A unified workflow to define multipotent progenitor hierarchies (HIVE)

Multipotent hematopoietic progenitors with coherent developmental transcriptional programs are defined by their ability to give rise to distinct cell lineages. While the identification of multilineage progenitors is aided by single-cell technologies, new techniques are required for their rigorous delineation and isolation. We describe a new approach to dissect and contrast well-defined and newly proposed immunophenotypically defined hematopoietic progenitors using integrative multimodal single-cell genomics and linked high-dimensional flow cytometry. The lineage potential of implicated multi-lineage progenitors could be resolved through their sequential isolation, lineage tracing, genetic reporters, in vitro differentiation and gene-regulatory prediction analyses. These analyses implicate coordinated transcription factors operating on composite elements for distinct multilineage cell states. A newly identified precursor of the neutrophil-monocytic bipotential (MultiLin-1) was confirmed to be transcriptionally reprogrammed by Th2 cytokines following infection, to produce the successive waves of neutrophils, basophils and eosinophils. This work supports a stepwise model of hematopoiesis in which lineage transitions occur as discrete, isolatable cell states. Overall design: A five-fold molecular titration of BioLegend antibodies was performed in murine bone marrow to develop an optimized CITE-Seq panel (10x Genomics) of 112 cell surface markers.