Non-Thermal Atmospheric Pressure Plasma Efficiently Promotes the Proliferation of Adipose Tissue-Derived Stem Cells by Activating NO-Response Pathways

Abstract

Non-thermal atmospheric pressure plasma (NTAPP) is defined as a partially ionized gas with electrically charged particles at atmospheric pressure. Our study showed that exposure to NTAPP generated in a helium-based dielectric barrier discharge (DBD) device increased the proliferation of adipose tissue-derived stem cells (ASCs) by 1.57-fold on an average, compared with untreated cells at 72 h after initial NTAPP exposure. NTAPP-exposed ASCs maintained their stemness, capability to differentiate into adipocytes but did not show cellular senescence. Therefore, we suggested that NTAPP can be used to increase the proliferation of ASCs without affecting their stem cell properties. When ASCs were exposed to NTAPP in the presence of a nitric oxide (NO) scavenger, the proliferation-enhancing effect of NTAPP was not obvious. Meanwhile, the proliferation of NTAPP-exposed ASCs was not much changed in the presence of scavengers for reactive oxygen species (ROS). Also, Akt, ERK1/2, and NF-κB were activated in ASCs after NTAPP exposure. These results demonstrated that NO rather than ROS is responsible for the enhanced proliferation of ASCs following NTAPP exposure. Taken together, this study suggests that NTAPP would be an efficient tool for use in the medical application of ASCs both in vitro and in vivo.

Figure 1. Helium-based dielectric barrier discharge type device used for non-thermal atmospheric pressure plasma (NTAPP) generation.

(A) Schematic description of the NTAPP-generating device used in this study (photographed by J. Park). (B) Inner components of the device that generate NTAPP (drawn by H. Lee).

Figure 2. Non-thermal atmospheric pressure plasma (NTAPP) accelerates the proliferation of adipose tissue-derived stem cells (ASCs) but induces apoptosis in HeLa cells.

(A–D) ASCs (A,B,E) and HeLa cells (C,D,E) were exposed to NTAPP for a total of 10 times, for 50 sec every h, and were further incubated for 72 h from the initial exposure. Cell viability was evaluated at each indicated incubation time-point. (A,C) Cell viability was measured by MTT assay, and all results were represented as mean ± SD. N = 4. P < 0.05 (*) indicates significant differences compared with the control. (B,D) Western blot analysis of ASCs (B) and HeLa cells (D) were performed to assess the expression of γ-H2AX and PARP following NTAPP exposure. Actin was used as the loading control. Cells exposed to UV were used as the positive control for DNA damage and cell death. (E) The mitochondrial membrane potential was monitored in NTAPP-treated HeLa and ASCs. Cells were stained with 2 μM JC-1 dye for 30 min at 37 °C, and both red and green fluorescence emissions were analyzed by flow cytometry. Cells treated with 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 4 h prior to JC-1 staining were used as a positive control for mitochondrial membrane disruption.

Figure 3. Non-thermal atmospheric pressure plasma (NTAPP)-exposed adipose tissue-derived stem cells (ASCs) maintain their stem cell properties.

(A) Reverse transcription-polymerase chain reaction (RT-PCR) was performed by using RNA extracted from ASCs. CD44 and CD105 were used as positive markers and CD45 was used as a negative marker for the analysis of ASCs. FABP4 was used as a differentiation marker of ASCs. (B) Expression of the markers of ASCs was analyzed by RT-PCR at 72 h after the first NTAPP exposure and compared to that in unexposed control cells. (C) SA-βGal assay was performed to evaluate senescence in ASCs at 72 h after exposure to NTAPP for a total of 10 times. ASCs treated with 100 μM H₂O₂ were used as the positive control. Scale bar, 100 μm. Senescent cells were counted, and the values were expressed as percentages. (D) Differentiation of NTAPP-exposed ASCs into adipocytes was induced by incubation for 28 days in adipogenic differentiation medium, and the differentiation of the ASCs was detected using Oil-red O staining. Scale bar, 50 μm. (E) ASCs were evaluated for the expression of an adipocyte marker, FABP4, by using RT-PCR. Random primers were used as negative controls for RT-PCRs.

Figure 4. NO plays a key role in non-thermal atmospheric pressure plasma (NTAPP)-induced proliferation of adipose tissue-derived stem cells.

(A) ASCs pretreated with culture medium alone (as the negative control) or 30 μM carboxy-PTIO were exposed to NTAPP for a total of 10 times. Cells were totally incubated for 72 h after the initial NTAPP exposure. Cell viability was measured by MTT assays, and the results were represented as mean ± SEM; N = 4. P < 0.05 (*) indicates significant differences among samples. (B) Different concentrations of DETA-NONOate (untreated, 10, 20, and 30 μM) were added to the medium containing ASCs. carboxy-PTIO (30 μM) was added to medium containing 30 μM DETA. Cell viability was evaluated by MTT assay, and the results were represented as mean ± SEM. N = 4. P < 0.05 (*) indicates significant differences compared with each sample. (C,D) The expression of (C) Akt, phospho-Akt, ERK1/2, and phospho-ERK1/2, and (D) NF-κB and phospho-NF-κB in NTAPP-exposed ASCs was analyzed by western blot at 0, 9, and 72 h from the initial exposure. Actin was used as the loading control.

Figure 5. Reactive oxygen species (ROS) are not responsible for non-thermal atmospheric pressure plasma (NTAPP)-induced proliferation of ASCs.

(A,B) ASCs were pretreated with culture medium alone, 100 μM butylated hydroxyl anisole (BHA; A), or 5 mM N-acetylcysteine (NAC; B) and exposed to NTAPP for a total of 10 times. Cells were further incubated for 72 h from the initial NTAPP exposure. The percentage of cell viability was measured by MTT assay, and the results were represented as mean ± SD. N = 4. P < 0.05 (*) indicate differences among each sample. (C) Untreated ASCs and those pretreated with 100 μM BHA or 5 mM NAC were exposed to NTAPP for a total of 10 times, and their intracellular ROS levels were monitored at 9 h from the initial exposure. Cells treated with 100 μM tert-butyl hydroperoxide (TBHP) were used as the positive control for ROS generation. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm.