Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90+CD34− BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.
Animals ; Bone Marrow* ; Coculture Techniques ; Extracellular Matrix* ; Fibrosis* ; Hepatic Stellate Cells* ; Immunohistochemistry ; In Vitro Techniques ; Liver ; Liver Cirrhosis ; Methods ; Rats ; Stem Cells* ; Tenascin

OBJECTIVE: This study aimed to examine the effect of vitrification on apoptosis and survival in human preantral follicles after thawing.METHODS: This experimental study was conducted at an acute tertiary care hospital from March 2012 to April 2013. Ovaries were sliced into 5×5×1-mm pieces and divided into the following three groups: preantral follicle isolation, ovarian tissue vitrification-warming followed by follicle isolation, and immunohistochemistry of fresh ovarian tissue. For statistical analyses, the Student t-test, chi-square test, Kruskal-Wallis test, and Kaplan-Meier survival analysis were used.RESULTS: A total of 161 preantral follicles (70% secondary) were collected from ovarian cortex tissue of six women between 30 and 37 years of age who underwent oophorectomy due to cervical cancer or breast cancer. There were no significant differences in the follicular morphology of fresh preantral follicles and vitrified follicles after thawing. The mean Fas ligand (FasL) mRNA expression level was 0.43±0.20 (relative to β-actin) in fresh preantral follicles versus 0.51±0.20 in vitrified follicles (p=0.22). The mean caspase-3 mRNA expression level in fresh preantral follicles was 0.56±0.49 vs. 0.27±0.21 in vitrified follicles (p=0.233). One vitrified-thawed secondary follicle grew and developed to an antral follicle within 6 days of culture.CONCLUSION: Vitrification did not affect preantral follicle morphology or mRNA expression of the apoptosis markers FasL and caspase-3. Further studies are required to establish whether vitrification affects the outcomes of in vitro culture and the maturation of preantral follicles.
Apoptosis ; Breast Neoplasms ; Caspase 3 ; Fas Ligand Protein ; Female ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Ovariectomy ; Ovary ; RNA, Messenger ; Tertiary Healthcare ; Uterine Cervical Neoplasms ; Vitrification

Objective: Ovarian tissue vitrification is widely utilized for fertility preservation in prepubertal and adolescent female patients with cancer. The current literature includes reports of successful pregnancy and live birth following autografting. However, the effects of the vitrification process on cumulus-mural granulosa cells (C-mGCs)—somatic cells in ovarian tissue crucial for oocyte maturation and early embryonic development—remain unclear. This study was conducted to explore the impact of vitrification on the cellular function of C-mGCs by quantifying the expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), connexin 37, survivin, and caspase 3. Methods: Mature and immature C-mGCs were obtained from 38 women with polycystic ovary syndrome who participated in an in vitro fertilization program. The C-mGCs were then divided into two groups: fresh and vitrified. The expression levels of target genes were assessed using real-time quantitative polymerase chain reaction. Results: After vitrification, GDF-9 expression was significantly decreased among both mature and immature C-mGCs, with 0.2- and 0.1-fold changes, respectively (p<0.01). Similarly, FSHR expression in the mature and immature groups was reduced by 0.1- and 0.02-fold, respectively, following vitrification (p<0.01). The expression levels of the other genes, including BMP-15, LHR, connexin 37, survivin, and caspase 3, remained similar across the examined groups (p>0.05). Conclusion Vitrification may compromise oocyte maturation through reduced GDF-9 and FSHR expression in C-mGCs after warming.

Objective: Ovarian tissue vitrification is widely utilized for fertility preservation in prepubertal and adolescent female patients with cancer. The current literature includes reports of successful pregnancy and live birth following autografting. However, the effects of the vitrification process on cumulus-mural granulosa cells (C-mGCs)—somatic cells in ovarian tissue crucial for oocyte maturation and early embryonic development—remain unclear. This study was conducted to explore the impact of vitrification on the cellular function of C-mGCs by quantifying the expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), connexin 37, survivin, and caspase 3. Methods: Mature and immature C-mGCs were obtained from 38 women with polycystic ovary syndrome who participated in an in vitro fertilization program. The C-mGCs were then divided into two groups: fresh and vitrified. The expression levels of target genes were assessed using real-time quantitative polymerase chain reaction. Results: After vitrification, GDF-9 expression was significantly decreased among both mature and immature C-mGCs, with 0.2- and 0.1-fold changes, respectively (p<0.01). Similarly, FSHR expression in the mature and immature groups was reduced by 0.1- and 0.02-fold, respectively, following vitrification (p<0.01). The expression levels of the other genes, including BMP-15, LHR, connexin 37, survivin, and caspase 3, remained similar across the examined groups (p>0.05). Conclusion Vitrification may compromise oocyte maturation through reduced GDF-9 and FSHR expression in C-mGCs after warming.

Objective: Ovarian tissue vitrification is widely utilized for fertility preservation in prepubertal and adolescent female patients with cancer. The current literature includes reports of successful pregnancy and live birth following autografting. However, the effects of the vitrification process on cumulus-mural granulosa cells (C-mGCs)—somatic cells in ovarian tissue crucial for oocyte maturation and early embryonic development—remain unclear. This study was conducted to explore the impact of vitrification on the cellular function of C-mGCs by quantifying the expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), connexin 37, survivin, and caspase 3. Methods: Mature and immature C-mGCs were obtained from 38 women with polycystic ovary syndrome who participated in an in vitro fertilization program. The C-mGCs were then divided into two groups: fresh and vitrified. The expression levels of target genes were assessed using real-time quantitative polymerase chain reaction. Results: After vitrification, GDF-9 expression was significantly decreased among both mature and immature C-mGCs, with 0.2- and 0.1-fold changes, respectively (p<0.01). Similarly, FSHR expression in the mature and immature groups was reduced by 0.1- and 0.02-fold, respectively, following vitrification (p<0.01). The expression levels of the other genes, including BMP-15, LHR, connexin 37, survivin, and caspase 3, remained similar across the examined groups (p>0.05). Conclusion Vitrification may compromise oocyte maturation through reduced GDF-9 and FSHR expression in C-mGCs after warming.