Safety and efficacy of platelet-derived mitochondrial transplantation in ischaemic heart disease.

BACKGROUND: Acute ST-elevation myocardial infarction (STEMI) remains a globally significant health challenge in spite of improvement in management strategy. Being aware that mitochondrial dysfunction plays a crucial role in ischaemia-reperfusion injury (IRI) modulation, empirical evidence suggests functional mitochondrial transplantation strikes as a reliable therapeutic approach for patients with acute myocardial infarction. METHODS AND RESULTS: We conducted a prospective, triple-blinded, parallel-group, blocked randomised clinical trial to investigate the therapeutic effects and clinical outcomes of platelet-derived mitochondrial transplantation in 30 patients with acute STEMI, such that the 15 subjects in the control group were given standard of care treatment, whereas the subjects in the intervention group received autologous platelet-derived mitochondria through the intracoronary injection. We observed that within 40 days, the intervention group had a slightly greater improvement in the left ventricular ejection fraction (LVEF) compared to the control group and experienced a significant enhancement in the exercise capacity (p < 0.001). Moreover, major adverse cardiac events (MACE), arrhythmia, fever, and tachycardia were compared between the groups and lack of significant difference marks the safety of mitochondrial transplantation (p > 0.05). Furthermore, the two groups were not significantly distinct as regards the average length of stay for a hospitalisation (p > 0.05). CONCLUSION: We suggest platelet-derived mitochondrial transplantation appears as a beneficial and highly promising therapeutic option for patients of ischaemic heart disease (IHD); however, we are aware that further in-depth studies with larger sample sizes along with longer follow-up periods are necessary for validating the clinical implications of our findings.

Lipocalin-2 Upregulation in Nasopharyngeal Carcinoma: A Novel Potential Diagnostic Biomarker.

Background: Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients. Methods: In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher's exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant. Results: Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients. Conclusion: Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.

Human umbilical cord-derived mesenchymal stem cells-harvested mitochondrial transplantation improved motor function in TBI models through rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation.

Each year, traumatic brain injury (TBI) causes a high rate of mortality throughout the world and those who survive have lasting disabilities. Given that the brain is a particularly dynamic organ with a high energy consumption rate, the inefficiency of current TBI treatment options highlights the necessity of repairing damaged brain tissue at the cellular and molecular levels, which according to research is aggravated due to ATP deficiency and reactive oxygen species surplus. Taking into account that mitochondria contribute to generating energy and controlling cellular stress, mitochondrial transplantation as a new treatment approach has lately reduced complications in a number of diseases by supplying healthy and functional mitochondria to the damaged tissue. For this reason, in this study, we used this technique to transplant human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)-derived mitochondria as a suitable source for mitochondrial isolation into rat models of TBI to examine its therapeutic benefit and the results showed that the successful mitochondrial internalisation in the neuronal cells significantly reduced the number of brain cells undergoing apoptosis, alleviated astrogliosis and microglia activation, retained normal brain morphology and cytoarchitecture, and improved sensorimotor functions in a rat model of TBI. These data indicate that human umbilical cord-derived mesenchymal stem cells-isolated mitochondrial transplantation improves motor function in a rat model of TBI via rescuing neuronal cells from apoptosis and alleviating astrogliosis and microglia activation, maybe as a result of restoring the lost mitochondrial content.

The reversibility of cancer radioresistance: a novel potential way to identify factors contributing to tumor radioresistance.

To understand the molecular mechanisms responsible for radioresistance in cancer cells, we previously established clinically relevant radioresistant (CRR) cell lines from several human cancer cell lines. These CRR cells proliferate even under exposure to 2 Gy/day of X-rays for more than 30 days, which is a standard protocol for tumor radiotherapy. CRR cells received 2 Gy/day of X-rays to maintain their radioresistance (maintenance irradiation; MI). Interestingly, CRR cells that did not receive MI for more than a year lost their radioresistance, indicating that radiation-induced radioresistance is reversible. We designated these CRR-NoIR cells. Karyotyping of the parental and CRR cells revealed that the chromosomal composition of CRR cells is quite different from that of the parental cells. However, CRR and CRR-NoIR cells were more similar compared with the parental cells because CRR cells repair X-ray-induced DNA damage with higher fidelity. To identify the factor(s) involved in tumor radioresistance, previously published studies including ours have compared radioresistant cells to parental cells. In this review, we conclude that a comparison between CRR and CRR-NoIR cells, rather than parental cells, is the best way to identify factors involved in tumor radioresistance.

Natural killer cell immunotherapy in glioblastoma.

Glioblastoma (GBM) is one of the most difficult cancers to treat because GBM has the high therapeutic resistance. Recently, immunotherapies for GBM have been used instead of conventional treatments. Among them, Natural killer (NK) cell-based immunotherapy has the potential to treat GBM due to its properties such as the absence of restriction by antigen-antibody reaction and deep penetration into the tumor microenvironment. Especially, genetically engineered NK cells, such as chimeric antigen receptor (CAR)-NK cells, dual antigen-targeting CAR NK cells, and adapter chimeric antigen receptor NK cells are considered to be an important tool for GBM immunotherapy. Therefore, this review describes the recent efforts of NK cell-based immunotherapy in GBM patients. We also describe key receptors expressing on NK cells such as killer cell immunoglobulin-like receptor, CD16, and natural killer group 2, member D (NKG2DL) receptor and discuss the function and importance of these molecules.

Combination Therapy of Metadichol Nanogel and Lipocalin-2 Engineered Mesenchymal Stem Cells Improve Wound Healing in Rat Model of Excision Injury.

Purpose: Currently, several disorders including burns, trauma, excisional and diabetic wounds, and bedsores threaten the human health. Application of mesenchymal stem cells (MSCs) is recommended for treatment of skin disorders. However, because of oxidative stress and inflammation after skin injury, survival of transplanted MSCs is low which in turn negatively affects the efficiency of the MSCs-based therapy. In an attempt to address the aforementioned challenge and introducing a novel potential therapeutic strategy, we employed combination therapy by lipocalin 2 (Lcn2)-engineered MSCs and a Metadichol (an inverse agonist of vitamin D receptor (VDR)) nanogel in a rat model of excisional wound. Methods: First, human umbilical cord MSCs (hUC-MSCs) was transfected by a recombinant plasmid encoding Lcn2 gene. Next, a combination of Metadichol nanogel and the engineered MSCs was co-applied on wound in rat model of excision injury. Finally the improvement of wound healing in experimental groups was evaluated by photography and histological assessments (hematoxylin and eosin staining). Results: Our findings revealed that the repair rate was higher in the group received combination therapy comparing to control groups. Notably, Metadichol+Lcn2-MSCs showed significantly higher wound contraction rate compared to control group at all time points (P value < 0.001). Furthermore, wound repair rate was 95% 14 days after surgery, and 100% after 21 days in the treatment groups. Our results also revealed that the combination therapy improved and accelerated the wound healing process. Conclusion: Our findings suggest a novel potential therapeutic strategy i.e. Lcn2-engineered MSCs and Metadichol for wound healing. However, further preclinical and clinical studies are required.

CRISPR/Cas9-mediated knockout of HO-1 decreased the proliferation and migration of T47D cells and increased cisplatin-induced apoptosis: an in vitro study.

Breast cancer is the most common type of neoplasm and the second cause of cancer-related death in women. Despite the development of novel therapeutic strategies and improved the clinical outcomes, the mortality rate for breast cancer is still high. Therefore, development of a new modality, particularly based on knocking out key genes, is under focus of investigation. Heme oxygenase-1 (HO-1) deregulation has been associated with various neoplasms-related behaviors of many types of tumor cells including breast cancer. In the current study, in order to evaluate the role of the HO-1 gene in breast cancer, we utilized the CRISPR/Cas9 technology to knock out HO-1 gene in T47D breast cancer cell line and studied its potential therapeutic effects in vitro. The cell proliferation and their sensitivity to Cisplatin were determined by CCK-8 kit. In addition, the apoptosis and the migratory potential of the cells were evaluated using Hoechst staining, and Transwell/Scratch methods, respectively. Our findings revealed that HO-1 suppression significantly reduced the proliferation ability of T47D cells (P < 0.001). Moreover, sensitivity to Cisplatin-induced toxicity increased significantly in KO-T47D cells compared to the control T47D cells. Furthermore, our findings indicated that Cisplatin-induced apoptosis increased in the KO-T47D cells. Moreover, the migratory capability of KO-T47D cells was abolished significantly (P < 0.001) as determined by Transwell migration assay. In a nutshell, our findings strongly suggest that HO-1 involved in breast cancer progression and metastasis and chemotherapy resistance. However, further comprehensive studies are required to clarify the precise role of the HO-1 gene on breast cancer cells.

SA/G hydrogel containing NRF2-engineered HEK-293-derived CM improves wound healing efficacy of WJ-MSCs in a rat model of excision injury.

AIM OF THE STUDY: Skin wounds are a major public health issue due to the lack of real effective remedies. Mesenchymal stem cells (MSCs) are considered as a promising therapeutic strategy for wound injuries; however, low survival rate following transplantation limited their application. In an attempt to introduce a novel potential wound dressing and improve wound healing properties, the current study was conducted. MATERIAL AND METHODS: we prepared conditioned medium (CM) harvested from HEK-293 cells overexpressing nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant genes expression. Then, the CM was loaded in a biodegradable hydrogel. Next, in an animal model of full-thickness excision wound, wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) were transplanted at the margins of the wound followed by application of the hydrogel on injury site. Finally, wound healing characteristics were evaluated by proper methods. RESULTS: Our findings revealed that, the NRF2-CM protected the WJ-MSCs against H2O2-induced toxicity in vitro. Furthermore, in vivo results showed that, SA/G hydrogel containing NRF2-CM significantly (P < 0.01) promoted WJ-MSCs survival, increased angiogenesis, accelerated wound contraction, and promoted wound healing compared to other groups. CONCLUSION: Though further preclinical and clinical studies regarding mechanisms behind the protection and also safety of the strategy are needed, our findings strongly suggest that the prepared wound dressing enhanced the efficacy of therapeutic potential of WJ-MSCs by providing an enriched/antioxidant niche support.

Retraction Notice: MS14 Down-regulates Lipocalin2 Expression in Spinal Cord Tissue in an Animal Model of Multiple Sclerosis in Female C57BL/6.

This retracts the article DOI: 10.6091/ibj.1375.2014.

Cell Survival Effects of Autophagy Regulation on Umbilical Cord-Derived Mesenchymal Stem Cells Following Exposure to Oxidative Stress.

BACKGROUND: Due to oxidative stress, hypoxia, and serum deprivation, a large percentage of mesenchymal stem cells (MSCs) die in the early stages of transplantation. The present study aimed to address whether induction or inhibition of autophagy would affect the viability of MSCs after exposure to oxidative stress. METHODS: MSCs were isolated from umbilical cord tissue using the Ficoll gradient method. pCMV-GFP-LC-3 plasmid containing GFP-tagged LC3 was transfected into MSCs to assay autophagy level in these valuable cells. The four study groups were: MSC-LC3-Rapa, MSC-LC3-3MA, MSCs without any transfection, and MSC-GFP-LC3 (control groups). To induce autophagy, the MSC-GFP-LC3 was treated with different concentrations of Rapa for 24 hours and named MSC-LC3-Rapa. To inhibit autophagy in MSC-GFP-LC3, these cells were cultured in the presence of 3MA for 24 hours and named MSC-LC3-3MA. Non-treated MSC-GFP-LC3 and MSCs were considered as control groups. MSCs were exposed to lethal doses of H2O2 followed by cell viability evaluation with the water-soluble tetrazolium salt assay method. The data were analyzed with SPSS version 18.0 using one-way ANOVA test. P<0.05 was considered statistically significant. RESULTS: The results revealed that the enhancement of autophagy in MSC-LC3-Rapa sensitized them against oxidative stress (P=0.0006) and inhibition of autophagy in MSC-LC3-3MA led to resistance against oxidative stress (P=0.0003). CONCLUSION: Inhibition of autophagy, as a non-genetic engineering method, in MSCs enhances cell viability following exposure to the oxidative stress. This may provide a novel strategy to promote the efficiency of MSC-based cell therapy for clinical applications.

Mesenchymal stem cell-based therapy for autoimmune diseases: emerging roles of extracellular vesicles.

In autoimmune disease body's own immune system knows healthy cells as undesired and foreign cells. Over 80 types of autoimmune diseases have been recognized. Currently, at clinical practice, treatment strategies for autoimmune disorders are based on relieving symptoms and preventing difficulties. In other words, there is no effective and useful therapy up to now. It has been well-known that mesenchymal stem cells (MSCs) possess immunomodulatory effects. This strongly suggests that MSCs might be as a novel modality for treatment of autoimmune diseases. Supporting this notion a few preclinical and clinical studies indicate that MSCs ameliorate autoimmune disorders. Interestingly, it has been found that the beneficial effects of MSCs in autoimmune disorders are not relying only on direct cell-to-cell communication but on their capability to produce a broad range of paracrine factors including growth factors, cytokines and extracellular vehicles (EVs). EVs are multi-signal messengers that play a serious role in intercellular signaling through carrying cargo such as mRNA, miRNA, and proteins. Numerous studies have shown that MSC-derived EVs are able to mimic the effects of the cell of origin on immune cells. In this review, we discuss the current studies dealing with MSC-based therapies in autoimmune diseases and provide a vision and highlight in order to introduce MSC-derived EVs as an alternative and emerging modality for autoimmune disorders.

Comparison of The Melatonin Preconditioning Efficacy between Bone Marrow and Adipose-Derived Mesenchymal Stem Cells.

OBJECTIVE: Mesenchymal stem cells (MSC) from various sources have the potentials to positively affect regenerative medicine. Furthermore, pre-conditioning strategies with different agents could improve the efficacy of cell therapy. This study compares the effects of an anti-inflammatory and antioxidant agent, melatonin, on protection of bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs). MATERIALS AND METHODS: In this experimental study, rat BMSCs and ADSCs were isolated and expanded. Pre-conditioning was performed with 5 µM melatonin for 24 hours. Cell proliferation and viability were detected by MTT assay. Expression of BAX, BCL2, melatonin receptors and osteocalcin genes were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Also, apoptosis was detected with tunnel assay. Osteogenic differentiation was analyzed using alizarin red staining. RESULTS: No significant increase was found in cell viability between BMSCs and ADSCs after melatonin preconditioning. Following melatonin preconditioning, BAX expression was significantly down-regulated in both ADSCs and BMSCs (P<0.05), with the difference being more significant in ADSCs compared to BMSCs. BCL2 expression was increased significantly in both cell types after preconditioning. Metalothionine 1 and Metalothionine 2 were both upregulated significantly in the two cell types (P<0.05). Melatonin increased osteogenesis capability through increasing osteocalcin expression. However, expression of osteocalcin in BMSCs before and after preconditioning was higher than that in ADSCs. On the other hand, melatonin expression in ADSCs was in higher levels than in BMSCs. Melatonin also improved alizarin red concentration significantly in both BMSCs and ADSCs (P<0.05). Alizarin red staining severity increased significantly in ADSCs after preconditioning compared to BMSCs (P<0.05). CONCLUSION: Here we have shown that the effects of preconditioning on melatonin expression in ADSCs are higher than those in BMSCs. These findings could be used in adoption of a proper preconditioning protocol based on the sources of MSCs in specific clinical applications, especially in bone regeneration.

Dual Preconditioning: A Novel Strategy to Withstand Mesenchymal Stem Cells against Harsh Microenvironments.

Purpose: Poor survival rate of mesenchymal stem cells (MSCs) following their transplantation is one of the major challenges in their therapeutic application. Therefore, it is necessary to augment the viability of the MSCs in order to improve their therapeutic efficacy. Several strategies have been used to overcome this problem. Preconditioning of MSCs with oxidative stresses has gained a lot of attention. Therefore, in the present study, we investigated the effects of simultaneous preconditioning of MSCs with hydrogen peroxide and serum deprivation stresses on their survival and resistance to stressful conditions. Methods: MSCs were isolated from human umbilical cord blood. To perform simultaneous preconditioning, the cells were cultured in DMEM medium containing 1, 2.5 and 5 percent FBS and different concentrations of H2O2 (5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80 and 100 µM) for 24 hrs. Then, the cells were cultured in recovery culture medium. Finally, one group of the cells was exposed to a lethal concentration of H2O2 (300µM), and the other cells were cultivated in FBS free DMEM medium as the lethal situation. In addition, the percentage of apoptotic cells was analyzed using Caspase 3 assay kit. Results: Simultaneous preconditioning of the MSCs with 15µM H2O2 plus serum deprivation, 2.5% FBS, significantly increased the resistance of the cells to the toxicity induced following their cultivation in FBS free DMEM medium. It exerted the protective effect on the cells after treating with the lethal dose of H2O2 as well. Conclusion: Simultaneous preconditioning of MSCs with oxidative and serum deprivation stresses enhances their survival against harsh conditions, which might increase the viability and stability of the MSCs following their transplantation.

Nucleosome remodelling, DNA repair and transcriptional regulation build negative feedback loops in cancer and cellular ageing.

Nucleosome remodelling (NR) regulates transcription in an ATP-dependent manner, and influences gene expression required for development and cellular functions, including those involved in anti-cancer and anti-ageing processes. ATP-utilizing chromatin assembly and remodelling factor (ACF) and Brahma-associated factor (BAF) complexes, belonging to the ISWI and SWI/SNF families, respectively, are involved in various types of DNA repair. Suppression of several BAF factors makes U2OS cells significantly sensitive to X-rays, UV and especially to cisplatin, and these BAF factors contribute to the accumulation of repair proteins at various types of DNA damage and to DNA repair. Recent cancer genome sequencing and expression analysis has shown that BAF factors are frequently mutated or, more frequently, silenced in various types of cancer cells. Thus, those cancer cells are potentially X-ray- and especially cisplatin-sensitive, suggesting a way of optimizing current cancer therapy. Recent single-stem cell analysis suggests that mutations and epigenetic changes influence stem cell functionality leading to cellular ageing. Genetic and epigenetic changes in the BAF factors diminish DNA repair as well as transcriptional regulation activities, and DNA repair defects in turn negatively influence NR and transcriptional regulation. Thus, they build negative feedback loops, which accelerate both cellular senescence and transformation as common and rare cellular events, respectively, causing cellular ageing.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Deregulation of miR-93 and miR-143 in human esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the second and third most common malignancy in Iranian males and females, respectively. Treatment of ESCC is largely ineffective due to lack of detection at early stages of the disease. In recent years, miRNA, a small RNA molecule, has drawn much attention to researchers as a potential biomarker for esophageal cancer. miR-93 and miR-143 are two miRNA molecules reported to be frequently deregulated in various cancers, including prostate, stomach, cervix, and etc. The purpose of this study was to investigate the expression levels of these miRNAs and evaluate their diagnostic and therapeutic potential in esophageal squamous cell carcinoma. In this study, total RNA was extracted from 30 tumor tissues and 30 nontumor tissues of esophageal tumor margins, using RNX-plus solution. After validating the quality and quantity of total RNA, cDNAs of interest were synthesized using microRNA-specific cDNA Synthesis Kit. The expression level of miR-93 and miR-143 was evaluated using quantitative real-time PCR with miRNA-specific primers. Finally, the obtained data was analyzed by SPSS ver.20 software and paired t test was performed to observe the significance of difference between groups. The expression level of miR-93 was significantly increased and of miR-143 was significantly decreased in most of the examined tumor tissues, compared to nontumor tissues. Also, our findings did not detect correlation between mir-93 and mir-143 expressions in regard to stage and grade of the samples. These findings suggest that the deregulation of these miRNAs may play an important role in esophageal squamous cell carcinoma. Both miR-93 and miR-143 might be used as potential biomarkers in esophageal squamous cell carcinoma. However, more studies with large population of samples are necessary.

The Lcn2-engineered HEK-293 cells show senescence under stressful condition.

OBJECTIVES: Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and. MATERIALS AND METHODS: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by ß-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. RESULTS: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence. CONCLUSION: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.

Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions.

BACKGROUND: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions. METHODS: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells. RESULTS: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001). CONCLUSION: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

Protective effects of restricted diet and antioxidants on testis tissue in rats fed with high-fat diet.

BACKGROUND: A high-fat diet (HFD) promotes the oxidative stress formation, which in turn has hazardous effects on reproductive system and fertility. The present study examines the potential positive effects of a restricted high-fat diet (RHFD) and antioxidants consumption on sperm parameters and testis tissue in rats. METHODS: Male rats (n = 48) were divided into four groups (12 in each group): control group (Cont), HFD group, RHFD, and RHFD with astaxanthin and vitamins E and C group (RHFDA). After 12 weeks, serum analysis and sperm parameters study were performed. Sections of fixed testes were stained with Hematoxilin and Eosin to study the histological changes. A one-way ANOVA was used to compare the data. RESULTS: HFD fed animals presented significant increase in weight load and serum low density lipoprotein (LDL-C) levels (P < 0.05). The sperm count in RHFD was lower than three other groups (P < 0.05) and sperm motility of RHFDA group was significantly higher than HFD and RHFD groups (P < 0.05). The histological study was showed a significant increase in spermatogonium number in RHFDA compared to three other groups (P < 0.05). The number of spermatocyte I and spermatid in RHFD was significantly (P < 0.05) lower than Cont and HFD groups. CONCLUSION: HFD and obesity can affect sperm parameters and spermatogenesis and antioxidants consumption may improve their quality. Although the RHFD is a benefit way in weight loss and decrease of LDL-C of serum, but it is suggested that is not effective on sperm quality improvement.

Positive selection of Wharton's jelly-derived CD105(+) cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells.

OBJECTIVE: Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. METHODS: CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. RESULTS: WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. DISCUSSION: In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. CONCLUSION: In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.