Heterogeneity in early lymphoid compartments [qPCR]

In order to understand the developmental trajectories of early lymphocyte development it is crucial to prospectively isolate stage and lineage specific cells. It has become clear that early lymphoid progenitor compartments in the bone marrow are molecularly and functionally heterogeneous which warrants further investigation to refine the marker combinations used to isolate these progenitors. An initial antibody screen revealed extensive surface marker heterogeneity amongst early lymphoid progenitors. This heterogeneity was resolved using single cell gene expression assays and single cell in vitro differentiation assays, identifying marker combinations that isolate functionally distinct populations. In addition, using reporter transgenic mice we were able to identify a set of surface markers that can be used alone or in combination with classical targets to identify specific stages of B-cell development. B-cell stages based on transgene expression which were used for screening purposes were verified by RNASeq. The data provides a greater resolution of the complexity of the lymphoid progenitor compartment within the bone marrow than has been understood to date and provides novel tools for the further identification of cell populations in B-lineage development. qPCR gene expression profiling of mouse common lymphoid progenitors (CLPs). Four 96-well plates with pre-amplified single CLP bone marrow cell cDNA (with 10 and 20 cell controls) were loaded on to a 96.96 Dynamic Array Chip for Gene Expression. Samples Ids follow where SS denotes well ID, and Pdenoted the plate. Controls include well where no RT is performed (noRT) and 10 and 20 cell controls.

为解析早期淋巴细胞发育的动态轨迹，前瞻性分离阶段特异性与谱系特异性细胞至关重要。现已明确，骨髓内的早期淋巴祖细胞区室在分子与功能层面均存在异质性，亟需开展进一步研究以优化用于分离此类祖细胞的标志物组合。初步抗体筛选实验揭示，早期淋巴祖细胞群体中存在广泛的表面标志物异质性。通过单细胞基因表达检测与单细胞体外分化实验，我们解析了该异质性，并确定了可分离功能迥异细胞群体的标志物组合。此外，借助报告基因转基因小鼠模型，我们鉴定出一组可单独使用，或与经典靶点联合应用的表面标志物，用于识别B细胞发育的特定阶段。本研究中用于筛选的、基于转基因表达的B细胞发育阶段，已通过RNA测序（RNA-seq）得到验证。本数据集相较此前的认知水平，更清晰地解析了骨髓淋巴祖细胞区室的复杂性，并为进一步鉴定B谱系发育中的细胞群体提供了全新工具。本数据集包含小鼠常见淋巴祖细胞（common lymphoid progenitor, CLP）的定量聚合酶链反应（qPCR）基因表达谱数据：将4块搭载经预扩增的单个CLP骨髓细胞cDNA（附带10细胞与20细胞对照样本）的96孔板，上样至96.96动态阵列基因表达芯片进行检测。样本ID遵循如下规则：SS代表孔位编号，P代表板号。对照样本包括未进行逆转录（noRT）的样本，以及10细胞、20细胞对照样本。