A unified workflow to define multipotent progenitor hierarchies (TEA-Seq)

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.