Name: directed differentiation of hemogenic endothelial cells from human pluripotent stem cells
Uploaded: 2021-03-31T14:00:00
Description: Watch this Scientific Journal Video about Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells at JoVE.com

这项工作得到了NIH拨款HL128064和U2EB017103的部分支持。CT Innovations 15-RMB-YALE-04赠款提供了进一步的支持。

1. 试剂和试剂制备
注意： 材料表中提供了试剂列表。
<ol><li>获得人类多能干细胞系：H1-hESC，H9-Fucci-hESC。 注意：在H1细胞系中，血源性EC的产生可能更有效。</li>
	<li>制备基质蛋白储备液：将基质蛋白等分到预冷的 1.5 mL 管（冰上）中，使每个试管含有 1 mg 基质蛋白。1mg基质蛋白足以包被两个6孔板的所有孔（总共12个孔）。将等分试样储存在-20°C直至使用。 ...

<ol>
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本文概述了使用无小鼠饲养层和无血清的2D培养系统在大约1周内从人胚胎干细胞生产血源性内皮细胞的步骤（图1）。该协议扩展了Sriram等人（2015）描述的方法，以获得原始ECs 38。通过在DLL4包被的平板上培养这些细胞并观察符合动脉规格的基因表达变化，证明了CD31 + CD45-EC的原始性质和规格潜力（图3）。此外，动脉身...

内皮细胞（ECs）是一个异质细胞群，在整个人体和工程组织中执行多种功能。除了支持和调节其他细胞类型（即心肌细胞1，成骨细胞2）外，这些功能还包括在血液和组织之间形成选择性屏障并协助组织形成3。成熟EC在正常发育过程中的分化需要多样化的信号通路。原始 EC 来源于中胚层祖细胞，然后针对成熟的动脉、静脉、毛细血管和淋巴表型4 进行指定。此外，胚胎外卵黄囊和胚胎主动脉-性腺-中胚层（AGM）区域中的一小部分EC也被指定为血源性EC，其产生造血干细胞和祖细胞（HSPC）迁移到胎儿肝脏和胎儿骨髓，在那里它们在出生后保留并在整个生命中产生所有血细胞类型4。EC表型的多样性对于所有组织发育和维护都是必不可少的。
因此，EC及其衍生物是旨在建模和阐明人类发育和/或疾病以及再生医学和组织工程应用机制的研究的关键组成部分5，6，7，8。然而，这些类型研究的主要局限性是缺乏所需数量的初级人类ECs。据估计，大多数治疗应用至少需要 3 x 108 个 EC6。为了解决这个问题，已经提出了使用人类胚胎干细胞（hESCs）和人诱导多能干细胞（hiPSCs），因为它们具有不同的谱系潜力和产生大量后代的能力6，9。
事实上，源自hESC或hiPSC的细胞的有用性已经在多项专注于疾病建模和药物筛选的研究中得到证实10，11，12。器官芯片（OOC）技术已被用于通过将细胞和组织整合到三维支架中来更忠实地概括人体的生理学。此外，多个单独的OOC（所谓的人体或人芯片，BOC / HOC）的连接可以通过微流体完成，以允许感兴趣的器官之间的串扰13，14，15。支持组织，如脉管系统，是 OOC 和 BOC/HOC 的关键组成部分;结合脉管系统允许营养物质，氧气和旁分泌因子在整个组织中运输，从而促进必要的组织特异性微环境3，12。因此，推导成熟人类EC的方法，如动脉，静脉，淋巴管和血源性ECs，对于推进这些组织工程方法至关重要。
已经发布了多个协议，详细说明了从hESC或hiPSC5，16，17，18，19，20，21，22，23，24，25，26衍生人类原始或祖EC的步骤.其中许多方案依赖于胚体（EB）形成或ESC / iPSCs与基质细胞的鼠饲养层共培养。这些策略往往既困难又耗时，EC产量低和/或人EC被鼠细胞污染。严格依赖2D培养而不使用基质细胞的方案通常需要长时间诱导，利用生长因子和/或抑制剂的复杂组合进行诱导，细胞分离后具有延长的扩增期，或这些因素的组合。推进对体内成熟EC类型衍生所涉及的信号通路和因素的了解，为简单而强大的体外分化方案奠定了基础。
以前，Notch和Retinoic Acid（RA）信号通路在发育过程中分别在小鼠动脉和血源性EC的规范中的关键作用被确定。Notch信号通路在动脉EC表型的规范和维持中起着多种作用。使用小鼠视网膜血管化模型的工作确定了流体剪切应力诱导Notch-Cx37-p27信号轴的途径，促进G1细胞周期停滞，从而使动脉EC规范27成为可能。据推测，细胞周期状态通过提供独特的机会窗口在细胞命运决策中发挥作用，在这些机会窗口中，细胞可以接受某些可以诱导基因表达和表型变化的信号28。这种Notch介导的G1停滞使富含动脉EC的基因表达，包括ephrinB2，Cx40，DLL4，Notch1和Notch 4（在29，30中审查）。还表明，通过RA信号传导31，32在体内促进血源性EC规范。其他研究发现，在RA信号传导的下游，c-Kit和Notch的表达上调p27，这使得鼠卵黄囊和AGM33中的产血性规范成为可能。小鼠血源性EC可以通过内皮（即CD31，KDR）和造血（即c-Kit，CD34）标志物的表达进行最低限度的鉴定4。最后，血源性EC经历内皮到造血的转变（EHT）形成HSPC，可以产生所有血细胞类型4，34，35。
最近的工作测试了这种相同的信号层次结构是否可以促进人类血源性EC规范。为此，开发了一种无血清和饲养层的2D培养方案，以从hESC中获取血源性EC，并且这些血源性EC在单细胞水平上表征为CD31 + KDR + c-Kit+ CD34 + VE-钙粘蛋白-CD45-。这项研究还利用了荧光泛素化细胞周期指示剂（FUCCI）报告基因，该报告基因使用表达FUCCI报告基因构建体（H9-FUCCI-hESC）的H9-hESC识别不同的细胞周期状态36。在这些细胞的研究中，证明RA促进EC中的早期G1细胞周期停滞，并且早期G1状态使体外血源性规范37成为可能。本文提供了用于分化这些人血源性内皮细胞的详细方案以及确认其身份的测定。这种简单的方法为未来研究人类血细胞发育的机制提供了一种有用的方法来生成这种专门的EC亚集。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 cm dishes</td>
			<td>Corning</td>
			<td>430599</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>35 mm dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>Antimicrobial reagent 
			Brand Name: Normocin</td>
			<td>Invitrogen</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D systems</td>
			<td>233-FB-025</td>
			<td>use at 50 ng/mL</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>BioLegend</td>
			<td>595202</td>
			<td>use at 25 ng/mL</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Detatchment Solution 
			Brand Name: vAccutase</td>
			<td>Stemcell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DLL4</td>
			<td>R&#38;D systems</td>
			<td>1506-D4/CF</td>
			<td>recombinant human; use at 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium 
			Brand Name: EGM-2 Endothelial Cell Growth Medium-2 BulletKit (EGM-2)</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Corning</td>
			<td>352235</td>
			<td>polystyrene round bottom with filter cap</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>ThermoFisher Scientific</td>
			<td>33016015</td>
			<td>use at 4 mg/cm2</td>
		</tr>
		<tr>
			<td>GSK3i/CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004-02</td>
			<td>10 mM stock; use at 5 &#956;M</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix protein&#160; 
			Brand Name: Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Growth factor reduced. Refer to the Certificate of Analysis for the lot to determine the protein (Matrigel) concentration. This concentration is required to calculate the volume of Matrigel that contains 1 mg of protein.</td>
		</tr>
		<tr>
			<td>Methylcellulose-based medium 
			Brand Name: MethoCult H4435 Enriched</td>
			<td>Stemcell Technologies</td>
			<td>4435</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluripotent stem cell differentiation medium 
			Brand Name: STEMdiff APEL 2</td>
			<td>Stemcell Technologies</td>
			<td>5270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluripotent stem cells: H1, H9, H9-FUCCI</td>
			<td>WiCell</td>
			<td>WA09 (H9), WA01 (H1)</td>
			<td>human; H9-FUCCI were obtained from Dr. Ludovic Vallier&#39;s lab at Cambridge Stem Cell Institute</td>
		</tr>
		<tr>
			<td>Protein-Free Hybridoma Medium (PFMH)</td>
			<td>Gibco</td>
			<td>12040077</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625-50mg</td>
			<td>use at 0.5 &#956;M</td>
		</tr>
		<tr>
			<td>Reverse transcription master mix 
			Brand Name: iScript Reverse Transcription Supermix</td>
			<td>BioRad</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction kit 
			Brand Name: RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell growth medium 
			Brand Name: mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green master mix 
			Brand Name: iTaq Universal SYBR Green Master Mix</td>
			<td>BioRad</td>
			<td>1725121</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25299956</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>VEGF165 (VEGF-A)</td>
			<td>PeproTech</td>
			<td>100-20</td>
			<td>use at 50 ng/mL</td>
		</tr>
		<tr>
			<td>&#945;-CD31-FITC</td>
			<td>BioLegend</td>
			<td>303104</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-CD34-Pacific Blue</td>
			<td>BioLegend</td>
			<td>343512</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-CD45-APC/Cy7</td>
			<td>BioLegend</td>
			<td>304014</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-c-Kit-APC</td>
			<td>BioLegend</td>
			<td>313206</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-Flk-1-PE/Cy7</td>
			<td>BioLegend</td>
			<td>359911</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-VE-Cadherin-PE</td>
			<td>BioLegend</td>
			<td>348506</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td colspan="4">* Antibody fluorescent conjugates should be optimized based on the cell sorter used. Presented here are the final concentrations utilized in this study.</td>
		</tr>
	</tbody>
</table>

directed differentiation of hemogenic endothelial cells from human pluripotent stem cells

血管普遍分布在身体的所有组织中，并执行不同的功能。因此，从人多能干细胞衍生出排列血管腔的成熟血管内皮细胞对于多种组织工程和再生应用至关重要。在体内，原始内皮细胞来源于中胚层谱系，并针对特定的亚型，包括动脉、静脉、毛细血管、血源性和淋巴管。血源性内皮细胞特别令人感兴趣，因为在发育过程中，它们会产生造血干细胞和祖细胞，然后在整个生命中产生所有血系。因此，创建一个在体外产生血源性内皮细胞的系统将为研究内皮到造血的转变提供机会，并可能导致人类血液制品的离体生产和减少对人类供体的依赖。虽然存在几种用于衍生祖细胞和原始内皮细胞的方案，但尚未描述从人类干细胞中产生特征明确的血源性内皮细胞。本文提出了一种在大约1周内从人胚胎干细胞衍生血源性内皮细胞的方法：分化方案，响应GSK3β抑制剂（CHIR99021）形成的原始条纹细胞，然后由bFGF介导的中胚层谱系诱导，然后是BMP4和VEGF-A促进的原始内皮细胞发育，最后是视黄酸诱导的血源性内皮细胞规格。该协议产生明确定义的血源性内皮细胞群，可用于进一步了解其分子调控和内皮到造血的转变，这有可能应用于下游治疗应用。

图1显示了概述hESC的原始EC和血源性EC的规格的示意图，以及电镀后24小时细胞的代表性图像。按照规范，原始EC和血源性EC分别在第5天和第8天纯化FACS。原始EC定义为CD31 + CD45-，血源性EC定义为CD31+ KDR + c-Kit+ CD34+ VE-钙粘蛋白-CD45-。用于原始EC和血源性EC纯化的代表性流式细胞术门控策略如图2</strong...

Watch this Scientific Journal Video about Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells at JoVE.com

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

这里介绍的是在大约 1 周内从人多能干细胞定向分化的血源性内皮细胞的简单方案。

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

该协议详细介绍了一种生成表征良好的人血源性内皮细胞群的方法。这些细胞可用于研究分子调控和内皮到造血的转变。首先培养人类胚胎干细胞四天，以将它们分化为原始内皮细胞。在第五天，吸出细胞上方的培养基。然后用每孔一毫升DMEM / F-12轻轻洗涤细胞。每孔加入一毫升新鲜制备的血源性内皮细胞分化培养基，并将细胞在37摄氏度和5%二氧化碳下孵育24小时。在第六天和第七天，用每孔一毫升新鲜制备的均质内皮细胞分化培养基替换细胞上方的培养基，并将细胞再孵育24小时。第八天。分离并洗涤细胞后，将它们均匀地分成冰上的微量离心管，每个含有至少600微升细胞，密度为每毫升10至第5个细胞。酌情将抗体添加到含有细胞的试管中，并在避光的冰上孵育30分钟。通过在1000倍G和4摄氏度下离心5分钟来沉淀细胞。除去上清液并将沉淀重悬于600微升冰冷的分选缓冲液中。通过五毫升FACS管的网状过滤器帽过滤样品，并将细胞储存在避光的冰上，以便立即进行细胞分选。要通过FACS分离血源性内皮细胞，首先对CD45阴性细胞群进行门禁。然后在CD45阴性群体中，门控CD31阳性细胞。接下来在CD31阳性群体中，VE-钙粘蛋白阴性细胞群的门。然后在VE-钙粘蛋白阴性群体中，对c-Kit阳性细胞进行门控。从c-Kit阳性群体中，门控CD34阳性细胞群。最后从CD34阳性细胞群中，鉴定并收集KDR阳性细胞。将血源性内皮细胞接种到为造血祖细胞生长而配制的甲基纤维素基基中后，在第八天计数集落形成单位-红系集落和原始细胞形成单位-红系菌落。集落形成单位-粒细胞-巨噬细胞和集落形成单位-粒细胞、红细胞、巨噬细胞和巨石菖。在第14天计数多能造血祖细胞菌落。每接种1000个血源性内皮细胞，产生约20个集落形成单位。具有内皮细胞形态的细胞在培养物中也可见。这些是血源性内皮细胞，在单细胞水平上产生多谱系造血祖细胞。该协议是一种2D血清和小鼠饲养层无饲养层培养系统，可在大约一周内产生人血源性内皮细胞。

Research

JoVE Journal

Developmental Biology

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

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

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 ...