Abstract
The hematopoietic system follows a hierarchical organization, with long-term repopulating hematopoietic stem cells (LT-HSCs) occupying the top tier. Application of systems-level single cell methods has revealed a remarkable functional and transcriptional heterogeneity within stem cells. To elucidate the molecular underpinnings of this functional heterogeneity, we developed a technique to barcode hematopoietic cells at the RNA level in order to simultaneously capture the lineage relationships and transcriptional states of HSCs with unprecedented robustness. Using a droplet-based single cell RNAseq platform, we analyzed the total fraction of engrafted hematopoietic stem cells together with a sufficiently significant representation of downstream progenitor cells to estimate HSC output. Inspection of the resulting stem cell state maps revealed a variety of clonal behaviors, including cell quiescence, asymmetric and symmetric divisions, and clonal competition. We also correlated these behaviors with some of the previously observed heterogeneity during transplantation, including lineage bias and clonal dominance. Importantly, comparison of single HSC expression profiles revealed significant differences in the transcriptional programs related with some of these behaviors, which illuminated the molecular machineries that operate at the stem cell level to define this heterogeneity. Finally, a loss-of-function in vivo screening for clonal quiescence/expansion regulators identified several novel candidates of this process. Thus, our work has harnessed the power of combined functional/transcriptional single cell readouts to discover novel mechanisms that orchestrate the in vivo complexity of stem cell activities.
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