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S. Singbrant et al.
As previously reported by us in fetal liver,16 exclusion of mature erythroid precursor cells in spleen was facilitated by inclusion of CD24a as a negative selection marker (Online Supplementary Figure S1B). Of the surface markers tested (CD9, CD11a, CD34, CD48, CD63, and CD79b), only CD9 could further fractionate the BFU-E-containing CD150+ population in stressed spleen (Online Supplementary Figure S1C). CD133 was used in a recent study together with CD34 to fractionate stress-progenitor potential of cultured Sca1+cKit+CD71/Ter119low cells.17 However, neither CD34 nor CD133 could further fraction- ate CD150+ stress-progenitors (Online Supplementary Figure S1C and Online Supplementary Figure S2A, respectively). To further discriminate putative multi-potent stress-progeni- tors from lineage restricted stress-BFU-E we also included Sca1, which in steady-state BM separates Sca1+ hematopoietic stem cells (HSC) and Sca1– myelo-ery- throid progenitors.9 Analysis of Lin–cKit+CD71low/CD24alow splenic stress-progenitors fractionated based on
CD150/CD9/Sca1 expression (Figure 1D), clearly demon- strated that the majority of multi-lineage, megakaryocytic and BFU-E colony forming potential resided in the CD150+CD9+ population (Figure 1E-F). Stress-progenitors negative for CD9 gave rise to more mature erythroid colonies (Figure 1E, G), which was also true for CD133+ cells (Online Supplementary Figure S2B), whereas CD150– cells mainly gave rise to myeloid colonies (Figure 1E). Only Sca1 expressing CD150+CD9+ cKit+ CD71low/CD24alow stress-progenitors (about 10%) gave rise to mixed and myeloid colonies (Figure 1E), while megakaryocytic/erythroid potential was retained in Sca1– stress-progenitors (Figure 1E-F). Stress-BFU-E are reported to form BFU-E colonies with Epo alone,1 but require hypoxia and additional cytokines for maximum expansion.3 Colony assays demonstrated that BFU-E- forming potential resided almost exclusively in the CD150+CD9+ population (Figure 1F). Importantly, as many as 21.4±2.2% of CD150+CD9+ cells gave rise to BFU-E
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Figure 2. Stress-BFU-E provide a transient wave of primarily erythroid cells, followed by multi-lineage reconstitution from stress-MPP. (A) Stress-erythropoiesis was induced using lethal irradiation followed by transplantation of unfractionated bone marrow (BM) from (B) wild-type or (C-K) transgenic Kusabira Orange (KuO) mice. Splenic stress-progenitor populations were FACS-sorted on day 8, transplanted into lethally irradiated secondary recipients (B) without support to evaluate spleen colony formation (CFU-S8) (n=3-7; 600 cells per recipient without support) or (C-K) together with 105 unfractionated wild-type BM support cells to monitor their repopulation capacity in vivo over time in (C) peripheral blood (PB), (D) spleen and (E) BM, as determined by KuO fluorescence and FACS (n=6 at 1-2 weeks and n=3 at 4 weeks; 500 sMPP or 5,000 of each stress-erythroid progenitor per recipient, all with 105 wild-type support BM cells). (F-J) Contribution of sorted KuO+ progen- itors in PB to (F) reticulocytes (whole PB), (G) Ter119+ erythroid cells, (H) CD41+ cells, (I) platelets (whole PB), and (J) Gr1+ myeloid cells in PB, as determined by FACS (after lysis of red blood cells if not stated otherwise). (K) Representative picture of whole spleens 2 weeks after transplantation assessing KuO contribution using epifluorescence microscopy and green filtered laser excitation. Data displayed as average ± standard error of the mean (SEM), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
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haematologica | 2020; 105(11)