Figure 3.

Prostaglandin E₂ Enables Efficient HSC Transduction with Shortened Ex Vivo Protocols

(A) CB CD34⁺ cells were transduced with LVs in the presence or absence of 10 μM PGE₂. VCN was measured in the progeny after >7 days culture (n = 5) or in the human CD45⁺ cell xenograft harvested from the PB (n = 2 experiments, blood from individual mice was pooled) or BM (2 experiments, n = 13 mice in the no PGE₂ group, n = 10 mice in PGE₂ group, statistics by Student's t test).

(B) BM CD34⁺ subpopulations (n = 3 donors) were transduced with GFP LV (MOI = 100) + 10 μM PGE₂ or DMSO, and VCN was measured in the myeloid progeny after 14 days of culture or on myelo-erythroid colonies obtained from clonogenic assay. Statistics by two-way ANOVA with Bonferroni's multiple comparison test.

(C) mPB CD34⁺ cells (n = 8 donors) were transduced with GFP LV (38 hr protocol) ± 10 μM PGE₂ added at the beginning of culture (t0) or during pre-stimulation, 120 min before LV addition (−120′). Transduction was evaluated after 14 days in culture. Left: VCN relative to the no PGE₂ group (statistics by Wilcoxon signed rank test). Right: transduction efficiency measured by flow cytometry (n = 6) (statistics by Student's t test).

(D) mPB CD34⁺ cells (n = 3 donors, with two replicates each) were transduced with GFP-expressing LV variants after adding PGE₂ or DMSO (−120′). IDLV, integrase-deficient, VSV-G pseudotyped LV; VSV LV, integration-competent, VSV-G-pseudotyped LV; P90A and N74D, VSV-G-pseudotyped LV with capsid mutants; BaEV-TR and RD114-TR, integration-competent LV pseudotyped with the BaEV-TR and RD114-TR envelopes, respectively. VCN were measured by specific droplet digital PCR assays. Statistics were performed by Mann-Whitney test.

(E) Quantification of late reverse-transcription DNA intermediates (late-RT) in mPB CD34⁺ cells (n = 6 donors), 6 hr after transduction (TD) in PGE₂ or DMSO.

(F–H) mPB CD34⁺ cells were transduced with gp91^phox-expressing LVs (MOI = 100) using a 24 or 46 hr ex vivo protocol ± 10 μM PGE₂, and xenotransplanted with the day 0 equivalent of 500,000 cells (n = 6–9 mice/group). (F) Human CD45⁺ cell engraftment in the PB. (G) Lineage composition of the human CD45⁺ cell graft in the BM at 20 weeks post-xenotransplantation. B cells, CD19⁺; myeloid cells: CD33⁺; T cells: CD3⁺; CD34⁺: HSPCs; Lin⁻CD34⁻: CD19⁻ CD33⁻CD3⁻CD34⁻ cells. (H) VCN in the human graft recovered from the BM at 20 weeks post-xenotransplantation. Statistics by one-way ANOVA with Bonferroni's multiple comparison test.

(I–K) mPB CD34⁺CD38⁻ cells were transduced with purified Globe LV (MOI = 100) using a 24 or 38 hr ex vivo protocol ±10 μM PGE₂, and xenotransplanted with the day 0 equivalent of 50,000 cells (experiment 1: 38 hr no PGE₂, n = 4; 38 hr + PGE₂, n = 4; 24 hr + PGE₂, n = 5; experiment 2: 38 hr no PGE₂, n = 4; 38 hr + PGE₂, n = 4; 24 hr no PGE₂, n = 5; 24 hr + PGE₂, n = 4). (I) Human CD45⁺ cell engraftment in the PB. (J) Lineage composition of the human CD45⁺ cell graft in the BM at 20 weeks post-xenotransplantation (representative mice from experiment 1). (K) VCN in the human graft recovered from the BM at 18 weeks (experiment 2) or 20 weeks (experiment 1) post-xenotransplantation. Statistics by one-way ANOVA with Bonferroni's multiple comparison test.

Error bars represent SEM. See also Figure S4.