Direct Reprogramming of Fibroblasts to Osteoblasts: Techniques and Methodologies.

Direct reprogramming (DR) is an emerging technique that can be applied to convert fibroblasts into osteoblast-like cells, promoting bone formation and regeneration. We review the current methodology of DR in relation to the creation of induced osteoblasts, including a comparison of transcription factor-mediated reprogramming and nontranscription factor-mediated reprogramming. We review the selection of reprogramming factors and delivery systems required. Transcription factor cocktails, such as the RXOL cocktail (Runx2, Osx, OCT3/4, and L-MYC), have shown promise in inducing osteogenic differentiation in fibroblasts. Alterations to the original cocktail, such as the addition of Oct9 and N-myc, have resulted in improved reprogramming efficiency. Transcription factor delivery includes integrative and nonintegrative systems which encompass viral vectors and nonviral methods such as synthetic RNA. Recently, an integrative approach using self-replicating RNA has been developed to achieve a longer and more sustained transcription factor expression. Nontranscription factor-mediated reprogramming using small molecules, proteins, inhibitors, and agonists has also been explored. For example, IGFBP7 protein supplementation and ALK5i-II inhibitor treatment have shown potential in enhancing osteoblast reprogramming. Direct reprogramming methods hold great promise for advancing bone regeneration and tissue repair, providing a potential therapeutic approach for fracture healing and the repair of bone defects. Multiple obstacles and constraints need to be addressed before a clinically significant level of cell therapy will be reached. Further research is needed to optimize the efficiency of the reprogramming cocktails, delivery methods, and safety profile of the reprogramming process.

Therapeutic Effects of Mesenchymal Stem Cells Expressing Erythropoietin on Cancer-Related Anemia in Mice Model.

BACKGROUND: Cancer-related anemia (CRA) negatively influences cancer patients' survival, disease progression, treatment efficacy, and quality of life (QOL). Current treatments such as iron therapy, red cell transfusion, and erythropoietin-stimulating agents (ESAs) may cause severe adverse effects. Therefore, the development of long-lasting and curative therapies is urgently required. OBJECTIVE: In this study, a cell and gene therapy strategy was developed for in vivo delivery of EPO cDNA by way of genetic engineering of human Wharton's jelly mesenchymal stem cells (hWJMSCs) to produce and secrete human EPO protein for extended periods after transplantation into the mice model of CRA. METHODS: To evaluate CRA's treatment in cancer-free and cancerous conditions, first, a recombinant breast cancer cell line 4T1 which expressed herpes simplex virus type 1 thymidine kinase (HSV1-TK) by a lentiviral vector encoding HSV1-TK was developed and injected into mice. After three weeks, all mice developed metastatic breast cancer associated with acute anemia. Then, ganciclovir (GCV) was administered for ten days in half of the mice to clear cancer cells. Meanwhile, another lentiviral vector encoding EPO to transduce hWJMSCs was developed. Following implantation of rhWJMSCs-EPO in the second group of mice, peripheral blood samples were collected once a week for ten weeks from both groups. RESULTS: Analysis of peripheral blood samples showed that plasma EPO, hemoglobin (Hb), and hematocrit (Hct) concentrations significantly increased and remained at therapeutic for >10 weeks in both treatment groups. CONCLUSION: Data indicated that rhWJMSCs-EPO increased the circulating level of EPO, Hb, and Hct in both mouse subject groups and improved the anemia of cancer in both cancer-free and cancerous mice.

Overexpression Effects of miR-424 and BMP2 on the Osteogenesis of Wharton's Jelly-Derived Stem Cells.

Recently, the translational application of noncoding RNAs is accelerated dramatically. In this regard, discovering therapeutic roles of microRNAs by developing synthetic RNA and vector-based RNA is attracting attention. Here, we studied the effect of BMP2 and miR-424 on the osteogenesis of Wharton's jelly-derived stem cells (WJSCs). For this purpose, human BMP2 and miR-424 DNA codes were cloned in the third generation of lentiviral vectors and then used for HEK-293T cell transfection. Lentiviral plasmids contained miR424, BMP-2, miR424-BMP2, green fluorescent protein (GFP) genes, and helper vectors. The recombinant lentiviral particles transduced the WJSCs, and the osteogenesis was evaluated by real-time PCR, Western blot, Alizarin Red staining, and alkaline phosphatase enzyme activity. According to the results, there was a significant increase in the expression of the BMP2 gene and secretion of Osteocalcin protein in the group of miR424-BMP2. Moreover, the amount of dye deposition in Alizarin Red staining and alkaline phosphatase activity was significantly higher in the mentioned group (p < 0.05). Thus, the current study results clarify the efficacy of gene therapy by miR424-BMP2 vectors for bone tissue engineering. These data could help guide the development of gene therapy-based protocols for bone tissue engineering.

A gene-based anti-angiogenesis therapy as a novel strategy for cancer treatment.

Angiogenesis-targeted therapy of cancer is considered a promising strategy for therapeutic management of cancer progression. Over the last two decades, a few anti-angiogenesis monoclonal antibodies (mAbs) blocking VEGF signaling have been developed and approved by the FDA. The most widely used anti-angiogenesis drug is bevacizumab which binds VEGFA and prevents its interaction with VEGF receptor leading to suppression of angiogenesis. Despite the remarkable success in development of angiogenesis inhibitory mAbs, their clinical application is limited by the high-cost of mAbs-based regimen which includes multiple doses of mAbs due to their short biological half-life. Antibody gene therapy is an alternative system of antibody production. In this study, we have developed a gene-based anti-VEGF mAb system which is expected to produce a high concentration of anti-VEGFA mAb upon a single administration in cancer patients. The full-length cDNA bevacizumab light and heavy chains joint with T2A sequence were cloned in pCDH lentivirus vector. The lentiviral particles expressing bevacizumab was produced in HEK-293T cells. Recombinant lentiviral particles containing bevacizumab (rLV-bev) efficiently transduced HEK-293cells and produced functional bevacizumab mAb. Bevacizumab expression in the transduced cell was assessed by qRT-PCR and western blot at both the mRNA and protein level, respectively. The functionality of the recombinant bevacizumab was confirmed using the tube formation assay in the co-culture system of endothelial cells and HT-29cells transduced with rLV-bev viral particles. Our results show that rLV-bev gene therapy can be useful for angiogenesis-targeted therapy of cancer.

Therapeutic targeting of angiogenesis molecular pathways in angiogenesis-dependent diseases.

Angiogenesis is a critical step in the progression of almost all human malignancies and some other life-threatening diseases. Anti-angiogenic therapy is a novel and effective approach for treatment of angiogenesis-dependent diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. In this article, we will review the main strategies developed for anti-angiogenic therapies beside their clinical applications, the major challenges, and the latest advances in the development of anti-angiogenesis-based targeted therapies.

Evaluating the role of silver nanoparticles on acrosomal reaction and spermatogenic cells in rat.

BACKGROUND: Nanoparticles have wide range of application while there are some reports regarding their probable effects on male reproductive system and spermatozoa. OBJECTIVE: The aim of this study was to evaluate the effect of different doses of silver nanoparticles (AgNPs) (70nm) on acrosome of rat spermatozoa and number of spermatogenic cells. MATERIALS AND METHODS: In this experimental study, in experimental group, 32 male wistar rats (8 rats/group) received oral feeding AgNPs every 12 hr in one spermatogenesis period (48 days) by means of gavages in 25, 50 , 100 and 200 mg/kg concentration (experimental groups 1-4, respectively). The control group (8 rats) was treated on schedule with distilled water. Spermatozoa were stained by triple staining protocol for acrosome reaction. Histological evaluation on testis sections was performed using tissue processing and hematoxylin-eosin (H&E) staining. RESULTS: There was significant difference between the control group and the experimental group 1 for acrosome reaction (11.00±0.00 and 24.25±3.68, respectively, p=0.01). There was only significant reduction in spermatogonia cells in experimental group 4. Experimental groups 2, 3 and 4 showed a significant reduction in the number of primary spermatocytes and spermatids as well as spermatozoa. But there were no significant differences between different groups for Sertoli cell number and seminiferous tubule diameter. CONCLUSION: It seems that Ag NPs have acute and significant effects on spermatogenesis and number of spermatogenic cells and also on acrosome reaction in sperm cells. More experimental investigations are necessary to elucidate better conclusion regarding the safety of nanoparticles on male reproduction system.

An appropriate plot area for analyzing canopy cover and tree species richness in Zagros forests.

In order to make the sampling procedure more efficient and more accurate to study the tree species richness and canopy cover, the appropriate plot size was calculated in the this study. The sampling was carried out using 48 four-hectare plots, each with 13 sub-plots of different plot sizes and 7 one-hectare plots, each with 7 sub-plots. The result of this study showed that 300 ARE plot size was determined as the best area for 1-5% density class, 125 ARE plots for 5-10% class, 150 ARE for 10-25% class, 100 ARE for 25-50% class and 75 ARE plot size to sample >50% density class, in 95% confidence level. Consequently, using 100 ARE sampling plots is suggested for all density classes in central Zagros forests.