**2. Results**

#### *2.1. Isolation and Growth Properties of ADSCs*

We have obtained lipoaspirates from three HIV-infected patients who are hemophilic and infected with HIV virus by unheated blood products, diagnosed as lipodystrophy after HAART (Table 1). Lipoaspirates from HIV-infected patients were obtained from the sites such as abdomen, thighs, and shoulders, where lipoatrophy was less severe. Lipoaspirates were also obtained from four patients, who are not related to HIV-infection (Table 1). Lipoaspirates obtained from one patient were mixed and used for ADSCs isolation.


**Table 1.** Origin of adipose tissue-derived cells.

Approximately 100 μL of processed lipoaspirates were plated onto type-I collagen-coated flasks and maintained in a serum-free medium. The cells, which clonally expanded were collected and stored as the primary ADSCs. We noticed that the number of colonies formed by the processed lipoaspirates obtained from the HIV-infected patients were about one-tenth of those observed in the lipoaspirates obtained from HIV-uninfected patients. ADSCs showed mesenchymal stem cell-like morphology (Figure 1).

ADSCs isolated from processed lipoaspirates were highly proliferative in serum-free medium, and a plenty of mitotic figures were discerned. We observed no detectable di fference in cell morphologies and growth patterns between the ADSCs obtained from the HIV-infected patients and HIV-unrelated patients. We also extensively compared the growth kinetics of ADSCs by cell growth assay, and there was no detectable di fference in the growth kinetics of both ADSCs (Table 2).

**Figure 1.** Morphology of adipose-derived stem/progenitor cells (ADSCs) cultures at passage 1 obtained from human immunodeficiency virus (HIV)-infected and HIV-unrelated patients. Exponentially growing ADSCs were cultured in serum free-medium in type I collage-coated flasks. Magnification ×100; the scale bar indicates 100 μm.


**Table 2.** Biological properties of ADSCs.

#### *2.2. Adipogenic Di*ff*erentiation of ADSCs*

Adipogenic differentiation was examined in confluent ADSCs by incubating them in a differentiation medium for 10 days. Multiple tiny lipid droplets became visible by day 5, and they gradually matured during 10 days' incubation. To quantify adipogenic differentiation, lipid droplets were stained with BODIPY 493/503, a highly sensitive lipophilic fluorescent dye. As shown in Figure 2, multiple lipid droplets were identified in the cytoplasm, whose morphology was similar to that observed in subcutaneous adipocytes. The frequency of differentiation-positive cells were approximately 97% or more in ADSCs from HIV-unrelated patients, and the same differentiation potential was confirmed in ADSCs derived from HIV-infected patients (Table 3). Average fluorescence per cell is compared in Table 3, and we detected significant difference between ADSCs derived from HIV-infected and HIV-unrelated patients.

Adipogenic differentiation was also confirmed by analyzing the expression of proteins associated with adipocytes. As shown in Figure 3, we identified significant induction of FABP4, adiponectin, and PPARγ in every ADSC differentiated into adipocytes, irrespective of HIV-infection. Average levels of protein expression also show any notable difference.

**Figure 2.** Adipogenic differentiation of ADSCs in culture. ADSCs were stained with 10 μg/mL BODIPY 493/503 and counterstained with 0.1 μg/mL of DAPI. Magnification ×100; the scale bar indicates 100 μm.

**Table 3.** Adipogenic differentiation of ADSCs.
