Heterogeneity in early lymphoid compartments [single-cell RT-PCR]
Ontology highlight
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.
Project description:In order to understand the developmental trajectories of early lymphocyte differentiation it is crucial to identify linage-restricted progenitors. It has become clear that the classical 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. Herein, 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, surface markers that could be used alone or in combination with classical targets to capture specific stages of B-cell development were identified in the screen. 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.
Project description:In order to understand the developmental trajectories of early lymphocyte differentiation it is crucial to identify linage-restricted progenitors. It has become clear that the classical 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. Herein, 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, surface markers that could be used alone or in combination with classical targets to capture specific stages of B-cell development were identified in the screen. 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.
Project description: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.
Project description: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.
Project description: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<X> denotes well ID, and P<X>denoted the plate. Controls include well where no RT is performed (noRT) and 10 and 20 cell controls.
Project description:Using different surface markers it has been possible to isolate lymphoid lineage-biased progentors and test their potential in vivo and in vitro. Here we apply single cell sequencing of lymphoid progenitors to obtain further insights into differentiation and commitment to the lymphoid lineage.
Project description:We identified a new type of bone marrow progenitors termed early innate lymphoid cell progenitor (EILP) using TCF-1 GFP reporter mice. We compared the transcriptomes of early innate lymphoid cell progenitors (EILP) with other early progenitors, including HSC, LMPP, CMP, CLP, ETP and DN3.
Project description:The functions of innate lymphoid cells (ILCs) in immune system are increasingly appreciated, whereas the early development of ILCs in human remains elusive. In this study, we sorted humanhematopoietic stem progenitor cells, lymphoid progenitors, presumed ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 PCW,for single-cell RNA-sequencing, followed by computational analysis and functional validation. We delineated the early phase of ILC development, from hematopoietic stem progenitor cells to multipotent lymphoid progenitors and to ILC progenitors, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor alpha (IL-3RA) as the surface marker for the lymphoid progenitors with T cell, B cell and ILC potentials. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the shared proliferating characteristics of the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2-CCR9+ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.
Project description:To characterize early human hematopoiesis on a single-cell level we developed an approach termed index-omics, which combines flow-cytometric, single-cell transcriptomic and single-cell lineage fate data. Healthy human bone marrow was labeled with a panel of up to 11 FACS surface markers commonly used to identify human hematopoietic stem and progenitor cells (HSPCs). Lin-CD34+38+ progenitors and Lin-CD34+CD38- stem cell enriched HSPCs were individually sorted, their surface marker fluorescence intensities recorded, and subjected to single-cell RNAseq or single-cell ex vivo cultures.
Project description:Tcf1 is necessary for optimal T lineage development. Tcf1 deficient progenitors fail to initiate the T lineage program in vitro and development is severely defective in vivo. We used microarrays to assess the overal global gene expression differences from Tcf1 wildtype and deficient lymphoid biased progenitors cultures on Notch-ligand expressing stroma to determine if Tcf1 deficient progenitors are able to intiate the T lineage specification program. Abstract of manuscript: The thymus imposes the T cell fate on incoming multipotent progenitors, but the molecular mechanisms are poorly understood. We show that transcription factor Tcf1 initiates T-lineage-specific gene expression. Tcf1 is downstream of Notch1 signaling and expressed in early T-cell progenitors. Progenitors deficient for Tcf1 are unable to initiate normal T-lineage specification. Conversely, ectopic expression of Tcf1 in hematopoietic progenitors is sufficient to induce expression of T-lineage specific genes in vitro. Thus, our study identifies Tcf1 as critically involved in the establishment T cell identity. Tcf1 wildtype and deficient bone marrow lymphoid primed progenitors (LMPPs, Lineage marker- Sca+kit+Flt3high) were harvested in triplicate and seeded onto OP9-DL4 expressing stroma for 4 days upon which highly pure lineage negative and Thy1+CD25+ T cells were cell sorted for expression analysis. The lineage negative populations represent three seperate mice from each genotype and the Thy1+CD25+T lineage population represents two replicates from the Tcf1 wildtype group. No Thy1+CD25+ T lineage cells develop from Tcf1 deficient progentiors.