Page 77 - 2020_11-Haematologica-web

Isolation and molecular signature of stress-BFU-Es
colonies, providing over 100-fold improved purity com- pared to the state-of-the-art (0.1-0.2%). Although both Sca1– and Sca1+ cells formed BFU-E colonies when stem cell factor (SCF) was present, only Sca1- cells formed BFU- E in erythropoietin (Epo) alone (Figure 1F). Furthermore, Sca1– progenitors generally gave rise to larger colonies
than Sca1+ progenitors (data not shown). A notable 27.4±3.2% of CD150+CD9– cells gave rise to CFU-E, which were mainly independent of hypoxia and SCF (Figure 1G). Although the number of colonies was not affected, hypoxia resulted in increased proliferation and larger colonies (data not shown). FACS-sorted
AB
CDE
Figure 3. Stress-BFU-E and stress-CFU-E display opposing expression patterns of BMP- and CBFA2T3-responsive genes. (A) Unsupervised clustering of RPKM nor- malized data on genes with a significantly different expression between any of the samples (FDR<0.05, log2 fold change>0.58) from RNA-sequencing of FACS sorted stress-progenitor populations as indicated (n=3, for further details see the Methods section). (B) Cellular processes for each of the clusters generated in (A), as ana- lyzed by GSEA. For a complete gene lists for each cluster see the Online Supplementary Table S1. (C) Experimental outline of competitive transplantation of wild-type or BMP receptor II (BMPRII) deficient bone marrow against KuO+ wild-type bone marrow. The stress recovery was analyzed in spleens of recipients on day 8 after transplantation (n=7 per group). (D) White blood cell count (WBC) per spleen before and after enrichment of mononuclear cells (MNC). (E) Detailed analysis of donor- derived (KuO-) MNC and stress-populations within MNC spleen cells as indicated. Data displayed as average ± standard error of the mean (SEM) of total number of cells/spleen (D) and normalized to wild-type (E), *P≤0.05, **P≤0.01.
haematologica | 2020; 105(11)
2565