Extracellular vesicles as a new horizon in the diagnosis and treatment of inflammatory eye diseases: A narrative review of the literature.

Extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Their cargos contain a diverse variety of lipids, proteins, and nucleic acids that are involved in both normal physiology and pathology of the ocular system. Thus, studying extracellular vesicles may lead to a more comprehensive understanding of the pathogenesis, diagnosis, and even potential treatments for various diseases. The roles of extracellular vesicles in inflammatory eye disorders have been widely investigated in recent years. The term "inflammatory eye diseases" refers to a variety of eye conditions such as inflammation-related diseases, degenerative conditions with remarkable inflammatory components, neuropathy, and tumors. This study presents an overview of extracellular vesicles' and exosomes' pathogenic, diagnostic, and therapeutic values in inflammatory eye diseases, as well as existing and potential challenges.

Differentiation of Human-Induced Pluripotent Stem Cells Into Insulin-Producing Clusters by MicroRNA-7.

OBJECTIVES: Diabetes results from inadequate insulin production from pancreatic ß-cells. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all diabetic patients. Thus, finding a new source with effective maturation of ß-cells is the major goal of many studies. MicroRNAs are a class of small noncoding ribonucleic acid that regulate gene expression through posttranscriptional mechanisms. MicroRNA-7 has high expression level during pancreatic islet development in humans, thereby playing a critical role in pancreatic ß-cell function. We study aimed to develop a protocol to differentiate human-induced pluripotent stem cells efficiently into isletlike cell clusters in vitro by using microRNA-7. MATERIALS AND METHODS: Human-induced pluripotent stem cell colonies were transfected with hsa-microRNA-7 by using siPORT NeoFX transfection agent. Total ribonucleic acid was extracted 24 and 48 hours after transfection. The expression of transcription factors which were important during pancreases development was also performed. On the third day, the potency of the clusters was assessed in response to high glucose levels. Diphenylthiocarbazone was used to identify the existence of the ß-cells. The presence of insulin and Neurogenin-3 proteins was investigated by immunocytochemistry. RESULTS: Morphologic changes were observed on the first day after chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The isletlike cell clusters were positive for insulin and Neurogenin-3 proteins in immunocytochemistry. The clusters were stained with Diphenylthiocarbazone and secreted insulin in a glucose challenge test. CONCLUSIONS: MicroRNA-7 transcription factor network is important in pancreatic endocrine differentiation. Chemical transfection with microRNA-7 can differentiate human induced pluripotent stem cells into functional isletlike cell clusters in a short time.

Different Methylation Patterns of RUNX2, OSX, DLX5 and BSP in Osteoblastic Differentiation of Mesenchymal Stem Cells.

OBJECTIVE: Runt-related transcription factor 2 (RUNX2) and osterix (OSX) as two specific osteoblast transcription factors and distal-less homeobox 5 (DLX5) as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein (BSP), osteopontin and collagen type Iα1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells (MSCs). MATERIALS AND METHODS: In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR (MSP). Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction (PCR). RESULTS: MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. CONCLUSION: The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role.

ROR2 Promoter Methylation Change in Osteoblastic Differentiation of Mesenchymal Stem Cells.

OBJECTIVE: Osteoblasts arise from multipotent mesenchymal stem cells (MSCs) present in the bone marrow stroma and undergo further differentiation to osteocytes or bone cells. Many factors such as proteins present in the Wnt signaling pathway affect osteoblast differentiation. ROR2 is an orphan tyrosine kinase receptor that acts as a co-receptor in the non-canonical Wnt signaling pathway. However, ROR2 has been shown to be regulated by both canonical and non-canonical Wnt signaling pathways. ROR2 expression increases during differentiation of MSCs to osteoblasts and then decreases as cells differentiate to osteocytes. On the other hand, research has shown that ROR2 changes MSC fate towards osteoblasts by inducing osteogenic transcription factor OSTERIX. Here we speculated whether ROR2 gene expression regulation during osteoblastogenesis is epigenetically determined. MATERIALS AND METHODS: MSCs from bone marrow were isolated, expanded and characterized in vitro according to standard procedures. ROR2 promoter methylation status was determined using methylation specific PCR in a multipotent state and during differentiation to osteoblasts. RESULTS: We determined that the demethylation process in ROR2 promoter occurs during the differentiation process. The process of demethylation begins at day 8 and continues until 21 days of differentiation. CONCLUSION: This result is in concordance with previous works on the role of ROR2 on osteoblast differentiation, which have shown an upregulation of ROR2 expression during this process.