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Introduction: The goal of the study described here, was to investigate the potential of umbilical cord derived mesenchymal stem cell (UC-MSCs) into hepatocyte like cells in a sequential 2D and 3D differentiation protocols as alternative therapy. Methods: Mesenchymal stem cells (MSCs) were isolated from the umbilical cord (UC) and CD markers were analyzed by flow cytometry. For hepatic differentiation of UC-MSCs, cells were induced with a sequential 4-step protocol in 3D and 2D culture system. Urea concentration and albumin secretion into the culture medium was quantified by ELISA. Gene expression levels of AFP, ALB, and CK18 were determined by RT-PCR. Data were statistically analyzed by the SPSS software. The difference between the mean was considered significant when p < 0.05. Results: Growth factor dependent morphological changes from elongated fibroblast-like cells to round epithelial cell morphology were observed in 2D culture. Cell proliferation analysis showed round-shaped morphology with clear cytoplasm and nucleus on the alginate scaffold in 3D culture. The mean valuses of albumin production and urea secretion were significantly higher in the 3D Culture system when compared with the 2D culture (p = 0.005 vs p = 0.001), respectively. Treatment of cells with TSA in the final step of differentiation induced an increased expression of CK18 and a decreased expression of αFP in both the 3D and 2D cultures (p = 0.026), but led to a decreased albumin gene expression, and an increased expression in the 2D culture (p = 0.001). Conclusion: Findings of the present study indicated that sequential exposure of UC-MSCs with growth factors in 3D culture improves hepatic differentiation.

Objective: Vitamin D receptor (VDR) is expressed in human spermatozoa. However, the role of vitamin D (VD) in human male reproduction has not yet been clarified. In this study, effects of VD on sperm parameters and its apoptosis in asthenozoospermic and healthy men were evaluated. Methods: The study was carried out on discharged semen samples of 80 asthenozoospermic and healthy men. The samples were divided into control and experimental groups (received 20 µMol of VD). This study assessed sperm motility using the Makler chamber, their morphology by Diff quick, apoptosis and necrosis by Annexin-V and TUNEL assays, and their chromatin integrity was assessed by Aniline blue and Toluidine blue staining, according to WHO guidelines. Results: The results revealed that: 1) the total number of motile sperms was increased by VD in both groups, but it was only significant in the asthenozoospermia group. 2) The progressive motility was increased with significant difference in both groups.3) Morphology of sperm did not show any changes due to VD in any of the groups. 4) Early apoptosis and necrosis of sperms were reduced in both groups, but the results of late apoptosis showed no statistical difference in these groups. 5) The percentage of positive toluidine blue was significantly decreased after using VD in the asthenozoospermia group. Conclusion: VD could improve motility, early apoptosis, and sperm necrosis, especially in asthenozoospermic men and it could be used for therapeutic opportunities.

Pancreatic and duodenal homeobox 1 (Pdx1) and Sonic hedgehog (Shh) are the key regulators of beta‐cell function. In vitro experiments have shown that there is significant cooperation between Pdx1 and Shh with regard to the production and maintenance of insulin‐producing cells ( IPC s). In this study, the combined effect of Pdx1 overexpression and Shh manipulation on the function of adipose tissue‐derived IPC s was determined. A eukaryotic expression vector ( Pdx1‐ pCDNA 3.1(+)) was constructed and transfected into a Chinese hamster ovary ( CHO ) cell line. Adipose tissue‐derived mesenchymal stem cells ( ADMSC s) obtained from rats were assigned to two groups [control (C) and manipulated (M)] and differentiated into IPC s. Manipulated cells were treated with a mixture of FGF ‐β and cyclopamine and recombinant Shh protein at days 3 and 11, respectively, and transfected with Pdx1‐ pCDNA 3.1(+) at day 10. The expression of multiple genes related to function of beta cells was analyzed using real‐time PCR . The functionality of IPC s in vitro was analyzed through dithizone ( DTZ ) staining and ELISA . IPC s were injected into the tail vein of diabetic rats, and blood glucose and insulin concentrations were measured. CHO cells transfected with Pdx1‐ pCDNA 3.1(+) showed a significantly higher expression of Pdx1 compared with nontransfected cells. Manipulated IPC s exhibited a significantly higher expression of MafA, Nkx2.2, Nkx6.1, Ngn3, insulin , and Isl1 and a higher insulin secretion in response to glucose challenge in relation to control cells. Rats that received manipulated IPC s exhibited a higher ability to normalize blood glucose and insulin secretion when compared to controls. Our protocol might be used for more efficient cell therapy of patients with diabetes in the future.

Inactivation of tumor suppressor genes, such as <i>RAP1GAP</i>, by hypermethylation of their regulatory region can give rise to thyroid tumors. The aim of this study was to investigate the expression of the <i>RAP1GAP</i> gene and the DNA methylation patterns of its CpG74_a, CpG74_b, and CpG24 in an Iranian population with differentiated thyroid cancer (DTC). In this study, 160 individuals who underwent thyroidectomy in the Tehran Erfan Hospital between 2018 and 2020 were selected. DNA methylation patterns of selected CpG islands (CpG74_a, CpG74_b, and CpG24) were determined using methylation-specific PCR. The mRNA expression and protein level of ­RAP1GAP were also evaluated. SW1736 and B-CPAP cells were treated with 5-aza-2′-deoxycytidine (5-Aza) to demethylate these regions. The hypermethylation rates of CpG74_a and CpG24 in DTC samples were significantly higher than in the control. The mRNA expression and protein level of ­RAP1GAP were significantly decreased in the DTC group. In the DTC group, hypermethylation in CpG74_a was correlated with decreasing <i>RAP1GAP</i> expression (<i>R</i>²: 0.34; <i>p</i> = 0.043). CpG74_a with a specificity of 86.4% has significant prediction power to distinguish between DTC and normal thyroid tissues. Additionally, hypermethylation of CpG74_a was significantly associated with higher tumor stages (stage III-IV: 77%; stage I-II: 23%; <i>p</i> = 0.012). Increasing expression of <i>RAP1GAP</i> after demethylation with 15 µM of 5-Aza was observed in both cell lines. These results indicate that DNA hypermethylation in CpG74_a can be considered as an epigenetic biomarker in DTC.

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