A novel apparatus applying long term intermittent cyclic hydrostatic pressure to in vitro cell cultures

A novel apparatus has been designed that allows for application of long term intermittent cyclic hydrostatic pressure to in vitro cell cultures, while maintaining cell viability on par with a standard incubator. The system has been tested with monolayer cultures, but could also accommodate three dimensional constructs.     

Germ cells from mouse and human embryonic stem cells

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.     

Available human feeder cells for the maintenance of human embryonic stem cells

Mouse embryonic fibroblasts (MEFs) have been previously used as feeder cells to support the growth of human embryonic stem cells (hESCs). In this study, human adult uterine endometrial cells (hUECs), human adult breast parenchymal cells (hBPCs) and embryonic fibroblasts (hEFs) were tested as feeder cells for supporting the growth of hESCs to prevent the possibility of contamination from animal feeder cells. Cultured hUECs, hBPCs and hEFs were mitotically inactivated and then plated. hESCs (Miz-hES1, NIH registered) initially established on mouse feeder layers were transferred onto each human feeder layer and split every 5 days. The morphology, expression of specific markers and differentiation capacity of hESCs adapted on each human feeder layer were examined. On hUEC, hBPC and hEF feeder layers, hESCs proliferated for more than 90, 50 and 80 passages respectively. Human feeder-based hESCs were positive for stage-specific embryonic antigen (SSEA)-3 and -4, and Apase; they also showed similar differentiation capacity to MEF-based hESCs, as assessed by the formation of teratomas and expression of tissue-specific markers. However, hESCs cultured on hUEC and hEF feeders were slightly thinner and flatter than MEF- or hBPC-based hESCs. Our results suggest that, like MEF feeder layers, human feeder layers can support the proliferation of hESCs without differentiation. Human feeder cells have the advantage of supporting more passages than when MEFs are used as feeder cells, because hESCs can be uniformly maintained in the undifferentiated stage until they pass through senescence. hESCs established and/or maintained under stable xeno-free culture conditions will be helpful to cell-based therapy.     

Effects of trans fatty acids on markers of inflammation in bovine mammary epithelial cells

Trans fatty acids (tFA) contribute to inflammation. The objective was to investigate the effects of tFA on mRNA expression of proinflammatory markers in cultured bovine mammary epithelial cells (MAC-T cell line). Bovine mammary epithelial cells were grown in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. Cells were then subcultured in a medium lacking fetal bovine serum, to which incremental concentrations (up to 90 μM) of elaidic acid (trans-9 C18:1) or linoleidic acid (trans-9, trans-12 C18:2) were added. Bovine serum albumin (fatty acid-free) solutions were added and cells were collected at specific time points over 48 h. Then, RNA was extracted and converted to complementary DNA for quantitative real-time PCR analysis of proinflammatory gene expression. Presence of elaidic acid caused increases in mRNA expression of interleukin (IL)-1β (3.4-fold; dose-independently over a 6-h period) and intercellular adhesion molecule (ICAM)-1 (up to 1.4-fold) relative to that for cells treated with no tFA, whereas expression of IL-6 and IL-8 was reduced 0.75- and 0.85-fold, respectively. Presence of linoleidic acid reduced mRNA expression of IL-6 and IL-8 relative to that for control (0.95- and 0.87-fold, respectively). Trans mono- and dienoic fatty acids upregulated mRNA expression of IL-1β and ICAM-1, whereas expression of IL-6 and IL-8 was downregulated in MAC-T cells. Because these genes are ultimately involved in inflammation, elaidic or linoleidic acid, either directly fed or formed in the rumen during biohydrogenation, may alter the risk for mastitis in vivo.     

尼古丁诱导人肠道上皮细胞自噬水平的研究

  目的  探讨尼古丁对人肠道上皮细胞自噬水平的影响和可能的分子机制。  方法  正常培养人肠道上皮细胞, 采用不同浓度(0、2.5、5、10μM)尼古丁处理细胞24h。倒置显微镜观察细胞形态变化; 通过CCK8法检测肠道上皮细胞的增殖情况; 通过Western blot检测自噬标志物LC3、beclin1和p62变化; 通过透射电子显微镜(TEM)检测自噬小体形成情况; 通过GFPLC3转染后激光共聚焦显微镜检测自噬斑点形成情况。同时, 也检测了内质网应和mTOR/AMPK/Akt信号通路变化。  结果  细胞增殖随着尼古丁浓度的增加受到明显抑制, 2.5μM尼古丁对细胞形态和增殖无显著影响。尼古丁能够上调LC3B-II和beclin1蛋白表达水平, 同时下调P62的表达, 且成浓度依赖性。TEM和激光共聚焦显示尼古丁作用后, 肠道上皮细胞自噬体数量显著增加。  结论  低浓度的尼古丁能够诱导肠道上皮细胞的自噬, 且对细胞增殖无影响, 机制与内质网应激和mTOR/Akt信号通路激活相关。      

The morphology of corneal endothelial cells in long term soft contact lens wearers in Kuala Lumpur

PurposeTo analyse and compare the alterations in corneal endothelium morphology induced by different materials and durations of wearing soft contact lenses (CL) among young adults living in Kuala Lumpur.MethodsHealthy soft CL wearers were invited to participate in this study. Visual acuity (VA) was measured using the Snellen chart, and subjective refraction was performed using cross-cylinder technique. Standard ocular assessments were conducted using a slit lamp biomicroscope and morphology of corneal endothelial cells (endothelial cell density, ECD, coefficient variation, COV, hexagonality, HEX and central corneal thickness, CCT) were evaluated using a non-contact specular microscope. Statistical analysis was conducted using ANOVA and data from the right eye only is included.ResultsA total of 72 subjects (32 SiHy and 40 HCL wearers) and 24 non-CL wearers (control) participated in this study. The gender distribution for study subjects was 13 males and 59 females, with a mean age 22.15 ± 1.84 years old. The mean refraction was −1.86 ± 1.25DS. The duration of wearing CL ranged from 1 to 9 years. Subjects were later divided into 2 groups following duration of CL wear: Group 1 (<5 years) and Group 2 (≥5 years) for analysis purposes. Statistical analysis showed significant alterations in ECD, COV and HEX of CL wearers (p < 0.05), with more changes found in HCL and Group 2 wearers. No significant change was found in CCT.ConclusionThis study concludes that soft CL wear induced alterations in the morphology of corneal endothelial cells. Contact lens material and duration of CL wear (in years) are factors that affect the alterations. Optometrists are recommended to regularly evaluate the morphology of corneal endothelial cells in CL wearers and provide necessary intervention when required.     

Anti-inflammatory and proliferative responses in human and ovine ovarian surface epithelial cells

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11betahydroxysteroid dehydrogenase (11betaHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1alpha. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1alpha stimulated a concomitant increase of 11betaHSD type 1 mRNA (31-fold; P <0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P <0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P <0.05) and >1.5-fold (P <0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.     

Derivation, growth and applications of human embryonic stem cells

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES cells.     

Myricetin inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells

Myricetin is a major flavonoid found in various foods that has antioxidant, anti-inflammatory, and anticancer effects. Although the functional effects of myricetin in various cell types are well characterized, it is not known whether myricetin has an effect on stem cell differentiation. In this study, we demonstrate that myricetin inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells, as indicated by decreased accumulation of intracellular lipid droplets. The mRNA levels of CCAATenhancer-binding proteins (C/EBP)-α, peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase, fatty acid binding protein (aP2), and adiponectin decreased significantly following treatment with 30 μM myricetin. C/EBP-α expression was inhibited from the beginning of differentiation in response to the myricetin treatment. PPAR-α was significantly inhibited beginning at day 9. These results suggest a novel effect of myricetin on adipocyte differentiation in human adipose tissue-derived mesenchymal stem cells and the possibility that myricetin might affect the differentiation of other types of stem cells.     

Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.     

Generation of human induced pluripotent stem cells from oral mucosa

Induced pluripotent stem (iPS) cells are one of the most promising sources for cell therapy in regenerative medicine. Using a patient's own genetically identical and histocompatible cells is the ideal way to practice personalized regenerative medicine. For personalized iPS cell therapy, the prerequisites for cell source preparation are a simple and safe procedure, no aesthetic or functional damage, and quick wound healing. Oral mucosa fibroblasts (OFs) may have high potential to fulfill these requirements. In this study, biopsy was performed in a dental chair; no significant incisional damage was recognized and rapid wound healing (within a week) was observed. We generated human iPS cells from the isolated OFs via the retroviral gene transfer of OCT4, SOX2, c-MYC, and KLF4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers such as OCT4, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Subsequent in vitro and in vivo analyses confirmed the pluripotency of resultant iPS cells, which matched the criteria for iPS cells. In addition, we found that the endogenous expression levels of c-MYC and KLF4 in OFs were similar to those in dermal fibroblasts. Taken together, we propose that OFs could be a practical source for preparing iPS cells to achieve personalized regenerative medicine in the near future.     

Chlorogenic acid promotes osteoblastogenesis in human adipose tissue-derived mesenchymal stem cells

High-fat diet (HFD)-induced obesity is associated with oxidative stress. The purpose of this study was to examine the antioxidant effect of Phaeodactylum tricornutum extract in mice with diet-induced obesity. Four-week-old C57BL/6J mice were fed a normal diet or HFD with and without 0.7% P. tricornutum lipid extract corresponding to 0.2% fucoxanthin for 8 weeks. P. tricornutum significantly decreased body weight and epidydimal white adipose tissue in mice fed the HFD. Serum triglyceride, glucose, insulin, and leptin levels, as well as homeostasis model assessment for insulin resistance (HOMA-IR) values, were significantly lower in the P. tricornutum group than in the HFD group. P. tricornutum significantly decreased thiobarbituric acid reactive substances (TBARS) and increased glutathione and the activities of superoxide dismutase, catalase, and glutathione peroxidase in the liver compared with the HFD group. Thus, P. tricornutum could exert antiobesity and antioxidant effects in mice fed a HFD.