Heterogeneity in early lymphoid compartments [single-cell RT-PCR]

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 and FrA). 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 10 and 20 cell controls. (Fluidigm) with 96 TaqMan assays and analysed on BioMark HD (Fluidigm)