我们感谢普华永在图准备方面的帮助。这项工作得到了中国国家自然科学基金 (81530004、31425016) 和中国科学技术部 (2016YFA0100500) 的资助。. 进行了实验并草拟了手稿;--编辑了手稿。两位作者阅读并批准了最后的手稿。

包括动物学科在内的所有程序都已获得中国科学院动物研究所伦理审查委员会的批准. 
 1. 材料准备 <ol> <li> 对0.65 和 #181 进行消毒; m 过滤器在紫外线灯下产生紫外线, 在清洁的长凳上为4小时, 并且在 2 h 标记以后转动滤光器。
	注意: 当使用 UV 光时, 请穿戴适当的保护. </li>
	<li> 121 和 #176 用高压灭菌器消毒不锈钢网格, 30 分钟. 
	注: 需要一定高度的不锈钢网格, 以支持气接口上的过滤器, 这可以用钢丝网 ( 图 1 A ) 实现. </li>
	<li> 将氟西汀稀释到10和 #181 的最终浓度; M 进入 M5300 长期培养基, 均匀混合. 
	注: 对于一个六井板, 2 毫升 M5300 长期培养基是适当的每一个井。氢化可的松在 10 -6 M 可以被选择性地稀释到培养基中. </li>
	<li> 将培养基与氟西汀加在10和 #181; M 成六孔板或碟, 并将灭菌不锈钢网放入介质中. </li>
	<li> 洗涤0.65 和 #181; m 过滤器与煮沸的水二次为3分钟每次在玻璃细胞培养盘为绝育。每次洗涤后, 将过滤器放入磷酸盐缓冲盐水 (PBS) 2 分钟, 并将湿过滤器放在气接口的不锈钢网格上, 如 图 1 A 所示 [示意图在中心]. 
	注: 用蒸压灭菌器产生的开水可实现无菌, 设备应及时取出灭菌器, 避免温度降低. </li>
</ol> 2。大会外植体培养 <ol> <li> 在 E10.5 或 E11 的胚胎中小心地将股东大会区域与镊子分开。
	<ol> <li> 用解剖剪刀将子宫从孕鼠中剥离出来, 并将胚胎从子宫中分离出来. </li>
		<li> 清除胚体中的卵黄囊、脐带和内脏. </li>
		<li> 小心移除背神经组织和周围肌肉组织. </li>
		<li> 用钳切断前、后肢部位的组织, 将其与胚体分离。 图 1 B-C 显示了解剖的周年大会的示意图表示和形态学. </li>
	</ol> </li> <li> 将被解剖的公司分别放在气接口 ( 图 1 ) 上的滤镜上, 并在 5% CO 2 中的区域性中, 37 和 #176; C 为 2-3 天. 
	注意: 不要将被解剖的周年大会放在钢网的钢丝上, 并确保过滤器在文化气界面。除了周年大会, 蛋黄囊和胎儿肝脏也可以培养与此系统 7 . </li>
</ol> 3。表达式分析 <ol> <li> mRNA 级别 <ol> <li> 将培养的公司收集到1毫升 PBS 和离心机在4和 #176; C, 310 x g 为 6 min. </li>
		<li> 除去上清液后, 根据制造商和 #39 的指示, 从收集的公司中提取总 RNA, 并采用单相苯酚溶液. </li>
		<li> 总 RNA (2 和 #181; g) 然后用 m-MLV 反转录酶反向转录, 以获得 cDNA 为模板。定量 real-time PCR 方法是用 SYBR 绿和 Gapdh 作为内部控制进行的. </li>
	</ol> </li> <li> 蛋白质级别 <ol> <li> 收集在步骤3.1.1 中如上所述的培养的年度股东大会。
		<li> 用细胞裂解缓冲液 (10 毫米的三盐酸盐, 10 毫米氯化钠, 0.5% NP-40) 制备含有蛋白酶抑制剂的蛋白质, 并用于西方印迹检测先前描述的蛋白质水平 13 . </li>
	</ol> </li> </ol> 4。菌落形成单位在文化 (CFU-C) 化验中 <ol> <li> 在培养 2-3 天后, 收集1毫升磷酸盐缓冲盐水 (pbs) 和离心机在4和 #176; C, 310 x g 为6分钟和离解与200和 #181; L 胶原酶 (0.1% 在 PBS) 在37#176; C. 
	注: 胶原酶产生单细胞悬浮液所需时间约20分钟。每5分钟摇晃一次含周年大会的管子可以加速消化过程。200和 #181; 0.1% 胶原酶用于每一个股东大会和200和 #181; l 10% 血清用于停止反应. </li>
	<li> 在超低附着的 M3434 介质中培养单细胞悬浮 24-井板. 
	注意: 为避免污染, 青霉素-链霉素溶液可选择性添加到培养基中。由于 M3434 介质是粘性的, 所以使用1.0 毫升无针头的注射器来混合细胞和培养基. </li>
	<li> 7-10 天后在37和 #176 培养; C 在 5% CO 2 中, 每种类型的菌落的数量, 包括爆裂形成单位-红 (BFU), CFU-granulo-单核细胞 (CFU-GM) 和 CFU-粒, 红细胞, 巨噬细胞, 巨 (CFU-GEMM)根据形态学进行区分, 并以倒置显微镜 ( 图 2 C ) 进行评分. </li>
</ol> 5。 在体内 移植试验 <ol> <li> 菌落形成单位-脾脏 (CFU S) 化验 <ol> <li> 2-3 天的外植体培养, 收集公司和准备单细胞悬浮通过孵化200和 #181; L 胶原酶 (0.1% 在 PBS) 为20最小 </li>
		<li> 在细胞注射前, 对8至10周的老雄性 C57BL/6 小鼠进行致死性照射 (9 X 射线), 4-5 小时. </li>
		<li> 将辐照的鼠标放入抑制剂以限制其活动. </li>
		<li> 用75% 酒精浸泡的棉签擦拭尾部, 以促进静脉舒张. </li>
		<li> 注射0.5 胚当量 (ee) 或 1 ee 单细胞悬浮剂, 静脉注射到经照射的小鼠尾静脉, 用1.0 毫升注射器. 
		注: 细胞悬浮与 pbs 和0.5 毫升 pbs 每接收者是一个适当的容量。25口径或26口径的针用于注射 1.0 cc 注射器。没有空气可以被引入到注射器。用消毒棉签按压注射部位止血. </li>
		<li> 十一天后移植, 收集并修复 Bouin 和 #39 中的受体小鼠的脾脏 (15 毫升1.22% 苦味酸酸饱和水溶液, 2 毫升40% 甲醇和1毫升乙酸) 1-2 天, 用80% 酒精清洗 1-2 天。从宏观上计算脾脏的可见菌落数, 并确定每个 ee 的菌落数量. 
		#8203; 注意: Bouin 和 #39 的固色剂, 穿戴适当的保护. </li>
	</ol> </li> <li> 长期移植化验 <ol> 2-3 #160;d 总是外植体培养, 收集公司, 并以200和 #181 孵化制备单细胞悬浮液; L 胶原酶 (0.1% 在 PBS) 为 20 min. <li>
		<li> 对8至10周的老雄性 C57BL/6 小鼠进行致死性照射 (9 x 射线), 在细胞注射前 4-5 小时. </li>
		<li> 将辐照的鼠标放入抑制剂以限制其活动. </li>
		<li> 用75% 酒精浸泡的棉签擦拭尾部, 以促进静脉舒张. </li>
		我注射0.5 胚当量 (ee) 或 1 ee 单细胞悬浮剂, 静脉注射到经辐照后的小鼠尾静脉, 用1.0 毫升注射器。每位获奖者注射 1 ee 2 和 #215; 10 5 骨髓细胞 (CD45.1 背景). 
		注: 注射器中不能引入空气。用消毒棉签按压注射部位止血. </li>
		<li> 四月后, 收集接受者的骨髓并将其用于重构试验。只有与 #8805 的收件人; 10% 捐助者派生的嵌被认为是成功重组的表现. </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有相互矛盾的财务利益。

众所周知, 成熟的干细胞在骨髓中可以重新填充受照射的接受者的血液系统。与这些功能干细胞不同的是, 在小鼠胚胎的周年大会上出现的干细胞是不成熟的。直接移植结果表明, I 型 (ve cad+ CD45- CD41+) 和类型 II (ve cad+ CD45+) 前干细胞具有没有重构能力20。利用股东大会的外植体培养体系, 在干细胞的新生中, 可以在移植试验中重建受体<sup clas...

造血干细胞 (干细胞) 是组织特异的成体干细胞, 具有多向的潜能, 包括红、髓细胞和淋巴细胞, 以及自我的能力。最近的研究表明, 最早的干细胞产生于一个专门的内皮细胞, 称为 hemogenic 内皮细胞 (他), 通过内皮到造血转移 (EHT) 在腹壁的背主动脉1,2 ,3,4。一旦从胚胎 (E) 10.5 到 E12.5 在小鼠胚胎中形成了主动脉-性腺-肾 (年会) 区域, 干细胞就会迁移到胎儿肝脏进行扩张, 最终在个体的生命中殖民骨髓以维持成人造血。5,6. 尽管这一研究已经进行了许多年, 但 HSC 产生和发展的基本机制仍未得到完全理解。
与体外不同的是, 斑马鱼胚胎的受精和发育, 小鼠胚胎的宫内发育使得在胚胎发生过程中的最终造血研究更不方便。虽然使用击倒 (ko) 小鼠的基因实验是常用的, 但某些高鼠的缺乏也限制了它们在造血研究领域的应用。此外,在体内抢救实验在 KO 小鼠中不易进行。自1996年以来, 在该领域的先驱在7中为造血研究开发了 "年度" 外植体培养。在这一文化体系的帮助下, 已经确定了股东大会区域的腹侧组织以促进 HSC 活动, 而背部组织则发挥相反的作用8,9。在 HSC 的发展中, 应用了 Mpl、蛋白、蛋白、BMP 和刺猬的信号, 确定了血清素、外植体培养系统的作用10、11、12、13、14. 重要的是, 它也是一种常用的方法来挽救突变体胚胎的造血缺损13,15。

<table style="border-collapse:collapse;width:458pt" width="610">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:81pt" width="108" />
		<col style="width:89pt" width="119" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl67" height="21" style="width:162pt;height:15.75pt" width="216">durapore 0.65 &#181;m filters</td>
			<td class="xl68" style="width:81pt" width="108">Millipore</td>
			<td class="xl69" style="width:89pt" width="119">R5BA63787</td>
			<td class="xl67" style="width:125pt" width="167">AGM explant culture</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl67" height="42" style="height:31.5pt">M5300 long-term culture medium</td>
			<td class="xl68" style="width:81pt" width="108">Stem Cell Technologies</td>
			<td class="xl69" style="width:89pt" width="119">5300</td>
			<td class="xl67">AGM explant culture</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Trizol&#160;</td>
			<td class="xl68" style="width:81pt" width="108">Tiangen</td>
			<td class="xl69" style="width:89pt" width="119">5829</td>
			<td class="xl67">RNA extration</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">M-MLV reverse transcriptase</td>
			<td class="xl68" style="width:81pt" width="108">Promega</td>
			<td class="xl69" style="width:89pt" width="119">90694</td>
			<td class="xl67">reverse transcription</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">SYBR Green</td>
			<td class="xl68" style="width:81pt" width="108">Qiagen</td>
			<td class="xl69" style="width:89pt" width="119">A6002</td>
			<td class="xl67">quantatitive real-time PCR</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">protease inhibitor</td>
			<td class="xl68" style="width:81pt" width="108">Roche</td>
			<td class="xl69" style="width:89pt" width="119">55622</td>
			<td class="xl67">Protein extration</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">collagenase</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl69" style="width:89pt" width="119">C2674</td>
			<td class="xl67">digestion of AGM tissues</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">MethoCult GF M3434 medium</td>
			<td class="xl68" style="width:81pt" width="108">Stem Cell Technologies</td>
			<td class="xl69" style="width:89pt" width="119">3434</td>
			<td class="xl67">CFU-C assay</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">ultra-low attachment 24-well plates</td>
			<td class="xl68" style="width:81pt" width="108">Costar</td>
			<td class="xl69" style="width:89pt" width="119">3473</td>
			<td class="xl67">CFU-C assay</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl67" height="63" style="height:47.25pt">C57BL/6 CD45.2 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">CFU-S assay</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">C57BL/6 CD45.1 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">Long-term transplantation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">phosphate buffered saline</td>
			<td class="xl68" style="width:81pt" width="108">Life Technologines</td>
			<td class="xl69" style="width:89pt" width="119">8115284</td>
			<td class="xl68" style="width:125pt" width="167">AGM Collection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Penicillin-Streptomycin solution</td>
			<td class="xl68" style="width:81pt" width="108">HyClone</td>
			<td class="xl68" style="width:89pt" width="119">SV30010</td>
			<td class="xl68" style="width:125pt" width="167">AGM explant culture</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45.2-PE-CY7</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">25-0454-80</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45-FITC</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">11-0451-81</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl68" height="105" style="width:162pt;height:78.75pt" width="216">UV lamp</td>
			<td class="xl68" style="width:81pt" width="108">Beijing jiangmorning yuan bio-technology co., LTD</td>
			<td class="xl68" style="width:89pt" width="119">039-14402</td>
			<td class="xl68" style="width:125pt" width="167">power: 30W</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">stainless steel mesh</td>
			<td class="xl68" style="width:81pt" width="108">AS ONE SHANGHAI Corporation</td>
			<td class="xl68" style="width:89pt" width="119">2-9817-10&#160;</td>
			<td class="xl68" style="width:125pt" width="167">aperture diameter: 2.5 mm</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">hydrocortisone</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl68" style="width:89pt" width="119">H0396</td>
			<td class="xl68" style="width:125pt" width="167">selectively diluted into M5300 medium</td>
		</tr>
	</tbody>
</table>

application of aorta-gonad-mesonephros explant culture system in developmental hematopoiesis

使用小鼠胚胎进行造血研究的局限性是在手术中增加了不便, 这主要是由于胚胎的宫内发育。尽管来自击倒 (ko) 小鼠的基因数据令人信服, 但在需要时为所有基因生成高鼠并不现实。此外, 执行在体内抢救实验, 以巩固从 KO 小鼠获得的数据是不方便的。为了克服这些局限性, 主动脉-性腺-肾 (年会) 外植体培养作为研究造血干细胞 (HSC) 发育的适宜系统而发展起来。特别是在抢救实验中, 可用于恢复高鼠的造血损伤。通过在培养基中加入适当的化学物质, 可以重新激活受损的信号, 或抑制上调的通路。利用这种方法, 可以进行大量的实验, 以确定 hsc 发展的关键调控因子, 包括 hsc 相关基因在 mRNA 和蛋白质水平、菌落形成能力和重构能力上的表达。这一系列的实验将有助于确定哺乳动物 HSC 发育所必需的基本机制。

最近发表的一篇报道说, 内皮细胞衍生的血清素通过抑制干细胞的亡途径, 促进了其生存.13。为了证实血清素对 HSC 发育的促进作用, 氟西汀被列入。作为一种选择性血清素再抑制剂 (SSRI), 氟西汀已被证明抑制血清素吸收在周围组织16,17,18。采用上述方法对氟西汀对 HSC 培养的影响进行了...

Watch this Scientific Journal Video about Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis at JoVE.com

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

本议定书描述使用培养的主动脉-性腺-肾的表达分析, 菌落形成单位在文化和脾脏, 并长期重建, 以确定影响的调节因子和信号通路的造血干细胞发育。这已被证明是一个有效的系统研究造血干细胞生物学和功能。

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

application-aorta-gonad-mesonephros-explant-culture-system

Research

JoVE Journal

Developmental Biology

8.3K 次观看.  Chinese Academy of Sciences. 本议定书描述使用培养的主动脉-性腺-肾的表达分析, 菌落形成单位在文化和脾脏, 并长期重建, 以确定影响的调节因子和信号通路的造血干细胞发育。这已被证明是一个有效的系统研究造血干细胞生物学和功能。

视频: Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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

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

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

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

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

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

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 ...

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 ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

从人类诱导多能干细胞诱导的体外神经肌肉结点

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

研究斑马鱼脊椎动物分割的三维尾部外植培养

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...