Adipose-derived human mesenchymal stem cells seeded on denuded or stromal sides of the amniotic membrane improve angiogenesis and collagen remodeling and accelerate healing of the full-thickness wound.

Several strategies have been proposed to enhance wound healing results. Along with other forms of wound dressing, the human amniotic membrane (HAM) has long been regarded as a biological wound dressing that decreases infection and enhances healing. This study investigates the feasibility and effectiveness of wound healing using decellularized HAM (dAM) and stromal HAM (sAM) in combination with adipose-derived human mesenchymal stem cells (AdMSCs). The dAM and sAM sides of HAM were employed as wound dressing scaffolds, and AdMSCs were seeded on top of either dAM or sAM. Sixty healthy Wistar rats were randomly divided into three groups: untreated wound, dAM/AdMSCs group, and sAM/AdMSCs group. The gene expression of VEGF and COL-I was measured in vitro. Wound healing was examined after wounding on days 3, 7, 14, and 21. The expression level of VEGF was significantly higher in sAM/AdMSCs than dAM/AdMSCs (P ≤ 0.05), but there was no significant difference in COL-I expression (P ≥ 0.05). In vivo research revealed that on day 14, wounds treated with sAM/AdMSCs had more vascularization than wounds treated with dAM/AdMSCs (P ≤ 0.01) and untreated wound groups on days 7 (P ≤ 0.05) and 14 (P ≤ 0.0001), respectively. On days 14 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), and 21 (P < 0.05 for sAM/AdMSCs, P < 0.01 for dAM/AdMSCs), the collagen deposition in the wound bed was significantly thicker in the sAM/AdMSCs and dAM/AdMSCs groups compared to untreated wounds. The study demonstrated that the combination of sAM and AdMSCs promotes wound healing by enhancing angiogenesis and collagen remodeling.

Post-Weaning Exposure to Sunset Yellow FCF Induces Changes in Testicular Tight and Gap Junctions in Rats: Protective Effects of Coenzyme Q10.

Studies on adverse health consequences of azo dyes are limited and conflicting. Coenzyme Q10 (CoQ10) supplementation has been shown to have benefits associated with antioxidant and anti-inflammatory characteristics on several body systems. This work investigates the possible toxic effects of the widely used food additive sunset yellow and the probable protective effects of CoQ10 on testicular tight and gap junctions in rats by assessing molecular, immunohistochemical, and histopathological changes. Sixty Sprague-Dawley male weanling rats were randomly divided into six groups (n = 10). The rats received their treatments via daily oral gavages for 6 weeks. The treatments included as follows: low dose of sunset yellow (SY-LD) (2.5 mg/kg/day), high dose of sunset yellow (SY-HD) (70 mg/kg/day), CoQ10 (10 mg/kg/day), CoQ10 with low dose of sunset yellow (CoQ10 + LD), CoQ10 with high dose of sunset yellow (CoQ10 + HD), and distilled water as the control treatment. At the end of the experiment, the rats were anesthetized, and the testes were removed for molecular (real-time quantitative PCR), immunohistochemical, and histopathological (H & E staining) assessments. Claudin 11 and occludin gene expression significantly decreased in HD and CoQ10 + HD groups compared with the controls. Connexin 43 (Cx43) expression in the control and CoQ10 groups was significantly higher than in the HD group. The immunohistochemical and histopathological data were largely in line with these findings. The results showed that exposure to a high dose of sunset yellow led to disturbances in cell-to-cell interactions and testicular function. Simultaneous treatment with CoQ10 had some beneficial effects but did not completely improve these undesirable effects.

Comparing the Effects of Two Cryoprotectant Protocols, Dimethyl-Sulfoxide (DMSO) and Glycerol, on the Recovery Rate of Cultured Keratinocytes on Amniotic Membrane.

Background: Off-the-shelf supply of viable engineered tissue is critical for effective and fast treatment of life-threatening injuries such as deep burns. An expanded keratinocyte sheet on the human amniotic membrane (KC sheet-HAM) is a beneficial tissue-engineering product for wound healing. To access an on-hand supply for the widespread application and overcome the time-consuming process, it is necessary to develop a cryopreservation protocol that guarantees the higher recovery of viable keratinocyte sheets after freeze-thawing. This research aimed to compare the recovery rate of KC sheet-HAM after cryopreservation by dimethyl-sulfoxide (DMSO) and glycerol. Methods: Amniotic membrane was decellularized with trypsin, and keratinocytes were cultured on it to form a multilayer, flexible, easy-to-handle KC sheet-HAM. The effects of 2 different cryoprotectants were investigated by histological analysis, live-dead staining, and proliferative capacity assessments before and after cryopreservation. Results: KCs well adhered and proliferated on the decellularized amniotic membrane and successfully represented 3 to 4 stratified layers of epithelialization after 2 to 3 weeks culture period; making it easy to cut, transfer, and cryopreserve. However, viability and proliferation assay indicated that both DMSO and glycerol cryosolutions have detrimental effects on KCs, and KCs-sheet HAM could not recover to the control level after 8 days of culture post-cryo. The KC sheet lost its stratified multilayer nature on AM, and sheet layers were reduced in both cryo-groups compared to the control. Conclusion: Expanding keratinocytes on the decellularized amniotic membrane as a multilayer sheet made a viable easy-to-handle sheet, nonetheless cryopreservation reduced viability and affected histological structure after thawing. Although some viable cells were detectable, our research highlighted the need for a better cryoprotectant protocol other than DMSO and glycerol, specific for the successful banking of viable tissue constructs.

An Update on Biochemical and Genomic Markers for Prostate Cancer.

PURPOSE: Detecting prostate cancer, developing therapeutic plans after negative biopsies, and prognosis-based patient counseling can be challenging for many urologists dealing with prostate cancer-specific antigens. New Biomarkers advances made improvement for prediction of responses to therapeutic option and can tell us about survival and recurrence. In this review, we have assessed current and upcoming biomarkers that are opening a new era in diagnosing the disease. MATERIALS AND METHODS: We conducted a comprehensive literature review of studies describing prostate cancer biomarkers. Two independent investigators searched PubMed, Embase, Web of Science, and Cochrane Databases to identify biomarkers in prostate cancer conducted a literature review. RESULTS: Recently, combining prostate cancer-specific biomarkers into a single test has gained increasing attention, especially since the introduction of genomic and molecular tools. The development of the Prostate Health Index (PHI), SelectMDx, and Confirm MDx have shown promising results for prostate cancer detection, in addition to risk stratification and biopsy avoidance. CONCLUSION: Despite major improvements and innovations in prostate cancer biomarkers, application in current clinical practice is limited. However, these biomarkers have an important role in determining risk, preventing unnecessary prostate biopsies, and predicting prognoses. Additional confirmatory studies will be needed to fully understand the impact of prostate cancer-specific biomarkers.

Regeneration and Repair of Skin Wounds: Various Strategies for Treatment.

Skin as a mechanical barrier between the inner and outer environment of our body protects us against infection and electrolyte loss. This organ consists of 3 layers: the epidermis, dermis, and hypodermis. Any disruption in the integrity of skin leads to the formation of wounds, which are divided into 2 main categories: acute wounds and chronic wounds. Generally, acute wounds heal relatively faster. In contrast to acute wounds, closure of chronic wounds is delayed by 3 months after the initial insult. Treatment of chronic wounds has been one of the most challenging issues in the field of regenerative medicine, promoting scientists to develop various therapeutic strategies for a fast, qualified, and most cost-effective treatment modality. Here, we reviewed more recent approaches, including the development of stem cell therapy, tissue-engineered skin substitutes, and skin equivalents, for the healing of complex wounds.

MicroRNA 155 Downregulation by Vitamin C-Loaded Human Serum Albumin Nanoparticles During Cutaneous Wound Healing in Mice.

This study focused on potential of vitamin C loaded human serum albumin (HSA) nanoparticles for treatment of wound. Nanocarrier were prepared and assessed for their effect on growth of 3T3 fibroblast cells, cell migration, wound healing rate and expression of miR-155, TGF-ß1 and SMAD 1,2 genes. Wound healing assay was done and wounds were treated with vitamin C loaded HSA nanoparticles. Nanoparticles were prepared with size and zeta potential of 180±6 and -29 mV, respectively. Vitamin C loaded HSA nanoparticles showed controlled release of vitamin C into the buffer solution. Also, yield and encapsulation efficacy of loaded nanoparticles were obtained as 70.6 and 52.1 %, respectively. MTT results showed that the growth of 3T3 fibroblast cells was promoted in culture medium with 20 µg/ml of vitamin C loaded HSA nanoparticles. Cell migration assay indicated the positive effect of loaded nanoparticles on wound healing. The in-vivo results showed that the rate of wound healing was increased after treatment with 20 µg/ml of vitamin C loaded HSA nanoparticles. The wounds were healed faster when treated with vitamin C loaded HSA nanoparticles in comparison with control group. The expression of miR-155 was downregulated after treatment. Furthermore, expression of TGF-ß1 and SMAD 1,2 were increased while the wounds were treated with these nanoparticles. In conclusion, these results showed for the first time that wounds were healed after treatment with albumin nanocarrier loaded with vitamin C. This nanocarrier changed expression of miR-155 and TGF-ß1 towards faster healing of wounds.

Investigation of antimicrobial effects of treated Lucilia sericata larvae extract on bacteria.

BACKGROUND AND OBJECTIVES: Larval therapy refers to the use of Lucilia sericata larvae on chronic wounds, which is a successful method of chronic wounds treatment. The secretions of these larvae contain antibacterial compounds and lead to death or inhibition of bacterial growth. MATERIALS AND METHODS: In this study, we investigated the antibacterial effects of Lucilia sericata larvae secretions which were in sterilized and multi antibiotic-resistant bacteria-treated forms on Gram-positive Bacillus subtilis bacteria and Gram-negative Escherichia coli bacteria. In the following, we evaluated changes in gene expression of lucifensin and attacin during treatment with multi antibiotic-resistant bacteria. Investigation of the antibacterial effect was carried out using optical absorption and antibiotic disk diffusion in order to study the expression of the aforementioned genes. RESULTS: The results of this study showed that E. coli-treated larvae were able to inhibit the growth of E. coli and secretions of B. subtilis-treated larvae and were also able to inhibit the growth of B. subtilis. Gene expression of antibacterial peptides in multi antibiotic-resistant bacteria-treated larvae was increased in comparison to non-treated larvae. CONCLUSION: Due to the significant antibacterial potency of bacteria-treated larvae secretions, the secretions can be a suitable candidate as a drug against antibiotic resistant bacteria, but additional tests are required. Since the antimicrobial peptides of insects have not yet produced any resistance in human pathogenic bacteria, they can be considered as a promising strategy for dealing with resistant infections.

Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro.

Although chicken spermatogonial stem cells (SSCs) have received considerable attention in recent years, only a few studies so far have focused on their derivation and characterization in vitro. Identification of specific molecular biomarkers and differentiation capacity of chicken SSCs would not only help us to understand cell and molecular biology of these cells, but also can contribute to their applications in biotechnology. In this regard, we found that colony-forming cells (SSCs) in newborn chicken testicular cell cultures were positive for alkaline phosphatase activity and also expressed specific markers including DAZL, STRA-8, CVH, PLZF, SPRY-1, GFRα1, GDNF, POU5F1, NANOG, GPR125, THY-1, c-KIT, and BCL6B, at mRNA level. Moreover, these cells expressed POU5F1 and GPR125 proteins as reliable intracellular and cell surface markers, respectively; whereas they were negative for SSEA-1. Furthermore, we showed that newborn chicken colony-forming cells had spermatogenesis potential and thus could be produced sperm-like cells in a three-dimensional matrix in vitro. In conclusion, this study reports novel insights into the molecular signature of newborn chicken SSCs in comparison with mammalian SSCs and for the first time we report a successful protocol for in vitro spermatogenesis and thus production of sperm-like cells from newborn chicken testicular cell cultures.

Construction and quantitative evaluation of a dual specific promoter system for monitoring the expression status of Stra8 and c-kit genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

A simple method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are expected to participate in male infertility therapy, endangered species preservation, and transgenic animal technology by their unique unipotency to differentiate into spermatozoa. The main challenges, however, remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive, culture, and maintain them in vitro. In the present study, the testicular tissues were isolated from 1-d-old male chickens to establish primary cell cultures. This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase (AP) activity assay and gene expression analysis. They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcription-polymerase chain reaction. These were indications of carrying characteristics of SSCs by these colonies. The cultures were also exposed to different concentrations of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) growth factors to seek optimum colony-forming conditions. Colony-forming activity assay indicated that they were able to propagate in vitro with an increased self-renewal property when cultured in the presence of 15 ng/mL of GDNF, 20 ng/mL of bFGF, and 15 ng/mL of LIF. The present work provides an easy and practical method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their self-renewal property based on supplying the necessary growth factors.

Human adipose-derived mesenchymal stem cells can survive and integrate into the adult rat eye following xenotransplantation.

BACKGROUND: Novel threads of discovery provide the basis for optimism for the development of a stem-cell-based strategy for the treatment of retinal blindness. Accordingly, achievement to suitable cell source with potential-to-long-term survival and appropriate differentiation can be an effective step in this direction. METHODS: After derivation of human adipose-derived mesenchymal stem cells (HAD-MSCs), they were stably transfected with a vector containing Turbo-green fluorescent protein (GFP) and JRed to be able to trace them after transplantation. Labeled HAD-MSCs were transplanted into the intact adult rat eye and their survival, integration, and migration during 6 months post-transplantation were assessed. RESULTS: The transplanted cells were traceable in the rat vitreous humor (VH) up until 90 days after transplantation, with gradual reduction in numbers, their adhesion and expansion capacity after recovery. These cells were also integrated into the ocular tissues. Nonetheless, some of the implanted cells succeeded to cross the blood-retina barrier (BRB) and accumulate in the spleen with time. CONCLUSIONS: The survival of the HAD-MSCs for a period of 90 days in VH and even longer period of up to 6 months in other eye tissues makes them a promising source to be considered in regenerative medicine of eye diseases. However, the potency of crossing the BRB by the implanted cells suggests that use of HAD-MSCs must be handled with extreme caution.