这项工作得到了欧洲血液学协会 2021 年研究高级资助、美国血液学会 2021 年全球研究奖以及欢迎信托基金和爱丁堡大学资助的内部战略支持基金 ISSF3 的支持。我们感谢流式细胞术设施的 Fiona Rossi 在流式细胞术分析方面的支持。出于开放获取的目的，作者已将知识共享署名 （CC BY） 许可应用于本次提交产生的任何作者接受的手稿版本。

hiPSC 来源的内皮细胞的分化和机械刺激

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作者没有要披露的利益冲突。

我们在这里描述的方案允许从人多能干细胞中产生机械敏感的内皮细胞，并研究它们对由受控剪切应力介导的机械刺激的反应。该方案完全基于细胞因子，与GMP试剂完全兼容，可潜在转化为细胞治疗的细胞生产。
hiPSCs的推导为科学家提供了胚胎发育早期阶段的仪器模型，使研究难以在体内研究的过程成为可能24。事实上，可用于研究的人类胚胎组织是从缺乏循环的胚胎中收集的，这可能对机械线索控制的分子特征产生重大影响。这里描述的方法能够对细胞对剪切应力的反应进行实时成像和实时研究。hiPSCs与流体的结合提供了一种研究模型，该模型克服了循环开始重塑和控制心血管和血液系统建立时发育中的胎儿组织的有限可用性和不可接近性3,9,10,25。
该方案的一个局限性是，从该方案衍生的内皮细胞可能无法反映发育组织中存在的不同内皮细胞的各种身份。为了克服这一局限性，在流体刺激之前的分化过程中可能需要特定的细胞因子组合，以获得所需的身份或组织特异性表型26。在分离步骤中，可以使用更精细的免疫表型获得内皮亚群的分离。该方案仅基于CD34的表达分离内皮细胞，从而允许柱分离而不是荧光激活细胞分选（FACS）;这减少了细胞死亡和污染风险。此外，该协议专门用于研究层流介导的剪切应力的作用。必须采用替代流体方法来研究其他机械线索的影响，例如拉伸或压缩，或其他类型的流动，例如扰动或扰动流动。
我们之前已经证明，iPSC 衍生的内皮细胞模仿异质动静脉细胞身份27，类似于在胎儿背主动脉28、29、30 中观察到的身份。这在已知由血液循环控制的血管发育和细胞规格方面尤为重要。不同模型的研究表明，缺乏循环会导致动静脉规格改变11,14,31。将机械线索与细胞规格联系起来的机制仍然未知，这里描述的管道允许进行无法在体内测试的精细功能研究。
该管道描述了使用市售流体通道生产和刺激源自 hiPSC 的内皮细胞，避免了像广泛使用的聚二甲基硅氧烷 （PDMS） 装置那样铸造装置的需要12。此外，PDMS芯片的使用使得刺激细胞的收集特别具有挑战性，而使用该协议，可以很容易地从通道中取出细胞。这显着提高了分析能力，允许后续分析，例如蛋白质组学和转录组学分析、流式细胞术和功能测定，这些可能需要进一步的培养或 体内 测定。

在胚胎发育过程中，心脏是第一个建立功能的器官1，从心内膜管形成的最早阶段2 开始就可检测到收缩。循环，以及由血管内血液流动介导的机械线索，控制着早期发育的许多关键方面。在胎儿循环建立之前，脉管系统被组织成一个初级毛细血管丛;心脏功能正常后，该神经丛重组为静脉和动脉脉管系统3。机械线索在动静脉规范中的作用反映在血流开始前动脉和静脉标志物的泛内皮表达4.
血流动力学不仅控制着脉管系统本身的发育，而且在控制血细胞形成中起着基础性作用。造血干细胞和祖细胞 （HSPC） 从称为造血内皮细胞5、6、7、8 的特殊内皮细胞中出现，仅在发育早期存在于胚胎的不同解剖区域。心脏缺陷模型与体外模型一起证明，机械线索指示并增加血液源性内皮细胞 HSPC 的产生 9,10,11,12,13,14。
不同类型的血流动力学已被证明可以差异控制细胞周期15，已知在血源性内皮 16,17 和动脉细胞规范18 中都很重要。总而言之，机械线索是发育过程中细胞身份和功能的关键决定因素。新型体外流体装置使我们能够克服在体内人类血液发育过程中研究发育机械生物学的局限性。
本手稿中方案的总体目标是逐步描述实验管道，以研究剪切应力对 体外 来源于人诱导多能干细胞（hiPSC）的人内皮细胞的影响。该方案包含有关将hiPSC分化为内皮细胞及其随后接种到用于刺激方案的流体芯片中的详细说明。使用这种方法，可以通过分析不同的 体外来源的内皮细胞响应流动的方向来测试它们感知剪切应力的能力。这将使其他实验室能够解决有关剪切应力的响应及其对不同内皮细胞身份的功能影响的问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.6 Luer uncoated slide</td>
			<td>ibidi</td>
			<td>IB-80186</td>
			<td></td>
		</tr>
		<tr>
			<td>25% BSA</td>
			<td>Life Technologies</td>
			<td>A10008-01</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Cited as Dissociation reagent</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Merck</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspiration pipette</td>
			<td>Sardtedt</td>
			<td>86.1252.011</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Cited as Neuronal cell culture supplement (50x)</td>
		</tr>
		<tr>
			<td>BD FACS DIVA</td>
			<td>BD Biosciences</td>
			<td>Version 8.0.1</td>
			<td>Cited as flow cytometry software</td>
		</tr>
		<tr>
			<td>BD LSR Fortessa 5 Laser</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 Microbead kit</td>
			<td>Miltenyil Biotec</td>
			<td>30-046-702</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 PE clone 4H11</td>
			<td>Invitrogen</td>
			<td>12-0349-42</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 PerCP-eFluor 710 clone 4H11</td>
			<td>Invitrogen</td>
			<td>44-0349-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar cell-repellent surface 6-well plates</td>
			<td>Greiner Bio-one</td>
			<td>657970</td>
			<td>Cited as cell-repellent plate</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemicals&#160;</td>
			<td>13122-1mg-CAY</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10&#160; cell cryopreservation</td>
			<td>Merck</td>
			<td>C2874-100ML</td>
			<td>Cited as Cryopreservation solution</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>VWR</td>
			<td>200-664-3</td>
			<td>Cited as DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DPB Ca2+ Mg2+</td>
			<td>Life Technologies</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY Strainer 40 &#956;m</td>
			<td>Greiner Bio-one</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Life technologies</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyil Biotec</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td>Version 1.53c</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Jo</td>
			<td></td>
			<td>Version 10.7.1</td>
			<td>Cited as flow cytometry sanalysis oftware</td>
		</tr>
		<tr>
			<td>FLT3L</td>
			<td>Peprotech</td>
			<td>300-19-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidic unit</td>
			<td>ibidi</td>
			<td>10903</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Cited as L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham F-12&#160;</td>
			<td>Life Technologies</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>Merk</td>
			<td>T0665-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Fujifilm UK LTD</td>
			<td>9988</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Pump system</td>
			<td>ibidi</td>
			<td>10902</td>
			<td>Cited as Pump system</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Life Technologies</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ioLight/Thisle Scientific</td>
			<td>IOL-IO-INVERT</td>
			<td>Cited as inverted in-incubator microscope</td>
		</tr>
		<tr>
			<td>Lyophilised BSA&#160;</td>
			<td>Merck</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td>Cited as Magnetic separator</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>Cited as Magnetic column</td>
		</tr>
		<tr>
			<td>MTG</td>
			<td>Merck</td>
			<td>M6145-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Notebook for pump system</td>
			<td>ibidi</td>
			<td>10908</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 37-41%</td>
			<td>Fisher Chemicals</td>
			<td>F/1501/PB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Pastette</td>
			<td>Greiner Bio-one</td>
			<td>612398</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Set YELLOW/GREEN: 50 cm, ID 1.6 mm, 10 mL reservoirs</td>
			<td>Ibidi</td>
			<td>IB-10964</td>
			<td>Cited as Perfusion set</td>
		</tr>
		<tr>
			<td>Polystyrene Round Bottom Tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td>Cited as Flow cytometry tubes</td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td></td>
			<td>Verison 9.4.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump control software</td>
			<td>ibidi</td>
			<td>version 1.6.1</td>
			<td>Cited as Pump software</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem cell tecchonologies</td>
			<td>5872</td>
			<td>Cited as Detaching solution</td>
		</tr>
		<tr>
			<td>rhBMP4</td>
			<td>R&#38;D</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>rhEPO</td>
			<td>R&#38;D</td>
			<td>287-TC-500</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIGF1</td>
			<td>Peprotech</td>
			<td>100-11-100uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL11</td>
			<td>Peprotech</td>
			<td>200-11-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL3</td>
			<td>Peprotech</td>
			<td>200-03-10uG</td>
			<td></td>
		</tr>
		<tr>
			<td>rhIL6</td>
			<td>R&#38;D</td>
			<td>206-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>rhLaminin-521</td>
			<td>Life technologies</td>
			<td>A29248</td>
			<td>Cited as Laminin</td>
		</tr>
		<tr>
			<td>rhSCF</td>
			<td>Life Technologies</td>
			<td>PHC2111</td>
			<td></td>
		</tr>
		<tr>
			<td>rhTPO</td>
			<td>R&#38;D</td>
			<td>288-TPN-025</td>
			<td></td>
		</tr>
		<tr>
			<td>rhVEGF</td>
			<td>R&#38;D</td>
			<td>293-VE-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT Lysis Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Serial Connector for &#181;-Slides: Sterile, Sterile</td>
			<td>ibidi</td>
			<td>IB-10830</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro-CD34 SFM media</td>
			<td>Life Technologies</td>
			<td>10639011</td>
			<td>Cited as Serum-Free media for CD34+ cells (SFM-34)</td>
		</tr>
		<tr>
			<td>StemPro-CD34 Nutrient Supplement&#160;</td>
			<td>Life Technologies</td>
			<td>10641-025</td>
			<td>Cited as 34 nutrient supplement</td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Life Technologies</td>
			<td>A1000701</td>
			<td>Cited as Culture media&#160;</td>
		</tr>
		<tr>
			<td>StemPro supplement</td>
			<td>Life Technologies</td>
			<td>A10006-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin (VTN-N) recombinant human protein, truncated</td>
			<td>Invitrogen</td>
			<td>A31804</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Cited as iRock</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model of fetal human vessel on-chip to study developmental mechanobiology

心脏是发育过程中第一个功能建立的器官，因此在妊娠早期就启动了血液循环。除了运输氧气和营养物质以确保胎儿生长外，胎儿循环还通过机械线索控制内皮层内发生的许多关键发育事件。生物力学信号诱导血管结构变化，建立动静脉规范，控制造血干细胞的发育。发育中的组织无法接近限制了对循环在人类早期发育中的作用的理解;因此， 体外 模型是研究船舶机械生物学的关键工具。本文描述了一种将内皮细胞与人诱导的多能干细胞区分开来的方案，并将其随后接种到流体装置中以研究它们对机械线索的反应。这种方法允许在机械刺激下长期培养内皮细胞，然后取回内皮细胞以进行表型和功能表征。这里建立的 体外 模型将有助于阐明细胞内分子机制，这些机制转导由机械线索介导的信号传导，最终协调人类胎儿生命中的血管发育。

During embryonic development, the heart is the first organ to establish functionality1, with detectable contractions from the earliest stage of endocardial tube formation2. Circulation, along with the mechanical cues mediated by the flow of blood within the vessel, controls many crucial aspects of early development. Prior to fetal circulation establishment, the vasculature is organized into a primary capillary plexus; upon cardiac functioning, this plexus reorganizes into venous and arterial vasculature3. The role of mechanical cues in arteriovenous specification is reflected by the pan-endothelial expression of arterial and venous markers before blood flow initiation4.
Hemodynamic forces not only control the development of the vasculature itself but also play a fundamental role in the control of blood cell formation. Hematopoietic stem and progenitor cells (HSPCs) emerge from specialized endothelial cells called hemogenic endothelium5,6,7,8, present in different anatomical regions of the embryos exclusively in the early stage of development. Heart-deficient models, together with in vitro models, have demonstrated that mechanical cues instruct and increase HSPC production from the hemogenic endothelium9,10,11,12,13,14.
Different types of flow dynamics have been shown to differentially control the cell cycle15, known to be important in both hemogenic endothelium16,17 and arterial cell specification18. Altogether, mechanical cues are critical determinants of cell identity and function during development. Novel in vitro fluidic devices allow us to overcome the limitations involved with studying developmental mechanobiology during human blood development in vivo.
The overall goal of the protocol in this manuscript is to describe, step-by-step, the experimental pipeline to study the effect of shear stress on human endothelial cells derived in vitro from human induced pluripotent stem cells (hiPSCs). This protocol contains detailed instructions on the differentiation of hiPSCs into endothelial cells and their subsequent seeding into fluidic chips for the stimulation protocol. Using this, different in vitro-derived endothelial cells can be tested for their ability to sense the shear stress by analyzing their orientation in response to the flow. This will allow other laboratories to address questions about the response to shear stress and its functional consequences on different endothelial cell identities.

作者聚焦：研究在流体介导的机械刺激下培养的 hiPSC 衍生的内皮细胞 

体外研究 胎儿人体血管芯片模型研究发育机械生物学

Our research explores how mechanical cues instruct the fate of endothelial cells during development, for which we combine human-induced pluripotent stem cell differentiation with fluidic-mediated mechanical stimulation. Endothelial cells are constantly exposed to the movement of blood, so important signals are missed out in static culture, which makes this the main challenge in studying endothelial cell biology using traditional culture systems. Mechanical cues, in fact, activate many crucial signaling pathways that might be overlooked in cells cultured in static conditions.Our protocol provides an easy, step-by-step guide on how to differentiate and mechanically stimulate induced pluripotent stem-cell-derived endothelial cells. This will help many laboratories in adopting a thermic culture to easily address questions about endothelial cell behavior while under flow.

我们在这里描述了一种用于分化和机械刺激源自hiPSC的内皮细胞的方案，该方案允许研究它们对机械线索的反应（图1）。该方案导致功能机械敏感内皮细胞的产生。我们在这里提供具有代表性的结果并描述预期的表型，以评估细胞在分化过程中对细胞因子刺激的反应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65492/65492fig01v2.jpg"> 图 1：分化和机械刺激方案的示意图。 分化方案示意图，显示了不同细胞因子混合物的时间、CD34+ 细胞分离、流控芯片接种和机械刺激细胞的最终分析。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig01largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
hiPSCs培养 重要的是从在自我更新条件下正确生长的hiPSC开始方案。文化质量的一个很好的指标是它们的生长速度。解冻后，细胞可能需要 2-3 周才能达到正确的生长阶段，以确保良好的分化。当细胞可以每周以 1：6 的比例传代两次，达到几乎完全汇合时，这是它们在需要传代的同一天准备分化的时间。
hiPSCs分化为内皮细胞 分化的第一步，包括胚状体（EBs）的形成，是细胞系依赖性的，可能需要对使用的特定细胞系进行一些优化。方案步骤1.3.2.2-1.3.2.4中描述的解离可以通过减少或延长与解离试剂的孵育以及随后与巴斯德移液管的解离来修饰。此外，除了使用切割工具或P100移液器吸头对菌落进行物理解离外，还可以使用其他解离试剂进行此步骤。质量好的EB在分化的第2天显示出明确的边缘，当使用显微镜观察时，它看起来清晰明亮;较暗的区域可能表明EB内的细胞死亡（图2）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65492/65492fig2v3.jpg"> 图2：胚状体形态 。 （A） 第 2 天胚状体显示轮廓分明的外缘和一致的大小。（B） 第 2 天，质量较差的胚状体显示广泛的细胞死亡，导致结构分解。比例尺 = 500 μm。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
在第 2 天，向 EB 中添加 CHIR99021 会抑制 GSK-3 蛋白，从而激活 Wnt 通路。不同的细胞系对CHIR处理有不同的反应，这应该通过使用不同的浓度量化第8天获得的CD34 + 细胞的数量来测试（图3）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65492/65492fig3v3.jpg"> 图 3：不同 CHIR 处理下的内皮细胞分化。 在第 2 天以 （A） 3 μM、（B） 5 μM 和 （C） 7 μM 进行 CHIR 处理后，通过流式细胞术在 CD34 膜表达分化第 8 天通过流式细胞术定量内皮细胞承诺。 使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
CD34+ 细胞分离 在洗脱色谱柱后，验证使用磁珠富集的CD34+是否至少提供80%的CD34+非常重要。为确保足够的纯度，可以通过流式细胞术分析从磁性分离中获得的细胞等分试样，确保使用与用于磁富集的抗体克隆不同的抗体克隆。在这里，使用4H11克隆，富集后纯度达到~85%（图4）。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65492/65492fig4v3.jpg"> 图4：磁分选富集前后CD34的膜表达。 对第 8 天解离的胚状体（灰色）和磁富集后的细胞（绿色）进行 CD34 表达染色，并通过流式细胞术进行分析，显示分选后成功富集。使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
将细胞接种到流体通道中 在流体通道中接种细胞时，跟踪内皮细胞的粘附和增殖至关重要。接种后，细胞需要~5小时才能完全粘附在通道上（图5A）。在此阶段，还可以测试替代涂层解决方案以提高附着力。为了确认被测细胞对机械敏感，因此能够对机械刺激做出反应，可以随时间推移测试细胞取向。刺激前的细胞显示随机取向（图5A和图5C），并且它们平行于流动方向重新定向（图5B，C）。这里描述的方案允许从通道中收集细胞以进行下游分析，例如流式细胞术，以研究其膜免疫表型，提供受刺激细胞的内皮特性（图5D，E）。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65492/65492fig5v3.jpg"> 图 5：hiPSCs 衍生的内皮细胞的机械反应性 。 （A） 接种后 48 小时分离的 CD34+ 细胞汇合层。（B） 动态培养 3 天的内皮细胞重新定向层。（C） 动态培养 5 天后内皮细胞的取向分析。（D） 流动培养 5 天的细胞的 CD34 表达谱。（E） 从流体通道中检索到的细胞群中 CD34+ 细胞的百分比。使用倒置培养箱显微镜拍摄图像;使用五激光流式细胞仪和专用软件获得流式细胞术数据（参见 材料表）。比例尺 = 100 μm （A，B）。 <a href="https://www.jove.com/files/ftp_upload/65492/65492fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>库存集中度</td>
			<td>添加的音量</td>
			<td>最终浓度</td>
		</tr><tr><td>Iscove 改良 Dulbecco 培养基 （IMDM）</td>
			<td>-</td>
			<td>333毫升</td>
			<td>-</td>
		</tr><tr><td>Ham's F-12 营养混合物 （F-12）</td>
			<td>-</td>
			<td>167毫升</td>
			<td>-</td>
		</tr><tr><td>N-2 补充剂 （100x）</td>
			<td>100 倍</td>
			<td>5毫升</td>
			<td>1 倍</td>
		</tr><tr><td>B-27 补充剂 （50x）</td>
			<td>50 倍</td>
			<td>10毫升</td>
			<td>1 倍</td>
		</tr><tr><td>抗坏血酸</td>
			<td>10毫克/毫升</td>
			<td>1.25毫升</td>
			<td>25微克/毫升</td>
		</tr><tr><td>α-单硫代甘油 （MTG）</td>
			<td>11.5 米</td>
			<td>19.5微升</td>
			<td>448.5微米</td>
		</tr><tr><td>人血清白蛋白</td>
			<td>100毫克/毫升</td>
			<td>2.5毫升</td>
			<td>0.5毫克/毫升</td>
		</tr><tr><td>全转铁蛋白</td>
			<td>100毫克/毫升</td>
			<td>0.75毫升</td>
			<td>150微克/毫升</td>
		</tr></tbody></table>表 1：500 mL 无血清分化 （SFD） 培养基的组成和配方。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>分化天数</td>
			<td>细胞因子混合物</td>
			<td>细胞因子</td>
			<td>最终浓度</td>
		</tr><tr><td>第 0 - 2 天</td>
			<td>混合 1</td>
			<td>BMP4型</td>
			<td>20纳克/毫升</td>
		</tr><tr><td>第2天</td>
			<td>混合 2</td>
			<td>CHIR99021</td>
			<td>7微米</td>
		</tr><tr><td>从第 3 天开始</td>
			<td rowspan="2">混合 3 和 4</td>
			<td>血管内生长因子（VEGF）</td>
			<td>15纳克/毫升</td>
		</tr><tr><td></td>
			<td>bFGF系列</td>
			<td>5纳克/毫升</td>
		</tr><tr><td>从第 6 天开始</td>
			<td rowspan="8">混合 4</td>
			<td>IL6型</td>
			<td>10纳克/毫升</td>
		</tr><tr><td></td>
			<td>FLT3L型</td>
			<td>10纳克/毫升</td>
		</tr><tr><td></td>
			<td>胰岛素样生长因子1</td>
			<td>25纳克/毫升</td>
		</tr><tr><td></td>
			<td>IL11型</td>
			<td>5纳克/毫升</td>
		</tr><tr><td></td>
			<td>云函数</td>
			<td>50纳克/毫升</td>
		</tr><tr><td></td>
			<td>欧洲专利局</td>
			<td>3 U/毫升</td>
		</tr><tr><td></td>
			<td>热塑性聚氨酯（TP</td>
			<td>30纳克/毫升</td>
		</tr><tr><td></td>
			<td>IL3型</td>
			<td>30纳克/毫升</td>
		</tr></tbody></table>表 2：用于内皮细胞分化的细胞因子混合物、将它们添加到 SFD 培养基中的天数和最终浓度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>剪切应力 （dyn/cm2）</td>
			<td>时间 （h）</td>
		</tr><tr><td>0.5</td>
			<td>1</td>
		</tr><tr><td>1</td>
			<td>1</td>
		</tr><tr><td>1.5</td>
			<td>1</td>
		</tr><tr><td>2</td>
			<td>1</td>
		</tr><tr><td>2.5</td>
			<td>1</td>
		</tr><tr><td>3</td>
			<td>1</td>
		</tr><tr><td>3.5</td>
			<td>1</td>
		</tr><tr><td>4</td>
			<td>1</td>
		</tr><tr><td>4.5</td>
			<td>1</td>
		</tr><tr><td>5</td>
			<td>直到实验结束</td>
		</tr></tbody></table>表 3：动态文化的剪切应力值及其应用时间。
补充图S1：用于该协议的芯片和管路的几何形状和尺寸。<a href="https://www.jove.com/files/ftp_upload/65492/SF1.pdf" target="_blank">请点击这里下载此文件。</a>
补充图 S2：控制气泵的软件的分步指南，并附有每个步骤的说明。<a href="https://www.jove.com/files/ftp_upload/65492/65492_Supp%20Figure%20S2%20FINAL.pdf" target="_blank">请点击这里下载此文件。</a>
补充图S3：使用FIJI进行取向分析的指南，显示了细胞形状的绘制、椭圆拟合和最终测量。<a href="https://www.jove.com/files/ftp_upload/65492/65492_SF3.pdf" target="_blank">请点击这里下载此文件。</a>
补充表S1：分化方案中使用的细胞因子的单位大小、重悬体积和储备液浓度。<a href="https://www.jove.com/files/ftp_upload/65492/Ventura%20et%20al,%202023%20-%20Supplementary%20Table%201.xlsx" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation at JoVE.com

这里描述的是将内皮细胞与人多能干细胞区分开来的简单工作流程，然后是其机械刺激的详细方案。这允许研究内皮细胞的发育机制生物学。该方法与机械刺激后从培养芯片收集的活细胞的下游测定兼容。

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides ...

我们的研究探讨了机械线索如何在发育过程中指导内皮细胞的命运，为此我们将人类诱导的多能干细胞分化与流体介导的机械刺激相结合。内皮细胞不断暴露在血液运动中，因此在静态培养中会遗漏重要信号，这使得使用传统培养系统研究内皮细胞生物学成为主要挑战。事实上，机械线索激活了许多关键的信号通路，这些通路在静态条件下培养的细胞中可能被忽视。我们的方案提供了关于如何分化和机械刺激诱导多能干细胞衍生的内皮细胞的简单、分步指南。这将有助于许多实验室采用热培养，以轻松解决有关内皮细胞在流动中行为的问题。

in-vitro-model-fetal-human-vessel-on-chip-to-study-developmental

Research

JoVE Journal

Developmental Biology

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

749 次观看.  University of Edinburgh. 我们的研究探讨了机械线索如何在发育过程中指导内皮细胞的命运，为此我们将人类诱导的多能干细胞分化与流体介导的机械刺激相结合。内皮细胞不断暴露在血液运动中，因此在静态培养中会遗漏重要信号，这使得使用传统培养系统研究内皮细胞生物学成为主要挑战。事实上，机械线索激活了许多关键的信号通路，这些通路在静态条件下培养的细胞中可能被忽视。我?...

视频: 作者聚焦：研究在流体介导的机械刺激下培养的 hiPSC 衍生的内皮细胞 

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

科研

发育生物学

﻿Differentiation of hiPSCs into Endothelial Cells

Differentiation of hiPSCs into Endothelial Cells  

To begin, on day zero, pre-warm serum-free differentiation medium or SFD medium containing Mix 1 cytokine to 37 degrees Celsius. Then, under a sterile tissue culture hood, add the Mix 1 cytokine-supplemented medium to each well of a cell repellent six-well plate. To prepare the cells, aspirate the culture medium from a six-well plate containing human-induced pluripotent STEM cells or human IPS cells.Wash them with DPBS followed by aspirating the DPBS. Then, add the dissociation reagent to the cells and incubate them at room temperature for a minute. After aspirating this dissociation reagent, incubate the cells for another three minutes at room temperature.Then, tap the plate containing the cells 10 times on each side to detach the cell clusters. Add one milliliter of pre-warmed Mix 1 cytokine-supplemented SFD medium to the cells per well. Using a pasture pipette, transfer the cell clusters from each well to one well of the cell repellent plate containing the medium with Mix 1 cytokine.After introducing the plate into the incubator, move it back and forth, as well as side to side, to disperse the content evenly and incubate it for embryoid bodies, or EB, formation. The next day, swirl the cell repellent plate containing the EBs to collect them into the center of the wells. Using a pasteur pipette, transfer the EB suspension from the wells to a 15-milliliter centrifuge tube.Wait five to 10 minutes for the EBs to settle at the bottom of the tube. Meanwhile, wash the cell repellent plates with sterile water or DPBS to remove single cells or debris. Add two milliliters of Mix 1 cytokine-supplemented SFD medium to each well of the plate.Now, gently aspirate the supernatant from the centrifuge tube without dislodging the EBs. Resuspend the EBs in the calculated volume of SFD medium containing Mix 1 cytokine. Add one milliliter of EB suspension to each well of the washed cell repellent plate already containing two milliliters of medium.Incubate the plate after dispersing the EBs by gently moving the plate as mentioned before. After gathering the EBs at the center of the wells, add the chiron on the side of each well, avoiding direct contact with the cells, Incubate the wells as demonstrated previously. On days three and six of differentiation, collect the EBs and perform media changes as demonstrated previously for day one.Use SFD medium supplemented with Mix 3 cytokines on day three and use SFD medium supplemented with Mix 4 cytokines on day six. After collecting and settling the EBs from the cell repellent plate into a 15-milliliter centrifuge tube, as demonstrated previously, aspirate the supernatant, then add one milliliter of cell dissociation reagent per well of EB collected in the tube to resuspend the EBs. Next, transfer one milliliter of this EB suspension to each well of the cell repellent plate washed with water.After incubating the plate for 10 minutes in the incubator, use a P1000 pipette to dissociate the EBs in each well by gently pipetting up and down no more than 10 times. Then, add five milliliters of washing buffer per well of dissociated EBs. Collect the cells into a 50-milliliter centrifuge tube by passing them through a 40-micron strainer.After counting the cells in the filtered suspension, spin the cells down for 10 minutes at 300g. Resuspend the cells in 300 microliters of washing buffer by gently pipetting a few times, ensuring no clumps are present. Proceed to isolate the CD34-positive cells using a CD34 microbead kit per the manufacturer's instructions.EBs of good quality showed a defined edge by day two of the differentiation and appeared clear and bright when observed under a microscope. The presence of darker areas indicated cell death within the EBs. Following chiron treatment with varying concentrations on day two, quantifying the number of CD34-positive cells at day eight of differentiation revealed the response of the cell line to the treatment.Flow cytometry showed that the CD34-positive enrichment using the magnetic beads provided about 85%CD34-positive cells.

hiPSCs分化为内皮细胞

Seeding CD34+ Cells into Fluidic Chips

Seeding CD34+ Cells into Fluidic Chips  

To begin, centrifuge the isolated CD34-positive cells and resuspend the cell pellet in 300 microliters of pre-warmed SFM-34 medium that is supplemented with mixed 4 cytokines, and count the cells using 10 microliters of the cell suspension. After counting and resuspending the cells in a final volume of 150 microliters cytokine-supplemented SFM-34, add 0.5 microliters of ROCK inhibitor to it. Slowly aspirate the laminin from fluidic chips by placing the tip of a P200 pipette inside the reservoir on the edge of the channel.Then add the cell suspension into the channel consistently, ensuring no bubbles are formed. Incubate the chip overnight at 37 degrees Celsius and 5%carbon dioxide to attach the cells to the channel completely. When the endothelial cells are fully attached, aspirate the medium as described previously.Add 200 microliters of cytokine-supplemented SFM-34 into the chip. Replace the medium daily until the cells reach 90 to 100%confluency.

将 CD34+ 细胞接种到流控芯片中

Application of Continuous Flow to Endothelial Cells: Aorta-on-a-Chip

Install the perfusion set into the unit per the manufacturer's protocol. Inside a hood, attach an empty fluidic chip to it and add cytokine supplemented SFM-34 medium to fill both reservoirs under sterile conditions. Transfer the fluidic unit to the incubator, connecting it to the pump for bubble removal and calibration.Carefully remove the fluidic unit with the connected set from the incubator and transfer it into the hood along with the chips containing the cells. Clamp the tubing on both sides of the test chip. Remove the clamped tubing from the test chip and connect the chip containing the cells to the tubing.After removing or opening the clamps, transfer the system to the incubator and connect the air pump to the fluidic unit. Remove the fluidic unit from the pump and move it into the hood. Clamp the tubing on both sides of the chip and remove the tubing from the reservoirs on the chip.After gently removing the medium from the chip, Wash the cells with Dulbecco's Phosphate-Buffered Saline or DPBS. Add 150 microliters of dissociation buffer and incubate for three minutes at 37 degrees Celsius. When the cells are detached, collect the dissociation buffer from one reservoir and wash the channel once with DPBS.Wash the reservoir with the washing buffer to collect the remaining cells from the chip. Finally, add one milliliter of washing buffer to the collected cells before using 10 microliters from it to count the cells. Before the stimulation, cells showed random orientation, but with stimulation, they reoriented parallel to the direction of the flow.CD34 expression profile of cells cultured under flow for five days and percentage of CD34 positive cells retrieved from the fluidic channel, showed the effectiveness of the protocol.