Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Natural Killer Cells Promote Differentiation Of Human Adipose-Derived Stem Cells In Vitro
Kameron S. Rezzadeh, BA, Akishige Hokugo, DDS,PhD, Anahid Jewett, PhD, MPH, Anna Kozlowska, PhD, Andres Segovia, MD, Patricia Zuk, PhD, Zheyu Zhang, BA, Reza Jarrahy, MD.
UCLA Ronald Reagan Medical center, Los Angeles, CA, USA.

Purpose: While adipocyte homeostasis has been associated with inflammatory processes, particularly in the setting of obesity, the potential immunomodulatory role of adipose derived stem cells (ASC) has not been fully elucidated. Prior studies have addressed the isolation and characterization of ASCs. However, examinations of the physiologic relationship between ASCs and inflammatory cells are lacking. We sought to determine the impact of Natural Killer (NK) cells on the differentiation of ASCs and to qualify and compare the properties of NK-differentiated adipocytes to those differentiated using traditional techniques.
Methods: Human NK cells were purified from healthy donors’ peripheral blood mononuclear cells. NK cells were activated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 18-24 h before the supernatants were removed and used in differentiation experiments. The amounts of IFN-γ produced by activated NK cells were assessed with IFN-γ ELISA (BioLegend, CA, USA). Differentiation of ASCs was conducted with gradual daily addition of increasing amounts of NK cell supernatant (NKS). On average, a total of 1000pg of IFN-γ containing supernatants obtained from IL-2 + anti-CD16 mAb-treated NK cells was added for 7 days to induce differentiation and resistance of ASCs to NK cell-mediated cytotoxicity. ASCs were isolated from raw human lipoaspirates from patients ranging 55-68 years. Cells were plated onto conventional tissue culture plates in control medium and harvested between passages 2 and 3. Cells were then incubated with growth media (GM), GM+NKS, adipogenic media (AM), and AM+NKS. We performed flow cytometric analysis using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC1. Gene expression analysis was used to assess transcriptional activity consistent with adipocyte differentiation and adipogenesis. Oil Red O staining was performed as a functional assay of adipogenesis.
Results: ASCs reliably express markers of “stemness” including CD 44, CD34, and CD29 at high levels, whereas they consistently lack markers of differentiation (e.g., B7H1, CD36). By contrast, cells treated with adipogenic media reliably express markers of differentiation, and lack expression of stemness markers. ASCs treated with NKS express B7H1 and MHC1 highly and also lack expression of stemness markers. ASCs differentiated with NKS showed lower expression of adipogenesis-related genes than cells treated with AM. Oil Red O staining showed significantly lower lipid accumulation in samples treated with both NKS and AM when compared to those that were AM-treated.
Conclusion: We demonstrate that, through secreted factors, NK cells are capable of differentiating ASCs. Stem cells differentiated with NKS were of a different immunophenotype than those treated with AM. ASCs differentiated with NKS showed lower expression of genes related to adipogenesis and less lipid formation than cells differentiated using traditional AM. This work prepares us for upcoming studies of the in vivo effects of NKS on ASCs.


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