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Single-cell RNA sequencing of EPLM subpopulations (152 Ly6D+ and 213 TN)

ABSTRACT: Single-cell RNA-sequencing has emerged as a powerful technology to assess heterogeneity within defined cell populations. Here, we comprehensively study the heterogeneity of a previously described B220+CD117intCD19-NK1.1- uncommitted hematopoietic progenitor with combined lymphoid and myeloid potential (EPLM). Using staining for surface markers together with functional assays, we describe four subpopulations of this progenitor with distinct lineage developmental potentials. Amongst them, the Ly6D+SiglecH-CD11c- fraction was lymphoid restricted exhibiting strong B-cell potential, whereas the Ly6D-SiglecH-CD11c- fraction showed mixed lympho-myeloid potential. Single-cell RNA-sequencing of these subsets revealed that the latter population is composed of a mixture of cells with distinct lymphoid and myeloid genetic signatures and identified a subgroup as the potential precursor of Ly6D+SiglecH-CD11c-. We observed a B-cell priming gradient within the Ly6D+SiglecH-CD11c- subset and propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Our study demonstrates that the multipotency of B220+CD117intCD19-NK1.1- progenitors is a result of underlying heterogeneity at the single-cell level. Moreover, it highlights the validity of single-cell transcriptomics to resolve cellular heterogeneity and identify developmental relationships in hematopoietic progenitors.

ORGANISM(S): Mus musculus

PROVIDER: GSE102455 | GEO | 2017/10/11

SECONDARY ACCESSION(S): PRJNA397781

REPOSITORIES: GEO

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Project description:microRNAs are assumed to fine-tune cellular mRNA expression levels predisposing them for the control of cell development and cell fates. However, despite increasing appreciation of the role of miRNAs in controlling myeloid cell functions and some evidence for their contribution in in vitro monocyte differentiation, the in vivo role of specific miRNAs in the development and homeostasis of myeloid cells remains to be investigated. Here we profiled the microRNA expression of 8 different myeloid cell population including myeloid precursors (MP), myeloid dendritic cell precursors (MDP), common dendritic cell precursors (CDP), plasmacytoid dendritic cells (PDC) and Gr1+ monocytes from the bone marrow and three different spenic denritic cells (DCs): CD4+ DCs, CD8+ DCs and CD4-CD8- DCs. The source of the cells were female C57BL/6 animals in an age of 6 weeks. The FACS isolation was done on a pooled cell suspension from a minimum of 5 animals. C57BL/6 mice in an age of 6 weeks were purchased from Harlan. For bone marrow (BM) precursor isolation, ACK (0.15M NH4Cl, 0.1M KHCO3, 1mM EDTA in PBS) lyzed BM cells from femur and tibia were pooled from 15 mice and enriched by MACS with biotinylated CD135 (A2F10) followed by anti-biotin MACS beads (Miltenyi). The enriched fraction was further stained with Streptavidin-PerCP, CD117 (2B8), CD115 (AFS98) and lineage antibody cocktail (CD11b (M1/70), CD3 (145-2C11), CD4 (GK1.5), CD8M-NM-1 (53-6.7), Gr1 (RB6-8C5), Sca-1 (D7), B220 (RA3-6B2), Ter-119, CD11c (N418), NK1.1 (PK136)). Splenic dendritic cells were pre-enriched from 8 mice by CD11c MACS beads (Miltenyi) and further stained for CD8M-NM-1, CD4, CD11c and CD86 (PO3). BM plasmacytoid DCs were isolated from 5 mice, followed by Ficoll enrichment and stained for CD11c, CD317 (927) and Siglec H (eBio440c). BM monocytes were isolated from Ficoll enriched BM cells from 5 mice. Staining markers were: CD11b, Gr1 and CD115. All antibodies were purchased from BioLegend or eBioscience if not indicated otherwise. A FACSAria cytometer (Becton-Dickinson) was used for sorting and duplets were excluded by their FSC-H vs. FSC-W appearance.

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Single-cell RNA sequencing of EPLM subpopulations (152 Ly6D+ and 213 TN)

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