Proliferation of human adipose tissue-derived stem cells stimulated by oil rich in thymol of Lippia origanoides

Purpose: To evaluate the effects of this thymol-rich oil in the proliferation of human adipose tissue-derived stem cells. Methods: Stem cells were isolated from human adipose tissue by liposuction. After the first passage, cells were cultivated in triplicate for three days in control medium and medium supplemented with three oil samples (1.0 μg/mL, 5.0 μg/mL, and 25.0 μg/mL). Cells were analyzed by the MTT assay at passage 1 (P1), and cell proliferation of control and 1 μg/mL groups was determined with a hemocytometer at P2 and P3. Results: Viability of the essential oil-treated cells was significantly higher than the control group at P1 (p = 0.0008). The treatment with the oil, at a concentration of 1 μg/mL, led to increases of 24.8% at P1 and 43.0% at P3 in the rate of cell proliferation compared with control cells. Conclusion: Supplementing culture medium with essential oil of Lippia origanoides increased cell proliferation, especially at later passages.

activity of this essential oil is attributed to the presence of phenol compounds such as thymol, carvacrol, and derivatives 13 .
Several biological activities have been reported for thymol.This phenolic compound plays an important role in the scavenging of free radicals such as DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid).In addition, at lower concentrations, it showed protective effects to cells against damage induced by the H 2 O 2 radicals 14,15 .Thus, the aim of this study was to assess the proliferation of human ADSCs in culture medium treated with natural antioxidants present in the L. origanoides Kunth essential oil.Introduction The isolation of mesenchymal stem cell (MSC) from human adipose tissue obtained from liposuction was described in 2001 1 .Liposuction is one of most common esthetic surgical procedures worldwide, and it generates an average of 100-3000 mL of adipose tissue that is routinely discarded, along with its incorporated stem cells 2 .In addition to being easily obtained, adipose-derived stem cells (ADSCs) also have significant therapeutic use 3 , as an acute myocardial infarction 4 and bone regeneration 5 .However, according to cellular therapy protocols used in clinical trials, more than one million stem cells are necessary per treatment, necessitating in vitro expansion 6 .
Repeated passaging of cells in vitro incurs the risk of replicative senescence, which primarily occurs due to oxidation caused by free radicals and can be identified by telomere shortening and fibroblast flattening with a concurrent increase in nuclear/nucleolar size that results in stalled cell proliferation 7 .These phenomena limit in vitro cell expansion and might hinder their therapeutic applications 8 .
Antioxidants are compounds that directly or indirectly inhibit production of reactive oxygen species 9 (ROS).In recent years, there is an increasing interest in finding antioxidant phytochemicals, because they can inhibit the propagation of free radical reactions 10 .Many essential oils have been reported as natural alternative for controlling of oxidative stress such as the essentials oils from Lippia species 11 .
Lippia origanoides Kunth (Verbenaceae) is an aromatic plant, popularly known in North Brazil as "erva-do-marajó" and is widely found throughout the Brazilian Amazon.The leaves are used as a condiment, as a substitute for oregano and its tea for treating gastrointestinal and respiratory diseases 12 .The antioxidant

Isolation and cultivation of MSCs from human adipose tissue
The lipoaspirate tissue (2mL) was washed with 2 mL of phosphate-buffered saline (PBS; Sigma Aldrich, St. Louis, Missouri, USA) and incubated with 200 U/mL collagenase II for 60 min at 37ºC.After incubation, the collagenase was neutralized by adding complete cultivation medium (Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich), 3.7 g/L NaHCO 3 (Sigma Aldrich), and 5 µg/ mL of gentamicin (Sigma Aldrich).Following centrifugation, the pellet was resuspended twice with DMEM and plated.After 24 hours, any cells still in suspension were removed and the cultivation medium was renewed.The remaining adherent cells, representing MSCs, were kept at 37°C in 5% CO 2 with high humidity, and the cultivation medium was changed every three days until the third passage.

Cellular and histological characterization of cell differentiation
Histology with tissue-specific dyes was used to characterize the ability of MSCs to differentiate into bone tissue, adipose tissue, and cartilage tissue.
After cultivation, the differentiation medium was removed, and the cells were washed once with PBS.Next, cell monolayers were fixed with paraformaldehyde (Electron Microscopy Sciences) for 30 minutes and then washed twice with PBS.To detect osteoblasts, cells were stained with Alizarin Red S (Life Technologies, Carlsbad, California, USA) for 3 minutes.To detect adipocytes, cells were stained with Oil Red O (Life Technologies) for 30 minutes.To detect chondroblasts, cells were stained with Alcian Blue (Life Technologies) for 30 minutes.After staining, cells were washed three times with distilled water and analyzed by an optical microscope (911873 AXIOLAB, Ziss, Germany) -osteoblasts and chondroblasts -and by an inverted microscope (TE-DH100W, Nikon, Japan) -adipocytes.

Plant collection and extraction and analysis of essential oil
The aerial parts of L. origanoides were collected in the National Forest of Carajás (Parauapebas-PA, Brazil), which occurs in endemic form in that area.The botanical material was identified by comparison with an authentic voucher (MG 133.921) existing in the Herbarium of Emílio Goeldi Museum (Belem-PA, Brazil).The essential oil was hydrodistilled using a Clevenger-type apparatus (Hanil Lab Tech Ltd, Incheon, Korea), dried with sodium sulfate and stored in a refrigerator at 5ºC.The oil was analyzed by gas chromatography coupled with mass spectrometry (GC-MS) and its volatile constituents identified by comparison with authentic standards existing in the Adams library 16 .The GC-MS analysis was carried on a Thermo Focus DSQ II, under the following conditions: DB-5ms (30 m x 0.25 mm; 0.25 mm film thickness) fused-silica capillary column; programmed temperature, 60-240°C (3ºC/min); injector temperature, 250 ºC; carrier gas, helium, adjusted to a linear velocity of 32 cm/s (measured at 100°C); injection type, split (1 μL), from 1:1000 hexane solution; split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 ml/min; EIMS, electron energy, 70 eV; temperature of the ion source and connection parts, 200°C.The quantitative data regarding the volatile constituents were obtained by peak area normalization using a FOCUS GC/FID operated under similar conditions of the GC-MS, except the carrier gas, which was nitrogen.The retention index was calculated for all the volatiles constituents using a homologous series of n-alkanes (C8-C32, Sigma-Aldrich) 17 .
For cell treatments, the essential oil was initially solubilized in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at concentration 10.0 μg/mL.This stock solution was diluted to concentrations of 25.0 μg/mL, 5.0 μg/mL and 1.0 μg/mL in DMEM.

MTT assay of cellular growth rate
The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed during the first cellular passage (P1) to determine cell viability in the treatment and control groups.Cells were plated at a concentration of 3x10 3 cells/well in a 96well plate (TPP, Trasadingen, Schaffhausen, Switzerland) and incubated with different essential oil concentrations (1.0 μg/mL, 5.0 μg/ mL, and 25.0 μg/mL) in triplicate.After the third day of cell cultivation, the culture medium was replaced with 100 μL of 5 mg/mL MTT solution in 900 μL in DMEM (without FBS).After 3 hours incubation at 37ºC, the MTT solution was removed and 100 μL of DMSO was added to each well and the plate was shaken for 10 minutes.The percentage of cellular growth rate (GR %) was based on absorbance at 490 nm (A 490 ) measured in spectrophotometer UV/ VIS and calculated according to the following equation.

Cell counting
At P2 and P3, 4x10 4 cells/mL were plated in two 35-mm plates, one with only DMEM and the other with 1.0 μg/mL essential oil (the concentration with the highest rate of cell proliferation).After 72 hours, cells were resuspended with trypsin and 1 µl of cell were placed and counted in a Neubauer Chamber.All the cells within each medium square were counted using x4 objective and multiplied for a correction factor of 10 4 (cell/ml).

Statistical analysis
Statistical analyses were performed using the GraphPad Prism version 5.0 (GraphPad Software, California, USA).A oneway ANOVA test was performed to assess the rate of cell proliferation using values obtained from the MTT assay followed by Tukey's post hoc test.Values of p ≤ 0.05 were considered significant.

■ Results
Characterization of tissues differentiated from ASCs are shown in Figure 1. Figure 1A-C exhibit the control of each histological characterization.Numerous fat drops formed in the adipocytic lineage (Figure 1D).We observed glycosaminoglycan accumulation in the chondrogenic lineage, as revealed by Alcian Blue (Figure 1E).The osteogenic lineage stained with Alizarin Red S, indicating an accumulation of extracellular calcium (Figure 1F).The proliferation rates cells treated with 1.0 μg/mL, 5.0 μg/mL, and 25.0 μg/ mL of essential oil were 24.8%, 14.7%, and 16.3% greater than control cells, respectively (Figure 3).At P2, control cells and cells treated with 1 μg/mL of essential oil were plated at a concentration of 4x10 4 cells/mL.After 72 h of cultivation and subsequent trypsin treatment, both groups had a concentration of 18x10 4 cells/mL (Figure 4).At P3, the cells were again plated at 4x10 4 cells/mL However, in this case the concentration of the essential oil-treated cells was approximately 43% greater than that of the control cells after cultivation and enzymatic digestion (20x10 4 cells/mL vs. 14x10 4 cells/mL) (Figure 5).The dried aerial parts of L. origanoides provide oil yield of 3.3%.Individual components were identified by comparison of both mass spectrum and GC retention data with authentic compounds, which were previously analyzed and stored in the data system.Seventeen volatile components were identified in the oil, comprising nearly 100% of its composition (Table 1).Thymol (88.2%) was the main constituent, followed by p-cymene (6.4%), its biogenetic precursor.

■ Discussion
Essential oils are one among the most valuable plant products used in medicine and complementary treatment strategies.However, this is the first study, which reports the use of essential oil from L. origanoides as a supplement in human stem cell culture.
Our results indicated that the cultivation medium supplemented with essential oil showed a proliferative effect, with the most significant result found at P3.The principal constituent of the oil of L. origanoides using in this study was thymol (88.2%),It is classified chemically as monoterpene.This compound exhibit different mechanisms of action because the presence of a phenolic ring in its structure enhance the antioxidant activity due to its ability for scavenging free radicals, donation of hydrogen atoms or electron, or chelation of metal cations 16 .We have hypothesized that the cell proliferation was due to Thymol's cytoprotective effect, as another monoterpene (1,8-Cineole, Camphor, α-Pinene and β-pinene) presents in oil of Salvia lavandulifolia Vahl showed a protective effect in cultured human astrocytoma too 18 .
Tavakolinejad et al. 19 cultivated human adipose tissue-derived stem cells in medium containing platelet-rich plasma (PRP) instead of FBS or L. origanoides.Cellular growth rates in the work of them (40%) were similar to this study at P3 (40 vs. 43%).However, even automated methods for obtaining PRP can result in variable product quality.On the other hand, essential oil from L. origanoides function as antioxidants, allowing extended cell cultivation in vitro without probable cell damage.
Cells become senescent with increasing passage number, thus reducing their growth 8 .Previous studies have found stem cells derived from senescent human adipose tissue at P2 based on a β-galactosidase assay.By P5, almost 20% of cells were derived from senescent tissue 20 .In our study, the antioxidant function of essential oil from Lippia origanoides may have inhibited the oxidation of free radicals produced in vitro, enabling cell growth even at P3. Future studies should be done to verify the maximum passage number that benefits from essential oil-treatment, as well as examine other basic cell characteristics such as cell senescence and telomere shortening.

■ Conclusions
Supplementing culture medium with essential oil of Lippia origanoides increased the proliferation of stem cells derived from human adipose tissue in vitro, especially at later cell passages.This essential oil has the potential to become a cheap and efficient mean for optimally expanding adult stem cells in vitro for cell therapy.

Figure 1 -
Figure 1 -(A-C) ASCs cultured control.(D) Adipogenic differentiation of the ASCs.Fat vesicles detected by staining with Oil Red O after 14 days of induction.(E) Chondrogenic differentiation of ASCs stained with Alcian Blue after 21 days of induction.(F) Osteogenic differentiation of ASCs stained with Alizarin Red S after 21 days of induction.The proliferation results obtained by the MTT assay are shown in Figure2.The absorbance values passed the Normality Test (p = 0.722) and the Equality of Variance Test (p = 0.24) in all treatments.Compared with the control, cell proliferation was higher in the three experimental groups treated with essential oil from L. origanoides (p = 0.0008).Cell proliferation did not significantly differ between the three experimental groups.

Figure 2 -
Figure 2 -Averages and standard deviation for absorbance related to cell proliferation (MTT assay) of stem cells derived from human adipose tissue cultivated with essential oil of Lippia origanoides in the 1 st passage.*indicates a significant difference (p < 0.05) in relation to the control group.

Figure 3 -Figure 4 -
Figure 3 -Growth rate of stem cells derived from human adipose tissue of each group treated (1.5 and 25 μg/mL) in relation to the control in the 1 st passage.

Figure 5 -
Figure 5 -Effect of the essential oil of Lippia origanoides (1 μg/mL) on multiplication of stem cells derived from human adipose tissue in the 3 rd passage after 3 days of cultivation.

Table 1 -
Chemical composition of the essential oil of Lippia origanoides.
a Retention index calculated using the homologous series of n-alcanes; b Retention index in the literature (ADAMS, 2007); c Percentage of compounds obtained by integrating the area in the flame ionization detector.