RESUMEN
Objective: The present study compared the efficiency of three mouse ovarian tissue culture methods - hanging drop, under mineral oil, and on the insert with regards to improving in vitro ovarian follicular development
Methods: Ovaries from 7-day-old old female NMRI mice sacrificed by cervical dislocation were collected and cultured in alpha-MEM medium supplemented with 5% fetal bovine serum for 7 days in 3 groups [hanging drop, under mineral oil and on the insert]. We evaluated and compared the morphology and surface area of the ovaries and percentage of normal follicles in all groups. After the 7-day culture, the ovaries cultured on the insert showed better growth compared to the other groups. Their preantral follicles were isolated and cultured for 12 days. We assessed the follicular diameter, survival and maturation rate of these oocytes at the end of the last culture period
Results: The percentages of normal follicles in cultured ovaries were 73.85 +/- 2.49% [insert], 51.63 +/- 3.93% [hanging drop], and 40.52 +/- 5.86% [mineral oil] after the 7-day culture. The percentage of preantral follicles significantly increased from 2.1 +/- 0.44 to 24.5 +/- 2.4 in the group cultured on insert [P<0.05], however there was no significant difference in the other groups [P>0.05]. There were significantly increased surface areas of the ovarian tissues after the 7-day culture in all groups [P<0.05]. Ovaries cultured on the insert had a diameter of isolated follicles after 12 days of culture of 410 +/- 7.07 microm and the MII rate was 30.26%
Conclusion: The ovarian growth and morphology were well preserved in tissues cultured on the insert compared to the other culture methods
RESUMEN
microRNAs [miRNAs] are noncoding RNAs that function as key regulators of diverse biological activities such as cellular metabolism, cell proliferation and cell cycle regulation. Recent studies have indicated the high potential of these small molecules to control stem cell differentiation into desired cells. The aim of present study is to investigate the possible effect of let-7f on expression of hepatic nuclear factor 4 alpha [HNF4a] and some hepatic specific factors such as albumin [ALB], alpha fetoprotein [AFP], cytokeratin18 [CK18] and cytokeratin19 [CK19] in human adipose tissue derived stem cells [hADSCs]. ADSCs were isolated from human adipose tissue using collagenase type I and were transduced by recombinant lentiviruses that contained human inhibitor let-7f and Scramble [negative control]. Afterward, the expressions of HNF4a, ALB, AFP, CK18 and CK19 were evaluated by Real-time PCR at different time points. Transduction efficiency of lentiviral vectors into ADSCs was more than 80% as judged by the expression of the GFP reporter gene. Real-time PCR analysis revealed that inhibition of let-7f in hADSCs resulted in significant up regulation of hepatic specific genes compared with the negative control. The expression level of HNF4a also increased in experimental cells at day 14, which supported the suppression of HNF4a expression by let-7f. The results of this study identified let-7f as a negative regulator of HNF4a expression in hADSCs and increased the expression of hepatocyte specific factors through silencing of let-7f. Therefore, suppression of let-7f could be a considerable tool for hepatic differentiation of hADSCs