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. 2017 Feb 17;45(3):1281-1296.
doi: 10.1093/nar/gkw1214.

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors

Jennifer Yang  1 , Yoshiaki Tanaka  2 , Montrell Seay  1 , Zhen Li  3 , Jiaqi Jin  1 , Lana Xia Garmire  4 , Xun Zhu  4 , Ashley Taylor  5 , Weidong Li  1   6 , Ghia Euskirchen  7 , Stephanie Halene  5 , Yuval Kluger  8 , Michael P Snyder  7 , In-Hyun Park  2 , Xinghua Pan  1   9 , Sherman Morton Weissman  1
Affiliations

Single cell transcriptomics reveals unanticipated features of early hematopoietic precursors

Jennifer Yang et al. Nucleic Acids Res. .

Abstract

Molecular changes underlying stem cell differentiation are of fundamental interest. scRNA-seq on murine hematopoietic stem cells (HSC) and their progeny MPP1 separated the cells into 3 main clusters with distinct features: active, quiescent, and an un-characterized cluster. Induction of anemia resulted in mobilization of the quiescent to the active cluster and of the early to later stage of cell cycle, with marked increase in expression of certain transcription factors (TFs) while maintaining expression of interferon response genes. Cells with surface markers of long term HSC increased the expression of a group of TFs expressed highly in normal cycling MPP1 cells. However, at least Id1 and Hes1 were significantly activated in both HSC and MPP1 cells in anemic mice. Lineage-specific genes were differently expressed between cells, and correlated with the cell cycle stages with a specific augmentation of erythroid related genes in the G2/M phase. Most lineage specific TFs were stochastically expressed in the early precursor cells, but a few, such as Klf1, were detected only at very low levels in few precursor cells. The activation of these factors may correlate with stages of differentiation. This study reveals effects of cell cycle progression on the expression of lineage specific genes in precursor cells, and suggests that hematopoietic stress changes the balance of renewal and differentiation in these homeostatic cells.

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Figures

Figure 1.
Figure 1.
Relationship among 18 murine bone marrow populations. Two and three dimensional plots of grouping 18 populations based on PC1, PC2 and PC3.
Figure 2.
Figure 2.
Classification of HSC and MPP1 single cells into three main subpopulations. (A) Dimensional reduction by tSNE separating HSC and MPP1 into three major clusters: U-, quiescent and active clusters. (B) Fraction of HSC and MPP1 cells falling into three major clusters in two biological replicates based on single cell RNA-seq. (C, D, E) GSEA analysis. (C) Quiescence-related genes (18). (D) Genes related to representative GO biological processes in the three major clusters. (E) Different lineages related genes among the three major clusters. (F) Comparison to (A) for the distribution of HSC and MPP1 cells displaying gene expression related to interferon response which mostly are in quiescent cluster. Average expression of ‘interferon gamma response-related’ genes (Supplementary Table S5) are shown. Red represents a higher level of expression and black a lower level. The dots are HSC, the triangles are MPP1, and two replicates are combined. (G) Comparison of the number of detected genes among three clusters.
Figure 3.
Figure 3.
Correlation between cell cycle phase and expression of lineage-related genes. (A) The tSNE patterns of G2/M (red), G1/S (orange) and early G1 states (purple) within the active cluster, along with the quiescent (blue) and U-cluster (black) cluster. (B) Comparative analysis of G1/S, S, G2/M and M phase-specific genes in U-, quiescent- and three subdivisions of the active cluster. (C) GSEA of lineage-specific genes in cells at various stages of the cell cycle in the active cluster. (D) Cells plotted according to increasing G2/M gene expression. The log of expression of red (erythroid) and cyan (G2/M) genes per cell is shown. (E) The relation between expression of S phase and G2/M phase genes in each cell. Cells are plotted according to their rank order of levels of G2/M genes. The lines are loess smoothed representations of the level of expression in each cell. The purple line represents the level of S phase genes and the red line the level of G2/M genes. The green line represents the ratio of S phase to G2/M genes.
Figure 4.
Figure 4.
Activation of HSC and MPP1 in anemic mice. (A) The tSNE patterns of HSC and MPP1 from control and anemic mice. Different subsets of cells from the same tSNE display are shown in each panel, with subsets labeled by the color scheme on the right side of the top lane. (B) Ratio of cells falling into each subpopulation in HSC and MPP1 from control and anemic mice. In each image several subsets of the cells are displayed, identified by the color code in the right side upper rectangle. bHSC and bMPP1 are HSC and MPP1 obtained from mice made anemic by bleeding. (C) Schematic graph of correlation between lineage specificity and the phase of the cell cycle. After induction of anemia by bleeding, HSC shift into the active cluster in map regions occupied by MPP1 cells in normal marrow.
Figure 5.
Figure 5.
Induction of anemia drives more cells into the active stage and especially the G2/M phase of the cell cycle. A, B and C are different tSNE runwith the same parameters and the appearance of the detailed pattern varies (111) (A) The tSNE patterns of the whole population of HSC (purple) and MPP1 (green). Unlike MPP1, the majority of HSC cells are quiescent cells (middle cluster). (B) Comparative analysis of cells expressing high levels of S (red) and G2/M (purple) phase-specific genes in the combined population of HSC and MPP1. The colored cells (red and purple) are 10 cells each expressing highest levels of S or G2/M genes. (C) Both anemic HSC (blue dots) ls shift to the active stage, and the anemic MPP1 (red dots) shift towards the G2/M stage. (D) Three lineage expression levels in HSC/MPP1 cells from the control mice. Cells plotted according to increasing G2/M gene expression. The red, black and purple curves respectively refer to the expression per cell of erythroid precursor genes, of megakaryocyte genes, and of granulocyte/macrophage precursor genes. The curves were generated by loess smoothing of scatter plots. The gene expression levels are plotted on a log scale. (E) Three lineage levels of expression of genes in HSC/MPP1 cells from the anemic mice. Cells plotted as in Figure 5D. Note that a larger fraction of the cells from anemic mice were in the S/G2/M fraction.
Figure 6.
Figure 6.
The expression of the transcription factors (TFs) specifically elevated in different lineages in mouse HSC and MPP1 single cells. The X axis presents the different TFs (Supplementary Table S9); the Y axis represents the log10 of the number of cells in which the TF mRNA was detected (plus 1 to avoid logs of numbers less than 0). In total 228 single cells were analyzed, combining replicate 1 and 2. (A) Klf1 is the least expressed among 55 TFs for the erythroid lineage although it is one of the most highly expressed factors in maturing erythroid cells. (B) Cebpa and Cebpe were depleted in contrast to the other 41 TFs for granulocyte lineage. (C) Seven TFs were not detected and several other TFs are very low among 55 TFs specific for lymphocyte lineage. (D) All of the list of 22 TFs expressed selectively in maturing megakaryocytes are expressed in a substantial fraction of the HSC/MPP1 cells.
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