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Isolation and molecular signature of stress-BFU-Es
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Figure 1. CD150, CD9 and Sca1 identify a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery. (A) Stress-erythropoiesis was induced using lethal irradiation followed by transplantation of unfractionated bone marrow (BM), and splenic stress-progenitors were assessed on day 8 using colony assays. (B) Gating strategy and (C) BFU-E potential of FACS-sorted stress-progenitors from spleen day 8 based on CD150 expression (n=5). (D) Gating strategy for further fractionation of stress-progenitors within lineage-Kit+ cells using CD150 and CD9 and Sca1. (D-G) Colony forming capacity of splenic day 8 stress-progenitors, FACS- sorted based on (D) CD150/CD9 (normoxia, n=4), and (E-G) CD150/CD9/Sca1 (n=7 for normoxia, n=4 for hypoxia) within the Lin-cKit+CD71lowCD24low fraction. The cells were incubated in normoxia (21% O2) or hypoxia (1% O2) as indicated, and scored on day 4 (CFU-E) or day 7-8 (mixed and BFU-E). (H) FACS sorted CD150+CD9+Sca1+ progenitors were cultured in vitro and analyzed at indicated time points for the formation of CD150+CD9+Sca1– cells (n=4). I) Expression pattern of novel surface markers within the Lin–cKit+CD71lowCD24low fraction separating multi-potent stem/progenitor cells (sMPPs), stress-BFU-Es (sBFU-Es) and stress-CFU- E (sCFU-Es), defining a stress-progenitor hierarchy in spleen during irradiation-induced stress recovery, where Sca1 and subsequently CD9 are downregulated with increased differentiation. Data displayed as average ± standard error of the mean (SEM), n.d: not detectable, n.a: not applicable, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
haematologica | 2020; 105(11)
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