Development of human B cells from HSPCs transduced with b12-IgA gene in vitro. (A) Schematic representation of the lentiviral constructs. Shown are pHAGE6-EEK-b12a-ZsGr and control vector pHAGE6-EEK-luc-ZsGr. The HIV-1 5′ LTR, b12 mAb heavy chain variable region (b12V_H), human IgA2 heavy chain constant region α1, α2, α3 (C_Hα1, C_Hα2, C_Hα3), picornavirus-derived self-cleaving 2A peptide sequences (F2A and T2A), b12 κ light chain variable and constant region (b12V_L-C_Lκ), human immunoglobulin J chain (J), the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and 3′ LTR are indicated. For control vectors, the b12-IgA sequence was replaced by the luciferase gene in the same lentiviral vector. The human κ light chain promoter preceded by enhancers and matrix association regions is denoted as iEκ. The vector, pHAGE6-EEK-b12a-ZsGr contains a b12-IgA tri-cistronic cassette and a reporter gene ZsGreen (ZsGr) linked via IRES for simultaneous expression. (B) Secretion of human monoclonal antibody b12-IgA in human cells. Secreted human IgA in culture supernatant from 293T cells, which were transfected with either the lentiviral vector carrying b12-IgA or control vector (FUGW), were measured by human IgA ELISA at multiple time points. (C) CD10⁺CD19⁺ pro–B-cell development from HSPCs. Lentiviral transduction and in vitro human B lymphopoiesis culture were performed as previously described.²²  CD34⁺ HSPCs transduced with either pHAGE6-EEK-luc-ZsGr (luc) or pHAGE6-EEK-b12a-ZsGr (b12-IgA) lentiviral constructs or left uninfected (none) cells were primed with IL-3, Flt3 ligand, thrombopoietin, SCF, and G-CSF for 5 days and then cocultured with MS5 stromal cells for indicated time points (C-E). Contour plots show CD10⁻CD19⁻ cell population at 14 days (left, D14) and emerging CD10⁺CD19⁺ pro–B-cell population after 21 days (right, D21) of culture. (D) Increased surface IgM expression. Data shown are gated on CD19⁺ cells from day 14 (D14) or day 32 (D32) culture. (E) Transgene expression during B-cell lymphopoiesis. Transferred luciferase or b12-IgA gene expression was assessed with the fluorescence of coexpressed ZsGreen protein using flow cytometry at indicated time points; day 14 (D14), day 21 (D21), day 32 (D32), and day 49 (D49) of HSPCs culture in the presence of MS5 cells. (F) Development to B-cell blast from mature B cells by stimulation. After being cocultured with MS5 cells for 8 to 9 weeks, CD19⁺ B cells were isolated and stimulated as previously described.²²  The 2 left panels of the contour plot show levels of IgM on CD19⁺ B cells before and after stimulation. The 2 right panels depict forward versus side scatter contour plots before and after stimulation. Data shown are gated on CD19⁺ cells.