A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice.This work was supported by grants from Bloodwise, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, the National Institute for Health Research Cambridge Biomedical Research Centre, and core support grants by Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. S.N. and F.K.H. are recipients of Medical Research Council PhD studentships. D.G.K. is the recipient of a Bennett Fellowship from Bloodwise, and E.L. is the recipient of a Sir Henry Dale Fellowship from the Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Society of Hematology via http://dx.doi.org/10.1182/blood-2016-05-71648