Cell Journal (Yakhteh)
Volume:21 Issue: 3, Autumn 2019

Proteomics is a powerful approach to study the whole set of proteins expressed in an organism, organ, tissue or cell resulting in valuable information on physiological or pathological state of a biological system. High throughput proteomic data facilitated the understanding of various biological systems with respect to normal and pathological conditions particularly in the instances of human clinical manifestations. The important role of proteins as the functional gene products encouraged scientists to apply this technology to gain a better understanding of extremely complex biological systems. In last two decades, several proteomics teams have been gradually formed in Iran. In this review, we highlight the most important findings of proteomic research groups in Iran at various areas of stem cells, Y chromosome, infertility, infectious disease and biomarker discovery.

The Lung is one of the most radiosensitive organs of the body. The infiltration of macrophages and lymphocytes into the lung is mediated via the stimulation of T-helper 2 cytokines such as IL-4 and IL-13, which play a key role in the development of fibrosis. It is likely that these cytokines induce chronic oxidative damage and inflammation through the upregulation of Duox1, and Duox2, which can increase the risk of late effects of ionizing radiation (IR) such as fibrosis and carcinogenesis. In the present study, we aimed to evaluate the possible increase of IL-4 and IL-13 levels, as well as their downstream genes such as IL4ra1, IL13ra2, Duox1, and Duox2. In this experimental animal study, male rats were divided into 4 groups: i. Control, ii. Melatonin- treated, iii. Radiation, and iv. Melatonin (100 mg/kg) plus radiation. Rats were irradiated with 15 Gy 60Co gamma rays and then sacrificed after 67 days. The expressions of IL4ra1, IL13ra2, Duox1, and Duox2, as well as the levels of IL-4 and IL-13, were evaluated. The histopathological changes such as the infiltration of inflammatory cells, edema, and fibrosis were also examined. Moreover, the protective effect of melatonin on these parameters was also determined. Results showed a 1.5-fold increase in the level of IL-4, a 5-fold increase in the expression of IL4ra1, and a 3-fold increase in the expressions of Duox1, and Duox2. However, results showed no change for IL-13 and no detectable expression of IL13ra2. This was associated with increased infiltration of macrophages, lymphocytes, and mast cells. Melatonin treatment before irradiation completely reversed these changes. This study has shown the upregulation of IL-4-IL4ra1-Duox2 signaling pathway following lung irradiation. It is possible that melatonin protects against IR-induced lung injury via the downregulation of this pathway and attenuation of inflammatory cells infiltration.

Polycystic ovarian syndrome (PCOS) is characterized by hormonal imbalance, oxidative stress and chronic anovulation. The present study was designed to assess ameliorative effect of auto-locating platelet-rich plasma (PRP), as a novel method, for inhibiting PCOS-induced pathogenesis in experimentally-induced hyperandrogenic PCOS. In this experimental study, 30 immature (21 days old) female rats were assigned into five groups, including control (sampled after 30 days with no treatment), 15 and 30 days PCOS-sole-induced as well as 15 and 30 days PRP auto-located PCOS-induced groups. Serum levels of estrogen, progesterone, androstenedione, testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), ovarian total antioxidant capacity (TAC), malondialdehyde (MDA), glutathione peroxidase (GSH-px) and superoxide dismutase (SOD) were evaluated. Expression of estrogen receptor α (Erα), β (Erβ) and c-Myc were assessed. Finally, the numbers of intact follicles per ovary and mRNA damage ratio were analyzed. PRP groups significantly (P<0.05) decreased serum levels of FSH, LH, testosterone and androstenedione and remarkably (P<0.05) increased estrogen and progesterone syntheses versus PCOS-sole groups. The PRP auto-located animals exhibited increased TAC, GSH-px and SOD levels, while they showed diminished MDA content (P<0.05) versus PCOS-sole groups. The PRP auto-located groups exhibited an elevated expression of Erα and Erβ versus PCOS-sole groups. Moreover, PRP groups significantly (P<0.05) decreased c-Myc expression and mRNA damage compared to PCOS-sole groups, and remarkably improved follicular growth. PRP is able to regulate hormonal interaction, improve the ovarian antioxidant potential as well as folliculogenesis and its auto-location could be considered as a novel method to prevent/ameliorate PCOS-induced pathogenesis.

The presence of a sex related metabolic difference in glucose utilization and, on the other hand, different developmental kinetic rates in human preimplantation embryos, has been previously observed, hawever, the correlation between these two events is unknown. Oxidative stress (OS) induced by higher glucose consumption appears to be a possible cause for the delayed development rate in female embryos. We examined the correlation between glucose consumption and total antioxidant capacity (TAC) concentration in individual embryo culture media for both male and female embryos. In this cross-sectional study, we evaluated high quality embryos from 51 patients that underwent intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD) at the Royan Institute between December 2014 and September 2017. The embryos were individually cultured in G-2TMmedium droplets at days 3-5 or 48 hours post PGD. We analysed the spent culture media following embryo transfer for total antioxidant capacity (TAC) and any remaining glucose concentrations through fluorometric measurement by chemiluminecence system which indirectly was used for measurement of glucose consumed by embryos. The results showed that female embryos consumed more glucose which was associated with decreased TAC concentration in their culture medium compared to male embryos. The mean of glucose concentration consumed by the female embryos (30.7 ± 4.7 pmol/embryo/hour) was significantly higher than that of the male embryos (25.3 ± 3.3 pmol/embryo/hour) (P<0.001). There were significantly lower levels of TAC in the surrounding culture medium of female embryos (22.60 ± 0.19 nmol/µl) compared with male embryos (24.74 ± 0.27 nmol/µl, P<0.01). This finding highlighted the utilization of sex dependent metabolic diversity between preimplantation embryos for non-invasive sex diagnosis and suggests the TAC concentration as a potential noninvasive biomarker for prediction of sex.

Ex vivo expansion is a promising strategy to overcome the low number of human umbilical cord blood hematopoietic stem cells (hUCB-HSCs). Although based on the obtained results in unnatural physiological condition of irradiated genetically immune-deficient mouse models, there has always been concern that the expanded cells have less engraftment potential. The purpose of this study was to investigate effect of common ex vivo expansion method on engraftment potential of hUCB-mononuclear cells (MNCs), using normal fetal mouse, as a model with more similarity to human physiological conditions. In this experimental study, briefly, isolated hUCB-MNCs were cultured in common expansion medium containing stem cell factor, Flt3 ligand and thrombopoietin. The unexpanded and expanded cells were transplanted to the fetal mice on gestational days of 11.5-13.5. After administration of human hematopoiesis growth factors (hHGFs), presence of human CD45+ cells, in the peripheral blood of recipients, was assessed at various time points after transplantation. The expanded MNCs showed 32-fold increase in the expression of CD34+38- phenotype and about 3-fold higher clonogenic potential as compared to the uncultured cells. Four weeks after transplantation, 73% (19/26) of expanded-cell recipients and 35% (7/20) of unexpanded-cell recipients were found to be successfully engrafted with human CD45+ cells. The engraftment level of expanded MNCs was significantly (1.8-fold) higher than unexpanded cells. After hHGFs administration, the level was increased to 3.2, 3.8 and 2.6-fold at respectively 8, 12, and 16 weeks of post transplantation. The increased expression of CXCR4 protein in expanded MNCs is a likely explanation for the present findings. The presented data showed that expanded MNCs compared to unexpended cells are capable of more rapid and higher short-term engraftment in normal fetal mouse. It could also be suggested that in utero transplantation (IUT) of normal fetal mice could be an appropriate substitute for NOD/SCID mice in xenotransplantation studies.

Liver transplantation is the gold standard approach for decompensated liver cirrhosis. In recent years, stem cell therapy has raised hopes that adjusting some clinical and laboratory parameters could lead to successful treatments for this disease. Cirrhotic patients may have multiple systemic abnormalities in peripheral blood and irregular cell populations in bone marrow (BM). Correcting these abnormalities before BM aspiration may improve the effectiveness of cell-based therapy of liver cirrhosis. In this controlled clinical trial study, 20 patients with decompensated liver cirrhosis were enrolled. Patients were randomly assigned to control and experimental groups. Blood samples were obtained to measure vitamin B12, folate, serum iron, total iron bonding capacity (TIBC) and ferritin before any intervention. Furthermore, the iron storage and fibrosis level in BM biopsies, as well as the percentage of different cell populations, were evaluated. Prior to cell isolation for transplantation, we performed palliative supplement therapy followed by a correction of nutritional deficiencies. Mononuclear cells (MNCs) were then isolated from BM aspirates and transfused through peripheral vein in patients in the experimental group. The model of end-stage liver disease (MELD) score, The international normalized ratio (INR), serum albumin and bilirubin levels were assessed at 0 (baseline), 3 and 6 months after cell transplantation. The MELD score (P=0.0001), INR (P=0.012), bilirubin (P<0.0001) and total albumin (P<0.0001) levels improved significantly in the experimental group after cell transplantation compared to the baseline and control groups. Moreover, the increase in serum albumin levels of patients in the experimental group was statistically significant 6 months after transplantation. We have successfully improved the conditions of preparing -BM-derived stem cells for transplantation. Although these cells are relatively safe and have been shown to improve some clinical signs and symptoms temporarily, there need to be more basic studies regarding the preparation steps for effective clinical use (Registration number: IRCT2014091919217N1).

Dendritic cells (DCs) as major regulators of the immune response in the decidua play a pivotal role in establishment and maintenance of pregnancy. Immunological disorders are considered to be the main causes of unexplained recurrent spontaneous abortions (RSAs). Recently, we reported that mesenchymal stem cells (MSCs) therapy could improve fetal survival and reduce the abortion rate in abortion-prone mice, although the precise mechanisms of this action are poorly understood. Since MSCs have been shown to exert immunomodulatory effects on the immune cells, especially DCs, this study was performed to investigate the capability of MSCs to modulate the frequency, maturation state, and phenotype of uterine DCs (uDCs) as a potential mechanism for the improvement of pregnancy outcome. In this experimental study, adipose-derived MSCs were intraperitoneally administered to abortion-prone pregnant mice on the fourth day of gestation. On the day 13.5 of pregnancy, after the determination of abortion rates, the frequency, phenotype, and maturation state of uDCs were analyzed using flow cytometry. Our results indicated that the administration of MSCs, at the implantation window, could significantly decrease the abortion rate and besides, increase the frequency of uDCs. MSCs administration also remarkably decreased the expression of DCs maturation markers (MHC-II, CD86, and CD40) on uDCs. However, we did not find any difference in the expression of CD11b on uDCs in MSCs-treated compared to control mice. Regarding the mutual role of uDCs in establishment of a particular immunological state required for appropriate implantation, proper maternal immune responses and development of successful pregnancy, it seems that the modulation of uDCs by MSCs could be considered as one of the main mechanisms responsible for the positive effect of MSCs on treatment of RSA.

During the cultivation of spermatogonial stem cells (SSCs) and their conversion into embryonic stem-like (ES-like) cells, transitional ES-like colonies and epiblast-like cells were observable. In the present experimental study, we aimed to analyze the efficiency of the multipotency or pluripotency potential of ES-like cells, transitional colonies and epiblast-like cells. In this experimental study, SSCs were isolated from transgenic octamer-binding transcription factor 4 (Oct4)-green fluorescent protein (GFP)-reporter mice. During cell culture ES-like, transitional and epiblast- like colonies developed spontaneously. The mRNA and protein expression of pluripotency markers were analyzed by Fluidigm real-time polymerase chain reaction (RT-PCR) and immunocytochemistry, respectively. Efficiency to produce chimera mice was evaluated after injection of ES and ES-like cells into blastocysts. Microscopic analyses demonstrated that the expression of Oct4-GFP in ES-like cells was very strong, in epiblast-like cells was not detectable, and was only partial in transitional colonies. Fluidigm RT-PCR showed a higher expression of the germ cell markers Stra-8 and Gpr-125 in ES-like cells and the pluripotency genes Dppa5, Lin28, Klf4, Gdf3 and Tdgf1 in ES-like colonies and embryonic stem cells (ESCs) compared to the epiblast-like and transitional colonies. No significant expression of Oct-4, Nanog, Sox2 and c-Myc was observed in the different groups. We showed a high expression level of Nanog and Klf4 in ES-like, while only a partial expression was observed in transitional colonies. We generated chimeric mice after blastocystic injection from ES and ES-like cells, but not from transitional colonies. We observed that the efficiency to produce chimeric mice in ES cells was more efficient (59%) in comparison to ES-like cells (22%). This new data provides more information on the pluripotency or multipotency potentials of testis-derived ES-like cells in comparison to transitional colonies and epiblast-like cells.

Human embryonic stem cells (hESCs) have the potential to give rise to all types of cells in the human body when appropriately induced to differentiate. Stem cells can differentiate spontaneously into the three-germ layer derivatives by embryoid bodies (EBs) formation. However, the two-dimensional (2D) adherent culture of hESCs under defined conditions is commonly used for directed differentiation toward a specific type of mature cells. In this study, we aimed to determine the propensity of the Royan hESC lines based on comparison of expression levels of 46 lineage specific markers. In this experimental study, we have compared the expression of lineage-specific markers in hESC lines during EB versus adherent-based spontaneous differentiation. We used quantitative real-time polymerase chain reaction (qRT-PCR) to assess expressions of 46 lineage-specific markers in 4 hESC lines, Royan H1 (RH1), RH2, RH5, and RH6, during spontaneous differentiation in both EB and adherent cultures at 0, 10, and 30 days after initiation of differentiation. Based on qRT-PCR data analysis, the liver and neuronal markers had higher expression levels in EBs, whereas skin-specific markers expressed at higher levels in the adherent culture. The results showed differential expression patterns of some lineage-specific markers in EBs compared with the adherent cultures. According to these results, possibly the spontaneous differentiation technique could be a useful method for optimization of culture conditions to differentiate stem cells into specific cell types such ectoderm, neuron, endoderm and hepatocyte. This approach might prove beneficial for further work on maximizing the efficiency of directed differentiation and development of novel differentiation protocols.

Recent achievements in stem cell biotechnology, nanotechnology and tissue engineering have led to development of novel approaches in regenerative medicine. Azoospermia is one of the challenging disorders of the reproductive system. Several efforts were made for isolation and culture of testis-derived stem cells to treat male infertility. However, tissue engineering is the best approach to mimic the three dimensional microenvironment of the testis in vitro. We investigated whether human testis-derived cells (hTCs) obtained by testicular sperm extraction (TESE) can be cultured on a homemade scaffold composed of electrospun nanofibers of homogeneous poly (vinyl alcohol)/human serum albumin/gelatin (PVA/HSA/gelatin). In this experimental lab study, human TCs underwent two steps of enzymatic cell isolation and five culture passages. Nanofibrous scaffolds were characterized by scanning electron microscopy (SEM) and Fourier- transform infrared spectroscopy (FTIR). Attachment of cells onto the scaffold was shown by hematoxylin and eosin (H&E) staining and SEM. Cell viability study using MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide] assay was performed on days 7 and 14. Visualization by H&E staining and SEM indicated that hTCs were seeded on the scaffold. MTT test showed that the PVA/HSA/gelatin scaffold is not toxic for hTCs. It seems that this PVA/HSA/gelatin scaffold is supportive for growth of hTCs.

Tilting the balance in favor of antioxidant agents could increase infertility problems in obese and diabetic individuals. The aim of this study was to evaluate oxidative stress status in semen of men with type 2 diabetes and obesity to investigate whether excessive amounts of oxidative stress, as a result of diabetes and obesity, influence infertility potential. A case-control study was conducted in men (n=150) attending the Infertility Center of Royan Institute between December 2016 and February 2017. Participants were categorized into four groups; normal weight (BMI<25 kg/m2) and non-type-2 diabetic (control=40), obese and non- type-2 diabetic (obese=40), non-obese and type- 2 diabetic (Nob-DM=35), and obese and type-2 diabetic (Ob-DM=35). The semen analysis was performed according to the World Health Organization criteria. Oxidative stress, DNA fragmentation, sperm apoptosis, and total antioxidant capacity (TAC) were evaluated in semen samples of men. Serum glucose, HbA1c, cortisol, and testosterone levels were determined using the enzyme-linked immunosorbent assay (ELISA) method. Compared with the control group, sperm motility, progressive motility, and normal morphology were significantly decreased in the obese, Nob-DM, and Ob-DM groups (P<0.01). The obese, Nob-DM, and Ob-DM groups showed significantly lower levels of TAC and higher amounts of oxidative stress, early apoptotic sperm, and the percentage of DNA fragmentation as compared with the control group (P<0.05). Testosterone concentration was decreased in the obese, Nob-DM, and Ob-DM groups when compared with healthy individuals (P<0.05), whereas the cortisol level was significantly increased in the Nob-DM and Ob-DM groups in comparison to the obese and control group (P<0.01). Increased amount of reactive oxygen species (ROS) levels and DNA fragmentation in men affected by either diabetes or obesity could be considered prognostic factors in sub-fertile patients, alerting physicians to an early screen of male patients to avoid the development of infertility in prone patients.

Phospholipase C zeta 1 (PLCζ) is one of the main sperm factor involved in oocyte activation and other factors may assist this factor to induce successful fertilization. Microinjection of recombinant tr-kit, a truncated form of c-kit receptor, into metaphase II-arrested mouse oocytes initiate egg activation. Considering the potential roles of tr- KIT during spermiogenesis and fertilization, we aimed to assess expression of tr-KIT in sperm of men with normal and abnormal parameters and also in infertile men with previous failed fertilization and globozoospermia. This experimental study was conducted from September 2015 to July 2016 on 30 normozoospermic and 20 abnormozoospermic samples for experiment one, and also was carried out on 10 globozoospermic men, 10 men with a history low or failed fertilization and 13 fertile men for experiment two. Semen parameters and sperm DNA fragmentation were assessed according to WHO protocol, and TUNEL assay. Sperm tr- KIT was evaluated by flow cytometry, immunostaining and western blot. The results show that tr-KIT mainly was detected in post-acrosomal, equatorial and tail regions. Percentage of tr-KIT-positive spermatozoa in abnormozoospermic men was significantly lower than normozoospermic men. Also significant correlations were observed between sperm tr-KIT with sperm count (r=0.8, P<0.001), motility (r=0.31, P=0.03) and abnormal morphology (r=-0.6, P<0.001). Expression of tr-KIT protein was significantly lower in infertile men with low/ failed fertilization and globozoospermia compared to fertile men. The significant correlation was also observed between tr-KIT protein with fertilization rate (r=-0.46, P=0.04). In addition, significant correlations were observed between sperm DNA fragmentation with fertilization rate (r=-0.56, P=0.019) and tr-KIT protein (r=-0.38, P=0.04). tr-KIT may play a direct or indirect role in fertilization. Therefore, to increase our insight regarding the role of tr-KIT in fertilization further research is warranted.

Human epidermal growth factor receptor 2 (HER-2), as a crucial factor involved in about 20% of breast cancer cases, is one of the most reliable tumor markers to determine prognosis and therapeutic trend of this disease. This marker is generally assessed by immunohistochemistry (IHC) technique. In the cases that result of IHC test cast doubt (+2), the test should be repeated or validated by applying in situ hybridization techniques, like chromogenic in situ hybridization (CISH). In this regard, the goal of current study was to figure out the link between different clinicopathological characteristics of patients suffering from invasive breast cancer, using tumor markers, hormone receptor (HR) and HER-2. Comparing IHC and CISH techniques for evaluating diagnostic value and usefulness of HER-2 were also the other objective of this study. Based on this retrospective study, histological markers of 113 individuals suffering from invasive breast cancer -such as estrogen receptor (ER), progesterone receptor, HER-2 receptor, E-cadherin, CK5/6, vimentin and Ki67 were examined by IHC technique. HER-2 amplification of all patients was also evaluated by CISH. Clinicopathological information of the patients was also extracted from medical documents and their associations with tumor markers were statistically evaluated. There is a significant relationship between tumor size, CK5/6 and tumor grade with HR status. Similar relationship was observed between HER-2 status and HR status, as well as vascular invasion (P<0.05). The comparison of HER-2 amplification showed no complete concordance of the result obtained from these two techniques, with score +3. Since the status of HER-2 is very important in decision making of the treatment process, CISH technique is recommended in the malignant conditions as the primary test, instead of IHC. In this study, we also determined that HER-2 expression is greatly correlated with ER- and PR- status. This might propose a better prognosis for HER-2+ patients.

To evaluate association of patients’ clinicopathological data with expression of nicotinamide nucleotide transhydrogenase (NNT) and naturally occurring antisense RNA of the same gene locus (NNT-AS1) in breast cancer samples. In the current case-control study, mean expressions of NNT and NNT-AS1 were assessed in 108 breast tissue samples including 54 invasive ductal carcinoma samples and 54 adjacent non-cancerous tissues (ANCTs) by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). NNT expression was not significantly different between tumor tissues and ANCTs. However, NNT-AS1 expression was significantly down-regulated in tumor tissues compared to ANCTs (expression ratio=0.51, P=0.01). NNT-AS1 expression was significantly higher in estrogen receptor (ER) negative samples, in comparison with ER positives (P=0.01). No considerable difference was found in the gene expressions between other subcategories of patients. Considerable correlations were detected between expression levels of these two genetic loci in both tumor tissues and ANCTs. In the current study, for the first time we simultaneously assessed expression of NNT and NNT-AS1 in breast cancer tissues. This study highlights association of ER status with dysregulation of NNT-AS1 in breast cancer tissues. Future researches are necessary to explore the function of this long non-coding RNA (lncRNA) in the pathogenesis of breast cancer.

Major birth defects are inborn structural or functional anomalies with long-term disability and adverse impacts on individuals, families, health-care systems, and societies. Approximately 20% of birth defects are due to chromosomal and genetic conditions. Inspired by the fact that neonatal deaths are caused by birth defects in about 20 and 10% of cases in Iran and worldwide respectively, we conducted the present study to unravel the role of chromosome abnormalities, including microdeletion/microduplication(s), in multiple congenital abnormalities in a number of Iranian patients. In this descriptive cross-sectional study, 50 sporadic patients with Multiple Congenital Anomalies (MCA) were selected. The techniques employed included conventional karyotyping, fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and array comparative genomic hybridisation (array-CGH), according to the clinical diagnosis for each patient. Chromosomal abnormalities and microdeletion/microduplication(s) were observed in eight out of fifty patients (16%). The abnormalities proved to result from the imbalances in chromosomes 1, 3, 12, and 18 in four of the patients. However, the other four patients were diagnosed to suffer from the known microdeletions of 22q11.21, 16p13.3, 5q35.3, and 7q11.23. In the present study, we report a patient with 46,XY, der(18)[12]/46,XY, der(18), +mar[8] dn presented with MCA associated with hypogammaglobulinemia. Given the patient’s seemingly rare and highly complex chromosomal abnormality and the lack of any concise mechanism presented in the literature to justify the case, we hereby propose a novel mechanism for the formation of both derivative and ring chromosome 18. In addition, we introduce a new 12q abnormality and a novel association of an Xp22.33 duplication with 1q43q44 deletion syndrome. The phenotype analysis of the patients with chromosome abnormality would be beneficial for further phenotype-genotype correlation studies.

This study was carried out to evaluate the relationship between mtDNA D-loop variations and the pathogenesis of a brain tumor. In this experimental study, 25 specimens of brain tumor tissue with their adjacent tissues from patients and 454 blood samples from different ethnic groups of the Iranian population, as the control group, were analysed by the polymerase chain reaction (PCR)-sequencing method. Thirty-six variations of the D-loop area were observed in brain tumor tissues as well as the adjacent normal tissues. A significant difference of A750G (P=0.046), T15936C (P=0.013), C15884G (P=0.013), C16069T (P=0.049), T16126C (P=0.006), C16186T (P=0.022), T16189C (P=0.041), C16193T (P=0.045), C16223T (P=0.001), T16224C (P=0.013), C16234T (P=0.013), G16274A (P=0.009), T16311C (P=0.038), C16327T (P=0.045), C16355T (P=0.003), T16362C (P=0.006), G16384A (P=0.042), G16392A (P=0.013), G16394A (P=0.013), and G16477A (P=0.013) variants was found between the patients and the controls. The results indicated individuals with C16069T [odds ratio (OR): 2.048], T16126C (OR: 2.226), C16186T (OR: 3.586), G16274A (OR: 4.831), C16355T (OR: 7.322), and T16362C (OR: 6.682) variants with an OR more than one are probably associated with a brain tumor. However, given the multifactorial nature of cancer, more investigation needs to be done to confirm this association.

Fermented garlic, often called black garlic, is a traditional food ingredient used in Asian cuisine and possesses various health benefits including anti-obesity activity. The anti-obesity effects of fermented garlic might, in part, might be mediated through direct actions of its components on adipocytes. To test this hypothesis, we examined whether fermented garlic extract might stimulate the metabolic activity of human adipose-derived stem cells (ADSCs) in culture. Cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay exhibited a complex dose- response relationship. The lowest concentration (0.4 mg/ml) reduced cell viability (P<0.05 compared to no extract, Bonferroni’s multiple comparison), whereas higher concentrations (0.8 and 1.0 mg/ml) resulted in higher cell viability (P<0.05 as compared to 0.4 mg/ml). However, the extract at concentrations >2 mg/ml markedly decreased cell viability. Higher cell viability observed following treatment with 0.8~1.0 mg/ml might be associated with raised oxygen consumption. Fluorescent dye-based measurement revealed that the garlic extract at 1.0 mg/ml significantly increased oxygen consumption. We also detected a significant increase in mRNA expression levels of uncoupling protein-1 (UCP- 1). These findings suggest that fermented garlic stimulates the basal metabolic activity of human ADSCs.