Effects of Cd47 or Thbs1 gene disruption on spleen cell numbers and content of cells expressing erythropoietic precursor markers or the proliferation marker Ki67.

(A) Total spleen cell numbers in WT, cd47−/− and thbs1−/− C57BL/6 mice determined after lysis of RBC (mean±SEM, n =3). Flow cytometry was performed to analyze gated singlet spleen cells stained with Ter119 antibody (B), CD34 antibody (C), Sca1 antibody (D), Ki67 antibody with the indicated gating for high and low expression (E), cKit antibody (F), Epor antibody (G), Ermap antibody (H), Gypa antibody (I), or Aqp1 antibody (J). Further analysis of Epor (K,O), Ermap (L,P), Gypa (M,Q), and Aqp1 expression (N,R) was performed after gating for Ter119 expression. The percentages of cells positive for the indicated surface markers are presented (n = 3 or 4) P-values were determined using a two-tailed t test for two-samples assuming equal variances in GraphPad Prism. * = p<0.05, ** = p<0.01, *** = p<0.001.

Expression of erythropoiesis-associated genes in lineage-depleted cd47−/− versus WT spleen cells

Effects of Cd47 or Thbs1 gene disruption on the percentage of Ter119+ and Ter119− spleen cells expressing markers of multipotent and committed erythroid precursors and cell proliferation.

Spleen cells isolated from WT, cd47−/− and thbs1−/− mice were costained with Ter119 antibody along with cKit, Ki67, Sca1 Ermap, Gypa, Epor or Aqp1 antibodies and acquired on an LSRFortessa SORP. After gating for singlet cells, the percentages of Ter119+ and Ter119− cells positive for stem cell markers cKit (A) and Sca1 (B), high or low levels of the proliferation marker Ki67 (C,D), were compared among WT, cd47−/− and thbs1−/− mouse spleens (n = 3-4). The proliferation of CD34+ and CD34− populations of Ter119+ spleen cells from WT, cd47−/−, thbs1−/− mice was evaluated by staining with CD34, Ter119 and Ki67 antibodies. Ter119+CD34+ cells (E), Ter119+CD34− cells (F), and Ter119−CD34+ cells (G) were also quantified (left panels) and analyzed for the proliferation marker Ki67 (center and right panels). P-values were determined using a two-tailed t test for two-samples assuming equal variances in GraphPad Prism. * = p<0.05, ** = p<0.01, *** = p<0.001

Effects of cd47 and thbs1 gene deletion on stem cell and erythroid precursor populations in mouse spleen identified using single cell RNA sequence analysis.

(A) tSNE clustering analysis of lineage-depleted spleen cells from WT, cd47−/− and thbs1−/−mice. The encircled area contains erythroid cells (clusters 9) and stem cells (cluster 11). (B) Distribution of WT, cd47−/− and thbs1−/− spleen cells in each cluster of the tSNE plot. (C) High resolution tSNE plots showing the distribution of the multipotent stem cell markers CD34 and Ly6a (Sca1) and Gata2, the erythropoietic markers Kit and Epor, the proliferation marker Mki67, erythroid differentiation transcription factors Klf1 and Gata1, and erythroid differentiation and extramedullary erythropoiesis markers Ermap, Aqp1, Tmem56, Trim10, Gypa1, Spta1, Sptb, Ebp42, Xpo1, and RanBP2 in clusters 9 and 11. (D) Co-expression of the indicated erythropoiesis related genes in WT, cd47−/− and thbs1−/− spleen cells in cluster 9 was quantified and expressed as a percentage of the total cell number of each genotype in cluster 9.

Differential effects of cd47 and thbs1 gene deletion on mRNA expression levels in cluster 9 erythroid precursor cells and proliferation of CD34−Ter119+Kit+ cells assessed by flow cytometry.

(A) Violin plots comparing mRNA expression levels of the indicated genes in cd47−/−, thbs1−/− and WT spleen cells in cluster 9. (B) Violin plots comparing mRNA expression levels of the indicated genes in cd47−/−, thbs1−/− and WT spleen cells in cluster 11. * = p<0.05.

Differential mRNA expression of erythropoietic, stem cell, and proliferation associated markers in WT, cd47−/−, and thbs1−/− cells in clusters 9 and 11.

Re-clustering of lineage-depleted spleen cells selected for expression of erythroid signature genes.

(A) tSNE plot showing the distribution of cells selected for expressing threshold levels of Gypa, Ermap, Klf1, and/or Aqp1. (B) Immgen and mouse RNAseq main cell type annotation of reclustered cells expressing the erythroid gene signature. Re-clustered cells are displayed in a UMAP projection. (C) Distribution of WT (blue), cd47−/− (red), and thbs1−/− cells (green) in the cluster UMAP projection. (D) Distribution of the multipotent stem cell markers CD34 and Ly6a (Sca1), erythropoietic markers Kit and Epor, the erythroid differentiation transcription factors Klf1 and Gata1, and erythroid differentiation and extramedullary erythropoiesis marker Aqp1, the proliferation marker Mik67, and the erythroid markers Ermap, Tfrc, Tmem56, Trim10, Gypa1, Spta1, Sptb, and Ebp42 in the T cell (lower left) and erythroid lineage cluster (upper right).