Figure 1.

Generation of anti-HIV induced pluripotent stem cells (iPSCs) and end-stage macrophages. (a) iPSCs were generated by transducing cord blood (CB) CD34⁺ human hematopoietic stem cells (HSCs) with four lentiviral vectors expressing the pluripotency factors octamer-binding transcription factor 4, sex determining region Y-box 2, Kruppel-like factor 4, and cytoplasmic Myc either alone [wild-type (WT)] or with an enhanced green fluorescent protein control vector (EGFP) or a combination anti-HIV vector (anti-HIV). iPSCs were further cocultured on OP9 stromal cells where cystic bodies developed. CD133⁺ HSCs were isolated from the cystic bodies and grown in semisolid methylcellulose media to form myeloid colony-forming units (CFUs). The CFUs were further cultured in media specific for macrophage development. EGFP and anti-HIV iPSCs and their differentiated progeny were visualized by both phase and EGFP fluorescence. H9 human embryonic stem cells (hESCs) and their differentiated progeny were used as controls. (b) CB CD34⁺ cells were used as a positive control and cultured in semisolid methylcellulose media and macrophage-specific media to derive myeloid CFUs and end-stage macrophages. (c) Representative fluorescence-activated cell sorting (FACS) plots displaying the EGFP percent of the EGFP and anti-HIV iPSCs at passage 21. WT iPSCs (EGFP negative) are displayed as the unshaded histogram. FACS analysis was performed in triplicate. (d) Representative karyotyping analyses of the WT and anti-HIV iPSCs derived from CB CD34⁺ HSCs. Karyotyping was performed in duplicate.