Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells.

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

Morphological changes during replicative senescence in bovine ovarian granulosa cells.

The objective of this study was to evaluate replicative senescence of bovine granulosa cells (bGCs) during in vitro long-term culture. WST-1 assay analysis showed that bGCs proliferation was reduced from primary culture to 14th passage. The several bGCs from the 3rd passage and 7th passage exposed the weak activity of beta-galactosidase, while a strongly positive staining of beta-galactosidase was observed in bGCs from 14th passage. Flow cytometry analysis showed that bGCs were induced to cell cycle arrest at G0/G1 phase through in vitro expansion. TERT transcript expression of bGCs was downregulated from primary culture to 14th passage. The cell and nuclear area of bGCs were dramatically increased from 14th passage to 25th passage. The nucleocytoplasmic ratio of bGCs was dramatically reduced in 22th passage (4.32%) and 25th passage (2.45%), comparing to previous passages: primary culture (10.67%), 7th passage (9.21%), or 14th passage (10.33%). The number of microfilament bundle of bGCs was increased in 22nd passage (67.42 ± 17.76) and 25th passage (56.31 ± 22.45). The diameter of microfilament bundle of bGCs in 25th passage was dramatically increased to 1.88 ± 0.32 µm comparing to the primary culture (1.15 ± 0.03 µm). In this study, we also assessed the nuclear form factor which illustrates the level of nuclear circular form. A reduction of nuclear form factor was observed in bGCs during long-term in vitro expansion. The changes of nuclear form factor were correlated to other senescent characteristics, especially the nucleocytoplasmic ratio.

Differentiating of banked human umbilical cord blood-derived mesenchymal stem cells into insulin-secreting cells.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent cells. They are able to differentiate into functional cells from not only mesoderm but also endoderm. Many researches showed that cells derived from fresh human UCB could transdifferentiate into insulin-secreting cells. In this study, transdifferentiating potential of cryopreserved human UCB-derived MSCs into insulin-secreting cell was investigated. Fresh human UCB was enriched the mononuclear cells by Ficoll-Paque centrifugation. The mononuclear cell population was cryopreserved in cryo-medium containing Iscove's modified Dulbecco's media (IMDM) with 10% DMSO at -196°C for 1 yr. After thawing, mononuclear cells were cultured to isolate MSCs in medium IMDM with 20% FBS supplemented with growth factors. At the fifth passages, MSCs were confirmed by flow cytometry about expression of CD13, CD14, CD34, CD45, CD166, and HLA-DR markers; after that, they were induced to differentiate into adipocytes and osteoblasts. After inducing with specific medium for islet differentiation, there were many clusters of cell like islet at day 14-28. Using real-time reverse transcription polymerase chain reaction (RT-PCR) to analyze the expression of functional genes, the result showed that Nestin, Pdx-1, Ngn3, Ils-1, Pax6, Pax4, Nkx2.2, Nkx6.1, Glut-2, Insulin genes expressed. The results showed that MSCs derived from banked cord blood can differentiate into functional pancreatic islet-like cells in vitro. If human MSCs, especially MSCs from banked cord blood of diabetes patients themselves can be isolated, proliferated, differentiated into functional pancreatic islet-like cells, and transplanted back into them (autologous transplantation), their high-proliferation potency and rejection avoidance will provide one promising therapy for diabetes.