*4.2. ADSCs and Culture*

ADSCs were isolated from lipoaspirates as described previously [36]. Briefly, lipoaspirates, obtained from the donor sites, such as lower abdomen, thighs, buttocks, and shoulders, were processed using a CelutionTM system (Cytori Therapeutics, Inc, San Diego, CA, USA) based upon the method reported previously [36]. Mixed lipoaspirates isolated from different sites in one patient were used. Approximately 100 μL of processed lipoaspirates were plated onto type I-collagen-coated culture flasks. Since a medium containing 10% FBS promoted cellular senescence of ADSCs, the lipoaspirates were cultured in a serum-free medium originally developed for primate embryonic stem cells (Primate ES medium, ReproCELL, Tokyo, Japan). Clonally expanded cells were collected and stored in liquid nitrogen as the primary ADSCs (passage 0). Exponentially growing ADSCs were maintained by subculturing when they reached to 80% confluence. ADSCs were treated with trypsin/EDTA solution, trypsin-neutralizing solution, and collected by centrifugation for 5 min at 1200 rpm. The cells were resuspended in a fresh medium and 3 × 10<sup>5</sup> cells were replated onto the T25 flasks (25 cm2), while rest of the cells were stored in liquid nitrogen at each passage.

ADSCs were obtained from three HIV-infected patients and four HIV-unrelated patients (Supplementary Table S1). The HIV-infected patient #1 and #3 are the same subject, and received treatments with indinavir, lamivudine (3TC), d4T, and lately, with atazanavir and Euzicom (3TC and abacavir combination) regimens for 15 years. The HIV-infected patient #2 received treatments with AZT, didanosine, 3TC, d4T, and lately, with atazanavir and Tenofovir/emtricitabine for 10 years. The patient #4 received treatments with AZT for 2 years, didaosine for 4 years, lamivudine (3TC) for 12 years, Nelfinavir for 3 years, d4T for 3 years, and Euzicom (3TC and abacavir combination) regimens for 10 years. All HIV-infected patients were diagnosed as HIV-negative by PCR-based assay at the time of isolating ADSCs. The study was approved by the Nagasaki University Hospital ethical committee (Internal Review Board approval No. 08070296, July 22, 2008, Nagasaki University Hospital), and all patients gave written informed consent.
