一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性
Summary
大体积基因表达测量在异构细胞种群中云个体细胞差异。在这里, 我们描述了一个关于如何通过花期活化细胞分类 (immunophenotypically) 的单细胞基因表达分析和指数排序的协议, 以描绘异质性和分子不同细胞种群的特征。
Abstract
免疫表型表征和分子分析长期以来被用来描绘异质性和定义不同的细胞种群。流式细胞法本质上是单细胞检测, 但在分子分析之前, 靶细胞通常是预先分离的, 从而失去单细胞分辨率。单细胞基因表达分析提供了一种方法来了解异质细胞种群中单个细胞之间的分子差异。在大容量细胞分析中, 不同细胞类型的过多会导致稀有细胞的信号偏倚和闭塞, 具有生物学重要性。利用流化指数分选与单细胞基因表达分析相结合, 可以在不损失单细胞分辨率的情况下研究种群, 同时捕获具有中间细胞表面标记表达的细胞, 从而使评估连续曲面标记表达式的相关性。在这里, 我们描述了一种方法, 结合单细胞逆转录定量 PCR (rt-pcr) 和流化酶指数排序, 同时表征细胞种群内的分子和免疫表型异质性。
与单细胞 RNA 测序方法不同的是, 使用具有特定靶向扩增的 qPCR, 可以对低丰度的转录物进行稳健的测量, 减少辍学量, 同时不混淆读深度。此外, 通过直接将单细胞分选成裂解缓冲液, 这种方法可以在一步内进行 cDNA 合成和特定靶向扩增, 以及随后的分子特征与细胞表面的相关性标记表达式。所描述的方法是研究造血单细胞, 但也被成功地用于其他细胞类型。
总之, 本文所述的方法允许对预先选择的基因组进行 mRNA 表达的敏感测量, 并有可能开发用于随后的分子不同亚群的未来分离的协议。
Introduction
每个单独的血细胞被认为驻留在细胞的层次结构中, 其中干细胞形成了一系列日益承诺的中间祖细胞顶部的顶点, 最终分化成具有特定的最终效应细胞生物功能1。许多关于干细胞系统是如何组织的知识是在造血系统中产生的, 这主要是因为能够前瞻性地隔离高浓度的干细胞或各种祖细胞的不同造血种群2的排序。这允许许多这些人口被分析功能或分子, 主要通过基因表达分析3,4。然而, 当分析大体积种群的基因表达时, 细胞间的个体差异平均为5, 损失较大。因此, 如果细胞的小子集占该种群6的推断生物学功能, 那么无法检测异质细胞分数内的细胞对细胞的变化可能会混淆我们对关键生物过程的理解, 7。相反, 在单细胞分辨率的基因表达特征的研究提供了一种可能性, 描绘异质性和规避遮蔽影响从8个以上的单元格子集。
迄今已开发出许多单细胞基因表达分析的协议;每种方法都有自己的注意事项。最早的方法是 rna 荧光原位杂交 (rna-鱼), 它一次测量有限数量的转录, 但它是唯一的, 它允许调查 RNA 本地化9,11。早期的方法使用 PCR 和 qPCR 检测单个或很少的转录, 也开发了12。然而, 这些最近被取代了基于微流体的方法, 可以同时分析数以百计的每细胞在数以百计的细胞通过 qPCR 的表达, 从而允许高维异质性分析使用预先确定的基因面板10,13。最近, 基于 RNA 测序技术已广泛用于单细胞分析, 因为这些理论可以测量细胞的整个转录组, 从而为异质性分析10添加探索维度, 14. 复用的 qPCR 分析和单细胞 RNA 测序具有不同的特征, 因此使用这两种方法的基本原理取决于所问的问题以及目标种群中的细胞数量。当未知细胞或大种群被调查时, 每个细胞的高通量和低成本以及无偏的、探索性的单细胞 RNA 测序特性是可取的。然而, 单细胞 RNA 测序也偏向于更频繁地测序高丰富的转录, 而低丰度的成绩单容易辍学。这可能导致相当复杂的数据, 使高要求的生物信息学分析, 以揭示重要的分子信号, 往往微妙或隐藏在技术噪声15。因此, 对于特征良好的组织, 使用预先确定的底漆面板选择用于功能重要基因或分子标记的单细胞 qPCR 分析可以作为一种敏感、直接的方法来确定人口。然而, 应该指出的是, 与单细胞 RNA 序列相比, 单细胞的 qPCR 方法的每个细胞的成本通常更高。在这里, 我们描述了一种结合单细胞 rt-pcr 的方法 (从 Teles J. 16), 将指数排序17和生物信息学分析18 , 以同时表征种群内的分子和免疫表型异质性。
在这种方法中, 细胞种群的兴趣被染色, 单细胞通过直接在96孔 PCR 板的裂解缓冲液中进行排序。同时, 在流式排序过程中, 为每个单个单元记录一组附加的单元表面标记的表达式级别, 该方法称为索引排序。随后用微流控平台对裂解细胞材料进行扩增, 并用 rt-pcr 分析了选定基因组的基因表达。该策略可对已排序的单细胞进行分子分析, 同时表征每个细胞的细胞表面标记表达。通过直接将分子不同的细胞子集映射到索引排序标记的表达式, 可以将亚群链接到可用于潜在隔离的特定免疫表型。图 1中逐步概述了该方法。预先确定的基因面板进一步有助于更高分辨率的靶向基因表达, 因为它绕过不相关的丰富的基因的测量, 否则可能咬合微妙的基因表达信号。此外, 具体的靶向放大、一步反转转录和扩增允许对低表达转录物 (如转录因子或非聚仍然 rna) 进行稳健测量。重要的是, qPCR 方法允许测量融合蛋白的 mRNA, 这在调查某些恶性疾病时非常重要19。最后, 被调查的基因的重点数量, 低辍学率, 和有限的技术差异的细胞使这种方法容易分析相比, 更高的维度方法, 如单细胞 RNA-通过遵循该协议, 可以在三天内完成整个实验, 从分拣细胞到分析结果, 使之成为敏感、高通量单细胞基因表达分析的一种简单而快速的方法。
Protocol
1. 裂解板的制备 使用 RNA/DNA 自由工作台, 准备足够的裂解缓冲液为96孔, 10% 额外, 通过混合390µL 核酸酶游离水, 17 µL 10% NP-40, 2.8 µL 10 毫米 dNTP, 10 µL 0.1 M DTT 和5.3 µL 核糖核酸酶抑制剂 (见材料表)。涡旋向下。 将4µL 裂解缓冲液分布到96孔 PCR 板的每个井中, 并用胶膜密封板。在盘子底部旋下管子以收集液体。保持板在冰上直到细胞分类 (最大24小时)。 <p class="jove_tit…
Representative Results
所描述的协议是快速、易于执行和高度可靠的。图 1给出了实验设置的概述。整个协议, 从单细胞的排序, 到特定的目标放大, 基因表达测量和初步分析可以在三天内进行。以热图的形式分析结果的一个例子, 它表示从单细胞基因表达分析中使用96引物和96个细胞从慢性髓性白血病 (CML) 患者或96细胞从老年匹配健康的初步分析数据控件如?…
Discussion
近年来, 单细胞基因表达分析已成为定义不同细胞种群23的异质性的重要补充。RNA 测序技术的出现在理论上提供了测量细胞的整个转录组的可能性, 然而这些方法由于细胞到细胞测序深度和辍学的变化而变得复杂。单细胞的 qPCR 提供了对数以百计的关键基因的表达的灵敏和稳健的分析, 其中所有细胞的处理类似, 减少技术噪音。有限数量的成绩单的集中分析还允许简化分析, 而不会…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了瑞典癌症协会、瑞典研究委员会、瑞典医学研究协会、瑞典儿童癌症基金会、拉格纳 Söderberg 基金会以及克努特和爱丽丝基金会的资助。
Materials
| CD14 PECY5 | eBioscience | 15-0149-42 | Clone: 61D3 |
| CD16 PECY5 | Biolegend | 302010 | Clone: 3G8 |
| CD56 PECY5 | Biolegend | 304608 | Clone: MEM-188 |
| CD19 PECY5 | Biolegend | 302210 | Clone: HIB19 |
| CD2 PECY5 | Biolegend | 300210 | Clone: RPA-2.10 |
| CD3 PECY5 | Biolegend | 300310 | Clone: HIT3a |
| CD123 PECY5 | Biolegend | 306008 | Clone: 6H6 |
| CD235A PECY5 | BD Pharma | 559944 | Clone GAR2 |
| CD34 FITC | Biolegend | 343604 | Clone: 561 |
| CD38 APC | Biolegend | 303510 | Clone: Hit2 |
| CD90 PE | Biolegend | 328110 | Clone: 5E10 |
| CD45RA BV421 | BD bioscience | 560362 | Clone: HI100 |
| CD49f Pecy7 | eBioscience | 25-0495-82 | Clone: eBioGOH3 |
| FBS | HyClone | SV30160.3 | |
| PBS | HyClone | SH30028.02 | |
| 96-well u-bottom Plate | VWR | 10861-564 | |
| SFEM | Stem cell technologies | 9650 | |
| Penicillin streptomycin | HyClone | SV30010 | |
| TPO | Peprotech | 300-18 | |
| SCF | Peprotech | 300-07 | |
| FLT3L | Peprotech | 300-19 | |
| Falcon Tube 15 mL | Sarstedt | 62.554.502 | |
| Eppendorph tube | Sarstedt | 72.690.001 | |
| CST beads | BD | 642412 | |
| Accudrop Beads | BD | 345249 | 6-µm particles |
| Adhesive film Clear | Thermo scientific | AB-1170 | |
| Adhesive film Foil | Thermo scientific | AB-0626 | |
| 96 well PCR plate | Axygen | PCR-96M2-HS-C | |
| PCR 1.5 mL tube | Axygen | MCT-150-L-C | |
| T100 PCR cycler | BioRad | 186-1096 | |
| 10% NP40 | Thermo scientific | 85124 | |
| 10mM dNTP | Takara | 4030 | |
| 0.1M DTT | Invitrogen | P2325 | |
| RNAsout | Invitrogen | 10777-019 | RNAse inhibitor |
| CellsDirect One-Step qRT-PCR Kit | Invitrogen | 11753-100 | |
| Neuclease free water | Invitrogen | 11753-100 | from CellsDirect kit |
| 2X Reaction Mix | Invitrogen | 11753-100 | from CellsDirect kit |
| SuperScript III RT/Platinum Taq Mix | Invitrogen | 11753-100 | from CellsDirect kit |
| Platinum Taq DNA Polymerase | Invitrogen | 10966026 | |
| TaqMan Cells-to-CT Control Kit | Invitrogen | 4386995 | |
| Xeno RNA Control | Invitrogen | 4386995 | From TaqMan Cells-to-CT Control Kit |
| 20X Xeno RNA Control Taqman Gene Expression Assay | Invitrogen | 4386995 | From TaqMan Cells-to-CT Control Kit |
| 96.96 Sample/Loading Kit—10 IFCs | Fluidigm | BMK-M10-96.96 | |
| 2X Assay Loading Reagent | Fluidigm | From 96.96 Sample/Loading Kit | |
| 20X GE Sample Loading Reagent | Fluidigm | From 96.96 Sample/Loading Kit | |
| Control line fluid | Fluidigm | From 96.96 Sample/Loading Kit | |
| TaqMan Gene Expression Master Mix | Applied Biosystems | 4369016 | |
| BioMark HD | Fluidigm | BMKHD-BMKHD | |
| 96.96 Dynamic Array IFC | Fluidigm | BMK-M10-96.96GT | |
| Excel | Microsoft | Microsoft | |
| FlowJo V10 | TreeStar | TreeStar | |
| Fluidigm real time PCR analysis | Fluidigm | Fluidigm | |
| CD179a.VPREB1 | Thermofisher scientific | Hs00356766_g1 | |
| ACE | Thermofisher scientific | Hs00174179_m1 | |
| AHR | Thermofisher scientific | Hs00169233_m1 | |
| BCR_ABL.52 | Thermofisher scientific | Hs03043652_ft | |
| BCR_ABL41 | Thermofisher scientific | Hs03024541_ft | |
| BMI1 | Thermofisher scientific | Hs00995536_m1 | |
| CCNA2 | Thermofisher scientific | Hs00996788_m1 | |
| CCNB1 | Thermofisher scientific | Hs01030099_m1 | |
| CCNB2 | Thermofisher scientific | Hs01084593_g1 | |
| CCNC | Thermofisher scientific | Hs01029304_m1 | |
| CCNE1 | Thermofisher scientific | Hs01026535_g1 | |
| CCNF | Thermofisher scientific | Hs00171049_m1 | |
| CCR9 | Thermofisher scientific | Hs01890924_s1 | |
| CD10.MME | Thermofisher scientific | Hs00153510_m1 | |
| CD11a | Thermofisher scientific | Hs00158218_m1 | |
| CD11c.ITAX | Thermofisher scientific | Hs00174217_m1 | |
| CD123.IL3RA | Thermofisher scientific | Hs00608141_m1 | |
| CD133.PROM1 | Thermofisher scientific | Hs01009250_m1 | |
| CD151 | Thermofisher scientific | Hs00911635_g1 | |
| CD220.INSR | Thermofisher scientific | Hs00961554_m1 | |
| CD24.HSA | Thermofisher scientific | Hs03044178_g1 | |
| NCOR1 | Thermofisher scientific | Hs01094540_m1 | |
| CD26.DPP4 | Thermofisher scientific | Hs00175210_m1 | |
| CD274 | Thermofisher scientific | Hs01125301_m1 | |
| CD276 | Thermofisher scientific | Hs00987207_m1 | |
| CD32.FCGR2B | Thermofisher scientific | Hs01634996_s1 | |
| CD33 | Thermofisher scientific | Hs01076281_m1 | |
| CD34 | Thermofisher scientific | Hs00990732_m1 | |
| CD344.FZD4 | Thermofisher scientific | Hs00201853_m1 | |
| CD352.SLAMF6 | Thermofisher scientific | Hs01559920_m1 | |
| CD38 | Thermofisher scientific | Hs01120071_m1 | |
| CD4 | Thermofisher scientific | Hs01058407_m1 | |
| CD41.ITGA2B | Thermofisher scientific | Hs01116228_m1 | |
| CD49f.ITGA6 | Thermofisher scientific | Hs01041011_m1 | |
| CD56.NCAM1 | Thermofisher scientific | Hs00941830_m1 | |
| CD9 | Thermofisher scientific | Hs00233521_m1 | |
| CD97 | Thermofisher scientific | Hs00173542_m1 | |
| CD99 | Thermofisher scientific | Hs00908458_m1 | |
| CDK6 | Thermofisher scientific | Hs01026371_m1 | |
| CDKN1A | Thermofisher scientific | Hs00355782_m1 | |
| CDKN1B | Thermofisher scientific | Hs01597588_m1 | |
| CDKN1C | Thermofisher scientific | Hs00175938_m1 | |
| CEBPa | Thermofisher scientific | Hs00269972_s1 | |
| CSF1r | Thermofisher scientific | Hs00911250_m1 | |
| CSF2RA | Thermofisher scientific | Hs00531296_g1 | |
| CSF3RA | Thermofisher scientific | Hs01114427_m1 | |
| E2A.TCF3 | Thermofisher scientific | Hs00413032_m1 | |
| EBF1 | Thermofisher scientific | Hs01092694_m1 | |
| ENG | Thermofisher scientific | Hs00923996_m1 | |
| EPOR | Thermofisher scientific | Hs00959427_m1 | |
| ERG | Thermofisher scientific | Hs01554629_m1 | |
| FLI1 | Thermofisher scientific | Hs00956711_m1 | |
| FLT3 | Thermofisher scientific | Hs00174690_m1 | |
| FOXO1 | Thermofisher scientific | Hs01054576_m1 | |
| GAPDH | Thermofisher scientific | Hs02758991_g1 | |
| GATA1 | Thermofisher scientific | Hs00231112_m1 | |
| GATA2 | Thermofisher scientific | Hs00231119_m1 | |
| GATA3 | Thermofisher scientific | Hs00231122_m1 | |
| GFI1 | Thermofisher scientific | Hs00382207_m1 | |
| HES1 | Thermofisher scientific | Hs01118947_g1 | |
| HLF | Thermofisher scientific | Hs00171406_m1 | |
| HMGA2 | Thermofisher scientific | Hs00171569_m1 | |
| HOXA5 | Thermofisher scientific | Hs00430330_m1 | |
| HOXB4 | Thermofisher scientific | Hs00256884_m1 | |
| ID2 | Thermofisher scientific | Hs04187239_m1 | |
| IGF2BP1 | Thermofisher scientific | Hs00198023_m1 | |
| IGF2BP2 | Thermofisher scientific | Hs01118009_m1 | |
| IKZF1 | Thermofisher scientific | Hs00172991_m1 | |
| IL1RAP | Thermofisher scientific | Hs00895050_m1 | |
| IL2RG | Thermofisher scientific | Hs00953624_m1 | |
| IRF8 | Thermofisher scientific | Hs00175238_m1 | |
| ITGB7 | Thermofisher scientific | Hs01565750_m1 | |
| KIT | Thermofisher scientific | Hs00174029_m1 | |
| Lin28B | Thermofisher scientific | Hs01013729_m1 | |
| LMO2 | Thermofisher scientific | Hs00153473_m1 | |
| LYL1 | Thermofisher scientific | Hs01089802_g1 | |
| Meis1 | Thermofisher scientific | Hs01017441_m1 | |
| mKi67 | Thermofisher scientific | Hs01032443_m1 | |
| MPL | Thermofisher scientific | Hs00180489_m1 | |
| MPO | Thermofisher scientific | Hs00924296_m1 | |
| NFIB | Thermofisher scientific | Hs01029175_m1 | |
| Notch1 | Thermofisher scientific | Hs01062011_m1 | |
| Pten | Thermofisher scientific | Hs02621230_s1 | |
| RAG2 | Thermofisher scientific | Hs01851142_s1 | |
| RPS18 | Thermofisher scientific | Hs01375212_g1 | |
| RUNX1 | Thermofisher scientific | Hs00231079_m1 | |
| Shisa2 | Thermofisher scientific | Hs01590823_m1 | |
| Spi1 | Thermofisher scientific | Hs02786711_m1 | |
| Sterile.IgH | Thermofisher scientific | Hs00378435_m1 | |
| TAL1 | Thermofisher scientific | Hs01097987_m1 | |
| THY1 | Thermofisher scientific | Hs00264235_s1 | |
| Tim.3.HAVCR2 | Thermofisher scientific | Hs00958618_m1 | |
| VWF | Thermofisher scientific | Hs00169795_m1 |
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Cite This Article
Sommarin, M. N., Warfvinge, R., Safi, F., Karlsson, G. A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations. J. Vis. Exp. (140), e57831, doi:10.3791/57831 (2018).