Maintenance of the blood system requires balanced cell fate decisions of haematopoietic stem and progenitor cells (HSPCs). Since cell fate choices are executed at the level of individual cells, new single cell profiling technologies offer exciting possibilities to map the dynamic molecular changes underlying HSPC differentiation. Researchers from the MRC Cambridge Stem Cell Institute have used single cell RNA-Seq to profile over 1,600 single HSPCs, where deep sequencing has enabled detection of an average of 6,558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed them to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. The researchers further show that independently generated single cell datasets 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-megakaryocytic and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. Using external spike-in controls, they estimate absolute 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, the researchers 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.
Generating linked transcriptional and surface marker profiles for over 1,600single HSPCs
Schematic of the sorting strategy used paired with index sorting data. Bone marrow cells were stained with 9 antibodies against various cell surface markers in order to isolate HSPCs (Lin- c-Kit+ Sca1+) and Progenitors (Lin- c-Kit+ Sca1-). Almost all cells in the Flk2-CD34 gate and the CD16/32-Flk2 gate were collected for HSPCs and Progenitors, respectively, within broad, all-encompassing gates. In addition, LT-HSCs were (Lin- c-Kit+ Sca1+ CD34- Flk2-) collected separately to ensure adequate numbers were collected. Each cell population retrospectively identified is shown in the table, colours and names remain consistent throughout the text. Letters indicate populations in the flow cytometry diagrams.
Availability – http://blood.stemcells.cam.ac.uk/single_cell_atlas.html