我们感谢位于印度班加罗尔的贾瓦哈拉尔·尼赫鲁高级科学研究中心的流通池设施使用流式细胞术核心设施。分化细胞沉淀培养物的冷冻切片在印度班加罗尔的 Neuberg Anand 参考实验室进行。这项工作得到了印度马尼帕尔高等教育学院（MAHE）的UC校内资助。AG感谢MAHE的T. M. A. Pai博士奖学金的支持。

人间充质干细胞的荧光激活单细胞分选

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作者声明，本文的发表不存在利益冲突。

在组织工程和再生医学领域，在出生后来源中，口腔组织来源的间充质干细胞因其最小的伦理义务和显着的多谱系分化潜力而引起了人们的极大兴趣21。来自受影响的第三磨牙和 SHED 的牙髓干细胞 （DPSC） 因其在神经退行性疾病和创伤性疾病中的治疗潜力而在牙科间充质干细胞中获得了最多的关注22.本手稿中描述的方案有助于使用多参数方法表征 SHED，并使用单...

间充质干细胞（MSCs）可以被视为适用于细胞疗法的可扩展细胞来源，可以被认为是再生医学的金标准系统。这些细胞可以从体内具有不同组织来源的各种来源中分离出来1.根据其来源组织，每种类型的间充质干细胞都表现出模棱两可的体外行为2。这在它们的形态和功能特性中得到了很好的观察3.多项研究表明，间充质干细胞的克隆内变异，包括成体组织分化、基因组状态以及代谢和细胞结构 2,4。
细胞免疫表型分析一直是流式细胞术鉴定干细胞的常见应用，国际细胞和基因治疗学会 （ISCT） 于 2006 年利用它来规定将细胞识别为 MSC 的最低标准列表。它指出，除了可塑性粘附和在体外分化为三个谱系（成骨性、成软骨性和产脂性）的能力外，≥95% 的细胞群必须表达 CD105、CD73、CD90，并且这些细胞必须缺乏 CD34、CD45、CD11b、CD14 和 HLA-DR 的表达（≤2% 阳性），如流式细胞术测量的那样5.尽管间充质干细胞是在ISCT的最低标准下由一组生物标志物定义的，但它们的免疫特性无法与这些生物标志物进行基准测试，并且需要超越这些标准，以使交叉研究比较和克隆变异更容易量化2。
尽管 ISCT 制定了指南，但对 MSC 的广泛研究表明，该群体中存在异质性，这可能是由于多种因素造成的，主要是由于 MSC 供体6、组织来源7、克隆群体中的单个细胞8 和培养条件2,9 之间出现的异质性无处不在，10.从各种组织来源中表征和纯化这些原代细胞，以确保质量和细胞命运是其生产的关键步骤。需要了解种群中显示的变异，需要一种有效的方法将其分解为可以单独划分和收集的亚种群11。单细胞水平分析有助于克服细胞间变异的挑战，减少异质性群体产生的生物噪声，并提供研究和表征稀有细胞的能力12。
根据目的和选择的参数，可以采用几种方法来对选定的种群进行排序和富集。细胞分选技术可以包括批量分选和单细胞分选方法。批量分选可以通过磁激活细胞分选 （MACS）13、分馏 14 和洗脱15 富集靶细胞群，而单细胞分选可以通过荧光激活细胞分选 （FACS）11 富集更均匀的细胞群。表1重点介绍了每种方法及其优缺点的比较分析。
表1：不同技术的比较分析：MACS、分馏、洗脱和FACS，突出了其原理的差异以及选择特定技术优于另一种技术的优缺点。 缩写：MACS = 磁活化细胞分选;FACS = 荧光激活的细胞分选。 <a href="https://www.jove.com/files/ftp_upload/65723/65723_Table_1_.xlsx" target="_blank">请按此下载此表格。</a>
自该技术问世以来，单细胞流式细胞术在异质样品17中特定细胞群的计数16、检测和表征中发挥了重要作用。Hewitt 等人于 2006 年奠定了自动细胞分选方法的基础，以增强分化人胚胎干细胞 （hESC） 的均质池的分离18。单细胞分选富集了GFP转导的hESCs群体，促进了转基因克隆的分离，为临床研究开辟了新的维度。为了提高分拣效率，一般采取两种方法;要么修改分选群体的收集培养基以维持分选后细胞的活力和增殖19，要么适当修改细胞分选算法/软件12。
随着技术的进步，商用流式细胞仪和细胞分选仪已经能够帮助解决在无菌分选脆弱和稀有细胞群（尤其是不同来源的干细胞）时遇到的挑战。干细胞生物学家面临的主要挑战之一是按照基因编辑研究所需的转染方案克隆分离人类多能干细胞19。通过将单细胞分选到涂有小鼠胚胎成纤维细胞 （MEF） 以及补充剂和商业小分子 ROCK 抑制剂的 96 孔板中来解决此问题。然而，细胞分离策略可以通过使用索引分选来在很大程度上改进，索引分选是分选算法的一个特征，用于识别分选的单个细胞的免疫表型12。这种改进的单细胞分选方式不仅有助于提高干细胞的分选效率，特别是在罕见的造血干细胞群方面，而且还有效地将单细胞克隆与其下游功能测定联系起来20。
本文重点研究对人脱落乳牙（SHEDs）的免疫表型干细胞进行单细胞分选，以富集亚群，研究其功能分化能力。使用两种 MSC 阳性标志物 CD90 和 CD73 以及阴性造血标志物 CD45 的组合，对 MSC 进行免疫表型分析，并鉴定出暗表达子和无效表达子。根据其免疫表型，将亚群鉴定为纯间充质干细胞、单阳性和双阴性群体。使用单细胞分选模式对它们进行分选，以获得纯度和富集的亚群，用于进一步的功能研究，以确定标记物的差异表达是否是 体外 培养条件的产物，或者它是否对功能特性也有任何影响。对不是“阳性MSC标志物”的均质表达者的细胞进行分类，以研究其功能特性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcian Blue Stain</td>
			<td>HiMedia</td>
			<td>CCK029-1KT</td>
			<td></td>
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			<td>Antibiotic-Antimycotic (100x)&#160;</td>
			<td>Gibco by ThermoFisher</td>
			<td>15240062</td>
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		<tr>
			<td>BD CompBead Plus Anti-Mouse Ig, &#954;/Negative Control (BSA) Compensation Plus (7.5 &#181;m) Particles Set</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Accudrop Beads&#160;</td>
			<td>BD Biosciences</td>
			<td>345249</td>
			<td>Used to set up the Laser delay when the sort module opens.</td>
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		<tr>
			<td>BD FACS Aria Fusion Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>---</td>
			<td></td>
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			<td>BD FACS Diva 9.4</td>
			<td>BD Biosciences</td>
			<td>---</td>
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			<td>BD FACS Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>Used as sheath fluid for both analysis and sorting experiments in the BD FACSAria Fusion</td>
		</tr>
		<tr>
			<td>BD FACSDiva CS&#38;T Research Beads</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td>Used for Instrument configuration depending on the nozzle size.</td>
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		<tr>
			<td>BD Horizon V450 Mouse Anti-Human CD44</td>
			<td>BD Biosciences</td>
			<td>561292</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Horizon V450 Mouse IgG2b, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>560374</td>
			<td>CD44-V450 isotype</td>
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		<tr>
			<td>BD Pharmingen APC Mouse Anti-Human CD105</td>
			<td>BD Biosciences</td>
			<td>562408</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>CD105-APC isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen DAPI Solution</td>
			<td>BD Biosciences</td>
			<td>564907</td>
			<td>DAPI Stock solution of 1 mg/mL</td>
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		<tr>
			<td>BD Pharmingen FITC Mouse Anti-Human CD90</td>
			<td>BD Biosciences</td>
			<td>555595</td>
			<td></td>
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		<tr>
			<td>BD Pharmingen FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>CD90-FITC isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse Anti-Human CD45</td>
			<td>BD Biosciences</td>
			<td>555483</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555749</td>
			<td>CD45-PE isotype</td>
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		<tr>
			<td>BD Pharmingen PerCP-Cy 5.5 Mouse Anti-Human CD73</td>
			<td>BD Biosciences</td>
			<td>561260</td>
			<td></td>
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		<tr>
			<td>BD Pharmingen PerCP-Cy 5.5 Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>550795</td>
			<td>CD73-PerCP-Cy 5.5 isotype</td>
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		<tr>
			<td>BD Pharmingen Purified Mouse Anti-Vimentin</td>
			<td>BD Biosciences</td>
			<td>550513</td>
			<td></td>
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		<tr>
			<td>Bovine serum albumin</td>
			<td>Hi-Media&#160;</td>
			<td>TC548-5G</td>
			<td></td>
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		<tr>
			<td>Crystal violet</td>
			<td>Nice chemical pvt ltd&#160;</td>
			<td>C33809</td>
			<td></td>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-aldrich&#160;&#160;</td>
			<td>D5652-50L</td>
			<td>dPBS used for culture work and maintenance.&#160;</td>
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		<tr>
			<td>Ethanol&#160;</td>
			<td>---</td>
			<td>---</td>
			<td>Used for general sterlization.</td>
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			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-21422</td>
			<td></td>
		</tr>
		<tr>
			<td>KO-DMEM</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>10829018</td>
			<td>Basal medium for undifferentiated hESCs, used in the preparation of culture media</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM (100x)</td>
			<td>Gibco by ThermoFisher</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, for Molecular Biology&#160;</td>
			<td>Hi-Media&#160;</td>
			<td>MB113</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O</td>
			<td>HiMedia&#160;</td>
			<td>CCK013-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>loba chemie</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (100x)</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>15140- 122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin (ActinGreen 488 ReadyProbes reagent)</td>
			<td>Invitrogen&#160;</td>
			<td>R37110</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Nitrate</td>
			<td>HiMedia&#160;</td>
			<td>MB156-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Thiosulphate pentahydrate</td>
			<td>Chemport</td>
			<td>10102-17-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphero Rainbow Fluorescent Particles, 3.0 - 3.4 &#181;m</td>
			<td>BD Biosciences</td>
			<td>556291</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining buffer&#160;</td>
			<td>Prepared in MIRM&#160;</td>
			<td>----</td>
			<td>It was prepared using 2% FBS in PBS&#160;</td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Differentiation Basal Media&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10410-01</td>
			<td>Basal media for Adipogenic media</td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10065-01</td>
			<td>Induction media for Adipogenic media</td>
		</tr>
		<tr>
			<td>StemPro Chondrogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10064-01</td>
			<td>Induction media for Chondrogenic media</td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10066-01</td>
			<td>Induction media for Osteoogenic media</td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis/Chondrogenesis Differentiation Basal Media&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10069-01</td>
			<td>Basal media for both Ostegenic and Chondrogenic media</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Hi-Media&#160;</td>
			<td>MB031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue&#160;</td>
			<td>Gibco by life technologies&#160;</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin - EDTA Solution 1x</td>
			<td>Hi-media&#160;</td>
			<td>TCL049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20&#160;</td>
			<td>MERCK&#160;</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell sorting of immunophenotyped mesenchymal stem cells from human exfoliated deciduous teeth

生物体的间充质干细胞 （MSC） 具有非凡的能力，可以在体内分化成多种成体细胞谱系，并以其免疫调节和抗炎特性而闻名。这些干细胞的使用对再生生物学领域来说是一个福音，但同时也是再生医学和治疗学的祸根，因为与它们相关的多种细胞模糊性。这些模糊性可能源于这些干细胞来源的多样性和它们的 体外 生长条件，这两者都反映了它们的功能异质性。
这就需要为治疗应用提供纯化、均质的间充质干细胞群体的方法。流式细胞术领域的进步使得使用多参数方法检测单细胞群成为可能。该协议概述了一种通过荧光辅助单细胞分选从人脱落乳牙（SHED）中鉴定和纯化干细胞的方法。同时表达表面标志物，即 CD90-异硫氰酸荧光素 （FITC）、CD73-peridinin-叶绿素蛋白 （PerCP-Cy5.5）、CD105-别藻蓝蛋白 （APC） 和 CD44-V450，使用多参数流式细胞术鉴定了 MSC 的“明亮”阳性表达基因。然而，从第 7 次传代到后来的传代，观察到这些阳性标志物的四重表达者的百分比显着下降。
使用单细胞分型模式对免疫表型亚群进行分选，其中只有两个阳性标记物和一个阴性标记物构成纳入标准。该方法确保了分选群体的细胞活力，并在分选后保持细胞增殖。这种分选的下游应用可用于评估门控亚群的谱系特异性分化。该方法可以应用于其他单细胞系统，以改善分离条件并获取多个细胞表面标记物信息。

Mesenchymal stem cells (MSCs) can be regarded as a scalable source of cells suitable for cell-based therapies and may be considered a gold standard system in regenerative medicine. These cells can be isolated from a variety of sources in the body with different tissue origins1. Depending on their source tissue, each type of MSC displays an ambiguous in vitro behavior2. This is well observed in their morphological and functional properties3. Multiple studies have shown intra-clonal variation in dimensions, including adult tissue differentiation, genomic state, and metabolic and cellular architecture of MSCs2,4.
Immunophenotyping of cells has been a common application of flow cytometry for the identification of stem cells and this was&#160;utilized by the International Society for&#160;Cell and Gene Therapy (ISCT) in 2006 to prescribe a list of minimal criteria to identify cells as MSCs. It stated that along with plastic adherence and the ability to differentiate into three lineages (osteogenic, chondrogenic, and adipogenic) in vitro, &#8805;95% of the cell population must express CD105, CD73, CD90, and these cells must lack the expression (&#8804;2% positive) of CD34, CD45, CD11b, CD14, and HLA-DR, as measured by flow cytometry5. Although the MSCs were defined by a set of biomarkers under the minimal criteria of the ISCT, their immune properties could not be benchmarked with these biomarkers and there was a need for more beyond these criteria to make cross-study comparisons and clonal variations easier to quantify2.
Despite the guidelines set by ISCT, extensive research on MSCs has shown that heterogeneity exists in this population, which could arise due to a multitude of factors, mainly due to the ubiquitous nature of heterogeneity that arises between MSC donors6, tissue sources7, individual cells within a clonal population8, and culture conditions2,9,10. Characterization and purification of these primary cells from a variety of tissue sources to ensure quality and cell fate are key steps in their production. The need to understand the displayed variations amongst the population requires an efficient method to resolve it into subpopulations that can be divided and collected separately11. Single-cell level analyses help overcome the challenges of cell-cell variation, reduce biological noise arising from a heterogeneous population, and offer the ability to investigate and characterize rare cells12.
Based on the purpose and chosen parameters, several methods can be employed to sort and enrich the selected populations. Cell-sorting techniques can comprise both bulk sorting and single-cell sorting methods. While bulk sorting can enrich target populations through Magnetic-activated cell sorting (MACS)13, fractionation14, and elutriation15, single-cell sorting can enrich more homogeneous populations by means of fluorescence-activated cell sorting (FACS)11. A comparative analyses&#160;of each of these methods with its own set of advantages and disadvantages is highlighted in Table 1.
Table 1: Comparative analyses of different techniques:&#160;MACS, Fractionation, Elutriation, and FACS highlighting the differences in their principle and the advantages and disadvantages of choosing a particular technique over another. Abbreviations: MACS = Magnetic-activated cell sorting; FACS = Fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/65723/65723_Table_1_.xlsx" target="_blank">Please click here to download this Table.</a>
Since the advent of the technique, single-cell flow cytometry has played a major role in enumeration16, detection, and characterization of a specific cell population in a heterogeneous sample17. Hewitt et al. in 2006 laid the foundation of automated cell sorting methodology to enhance the isolation of homogenous pools of differentiated human embryonic stem cells (hESCs)18. Single-cell sorting enriched the population of GFP-transduced hESCs facilitating the isolation of genetically modified clones, which opened a new dimension in clinical research. To improve the sort efficiency, two approaches have generally been taken; either the collection media of the sorted populations are modified to sustain viability and proliferation of post-sorted cells19 or the cell-sorting algorithm/software is appropriately modified12.
With the advancement of technology, commercial flow cytometers and cell sorters have been able to help address challenges that were met while aseptically sorting fragile and rare cell populations, especially stem cells of different origins.&#160;One of the major challenges of stem cell biologists has been the clonal isolation of human pluripotent stem cells following transfection protocols required in gene editing studies19. This was addressed by sorting single cells into 96-well plates that were coated with Mouse Embryonic Fibroblasts (MEFs) along with supplements and commercial small molecule ROCK inhibitors. However, cell isolation strategies could be largely refined with the use of index sorting, a feature of the sorting algorithm that identifies the immunophenotype of individual cells sorted12. This refined modality in single-cell sorting helped not only in enhancing sort efficiency for stem cells, especially with regard to rare hematopoietic stem cell populations, but also efficiently linked single-cell clones to their downstream functional assays20.
This paper focuses on single-cell sorting of immunophenotyped&#160;stem cells from human&#160;exfoliated deciduous teeth (SHEDs) for the enrichment of sub-populations to study their functional differentiation capacities. Using a combination of two MSC-positive markers, CD90 and CD73, and a negative hematopoietic marker CD45, the MSCs were immunophenotyped and the dim and null expressors were identified. Based on their immunophenotype the subpopulations were identified as pure MSCs, single positive and double negative populations. They were sorted using the single-cell sort mode to obtain pure and enriched subpopulations for further functional studies to identify whether the differential expression of markers was an artifact of in vitro culture conditions or whether it has any effect on the functional properties as well. Cells that were not homogeneous expressors of the &#34;positive MSC markers&#34; were sorted to study their functional properties.

作者聚焦：单细胞分选在分离间充质干细胞纯化群体中的重要性 

人脱落乳牙免疫表型间充质干细胞的单细胞分选

This protocol details a crucial method for accurately characterizing and purifying human mesenchymal stem cells on a single platform, enabling efficient downstream applications like stem cell transplantation. It is particularly significant for clinical labs enhancing molecular and cytogenetic assays through precise single cell sorting of engineered or natural stem cells. The commonly used methods of isolation of purified stem cell populations refer to techniques such as immunomagnetic separation, density gradient, or microfluidic cell sorting.A technique like index sorting allows it to be linked to single cell RNA profiling that can extract transcriptomic information of each sorted cell. The key challenges involve maintaining sample quality, optimization of cell culture conditions, including cell density and viability during and after sorting is required. Additionally, choosing suitable nozzle sizes for the instrument and ensuring timely access to the sorters would also be considered.This protocol illustrates a precise staining panel for immunophenotyping and sorting the desired cell population. It ensures standardized sample quality, optimal cell viability, density for cell population sorting and define sorting experimental parameters on the FACSDiva software. This method enables a straightforward selection and immunophenotyping of stem cell population expressing specific biomarkers.This simple platform allows efficient sorting based on the target population aiding and obtaining purified samples for subsequent downstream applications.

SHED通过标准免疫荧光测定法表征波形蛋白（红色，III型中间丝），肌动蛋白丝（Alexa fluor 488鬼笔环肽探针）和用DAPI染色的细胞核的表达（图1A）。为了估计它们的增殖和集落形成能力，进行了标准的短期细胞生长测定。从第2天到第8天，增殖速率增加了14.3倍，如图1B所示。SHED的克隆发生特性是通过图1C-E所示的C...

Watch this Scientific Journal Video about Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells at JoVE.com

该协议描述了使用单细胞分选方法对人间充质干细胞进行荧光激活细胞分选的使用。具体而言，当与基于多参数流式细胞术的方法相结合时，使用单细胞分选可以实现来自异质群体的免疫表型细胞的 99% 纯度。

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and ...

该协议详细介绍了在单个平台上准确表征和纯化人类间充质干细胞的关键方法，从而实现干细胞移植等高效的下游应用。对于通过对工程干细胞或天然干细胞进行精确的单细胞分选来增强分子和细胞遗传学检测的临床实验室来说，这一点尤为重要。分离纯化干细胞群的常用方法包括免疫磁分离、密度梯度或微流控细胞分选等技术。像索引分选这样的技术允许它与单细胞RNA谱联系起来，该分析可以提取每个分选细胞的转录组信息。关键挑战包括保持样品质量、优化细胞培养条件，包括分选期间和分选后的细胞密度和活力。此外，还将考虑为仪器选择合适的喷嘴尺寸，并确保及时接触分选机。该协议说明了用于免疫表型和分选所需细胞群的精确染色panel。它确保了标准化的样品质量、最佳的细胞活力、细胞群分选的密度，并在 FACSDiva 软件上定义了分选实验参数。该方法能够对表达特定生物标志物的干细胞群进行直接选择和免疫表型分析。这个简单的平台允许根据目标人群进行高效分选，帮助和获得纯化的样品，用于后续的下游应用。

single-cell-sorting-immunophenotyped-mesenchymal-stem-cells-from

Research

JoVE Journal

Biology

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

1.1K 次观看.  Manipal Academy of Higher Education, Manipal. 该协议详细介绍了在单个平台上准确表征和纯化人类间充质干细胞的关键方法，从而实现干细胞移植等高效的下游应用。对于通过对工程干细胞或天然干细胞进行精确的单细胞分选来增强分子和细胞遗传学检测的临床实验室来说，这一点尤为重要。分离纯化干细胞群的常用方法包括免疫磁分离、密度梯度或微流控细胞分选等技术。像索引分选这样的技术允许它与单细胞R...

视频: 作者聚焦：单细胞分选在分离间充质干细胞纯化群体中的重要性 

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.
This warrants methodologies to provide purified, homogeneous populations of MSCs&#160;for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify&#160;stem cells from&#160;human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.
The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

科研

生物学

A Step-by-Step Guide for Seeding and Differentiation of Mesenchymal Stem Cell Cultures for Characterization

To begin, seed mesenchymal stem cells into T-75 flasks, 100 mm dishes, and two 48-well plates for all the experiments. Add 200 microliters of 10%MSC culture media to the wells of the 48-well plates. When the cell monolayer reaches 90%confluency, replace the existing media of the designated control wells with 2%serum media.Add differentiation media of either adipogenic or osteogenic lineage into the last two wells labeled Test. On the same day, trypsinize a confluent 100 mm dish and transfer the cell suspension into two labeled 15-milliliter tubes. Centrifuge the tubes at 300 x g for six minutes.Then pipette 2 milliliters of the respective media into the control and test tubes. Place the loosely capped tubes in the incubator at 37 degrees Celsius under 5%carbon dioxide for 21 days with media replacement every three days. The SHEDs were characterized with standard immunofluorescence assays, showing the expression of vimentin, actin filaments, and nuclei stained with DAPI.Short-term cell growth assays showed an increased proliferation rate from day two to day eight. Colony-forming unit assay and crystal violet staining confirmed the colony-forming ability of SHEDs after 14 days.

间充质干细胞培养物的接种和分化分步指南，用于表征

Lineage-Specific Cytochemical Staining of Mesenchymal Stem Cells for Characterizing Differentiated Cells

To stain the 2D adipocyte lineage, add 200 microliters of permeable solution from kit to the plate. Next, incubate the cells in 200 microliters of Oil Red O Working solution for 10 minutes. Subsequently, wash the cells five times with 200 microliters of distilled water after removing the stain.To stain the 2D osteoblast lineage, add 200 microliters of 5%fresh silver nitrate to each well, then place the plate under ultraviolet light for an hour. Incubate the cells in 200 microliters of 2.5%sodium thiosulfate for five minutes before a distilled water rinse. For the 3D cultures, first, obtain the cryo sections of the pelleted cultures.To stain the cells of the chondrocyte lineage, add one to two milliliters of washing solution to the air dried sections. Next, incubate them in fixing solution for 30 minutes. After washing with distilled water, place the cells in one to two milliliters staining solution for 30 minutes.Rinse the cells three times with 0.1 normal hydrochloric acid before rinsing with distilled water. After 21 days, the MSCs under in vitro conditions differentiated into adipocytes, osteoblasts, and chondrogenic lineages.

间充质干细胞的谱系特异性细胞化学染色，用于表征分化细胞

Mesenchymal Stem Cell Surface Staining for Immunophenotyping

To begin, centrifuge the trypsinised mesenchymal stem cells at 300 g for six minutes to obtain the cell pellet. Next, resuspend the pellet in one milliliter of MSC media before cell counting. Centrifuge the cell suspension again, then resuspend the pellet in an appropriate volume of the staining buffer after discarding the supernatant.For the preparation of compensation controls, label seven fax tubes as unstained, DAPI, V450, FITC, PE, PerCP-Cy5.5, and APC. Next, prepare the single stain tubes for compensation and incubate them in the dark for 30 minutes. Following incubation, wash the cells in each tube two times and the beads one time with one milliliter of staining buffer.Next, centrifuge the vortexed tubes at 200 g for 10 minutes at room temperature. After discarding the supernatant, resuspend the pellet in 500 microliters of staining buffer. Incubate the DAPI tube in a 60 degree Celsius water bath for five minutes followed by a 15-minute incubation on ice to induce the inclusion of the dye.Add five microliters of DAPI to the suspension and incubate until acquisition. Prepare the cell and antibody suspension in five labeled fax tubes as per the table. Gently vortex the tubes before incubation.Then, wash each tube two times with one milliliter of the staining buffer. Centrifuge the vortex tube at 200 g for 10 minutes at room temperature, then resuspend the remaining pellet in 500 microliters of staining buffer.

用于免疫表型分析的间充质干细胞表面染色

Mesenchymal Stem Cell Sample Preparation, Flow Cytometric Analysis, and Cell Sorting

To begin, label two new fax tubes as mixed tubes one and two. Prepare the cell and antibody suspension as per the given table for flow cytometric analysis. After incubating the tubes for 30 minutes, wash each tube two times with one milliliter of staining buffer.Centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. Then re-suspend the pellet in 500 microliters of staining buffer. Coat the wells of a 48 well plate with 200 to 500 microliters of FBS, and keep for one hour.Then pipette 200 to 500 microliters of 10%MSC media after removing the residual FBS. To prepare the samples for single cell sorting, pipette one milliliter of FBS into two new fax tubes. Roll the tubes to create an even coat of the FBS on the tube's inner surface.Prepare the cell and antibody suspension in mixed tubes one and two, as per the table, for the sorting experiment. Then incubate the tubes in the dark for 30 minutes at room temperature. After washing with staining buffer, centrifuge the tubes at 200 G for 10 minutes at room temperature.Then re-suspend the pellet in 500 microliters of staining buffer and incubate for 15 minutes in five microliters of DAPI before sorting. To set up the compensation matrix, use the single stain compensation tubes. Click on experiment from the toolbar in the instrument software.Then click on compensation setup, followed by create compensation controls. Next, click on unstained tube, followed by parameters in the cytometer dialogue box. Adjust the voltages by editing the values for each parameter.Click on load in the acquisition dashboard and then click acquire. Drag the P-1 gate to the cell population and apply it across all compensation controls to set the voltage and negative gate for each parameter. Now load the single stain compensation tubes individually.Record the data and save it. Select the current experiment, right click on it, and select calculate compensation values, followed by link and save. Run the mixed tube one and record 10, 000 cells.Set the gates on the population of interest to be sorted with the proper gating strategy. Next, load a collection plate into the instrument and set a target cell number, ranging from 2, 500 to 5, 000 cells per well. Then select the single cell sort purity mask.Collect the sorted cell populations in a collection device, keeping them on ice for the duration of the sorting experiment. Finally, shift the plates into a 5%carbon dioxide incubator to maintain the cultures at 37 degrees Celsius. The multi-parametric flow cytometry assays plots showed no expression of the FITC isotype of CD 90 FITC antibodies.Positive expression of the markers CD-90 and CD-73 and negative expression of CD-45 are comparable with that of their unstained and FMO controls. The immuno phenotypes showed the marker distribution from one of the early passages with the ISCT recommended markers and CD-44, determining the parent populations as MSCs. The late passage sheds were used for single cell sorting of high and dim expressors.The post sorted cells collected in the 48 well plate showed adherence and proliferation, like their parent population. The post sorted cells differentiated in the presence of adipogenic growth factors.