我们感谢 Jolanta Chmielowiec 就该协议和手稿提供了有益的讨论。我们还感谢本杰明 Arenkiel 获得共聚焦显微镜。这项工作得到了 NIH (P30-DK079638 到手机和 T32HL092332-13 到 M.A.S. 和手机)、捷尔医学基金会 (手机) 以及在医疗知识和发展残疾研究中心的共焦核心 (nih U54 HD083092 的支持)。肯尼迪国家儿童健康和人类发展研究所)。

该方法所描述的所有动物实验均经贝勒医学院机构动物护理和使用委员会批准。
1. 小鼠胚胎日10.5 胰祖细胞的制备
注: 本协议不需要在无菌条件下遵循, 直到步骤 2, 但它是最佳的消毒解剖工具和喷雾与70% 乙醇之前使用。
<ol><li>为解剖, 填充冰桶和放置一个容器磷酸盐缓冲盐水 (PBS) 在冰。清洁两个细尖钳和解剖剪刀与70% 乙醇。设置在解剖区附近, 一个容器至少有三硼硅酸盐毛细管, 口吸管, 打火机, 过滤1000µL 吸管提示。<ol>
<li>准备至少1毫升的1.25 毫克/毫升冷酶稀释在消毒水和地方在第二井的12井板冰。将 PBS 放在12井板的第一、第三和第四口井中。在冰上放置1毫升0.05% 胰蛋白酶在1.5 毫升管。将震动孵化器预热至 37 oC。</li>
	</ol>
</li>
	<li>弄死 e10.5 符合 IACUC 准则的定时怀孕小鼠。用70% 乙醇喷雾腹部以清洁区域, 然后用剪刀和镊子在小鼠生殖器上做 V 形切口, 然后继续到隔膜。 注: 此协议中的阴道插头外观显示为0.5 天。使用 5-6 周大的老鼠, 体重增加超过2克的行为作为一个次要措施成功浸渍。<ol>
<li>通过在子宫最上部的一侧切开切口, 仔细切除子宫, 将器官从鼠标向上拉出, 然后沿着这个切口向下到生殖器区域, 然后再重复另一侧。把它放在一个10厘米培养皿与冷 PBS。 注意: 从 e11.5 分离细胞中获得 organoids 是可能的, 然而不同于 e10.5 pancreatoids, 这些 organoids 还没有被描绘出来, 因此可能无法保留区分成所有三种主要胰血统的能力。</li>
	</ol>
</li>
	<li>在光解剖显微镜下, 放置培养皿, 并小心地打开子宫组织通过放置两个钳在每个胚胎和剥离组织远离蛋黄囊。用镊子轻轻抓住蛋黄囊, 在10厘米培养皿中放入新鲜的冷 PBS, 转移胚胎。把培养皿放在冰上。 注意: 重要的是 heedfully 删除胚胎, 最好保持在蛋黄囊中, 因为强力去除可以拉脐带, 撕裂组织的胚胎和损害结构完整性。</li>
	<li>将多个 PBS 滴放在10厘米的培养皿盖上。使用这些水滴转移胚胎在解剖时, 胚外组织被删除, 以确保能见度。将胚胎转移到 pbs 上, 用镊子轻轻地从蛋黄囊中取出, 其余的胚胎留在 pbs 上 (图 1a)。把胚胎移到新的 PBS 上, 用镊子轻轻舀起胚, 不损伤组织 (图 1B)。 注意: 组织可能需要更频繁地转移到新鲜的 PBS 水滴比这里描述, 取决于液体的不透明度。<ol>
<li>在一个新的 PBS 下降, 删除前肢芽使用钳 (图 1B)。将镊子放入开口处, 肢体芽出现, 轻轻撕裂最外部组织前方 (图 1B)。旋转胚胎并在后方向重复, 停止在后肢芽。此时, 胃肠道应该可见 (图 1B)。</li>
		<li>胃肠道后区有轻微弯曲 (图 1F)。在胃肠道和体壁脊柱区之间的开口处插入镊子。分离胃肠道, 慢慢向上工作, 直到最前面的区域到达心脏区域连接的地方 (图 1B-E)。 可选: 从胃肠道的腹部切除原始心脏和肝芽, 只留下连续的肠道管。前几次这个协议是执行它可能会更容易离开心脏和肝脏的腹部标志性, 直到胃肠道和背胰芽的形态学更熟悉 (图 1C和D).</li>
		<li>将胃肠道转移到新鲜的 PBS 液滴上。</li>
		<li>取镊子, 轻轻捏起凸出芽和小肠下方的组织, 稍微抬起外层组织 (图 1d-F)。 注意: 背部的胰芽位于胃和肠道之间 (图 1c-G)。下面的胰芽应该看起来像一个圆形的, 诺比结构 (图1 G).</li>
		<li>放置钳, 芽连接到肠道和捏向上松开芽 (图 1G)。洗一次在一个新的 PBS 泡沫之前, 将芽转移到12井板的第一井, 在寒冷的 pbs 冰。重复直到所有芽从胚胎中收集, 并放置在12井板块的第一井。</li>
	</ol>
</li>
	<li>将12井盘放在光夹层显微镜下。计数成功解剖的胰芽的数量, 以后计算分裂比率。<ol>
<li>将过滤后的1000µL 吸管尖端放入口管中, 并附上毛细管。火焰对毛细管进行杀菌, 并在管子中产生弯曲, 便于使用。使用毛细管将芽转移到含有冷酶溶液的第二井, 2 分钟, 然后用干净的 PBS (图 1G) 向第三井传输。 注意: 将芽从酶转移到 PBS 时, 避免接触毛细血管管尖端的组织。如果组织被卡在管子的尖端, 吸管向上和向下或在干净的 PBS 溶液中摇动, 直到松开。</li>
		<li>吸管芽向上和向下, 并转移到第四井与清洁 PBS。最后, 使用毛细管装置转移芽到1.5 毫升管与0.05% 胰蛋白酶。将管置于预热后的 37 oC 振动筛中, 并在 1500 rpm 处晃动4分钟。</li>
		<li>漩涡为大约十年代然后立刻安置管子在离心机并且旋转5分钟在 200 g. 谨慎地删除所有但大约50µL 的解决方案, 以不打扰离心单元格 (图 1g)。</li>
	</ol>
</li>
</ol>2. 培养游离祖细胞形成 Pancreatoids
注: 使用标准的无菌程序时, 应在标准组织培养罩的无菌气氛下进行。
<ol><li>对于收集的每个芽, 请将400µL 器官介质16,17 (表 1) 添加到离心单元格中。脉冲涡三倍和板100µL 每井低附件96井板, 分裂芽在1:4 比率 (图 1G)。</li>
	<li>检查显微镜下以可视化分离的细胞自由浮动 (图 1H)。将养殖盘放在 37 oC 孵化器中, 以中等速度在摇杆上使用 5% CO2 。</li>
	<li>监控 pancreatoids 的进度。每3天更换100µL 的新鲜媒体, 仔细吹打媒体下的控制罩, 在离开 pancreatoid 在井中删除。另外, 新鲜的100µL 的介质可以添加到一个新的井和 pancreatoid 转移使用硼硅毛细管连接到嘴吸管和1000µL 过滤吸管提示。</li>
</ol>3. 荧光成像的处理 Pancreatoids
<ol><li>要处理 pancreatoids 的免疫荧光图像, 首先准备4% 多聚甲醛/PBS, pH7.4 (粉煤灰) 解决方案。在96井板井中放置50µL 4% 粉煤灰。<ol>
<li>采用硼硅酸盐毛细管和口吸管与1000µL 过滤吸管尖端, 转移 pancreatoids 从培养基尽可能少的培养基, 以尽量小的介质到新鲜的井与4% 粉煤灰。设置在摇杆在室温下15分钟。</li>
		<li>将 pancreatoids 从4% 粉煤灰输送到井中, 100 µL 的新鲜 pbs, 室温下的岩石 5-10 分钟. 重复共三个新鲜 pbs 洗涤。 注意: 该协议可以在这里暂停, pancreatoids 存储在 4 oC 的 PBS 中100µL, 最多一周。</li>
	</ol>
</li>
	<li>对于 wholemount 成像 (建议如果抗体是适当的, 在3.3 节的替代方法. 显微镜 3.4.), 从 pbs 转移到50µL 5% 驴血清在 1x PBS 与0.1% 非离子洗涤剂 (PBST) 阻拦。在 4 oC 或在室温下以4小时为一夜摇滚。<ol>
<li>将 pancreatoids 转移到一个新的含原抗体的井中, 在 1x PBST 中稀释50µL 5% 驴血清。优选地, 岩石为 24 h 在 4 oC (至少 12 h, 但至多 36 h)。 注: 在材料表中列出的抗体对 Chga (1:100)、DBA (1:400)、Vim (1:400) 和 Pdx1 (1:100) 适用于 wholemount 染色;其他抗体可能需要优化。</li>
		<li>将 pancreatoids 转移到含有100µL 新鲜 PBST 的井中, 在室温下清洗和摇动30分钟。重复这一步骤, 共三新鲜 PBST 洗涤。</li>
		<li>将 pancreatoids 转移到一个新的含二级抗体的井中, 在 1x PBST 中稀释50µL 5% 驴血清。在 4 oC 中保护板, 使其处于最佳的 24 h (至少12小时, 但高达36小时)。 注: 在材料表和 DAPI 核 counterstain 中列出的所有次级抗体均适用于 wholemount 染色。</li>
		<li>将 pancreatoids 转移到含有100µL 1x PBST 和岩石室温30分钟的新井中, 在 1x PBST 中重复三洗涤。在第三和最后的洗涤, 添加 300 nM DAPI。</li>
		<li>最后, 将100µL 的清洁 PBS 和存储在4个oC 上, 在通过共聚焦显微镜进行成像前长达一周的时间内进行保护, 不过最好的结果来自于染色后立即进行成像。为防止 pancreatoids 在成像过程中的移动, 但防止干燥, 将每个 pancreatoid 放入20µL 的 PBS 中。如果 pancreatoids 不保持静止, 则减少 PBS 音量。</li>
	</ol>
</li>
	<li>对于冰冻切片, 在 4 oC 上隔夜将 pancreatoids 从 PBS 转移到30% 蔗糖溶液中。<ol>
<li>在夹层显微镜下的气泡中添加嵌入介质。在显微控制下使用镊子, 通过在嵌入介质中轻轻地用力挤压 pancreatoids, 将残留蔗糖去除, 然后再用冷冻嵌入介质转移到组织块。</li>
		<li>将组织块放在干冰上, 直到冷冻, 恒温器在8µm 的部分。 注意: 节可以立即存储在-80 oC 或 immunostained 中。</li>
		<li>允许从-80 oC 中的节在室温下解冻约5分钟, 直到干燥。使用疏水性的 pap 笔在组织和块使用5% 驴血清稀释 1x PBST 30 分钟在室温下, 绘制疏水性屏障。</li>
		<li>在 4 oC 中, 在 1x PBST 的一夜之间, 用5% 驴血清稀释的原抗体去除介质并立即更换。</li>
		<li>取出主要溶液, 用 1x PBST 三次, 每10分钟清洗组织。在此之后, 添加二次抗体溶液稀释5% 驴血清稀释在 1x PBST 1 小时的室温和保护幻灯片免受光照。</li>
		<li>删除第二个解决方案, 并在 1x PBST 中清洗幻灯片三次, 每10分钟。在最后的洗涤, 添加 300 nM DAPI。</li>
		<li>删除介质并添加安装介质和盖玻片。在 4 oC 中存储幻灯片, 直到准备好映像。</li>
	</ol>
</li>
</ol>4. 从 Pancreatoids 中分离 RNA 进行转录分析
<ol><li>收集 pancreatoids 使用含硼硅酸盐毛细管与过滤1000µL 吸管提示不育, 并放入500µL 酸胍硫氰酸盐-苯酚-氯仿溶液在1.5 毫升管。存储在-80 oC 或立即进行 RNA 隔离。</li>
	<li>对于 RNA 分离, 在冰上解冻样品, 如有必要, 加入100µL 氯仿。漩涡井和地方在 4 oc 为15分钟前冷却离心机到 4 oc。<ol>
<li>将管子放入离心机中, 在 4 oC 的1.2万克处旋转20分钟。</li>
		<li>小心移除管子, 以免干扰层的分离。使用200µL 吸管, 收集明确的水溶液, 并放置到一个新的1.5 毫升管, 留下少量的缓冲从白色蛋白层和粉红色的 DNA 层。不要触及这个过程中的任何东西的吸管的尖端, 不要触摸1.5 毫升管的墙壁。</li>
		<li>添加500µL 100% 异丙醇的新管含有水层和漩涡。让我们在室温下坐20分钟, 在冰上再长一点, 或者在-20 oC 上过夜的最大降水。</li>
		<li>离心机以1.2万克为15分钟, 在 4 oC 上颗粒状 RNA。除去异丙醇, 不干扰颗粒。 注: 由于 pancreatoids 小, 很可能颗粒不会被看到。</li>
		<li>加入冷, 75% 乙醇和反转管几次。放置在离心机和旋转在 9000 x g 10 分钟在 4 oc 去除乙醇和旋转2分钟在 9000 x g。</li>
		<li>使用200µL 吸管去除管中残留的乙醇, 而不接触颗粒所在的区域。在室温下将瓶盖打开 5-10 分钟, 以确保剩余乙醇的蒸发。</li>
		<li>并用重悬颗粒由涡流在20µL 清洁, 核酸酶水。 可选: 将 RNA 加热 65 oC 以3分钟, 然后立即返回到冰上。RNA 可以存储在-80 oC 或立即用于反向转录和 qPCR。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

细胞培养模型的进展对于正确的模型开发, 产生临床相关的细胞类型, 测试药物疗效, 甚至移植到病人来说是至关重要的。然而, 人工综述在菜肴中的发展是有挑战性的, 因为我们还远未了解器官和生理的机制在体内.因此,体外细胞的生成效率低下, 不能充分发挥作用, 不能长时间维持, 或使体内可比细胞的其他异常发生。这是因为许多不同的细胞类型相互作用, 而复杂的形态发生变化影响发展和生理学。通过结合体外系统的方便性, 同时保留体内开发的复杂性, 了解开发如何进行, 这将为生物医学研究提供一个宝贵的工具。
organoids 的发展是对发展复杂性进行建模的一个有希望的途径。在本议定书中, 我们概述了如何使胰腺 organoids, 称为 pancreatoids, 它保留本地间质和强健的产生内分泌细胞, 虽然 pancreatoids 不显示葡萄糖反应。此工具可用于研究开发机制以及在保持体内中发现的组织的异质性的区域性系统中的功能。此外, 这可以用于基因屏幕或筛选小分子或药物。这是特别有趣的, 因为测试候选药物在相对均匀的细胞培养系统可能绕过这些化合物对其他密切相关的细胞类型的影响。
在此协议中有几个关键步骤。首先, 仔细去除子宫中的胚胎是重要的, 因为将胚胎强行拉出可以撕裂腹部区域, 使其难以辨别胃肠道获得胰芽 (步骤 1.3)。第二, 从胃肠道清除胰腺芽是至关重要的, 因为服用过量的组织可能会导致其他细胞类型的分化 (步骤 1.4.4)。要做到这一点, 重要的是捏在胰芽下面, 并在分离胰芽之前提起覆盖组织。最后, 当将芽从酶移回 PBS 冲洗时, 必须使用硼硅酸盐毛细管, 不要让组织接触管开口的边缘, 否则, 组织会粘在管子的尖端 (步骤 1.5.1)。为了避免这种情况, 在向芽前开始口吹打溶液, 让芽进入管而不是在外面。
这种方法允许在 3D pancreatoid 中形成内分泌细胞, 但是, 上皮分支比其他现有协议16,17更受限制。此外, 当胰岛素分泌的内分泌细胞形成时, 它们并不表现出葡萄糖反应。因此, 进一步调查这些细胞的成熟将提供有价值的信息, 无论是在产生功能性 pancreatoids, 以及对β细胞生成领域的一般。
生成内分泌细胞的 pancreatoids, 以及将来获得葡萄糖反应能力的人, 对糖尿病的治疗有潜在的影响。糖尿病是再生治疗的首要候选者, 因为胰腺β细胞丢失或功能失调, 替换这些细胞可能会减轻疾病并发症。在将 hPSCs 分化为β细胞方面取得了进展, 但在糖尿病方面, 胰腺中其他细胞类型和β细胞, 包括阿尔法细胞或腺泡细胞28,29,,经常出现功能障碍.30,31,32。因此, 产生新的胰腺组织移植可以完全取代受折磨的组织。通过对小鼠 pancreatoid 形成和功能的进一步调查, 可以模拟发育轨迹, 以产生 hPSCs 的人 pancreatoids。这些人类 pancreatoids 可以用于个性化的药物, 以筛查在病人特定的情况下对药物的反应, 并为再生治疗。

界定正常发展和生理学的机制, 对于了解疾病病因, 最终培养治疗方法至关重要。虽然培养和区分干细胞能够快速和高通量地分析发展, 但它受现有的知识机构的限制, 这些机制调控细胞命运和人为概括发展在一个相对同构, 二维状态1,2。不仅是受外部影响影响的 "体内" 开发, 在利基和环境中提供分泌信号和组织支持的不同单元格类型也可用于引导器官, 但这些细胞的功能也依赖于其用于指导的环境3,4,5。鉴于这些外部线索的重要性, 差异化协议的局限性, 以及体内老鼠模型的费力性质, 需要新的系统来实验研究基本的发育过程和生理学。
生成三维复杂 organoids 的协议的出现, 提供了一个方便和一致的系统来研究器官发生、生理学、药物功效, 甚至发病机制.建立小鼠 organoids 为不同的系统, 如胃6和肠道7已经扩大了我们对器官的理解, 提供了一个工具来研究发展复杂性较少的限制比在体内和体外模型。由于这些进展在小鼠 organoid 形成和人类多潜能干细胞的出现, 人类肠道8, 视网膜9, 肾脏10,11, 和大脑12 organoids 已经产生, 这《汇辑》仅限于现有关于发展机制的知识。
特别感兴趣的是胰腺 organoids 的产生, 因为无数的疾病瘟疫不同的胰腺细胞类型, 包括腺泡细胞和导管在外分泌的胰腺功能不全13, 胰腺炎的腺泡细胞14, 并β细胞在糖尿病15。获得有关这些不同细胞类型发展的知识有助于理解其病理学, 也可以作为个性化药物筛选或移植的平台。以前, Greggio et开发了一种方法来创建小鼠胰腺 organoids, 它概括了体内形态发生并发展了由所有主要的胰腺上皮细胞组成的三维复杂结构。类型16,17。这是胰腺领域向前迈出的重要一步, 特别是使细胞体外能够使β细胞的生物研究得以发展。然而, 在本议定书中形成的内分泌细胞的匮乏, 除非 organoids 移植到组织, 在那里利基可以互动和提供指导提示17。间质构成利基的最大部分, 严重包裹发育上皮从器官的早期阶段到以后阶段, 包括内分泌分层和分化 3, 4, 18。间质与发展中的胰腺的相互作用又是外部信号的另一个例子, 也是维持体内细胞复杂性来研究器官的重要性. 
在这里, 我们描述如何产生三维胰腺 organoids, 称为 pancreatoids, 从分离 e10.5 小鼠胰腺祖细胞。这些 pancreatoids 保留本机间质, 自组装在自由漂浮条件, 并产生所有主要的胰腺细胞类型, 包括强健的数量的内分泌β细胞 19.这种方法最适合分析内分泌的发展, 因为以前的协议缺乏强健的内分泌分化。然而, 使用 Greggio et所描述的胰 organoids 的协议, 更适合于分析胰腺上皮分支和形态发生, 因为 pancreatoids 中分支更受限制。

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">M6250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Aspirator Tube Assemblies for Calibrated Microcapillary Pipettes</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">A5177</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">BarnStead NanoPure Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td>D119</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Borosilicate Capillary Tubes</td>
			<td style="width:123px;">Sutter Instruments</td>
			<td style="width:344px;">GB1007515</td>
			<td style="width:167px;">O.D. 1mm, I.D. 0.75mm, 1.5cm length</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">CaCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">C5080</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cell-Repellent 96-Well Microplate</td>
			<td style="width:123px;">Greiner Bio-One</td>
			<td style="width:344px;">650970</td>
			<td style="width:167px;">U-bottom</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge 5424 R</td>
			<td>Eppendorf</td>
			<td>5401000013</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chloroform</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">233306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chromogranin-A antibody</td>
			<td>Abcam</td>
			<td>ab15160</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Compact, Modular Stereo Microscope M60</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Automated Cell Counter</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Countess Cell Counter Slides</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">C10312</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CryoStar NX70</td>
			<td>ThermoFisher</td>
			<td>957000L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">G7528</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAPI (4&#39;,6-Diamidine-2&#39;-phenylindole-dihydrochloride)</td>
			<td style="width:123px;">Roche</td>
			<td style="width:344px;">10 236 276 001</td>
			<td style="width:167px;">Powder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DBA antibody</td>
			<td>Vector Lab</td>
			<td>RL-1032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dispase II, Powder</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">17105041</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DMEM/F-12, HEPES</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:344px;">11330032</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dnase I</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">18068-015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont #5 Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:344px;">11251-10</td>
			<td style="width:167px;">0.05 x 0.02 mm; Titanium; Biology tip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">EGF (Epidermal growth factor)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">E9644</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ethanol, 200 Proof</td>
			<td style="width:123px;">Decon Laboratories</td>
			<td style="width:344px;">2716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Forma Steri Cycle CO2 Incubators</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">370</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fluoromount-G</td>
			<td style="width:123px;">Southern Biotech</td>
			<td style="width:344px;">OB10001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">H3149-10KU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">INSM1 Antibody</td>
			<td style="width:123px;">Santa Cruz BioTechnology</td>
			<td style="width:344px;">sc-271408</td>
			<td style="width:167px;">Polyclonal Mouse IgG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Isopropanol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:344px;">a4164</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isothesia Isoflurane, USP</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Insulin Antibody</td>
			<td style="width:123px;">Dako</td>
			<td style="width:344px;">A056401</td>
			<td style="width:167px;">Polyclonal Guinea Pig</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KAPA SYBR FAST Universal</td>
			<td style="width:123px;">KAPA Biosystems</td>
			<td style="width:344px;">KK4618</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KCl</td>
			<td style="width:123px;">KaryoMax</td>
			<td>10575090</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">KnockOut Serum Replacement</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">10828028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Leica TCS SPE High-Resolution Spectral Confocal</td>
			<td style="width:123px;">Leica</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MgCl2</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">442615</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse C-Peptide ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-CPTMS-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse Ultrasensitive Insulin ELISA</td>
			<td style="width:123px;">ALPCO</td>
			<td style="width:344px;">80-INSMSU-E01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">MX35 Microtome Blades</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:344px;">3052835</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaCl</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S7653</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaHCO3</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S3817</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NaH2PO4</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Donkey Serum</td>
			<td style="width:123px;">Jackson Immuno Research</td>
			<td style="width:344px;">017-000-121</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Paraformaldehyde</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">158127</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PBS 1X</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">21-040-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pdx1 antibody</td>
			<td>DSHB</td>
			<td>F6A11</td>
			<td>Monoclonal Mouse MIgG1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Peel-A-Way Disposable Embedding Molds</td>
			<td style="width:123px;">VWR</td>
			<td style="width:344px;">15160-157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Penicillin-Streptomycin Solution</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">MT30002CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">PMA (Phorbol 12-Myristate 13-Acetate)</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">P1585</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein LoBind Microcentrifuge Tubes</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">22431081</td>
			<td style="width:167px;">1.5mL Capacity</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-10 Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">345-FG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human FGF-Acidic</td>
			<td style="width:123px;">Peprotech</td>
			<td style="width:344px;">100-17A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Recombinant Human R-Spondin I Protein</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td style="width:344px;">4546-RS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">BenchRocker 2D</td>
			<td style="width:123px;">Benchmark</td>
			<td style="width:344px;">BR2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sucrose 500g</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">S0389</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SuperFrost Plus Microscope Slides</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Super Pap Pen</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Thermomixer R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:344px;">05-412-401</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Tissue Tek O.C.T. Compound</td>
			<td style="width:123px;">Sakura</td>
			<td style="width:344px;">4583</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T8787</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TRIzol Reagent</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15596018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">TrypLE Express</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">12604039</td>
			<td style="width:167px;">(1x), no Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypan Blue Stain</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:344px;">15250061</td>
			<td style="width:167px;">For cell counting slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:344px;">25-052-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:123px;">Gibco</td>
			<td>25200072</td>
			<td style="width:167px;">Phenol Red</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment 24-Well Plate</td>
			<td style="width:123px;">Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultra-Low Attachment Spheroid Plate 96-Well</td>
			<td style="width:123px;">Corning</td>
			<td>4520</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vimentin Antibody</td>
			<td style="width:123px;">EMD Millipore</td>
			<td style="width:344px;">AB5733</td>
			<td style="width:167px;">Polyclonal Chicken IgY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Vortex Genie</td>
			<td style="width:123px;">BioExpres</td>
			<td style="width:344px;">S-7350-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Y-27632 Dihydrochloride</td>
			<td style="width:123px;">R&#38;D Systems</td>
			<td>1254</td>
			<td style="width:167px;">Also known as ROCK inhibitor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Zeiss 710 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of scaffold-free, three-dimensional insulin expressing pancreatoids from mouse pancreatic progenitors in vitro

胰腺是由许多不同细胞类型组成的复杂器官, 它们协同工作来调节血糖的稳态和消化。这些细胞类型包括分泌酶的腺泡细胞, 一种 arborized 导管系统, 负责将酶输送到肠道, 以及产生激素的内分泌细胞。
内分泌β细胞是体内唯一的细胞类型, 它能产生胰岛素以降低血糖水平。糖尿病是一种以丧失或β细胞功能障碍为特征的疾病, 正达到流行的程度。因此, 必须建立一些协议来调查β细胞的发展, 它可以用于筛选目的, 以获得药物和细胞的治疗方法。虽然实验性研究老鼠的发展是必不可少的,在体内研究是费力和耗时的。培养细胞为筛查提供了更方便的平台;然而, 他们无法维持细胞多样性, 建筑组织, 和细胞之间的相互作用发现在体内.因此, 有必要开发新的工具来调查胰腺器官和生理。
胰腺上皮细胞在与间质的密切联系中发展, 从器官发生开始, 细胞组织并分化成复杂的、生理上有能力的成体器官。胰腺间质为内分泌的发展提供了重要信号, 其中许多还没有得到很好的理解, 因此在体外文化中很难重述。在这里, 我们描述一个协议, 文化三维, 细胞复杂的老鼠 organoids 保留间质, 称为 pancreatoids。e10.5 小鼠胰芽被解剖, 分离, 并培养在一个无支架的环境。这些漂浮细胞自组装与间质包络的发展 pancreatoid 和强健的数量的内分泌β细胞与腺泡和导管细胞一起发展。该系统可用于研究细胞的命运决定, 结构组织, 和形态发生, 细胞间的相互作用, 在器官发生, 或为药物, 小分子, 或基因筛选。

仔细解剖小鼠胚胎在 e10.5 从子宫角应该产生未损坏的胚胎在 PBS 进一步解剖 (图 1A)。胃肠道可以有效地从胚胎中移除 (图 1B), 允许辨别肠道和胃交界处的背胰芽 (图 1C-F)。e10.5 的胰芽以前有特征;祖祖先应表达Pdx1、Sox9、Ptf1a、和Hes120、21、22、23。在组织处理步骤后, 分离的胰单细胞或小组细胞可以通过光显微术 (图 1G H) 进行可视化。
在器官化介质中, 这些自由漂浮的无支架细胞自组装并组织成三维 pancreatoids, 在文化中生长并持续至少十天 (图 2A)。pancreatoids 与体内胰腺有形态学相似之处, 出现分支形态发生。利用转基因小鼠, 可以实时监测不同细胞类型或过程。例如, 使用 Ins1-eGFP24小鼠来标记内分泌β细胞的形成, pancreatoids 可以每天成像, 以可视化β细胞开发 (图 2B)。此外, 通过改变培养条件, 添加小分子, 药物, 或操纵基因组, 不仅可以评估细胞命运的决定, 但结构和形态学的变化也可以研究。在这里, 我们展示了蛋白激酶 C 激活剂佛波 12-酯 13-醋酸盐 (PMA) 在高浓度 (160 nM), 这改变了发展 pancreatoids 的形态学, 导致松散的相关上皮细胞和增加分支19 (图 2C)。
为了评估胰腺间质组织与胰腺上皮细胞的关系, 染色可以对每个组织的标记进行。染色的管道标记, DBA25,26, 具有内分泌标记, Chga27, 和 DAPI 标记的细胞核显示 pancreatoids 中的多血统形成 (图 3A)。一个间质标记, 波形, 与一个胰腺祖标记 (从 e9.0 到大约 e15.5) Pdx1, 显示间质信封 pancreatoid(图 3 B).染色也可以用来检查形态学, Pdx1 揭示分支结构, 以及不同的细胞类型标记的兴趣。在图 3C中, 我们通过对上皮标记 Pdx1 和β细胞标记外接的染色来可视化β细胞的发育, 使用 qPCR 分析, 祖的转录和分化细胞 a 被评估, 如祖基因Pdx1和Hes1、和区分标记Prss1、Prss3、Hnf6、Isl1、Nkx6-1、和Ins1 (图 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57599/57599fig1.jpg"> 图 1: pancreatoid 生成的 e10.5 胰祖细胞的解剖、离解和电镀概述。(A) e10.5 胚的 brightfield 图像。(B)解剖过程示意图, 从左上开始。红色虚线表示区域要切割, 而绿色区域则是胃肠道。首先, 把头移除, 然后除去前肢芽和机体一侧的开口。(C)胃肠道被仔细切除, 心脏和肝芽从该道的腹区突出。胃、背胰芽和肠道可以可视化, 如 brightfield 图像所示。(D)删除心脏和肝芽。示意图在左边, brightfield 图像在右边。(E)捏紧背胰芽周围的组织以暴露芽进行解剖。示意图在左边, brightfield 图像在右边。(F) brightfield 显微镜下的胃肠道与背胰芽。在左侧的图像中, 肠道中的弯曲可见, 在从脊柱区分离胃肠道时可以放置镊子。在右侧, 高放大 brightfield 图像显示背部和腹侧的胰芽。(G)切除后的胰芽和组织的处理, 分离前, 泥沙在器官的培养基和电镀。(H) brightfield 图像在电镀后立即显示离解细胞。<a href="/files/ftp_upload/57599/57599fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57599/57599fig2.jpg"> 图 2: 随着时间的推移监视 pancreatoids。(A) Brightfield pancreatoids 在1、2天和3天的图像。缩放 bars=100 嗯。(B)操作文化条件以调查胰腺形态发生。在这里, 加入高水平的可转移性导致松弛的上皮结构和增加分支, 如可视化的 brightfield 显微镜在1和2天。刻度条 = 100 µm (C) Ins1-eGFP 鼠标 pancreatoids 在4天、5天、6天、7天和9天, 并由 eGFP 在绿色中表示的β细胞的发展。刻度条 = 100 µm.<a href="/files/ftp_upload/57599/57599fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57599/57599fig3.jpg"> 图 3: 染色 pancreatoids.(A)染色 pancreatoid 在5天, 用红色的 DBA 标记管道, 由 Chga 在绿色中的内分泌细胞, 以及以蓝色 DAPI 标记的细胞核。(B)染色 pancreatoid 在7天, 间质由波形在红、胰祖细胞中标记为绿色 Pdx1。(B)染色 pancreatoid 在7天, 以绿色的 Pdx1 在红色和胰腺祖细胞中标记β电池。<a href="/files/ftp_upload/57599/57599fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57599/57599fig4.jpg"> 图 4: 成绩单分析.7天 pancreatoids 的定量 PCR 方法, 用于对祖细胞和分化单元的标记。与体内小鼠组织的比较显示在 Scavuzzo et . 中。(2017). 结果被规范化为Gapdh。N = 2, 刻度条是 SEM.<a href="/files/ftp_upload/57599/57599fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>组件</td>
			<td>缩写</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>青霉素-链霉素</td>
			<td>P/秒</td>
			<td>1%</td>
		</tr>
<tr>
<td colspan="2">无血清培养基补充剂</td>
			<td>10%</td>
		</tr>
<tr>
<td>β-巯基乙醇</td>
			<td>bME</td>
			<td>0.1 毫米</td>
		</tr>
<tr>
<td>佛波 12-酯13乙酸酯</td>
			<td>Pma</td>
			<td>16毫微米</td>
		</tr>
<tr>
<td>Y-27632 或岩石抑制剂</td>
			<td>日</td>
			<td>10 uM</td>
		</tr>
<tr>
<td>表皮生长因子</td>
			<td>Egf</td>
			<td>25 ng/毫升</td>
		</tr>
<tr>
<td>R-Spondin1</td>
			<td>-</td>
			<td>500 ng/毫升</td>
		</tr>
<tr>
<td>酸性成纤维细胞生长因子, 成纤维细胞生长因子1</td>
			<td>aFGF, FGF1</td>
			<td>25 ug/毫升</td>
		</tr>
<tr>
<td>肝素钠盐</td>
			<td>肝 素</td>
			<td>2 U/毫升</td>
		</tr>
<tr>
<td>成纤维细胞生长因子10</td>
			<td>FGF10</td>
			<td>100 ng/毫升</td>
		</tr>
<tr>
<td>Dulbecco 改性鹰培养基/营养混合物 F-12</td>
			<td>DMEM/F12</td>
			<td>到5毫升</td>
		</tr>
</tbody></table>表1。器官的媒介。

Watch this Scientific Journal Video about Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro at JoVE.com

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

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

在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。

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

generation-scaffold-free-three-dimensional-insulin-expressing

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

9.0K 次观看.  Baylor College of Medicine. 在这里, 我们提出了一种生成胰岛素表达3D 小鼠 pancreatoids 从游离漂浮 e10.5 分离的胰祖细胞和相关间质的协议。

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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 ...

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 ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

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 ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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

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

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

从多能干细胞中无血清自由生产三维人类肝球

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