Cryopreservation of Iranian Markhoz goat fibroblast cells as an endangered national genetic resource.

BACKGROUND: The continuous accessibility of local animals for sustainable use is being eroded annually. Thus, a strategic vision for the conservation of biodiversity is of far-reaching emphasis to deal with unprecedented challenges in the local population extension facing in the future. This study aimed to establish and cryopreserve endangered Markhoz goat (Capra hircus) fibroblast cell lines in vitro. METHODS AND RESULTS: These primary fibroblast cells were isolated from 58 Iranian Markhoz goats and individually cultured by explant technique in DMEM medium supplemented with 10% FBS and 2 mM L-Glutamine, in the presence of Penicillin (200 U/ml)-Streptomycin (200 mg/ml) during the first passage number. The extracted cell lines were confirmed morphologically as fibroblast cells. The population doubling time for DMEM-cultured cells was 23 ± 0.5 h. Chromosomal analysis indicated a total chromosome number of 2n = 60 with > 95% frequency. The cultured cells were checked for bacteria, fungi, yeast, and mycoplasma contaminations and the results were reported negative. The efficiencies of the fluorescent protein encoded by VSV-G (pMDG) and lentiviral pCSGW vectors reported in a range of 65% value. According to the species identification analysis, the goat cell lines were banked and confirmed without any miss- and cross-contamination. CONCLUSIONS: The significant issue in this paper can be concluded about the first report of the establishment of endangered Markhoz goat cell banking inside the country. This study demonstrated the successful establishment of a genetically stable fibroblast bank as a valuable genetic resource for the endangered Iranian Markhoz goat breed.

The extracellular vesicles-derived from mesenchymal stromal cells: A new therapeutic option in regenerative medicine.

Mesenchymal stem cells (MSCs) are adult multipotent cells that due to their ability to homing to damaged tissues and differentiate into specialized cells, are remarkable cells in the field of regenerative medicine. It's suggested that the predominant mechanism of MSCs in tissue repair might be related to their paracrine activity. The utilization of MSCs for tissue repair is initially based on the differentiation ability of these cells; however now it has been revealed that only a small fraction of the transplanted MSCs actually fuse and survive in host tissues. Indeed, MSCs supply the microenvironment with the secretion of soluble trophic factors, survival signals and the release of extracellular vesicles (EVs) such as exosome. Also, the paracrine activity of EVs could mediate the cellular communication to induce cell-differentiation/self-renewal. Recent findings suggest that EVs released by MSCs may also be critical in the physiological function of these cells. This review provides an overview of MSC-derived extracellular vesicles as a hopeful opportunity to advance novel cell-free therapy strategies that might prevail over the obstacles and risks associated with the use of native or engineered stem cells. EVs are very stable; they can pass the biological barriers without rejection and can shuttle bioactive molecules from one cell to another, causing the exchange of genetic information and reprogramming of the recipient cells. Moreover, extracellular vesicles may provide therapeutic cargo for a wide range of diseases and cancer therapy.

Simple and fast multiplex PCR method for detection of species origin in meat products.

Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I (COX1) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

Extracellular Vesicles as a Neprilysin Delivery System Memory Improvement in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative brain disorder which has no effective treatment yet due to the blood barrier in the brain that limits the drugs with the potential of disease improvement. Extracellular vesicles (EVs) are biocompatible nanoparticles with a lipid membrane. These vesicles are secreted from various cells such as mesenchymal stem cells (MSCs) and can pass through biological barriers for transfer of information such as signals or be used as carriers for various proteins like Neprilysin (NEP). NEP is an active enzyme in the clearance of abnormal aggregated beta-amyloid sheets in the brain. In the present study, we used EVs to carry NEP for memory improvement in Alzheimer's disease. For this purpose, bone marrow MSCs were isolated from rat femur. Stemness evaluation of established cells was characterized by differentiation potency and specific markers with flowcytometry. EVs were isolated from MSCs supernatant by ultracentrifugation and analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS) and western blotting. EVs were loaded with NEP by freeze-thaw cycle and then administrated intranasally in a rat model of the AD for 14 days. Our findings showed EV-loaded NEP caused a decrease in IL-1beta and also BAX but an increase in BCL2 expression level in the rat brain. Altogether, these data showed that EV-loaded NEP can improve brain-related behavioural function which may be mediated through the regulation of inflammation and apoptosis. These findings suggest that EV-loaded NEP can be considered as a potential drug delivery system for the improvement of AD.

Establishment and Preservation of Lymphoblastoid Cell Lines from Fresh and Frozen Whole Blood and Mononuclear Cells.

Although blood cells are interesting sources for genome investigations, one of the main problems in obtaining genomic DNA from blood is the restricted amount of DNA. This obstacle can be avoided by generating Epstein-Barr virus (EBV)-induced B cell lines. This study investigates the efficiency of four different methods to generate lymphoblastoid cell lines (LCLs). Blood samples (n = 120) were obtained from donors and categorized into four groups: fresh whole blood, frozen whole blood, fresh peripheral blood mononuclear cells (PBMCs), and frozen PBMCs. The samples were followed by EBV transformation to generate LCLs. Quality control and authentication of the cells were performed using multiplex PCR and short tandem repeat (STR) analyses. Finally, we assessed the success rate and amount of time to establish the cell lines in each group. The results showed that the cells were not contaminated nor were they misidentified or cross-contaminated with other cells. The success rate of LCLs generated from the whole blood groups was lower than the PBMC groups. The freezing procedures did not have any considerable effect on the establishment of lymphoblastoid cells. These established cells have been preserved in the human and animal cell bank of the Iranian Biological Resource Center (IBRC) and are available for researchers. Due to the management and transformation of a substantial number of blood samples, we recommend that researchers freeze PBMCs for further use with high efficiency and time-saving. We suggest that whole fresh blood should be directly transformed when the volume of the blood sample is less than 0.5 ml.

A systematic study of the function of the human beta-globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells.

BACKGROUND: Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human beta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. METHODS: Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. RESULTS: All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. CONCLUSIONS: Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.

Potential of Extracellular Vesicles in Neurodegenerative Diseases: Diagnostic and Therapeutic Indications.

Extracellular vesicles (EVs) are membrane-bound vesicles, including exosomes and microvesicles. EVs are nanometer sized, found in physiological fluids such as urine, blood, cerebro-spinal fluid (CSF), with a capacity of transferring various biological materials such as microRNAs, proteins, and lipids among cells without direct cell-to-cell contact. Many cells in the nervous system have been shown to release EVs. These vesicles are involved in intercellular communication and a variety of biological processes such as modulation of immune response, signal transduction, and transport of genetic materials with low immunogenicity; therefore, they have also been recently investigated for the delivery of therapeutic molecules such as siRNAs and drugs in the treatment of diseases. In addition, since EV components reflect the physiological status of the cells and tissues producing them, they can be utilized as biomarkers for early detection of various diseases. In this review, we summarize EV application, in diagnosis as biomarker sources and as a carrier tool for drug delivery in EV-based therapies in neurodegenerative diseases.

Establishment and characterization of rough-tailed gecko original tail cells.

Some of lizard species have the ability to lose their tail in order to defend against predators and regenerate the new tail. Lizard's regenerated tail has attracted scientists' attention for unraveling the regeneration process, but less information is known about the cellular characterization and cell growth properties of original tail. This research aimed to report cell culture and banking process of rough-tailed gecko or Cyrtopodion scabrum's original tail cell sample from inner tissue without skin using tissue explant technique. For banking reports, it is essential to analyze this cells' potential to proliferate, to investigate biological aspects such as cell culture features, differentiation and chromosome number and to report its species identification and quality control. To achieve optimal growth conditions, three different temperatures for incubation including 18, 23 and 37 °C and two different media including DMEM and L-15 were applied. The expanded cells were studied for their potential to adipose and osteoblast differentiation. Results indicated that lizard's original tail cells could be successfully obtained by explant technique. The cells demonstrated fibroblast like morphology with population doubling times of approximately 24 ± 0.5 h. Karyotyping analysis showed a distribution of 2n = 40 chromosome number for this cell line. The comparison of different incubation media and temperatures showed that cell growth is equally optimal in all mentioned conditions according to growth curves. Adipose and osteoblast differentiation was obviously observed in these cells which confirms the hint of stem-ness in the produced mixed cells. According to cell banking policies, produced cells were also checked for bacterial, fungal, yeast and mycoplasma contaminations and no contamination was observed. Multiplex PCR for identification of species confirmed the species of lizard with no cross-contamination with other cells in the cell bank. Establishment of authenticated and well-characterized lizard's original tail cell line will provide a valuable source for subsequent in vitro regenerative research and molecular studies which are not feasible in in vivo methods. This finding will allow us to get an opportunity to create and preserve a new collection of lizard cell lines in the future.

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23 ± 0.5 h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

Establishment and Characterization of Primary Cultures from Iranian Oral Squamous Cell Carcinoma Patients by Enzymatic Method and Explant Culture.

OBJECTIVES: Oral Squamous Cell Carcinoma (OSCC) is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population. MATERIALS AND METHODS: The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS) methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR) profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers. RESULTS: Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis. CONCLUSIONS: Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.