Single cell assays unveil functional and transcriptional heterogeneity of human hemopoietic lympho-myeloid progenitors

Human lympho-myeloid progenitors (LMPs) downstream of hemopoietic stem cells remain poorly defined and incompletely purified. Here we purify, functionally (by in vivo transplantation, in vitro single cell, limit dilution and colony assays) and transcriptionally (by population and single cell RNA analysis) characterize 7 human hemopoietic stem and progenitor populations. Focusing on the earliest LMPs, the lymphoid-primed multipotent progenitor (LMPP), the granulocyte-macrophage progenitor (GMP) and the multi-lymphoid progenitor (MLP) we show:1.The LMPP and MLP are very rare within the mononuclear fraction (1 in 10ˆ4 - 1 in 10ˆ5 cells).2.In vitro culture of 3806 single cells in 3 different culture conditions and limit dilution analysis shows that LMPP and GMP are the only progenitors with clonal lympho-myeloid potential (9-15%). The minority of single cells within the populations are bipotential progenitors but the majority are unipotential (50-80%). The GMP has mainly myeloid and LMPP and MLP mainly lymphoid potential.3.When transplanted in vivo using a novel humanized ossicle assay, the LMPP gives robust myeloid and B cell engraftment, the GMP principally myeloid engraftment and the MLP mainly B cell output.4.RNA sequencing shows that all progenitors have distinct transcriptional signatures with unique expression patterns of transcription factors.5.Linking single cell gene expression to FACS indexing we are able to purify exclusively lymphoid and myeloid only progenitors from the current LMP populations. In summary, these data define the heterogeneity within existing LMPP, GMP and MLP populations and suggest a radically new model of human lympho-myeloid progenitor specification.