Figure 5.

Reduced apoptosis and ROS levels by hypoxia during DXI-mediated expansion of human LTR-HSCs

(A) Enrichment plots showing the relative expression of genes associated with the apoptosis pathway for all six cell clusters identified by scRNA-seq of CD34⁺ cells treated in vessels coated with optimized densities of DXI under normoxic or hypoxic conditions. NES, normalized enrichment score; FDR, false discovery rate.

(B) Representative flow-cytometry plots obtained by dual annexin V/7AAD immunostaining of human CD34⁺ cells treated as described in (A).

(C) Summary of percentages of annexin V⁺ cells in CD34⁺ cell subpopulations comprising hematopoietic CD34⁺CD38⁺ progenitors (PROGs) or phenotypically defined CD34⁺CD38⁻CD90⁺CD45RA⁻ LTR-HSC populations (pHSCs) (n = 3 independent donors). Addition of N-acetyl-L-cysteine (NAC) ROS scavengers in culture rescued pHSC apoptotic death under normoxic conditions.

(D) Quantification of ROS levels in human PROGs and pHSCs after culture for 24 h with optimized densities of DXI under normoxic or hypoxic conditions using a CellROX green assay. Representative flow-cytometry plots are shown. Cells treated with menadione were included as a positive control.

(E) Summary of relative mean fluorescence intensity (MFI) of ROS (n = 3 independent donors).

Data are displayed as mean ± SEM. Ordinary one-way ANOVA was used. ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001; ns, not significant.