Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors

Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220(+)CD117(int)CD19(-)NK1.1(-) uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among 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 comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D(+)SiglecH(-)CD11c(-). Subsequent functional assays confirmed that B220(+)CD117(int)CD19(-)NK1.1(-) single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D(+)SiglecH(-)CD11c(-) subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220(+)CD117(int)CD19(-)NK1.1(-) progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors.