Hematopoietic reconstitution of transduced human CD34+ HSPCs in immunodeficient NBSGW mice
Animals were euthanized 16 weeks after CD34+ HSPCs transplantation with cells derived from 3 different healthy donors, and bone marrow (BM) was collected and sorted into various subpopulations by flow cytometry. (A) Human chimerism in the BM of transplanted animals as determined by expression of hCD45. Data represent mean ± SD; each dot represents 1 animal. (B) Lineage distribution of human BM cells; data represent mean ± SD. (C) Vector copy number (VCN) determined in BM and multilineage cells; data represent mean ± SD; each dot represents 1 animal. Gene expression analysis by qRT-PCR in human cells from BM of engrafted mice. BCL11A and ZNF410 expression normalized to GAPDH in human B cells (D) or human erythroid cells (E) sorted from the BM of engrafted mice, and γ-globin expression (F) by qRT-PCR in human erythroid cells sorted from BM. Data represent mean ± SD. Each data point represents an individual mouse. ns, not significant; ∗p < 0.05; ∗∗∗p < 0.001. (G) γ-Globin shown in relation to VCN for SS BCL11A and DS BCL11A/ZNF410. (H) HbF by HPLC analysis of CD34+ cells isolated from the BM and subjected to in vitro erythroid differentiation. (I) Percentage of HbF+ cells by flow cytometry analyses in human CD235a+ erythroblasts isolated from BM. Data represent mean ± SD. Each data point represents an individual mouse, n = 10; ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.