织学分析表明，培养7天之后，所产生的微型器官是由胰腺祖细胞（SOX9 + / HNF1B + / 的Pdx1 +细胞: 图3B）和分化细胞表达两种外分泌（淀粉+）或内分泌（胰岛素+或胰高血糖素+）标记（ 图3A，C）。分化为内分泌细胞比内源性胰腺（约0.1％）低，但提高到1％时，FGF1不会被添加到培养液中28。值得注意的是，不仅不引晶的祖细胞分化为胰腺预期谱系，但它们也自发地采取正常胰腺架构。尽管电子10.5多能胰腺祖细胞不极化，细胞在培养分化就证明了Mucin1和的aPKC在面对中央管腔膜的分离和它们组织到一个支管网络。区域化的"塞尖和躯干"域出现:HNF1B +祖细胞和内分泌细胞被定位在中央区域，而腺泡细胞都位于周边的细胞的部分或完整的冠。的组织体可保持在培养10天;此期间之后，他们一般失去他们的建筑机构，成为囊状（未示出）。传代可以部分解离后进行，但很快导致囊肿形成，这种现象是减少了另外的BMP抑制剂头蛋白28。 与球体介质，膨胀是更频繁和从单细胞的2％可见;然而的球体形成的效率相关联的seede的大小d磁簇28。在天2/3，内腔被检测到的小簇和膨胀之后，导致基本上单层空心球，偶尔本地多层区域（ 图2B）。从基质胶（ 图3D-H）中检索时，这些球体崩溃。在这些条件下，所得到的结构主要由胰腺祖细胞，具有分化外分泌和内分泌细胞的小的百分比在第7天（ 图3D-H）。在该领域祖也成为顶部极化，就证明了的aPKC在面对所有细胞（ 图3F）的中央管腔膜的隔离。 Pancreatospheres可至少两次（未示出）传代。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig1highres.jpg" width="500" /> 图1的procedur的示意图（五）本胃肠道最初是从胚胎解剖，随后背胰芽是孤立的。间质被删除，胰腺祖细胞用胰蛋白酶解离。所得到的部分分散细胞，然后以低密度接种于生长因子耗尽基质胶。比例尺:1.00毫米除了导致类器官图片所在的比例尺为200μm的<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig1highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig2highres.jpg" width="500" /> 图2。进步的文化随着时间的推移，（A）小簇生长的类器官中，接着从3小时时间推移显微镜电镀，60小时后胰腺祖细胞。在博TTOM板，培养7天后的类器官。比例尺:200微米;适用于所有的面板A中的一个球体（B）的实施例随后在一个60小时的时间推移，并在培养的第7天拍摄的。比例尺:200微米;适用于所有面板B 中 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51725/51725fig3highres.jpg" width="500" /> 图3。组织学。 （AC）串行为期7天的部分组织体染色祖（B - HNF1B）和分化（A -淀粉酶，C -胰岛素）标记。该组织体是由上皮（A - E-cadherin蛋白）和顶部极化（B - mucin1）细胞。虚线对应于非腺泡中心区域（A），其中被检测HNF1B（B）和内分泌（C）的细胞染色祖7天球体（DH）的部分。（G - HNF1B，H - SOX9）和内分泌（D -胰岛素和胰高血糖素）标记。球体是由顶部极化（D，F - 的aPKC）上皮（E - E-cadherin蛋白）细胞。比例尺:50微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51725/51725fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

在本协议中所述的三维培养方法概括胰腺发育从胚胎分散小鼠胰腺祖细胞，包括他们的大幅扩张，分化和形态发生成支器官。这种方法适合于成像，利基的功能的干扰和操纵。

体外胰腺器官从分散的小鼠胚胎祖细胞

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Nanyang Technological University

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K 次观看.  Swiss Institute for Experimental Cancer Research. 在本协议中所述的三维培养方法概括胰腺发育从胚胎分散小鼠胰腺祖细胞，包括他们的大幅扩张，分化和形态发生成支器官。这种方法适合于成像，利基的功能的干扰和操纵。 

视频: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

科研

发育生物学

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

医学

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

高效生成粘附培养中 Hpsc 的胰腺-二叶多布蛋白 1+后后福格内胰腺祖细胞

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

生物学

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

体外胰腺器官从分散的小鼠胚胎祖细胞

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