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

Sonia Nestorowa, Fiona K. Hamey, Blanca Pijuan Sala, Evangelia Diamanti, Mairi Shepherd, Elisa Laurenti, Nicola K. Wilson, David G. Kent and Berthold Göttgens

Key Points

  • An expression map of HSPC differentiation from single-cell RNA sequencing of HSPCs provides insights into blood stem cell differentiation.

  • A user-friendly Web resource provides access to single-cell gene expression profiles for the wider research community.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Abstract

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.

  • Submitted May 12, 2016.
  • Accepted June 28, 2016.
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