Name: immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors
Uploaded: 2021-08-07T15:00:00
Description: Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

我们感谢马科斯·佩雷斯·巴斯特雷切亚、洛雷纳·罗德里格斯·洛伦佐和贝戈尼亚·加西亚·门德斯（胡卡）和帕洛玛·塞雷佐、阿尔穆德纳·帕耶罗和玛丽亚·德拉波韦达-科洛莫（HCSC）的技术支持。这项工作部分得到了医疗赠款（罗氏SP200221001）对A.B，RYC奖学金（RYC-2013-12587;西班牙经济部长和竞争组织）和2017年国际发展基金赠款（SAF2017-85489-P;西班牙新星大学和丰多斯联邦大学部长向L.G.提供塞韦罗·奥乔亚赠款（PA-20-PF-BP19-014）：西班牙阿斯图里亚斯新星大学）至P.M-B。和2018年博士后资助（阿斯图里亚斯生物萨尼塔里亚基金会 - FINBA，西班牙奥维耶多）给A.A.-H。我们感谢雷尼尔·范德林登分享他的知识（和时间），特别是他在多色标记抗体面板组合和单色珠控制准备方面的明智建议。

根据1964年《赫尔辛基宣言》获得和处理了全血和骨髓样本。在我们的机构医学伦理委员会（阿斯图里亚斯中央大学-HUCA-）批准的一项研究中，从健康献血者那里获得了全血样本。骨髓样本是从圣卡洛斯医院血液学部管理的患者的骨髓吸附丢弃材料中获得的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62569/62569fig01.jpg"> 图<stron...

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迄今为止，大部分以流细胞学研究巨型卡罗波西斯的研究为重点，仅限于仅使用血统特定表面标记（即CD42A/CD42B、CD41/CD61）识别MK子集，而早期的MK分化阶段则检查不力。在本文中，我们展示了一种免疫细胞学策略，以解决人类巨细胞体的综合流动细胞特征。总体而言，我们想强调将MK特定和非特定表面标记（表1和2）组合在同一流动细胞测定抗体面板中的效用，以便更详细地研究巨型细胞分...

巨细胞（MKs）是按照一种称为巨细胞的复杂过程从造血干细胞（HSCs）中发育的，这个过程主要由激素血栓素（TPO）编排。巨型卡洛波伊斯的经典观点描述了从HSC到承诺祖先和前体细胞的一系列分层阶段的细胞之旅，最终导致一个成熟的MK。在成熟过程中，MKs 经历多轮内分泌，开发一个复杂的细胞内分界膜系统 （DMS），该系统为血小板生产提供足够的膜表面，并高效生产和包装成熟血小板1、2、3遗传的不同颗粒中所含的过多因子。因此，成熟的MK是大细胞（40-60微米），其特征是高度多倍体细胞核（达到甚至>64N）。最近的研究表明，HSC绕过传统的血统承诺检查站，根据某些生理病理条件，将HSC区分为MK的替代途径4、5、6、7、8、9、10、11。这些发现突出表明，对成熟MK的造血分化是一个对生物需求作出反应的连续体和自适应过程。
随着对细胞生物学和巨型细胞学12特征分子方面的了解日益增多，大部分致力于流细胞学过程研究的研究仅限于使用血统特定表面标记（即CD42A/B、CD41/CD61）识别成熟MK，而早期的MK分化阶段尚未探索。我们以前记录了在小鼠骨髓和骨髓衍生的MK培养13，14中上演巨细胞化策略，我们已经适应并应用于人类15。在本文中，我们展示了一种免疫异种策略，允许对巨型胡萝卜素进行定性， 从 HSC 到成熟的 MK，在人类主要来源（骨髓 -BM-和外周血液 -PB-）或体外培养物中使用集成 MK 特定和非特定表面标记（CD61、CD42B、CD49B、CD31、KIT 和 CD71 等）的面板。尽管MKs体积大且脆弱，但可以使用上述细胞表面标记进行免疫异种化，并通过在特定压力和喷嘴直径条件下的荧光激活细胞分类来丰富MKs，以尽量减少细胞破裂和/或损伤。这项技术促进多分子方法，目的是更好地了解巨细胞和血小板生产在人类健康和疾病中的复杂性。值得注意的是，它将构成一个有用的工具，以帮助诊断和预后在不断增长的需求的临床背景下。
在这份手稿中，我们记录了一个战略，以阶段人类巨型卡约波伊斯与面板集成MK特异性和非特定的表面标记从主要来源或产生体外。此外，我们提供一个协议来排序，与荧光激活的细胞分拣机，首选分数和成熟的MKs（图1）。这一步骤并不受欢迎，因为由于 MK 的体积大且脆弱，在技术上很难。然而，它以前在小鼠和人体骨髓样本中都使用过，由于技术的进步，每次16、17、18年都有更好的结果。可以研究MK或MK前体的人类主要来源包括骨髓、脐带血和外周血等。适当的样品处理，以隔离相关的细胞分数，对每个样本进行分析是重要的。纳入了标准程序，在研究巨型卡洛波伊斯时应考虑到一些考虑。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>130 micron Nozzle</td>
			<td>BD</td>
			<td>643943</td>
			<td>required for MK sorting</td>
		</tr>
		<tr>
			<td>5810R Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>A-4-62 Swing Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>Aerospray Pro Hematology Slide Stainer / Cytocentrifuge</td>
			<td>ELITech Group</td>
			<td></td>
			<td>Automatized cytology devise, where slides are stained with Mat-Gr&#252;nwald Giemsa</td>
		</tr>
		<tr>
			<td>CO2 Incubator Galaxy 170 S</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell Incubation</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>To prepare cytospins</td>
		</tr>
		<tr>
			<td>FACSAria IIu sorter</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm and 633-nm</td>
		</tr>
		<tr>
			<td>FACSCanto II flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm , 633-nm and 405-nm</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 41</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 61</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Zoe Fluorescent Cell Imager</td>
			<td>BioRad</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>To obtain PBMCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipids Cholesterol Rich from adult bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>L4646</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stem Cell Technologies</td>
			<td>#07801</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human Erythropoietin (EPO)</td>
			<td>&#160;R&#38;D Systems</td>
			<td>287-TC-500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human stem cell factor (SCF)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC2115</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human thrombopoietin (TPO)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC9514</td>
			<td>or TPO receptor agonists</td>
		</tr>
		<tr>
			<td>StemSpan SFEM</td>
			<td>Stem Cell Technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck</td>
			<td>A7906-100G</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD CompBead Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD</td>
			<td>552843</td>
			<td>Antibodies for human cells are generally from mouse.</td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD FACS Accudrop Beads</td>
			<td>BD</td>
			<td>345249</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 AF-647</td>
			<td>BD</td>
			<td>561654</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD31 FITC</td>
			<td>Immunostep</td>
			<td>31F-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 FITC</td>
			<td>BD</td>
			<td>555821</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PE</td>
			<td>BD</td>
			<td>555467</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PerCP-Cy5.5</td>
			<td>BD</td>
			<td>333148</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42A APC</td>
			<td>Immunostep</td>
			<td>42AA-100T</td>
			<td>We observed unspecific binding... that needs to be assessed</td>
		</tr>
		<tr>
			<td>CD42A PE</td>
			<td>BD</td>
			<td>558819</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42B PerCP</td>
			<td>Biolegend</td>
			<td>303910</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD49B PE</td>
			<td>BD</td>
			<td>555669</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD61 FITC</td>
			<td>BD</td>
			<td>555753</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD71 APC-Cy7</td>
			<td>Biolegend</td>
			<td>334109</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD</td>
			<td>564219</td>
			<td>Fc blocking - control</td>
		</tr>
		<tr>
			<td>KIT PE-Cy7</td>
			<td>Biolegend</td>
			<td>313212</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Lineage Cocktail 2 FITC</td>
			<td>BD</td>
			<td>643397</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Merck</td>
			<td>R6513</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Triton X-500</td>
			<td>Merck</td>
			<td>93443-500ML</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cell strainers for sorting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics Filters 100 micrometers</td>
			<td>Sysmex</td>
			<td>04-004-2328</td>
			<td>Cell strainers</td>
		</tr>
		<tr>
			<td colspan="4">Note: we do not specify general reagents/chemicals (PBS, EDTA, etc) or disposables (tubes, etc), or reagents specified in previous published and standard protocols&#160; - unless otherwise specified.</td>
		</tr>
	</tbody>
</table>

immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors

巨型胡萝卜素 （MK） 分化包括多个内分泌周期，导致高度多倍体（达到甚至>64N）和超大细胞 （40-60 μm）。与细胞生物学和分子水平上对巨细胞测定知识的快速增加相反，流细胞学对巨型卡罗波测定型的定性仅限于使用血统特定表面标记识别成熟的MK，而早期的MK分化阶段尚未探索。在这里，我们提出了一个免疫噬菌体策略，允许识别连续的MK分化阶段，随着策略地位的提高，在人类的主要来源或 体外培养物与 一个面板集成MK特定和非特定表面标记。尽管 MKs 的体积和脆弱性，但可以使用上述面板进行免疫型，并在压力和喷嘴直径的特定条件下通过荧光激活细胞分拣而丰富。这种方法有助于多奥米学研究，目的是更好地了解人类巨细胞和血小板生产的复杂性。更好地描述巨无霸病症可能对与血统有关的病理学和恶性肿瘤的诊断或预后构成根本。

骨髓和普洛伊迪 在 图4中， 我们展示了对BM样本（渴望）中巨型胡萝卜素的代表性免疫异种分析。在绘制针对 CD71 和 CD31 的蜂窝分数时，我们已对六个主要人群进行了限制：CD31- CD71- （红色）、CD31- CD71+ （蓝色）、CD31+ CD71- （橙色）、CD31+ CD71中 （浅绿色）、CD31+ CD71+</s...

Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

在这里，我们提出了一个免疫嗜血策略，用于巨型胡萝卜细胞分化的特征，并展示该策略如何允许使用荧光激活细胞分拣器在不同阶段对巨型胡萝卜细胞进行排序。该方法可以应用于人体原生组织，也适用于 体外培养产生的巨型核细胞。

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因 的体外 分化

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

你好，我是安德里亚·阿塞贝斯·韦尔塔。ISPA阿斯图里亚斯公国健康研究所血小板研究实验室的博士后研究员。位于西班牙奥维耶多。你好，我是劳拉·古蒂

Research

JoVE Journal

Biology

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Human ES cells: Starting Culture from Frozen Cells

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Human Bronchial Epithelial Cells Grown from Explants

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因 的体外 分化

人类MK从人类主要来源的免疫细胞分拣或血细胞遗传基因的体外分化