Generation of HIV-1 infected patient's gene-edited iPSC using feeder free culture conditions.

Objectives: The discovery of induced pluripotent stem cells (iPSC) has brought promise to regenerative medicine as it breaks the ethical barrier of using embryonic stem cells. Such cell culture-derived patient-specific autologous stem cells are needed for transplantation. Here we report deriving HIV-1-infected patients' iPSC lines under transgene-free methods and under feeder-free and xeno-free culture conditions to meet the requirement for clinical application. Methods and results: We have reprogrammed patients' peripheral blood mononuclear cells with EBNA1/OriP episomal vectors, or a defective and persistent Sendai virus vector (SeVdp) to ensure a nonintegrating iPSC generation. Both single picked and pooled iPSC lines demonstrated high pluripotency and were able to differentiate into various lineage cellsin vivo. The established cell lines could be modified by genetic editing using the TALENs or CRISPR/Cas 9 technology to have a bi-allelic CCR5 Delta 32 mutations seamlessly. All generated iPSC lines and modified cell lines had no evidence of HIV integration and maintained normal karyotype after expansion. Conclusions: This study provides a reproducible simple procedure for generating therapeutic grade iPSCs from HIV-infected patients and for engineering these cells to possess a naturally occurring genotype for resistance to HIV-1 infection when differentiated into immune cells.