Figure 7.

Erythroid differentiation and RBC parameters were not impaired in cells derived from LV AS3m.C-transduced, edited SCD HSPCs

(A–E) Flow cytometry analysis of the enucleation rate and of the early (CD71, CD36, and CD49d) and late (CD235A and Band3) erythroid markers at day 13, 16, and 19 or 20 of erythroid differentiation of untreated HD (n = 1) and SCD (n = 3) cells (Ctr UT), gR-C-treated HD (n = 1) and SCD (n = 1) cells (gR-C), and LV AS3m.C-treated SCD HSPCs that were mock transfected or transfected with Cas9-GFP protein (n = 3). VCN and indel values are reported below the graph as mean ± SEM. (A–D) Data are expressed as mean ± SEM. (A) Enucleation rate measured using DRAQ5 nuclear staining. (B–D) Proportion of CD71⁺, CD36⁺, and CD235A⁺ cells during erythroid differentiation. (E) Expression of CD49d and Band3 during the erythroid differentiation. During terminal erythroid differentiation, cells lose CD49d expression. (F–I) RBC parameters extracted using the BIO-Data software. RBCs were obtained after 19 days of differentiation from SCD HSPCs transduced with LV AS3m.C and either mock or Cas9 transfected. As controls, we used RBCs obtained from SCD/HD HSPCs transfected with RNPs containing gR-C For each population; data were normalized to the total number of RBCs and are reported as overlaid histograms. Darker colors represent controls and lighter colors edited samples. From top to bottom: control HD RBCs compared with HD RBCs derived from HSPCs treated with gR-C; control SCD RBCs compared with SCD RBCs derived from HSPCs treated with gR-C; control SCD RBCs derived from HSPCs transduced with LV AS3m.C and mock transfected compared to RBCs derived from HSPCs transduced with LV AS3m.C and transfected with Cas9 protein. (F) Dry mass (pg). (G) Surface (μm²). (H) Perimeter (μm). (I) Ellipticity.