Unknown,Transcriptomics,Genomics,Proteomics

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Heterogeneity in early lymphoid compartments [qPCR]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Shamit Soneji

PROVIDER: E-GEOD-74606 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by âbulkâ assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) âPre-MEPâ, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) âE-MEPâ, strongly biased towards erythroid differentiation, and (3) âMK-MEPâ, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.

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Heterogeneity in early lymphoid compartments [qPCR]

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