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HbF rescues dyserythropoiesis in SCD
(version 7). The data was analyzed using Mann-Whitney unpaired test and Wilcoxon paired test, as indicated in the figure legends.
Results
Hypoxia-induced cell death during in vitro terminal erythroid differentiation
The bone marrow environment has been well docu- mented to be hypoxic (0.1-6% O2).16-18 As hypoxia induces HbS polymerization, we hypothesized that cell death may occur in vivo because of HbS polymer forma- tion in the late stages of differentiation characterized by high intracellular hemoglobin concentration. In order to test our hypothesis, we performed in vitro erythroid dif- ferentiation using CD34+ cells isolated from SCD patients and from healthy donors. A two-phase ery- throid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e., normoxia and partial hypoxia (5% O2), starting at D3 of the second phase, at which time hemoglobin synthesis begins to increase markedly (Online Supplementary Figure S1). The choice of 5% O2 was made since it falls at the high end of the reported oxygen tension range of the hematopoietic niche (0.1-6% O2),17 and because it drives HbS polymerization and cell sickling, as we previously reported.19 First, we performed video microscopy experi- ments with nucleated SCD erythroblasts at D9 of culture and confirmed their ability to sickle at 5% O2 (Figure 1A; Online Supplementary Video S1). Differentiation of control erythroblasts showed no difference in the general water- fall pattern between normoxia and hypoxia (Figure 1B), although hypoxia translated into a consistently higher but not statistically significant increase in total cell count (Figure 1C). Under normoxia, SCD differentiation showed a mild deceleration till D9 as compared to con- trol (Figure 1B), with a proliferation that was negatively impacted by hypoxia (Figure 1C). Under both oxygen conditions, cell proliferation was significantly higher in the control than in the SCD cultures, starting from D7 (Figure 1C). May Grünwald-Giemsa (MGG) staining confirmed the differentiation delay of SCD cells, espe- cially under hypoxia where cells seemed to accumulate at the polychromatic stage when compared to control cells (Figure 1D). In addition, higher proportions of enu- cleated cells were found in control cells at D9 (Figure 1D) and D11 (Figure 1E). Enucleation was improved under hypoxia for control erythroblasts while it was signifi- cantly diminished for SCD cells (Figure 1E), indicating a negative impact of hypoxia at this critical maturation step in the context of SCD.
In order to assess if the decrease in proliferation in SCD was due to cell death, we measured the percentage of apoptotic cells in the cultures by staining the gly- cophorin A (GPA)-positive cells with annexin V (Figure 1F). The percentage of annexin V+ cells was higher in SCD than in control cultures under both oxygen condi- tions at D7 and D9, with a greater variability among SCD than in control cells (Figure 1G). Furthermore, the extent of apoptosis of SCD cells was higher under hypoxia than under normoxia at both time points while no difference was noted for control cells (Figure 1G). Altogether, these findings imply that even under a con- servative choice of 5% O2 to mimic hypoxia in bone mar-
row there is increased cell death during the terminal dif- ferentiation stages in SCD cells only.
F-cells are enriched during sickle cell disease erythroid differentiation
Using flow cytometry, we measured the percentage of cells expressing HbF (F-cells) during in vitro differentiation (Figure 2A). The percentage of F-cells (%F-cells) in control cultures fell within the reported range of 20-40%,20,21 while it was very variable for SCD cells reaching more than 70% at D9 (Figure 2B). Interestingly, there was no difference of %F-cells between normoxia and hypoxia for control cells, while for SCD, %F-cells was higher under hypoxia than under normoxia for all of the six independent primary cell samples (Figure 2B). Taken together with the apoptosis data, these findings imply that F-cells were positively selected under hypoxia in SCD. This inference was sup- ported by flow cytometry data showing higher percent- ages of dead cells, based on the fixable viability stain (FVS), within the non-F-cell population as compared to the F-cell population for SCD cells (Figure 2C and D). In contrast, these percentages were similar between both cell popula- tions in the control (Figure 2C and D), confirming preferen- tial apoptosis of the cells with low/no HbF expression in the SCD context only.
HSP70 is sequestered in the cytoplasm of non-F-cells
As cytoplasmic sequestration of the chaperone protein HSP70 by α-globin aggregates is associated with cell death during erythropoiesis in b-thalassemia major,5 we investi- gated if apoptosis of SCD erythroblasts might be due to cytoplasmic trapping of HSP70 by HbS polymers. We per- formed western blot analyses to quantify HSP70 in the cytoplasmic and nuclear extracts of erythroblasts at D7 of phase II of culture (Figure 3A). There was less HSP70 in the nucleus and more in the cytoplasm of SCD erythroblasts under hypoxia compared to normoxia (Figure 3A), indicat- ing mislocalization of HSP70 in SCD cells under hypoxia. Importantly, these lower amounts of nuclear HSP70 were associated with lower amounts of GATA-1 (Figure 3A), suggesting that cell death under these conditions was likely due to altered protection of GATA-1 by HSP70.
The cells used in these assays were pools of live and apoptotic, F-cells and non-F-cells. In order to better address HSP70 localization in each of these subpopulations, we performed imaging flow cytometry experiments with cells stained for multiple markers. Only Hoechst-positive cells with high GPA expression were taken into consideration to exclude both reticulocytes and proerythroblasts. HSP70 fluorescence intensity was measured in the cytoplasm and the nucleus of live (FVS-) and dead (FVS+) cells at D7. HSP70 nuclear intensity was lower in dead than FVS- cells, for both control and SCD cells under hypoxia (Figure 3B). Moreover, SCD FVS+ cells showed higher HSP70 cytoplas- mic levels than FVS- cells, while no difference was detected between both subpopulations for control cells (Figure 3B). These results suggest that hypoxia-induced cell death in the SCD context is likely due to HSP70 entrapment in the cytoplasm. Next, we measured HSP70 intensity in F-cells and non-F-cells. Under hypoxia at D7, HSP70 nuclear intensity was higher in SCD F-cells than non-F cells, while there was no difference between both cell types in control (Figure 3C; Online Supplementary Figure S2A). We then measured the nucleus/cytoplasm (N/C) ratio of HSP70 intensity in polychromatic and orthochromatic cells. The
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