Figure 4.

The transient overexpression of CXCR4 increases chimerism in the humanized context, following M-HSCT

(A and B) Fold change of CXCR4^high cells (A) and CXCR4^high MFI (B) in bulk CD34⁺ cells, electroporated with CXCR4 mRNA (pVAX) or optimized CXCR4 mRNA (pVAXi), normalized to electroporated-only (EO) cells.

(C and D) Fold change of CXCR4^high cells (C) and CXCR4^high MFI (D) in CD34⁺CD133⁺CD90⁺ HSPCs, electroporated with CXCR4 mRNA or optimized CXCR4 mRNA, normalized to EO cells.

(E and F) The percentage of migrating bulk CD34⁺ cells (E) and HSPCs (F), electroporated with GFP, CXCR4, or optimized CXCR4 mRNA.

(G and H) Fold change of CXCR4^high cells (G) and CXCR4^high MFI (H) in HSPCs, electroporated with CXCR4 isoform1 or isoform2 mRNA, normalized to EO cells.

(I) The percentage of migrating HSPCs, electroporated with GFP, CXCR4 isoform1, or isoform2 mRNA.

(J) Long-term follow-up of human CD45⁺ in the PB of NSG mice, following the transplantation of CD34⁺ cells electroporated with GFP or CXCR4 mRNA.

(K) The reconstitution of myeloid and lymphoid lineages over time within total CD45⁺ cells in the PB of NSG mice, transplanted with CD34⁺ cells electroporated with GFP or CXCR4 mRNA.

(L) Myeloid and lymphoid cell composition of human CD45⁺ cells in the BM of NSG mice, transplanted with CD34⁺ cells electroporated with GFP or CXCR4 mRNA.

(M) CD34⁺ subpopulation in the BM of NSG mice, transplanted with CD34⁺ cells electroporated with GFP or CXCR4 mRNA.

(N) Schematic illustration of competitive transplantation, after G7AB mobilization, between human resident cells and newly transplanted CD34⁺ G-CSF mPB GFP-transduced and electroporated cells in NSGW41 mice.

(O and P) Long-term follow-up of human CD45⁺ (O) and CD45⁺/GFP⁺ (P) cells in the PB after M-HSCT with CD34⁺ cells transiently overexpressing GFP or CXCR4 mRNA, in NSGW41 mice.

(Q) Counts of GFP⁺ cells per mL in the PB after M-HSCT with CD34⁺ cells transiently overexpressing GFP or CXCR4 mRNA, in NSGW41 mice.

(R) Myeloid and lymphoid lineage composition of human CD45⁺ (left panel) and CD45⁺/GFP⁺ cells (right panel) in the PB after M-HSCT, with CD34⁺ cells transiently overexpressing GFP or CXCR4, in NSGW41 mice.

(S) Chimerism of GFP⁺ cells was observed within CD19⁺, CD13⁺, and CD3⁺ cells in the PB after M-HSCT, with CD34⁺ cells transiently overexpressing GFP or CXCR4, in NSGW41 mice.

(T) The percentage of human CD45⁺ cells in the PB of NSG mice at 16 weeks, following the secondary transplant of cells collected from groups described in Figure 4O.

(U) Percentage of human CD45⁺/GFP⁺ cells in PB of NSG mice at 16 weeks, following the secondary transplant of cells collected from groups described in Figure 4O.

The results are mean ± SEM, with n ≧ 10 biological replicates for data collected in vivo, n ≧ 4 biological replicates for secondary transplant experiments, and n ≧ 5 for data collected in vitro (3 biological replicates and 2 technical replicates). In all the analyses, p less than 0.05 were considered significant (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. “ns” means non-significance).