Cell biology international reports最新文献

Pluripotent mouse embryonic stem (ES) cells have the ability to generate completely ES cell-derived mice by tetraploid (TE-4N) complementation although; derivation of ES cells from TE-4N animals has not been well defined. In this study, we generated TE-4N mice from high passage number ES cells and, in turn, determined the possibility of deriving ES cells from the TE-4N mice. The results showed that adult fertile TE-4N mice could be generated by aggregation of tetraploid embryos using ES cells passaged 23 times. Furthermore, ES cells could be derived from the TE-4N male mice and as judged by molecular characterisation and a study of chimeras, these ES cells were pluripotent. The findings demonstrate that tetraploid complementation is an efficient way to produce fertile mice, which can then give rise to pluripotent ES cells.

Oxidative stress has been progressively recognised as a key player in the development of osteoporosis. Although oxidative stress induces osteoporosis partly through inhibition of hedgehog signalling, the details of the signal interaction remain unclear. To elucidate this mechanism, we investigated osteoblast differentiation in C3H10T1/2 cells, murine embryonic mesenchymal cells, with recombinant sonic hedgehog (shh) in the presence or absence of hydrogen peroxide, and examined Gli transcriptional activity, with a focus on the interaction with mitogen-activated protein kinases (MAPKs) as possible mediators. The transcription of Gli1, Ptch1b, Alp and Bsp was facilitated by shh and strongly inhibited by oxidative stress with the hydrogen peroxide treatment; however, the other Gli isoforms, Bmp2, Runx2 and Osx, were unaffected. Oxidative stress also facilitated the phosphorylation of JNK1 and p38. Anisomycin treatment as a JNK activator or transient overexpression of the constitutive JNK1 active form inhibited hedgehog activity. Interestingly, the inhibitory effect of hydrogen peroxide on hedgehog signalling was blocked by a JNK inhibitor, but not by a p38 inhibitor. Osteoblast differentiation determined by calcium staining was, in part, rescued from oxidative stress by the JNK inhibitor. These findings suggest that oxidative stress inhibits hedgehog signalling and subsequent osteoblast differentiation through an interaction between phospho-JNK1 and GLI1.

Tissue engineering and regenerative are aiming at generating tissues to replace damaged and deteriorated organs. Recently, tissue engineering was applied to generate artificial skin for burn patients, tissue engineered trachea, cartilage for knee-replacement procedures, urinary bladder, urethra substitutes and offered cellular therapies for the treatment of urinary incontinence. The major advantage of tissue engineering approach over traditional organ transplantation is to circumvent the problem of organ shortage. Tissues reconstructed from readily available patents' stem cells induced no immunogenicity when reimplanted in the patient. However, pluripotent stem cells are major limited factors in regenerating new tissues. To overcome these problems, we developed a new technology called as ‘invivo interspecies tissue engineering’ (INVITE) and used the potential of induced pluripotent stem cells (iPSCs) to regenerate new tissues in a host organism. As a model, we used the mouse pluripotent stem cells to assess the potential of these cells to regenerate mouse tissues in rat. Three chimeric rats have been generated by mouse-induced pluripotent stem cells assessed by monitoring of green fluorescence protein (GFP) and polymerase chain reaction (PCR) assays. This study shows clearly that mouse-induced pluripotent stem cells are able to engraft in rat embryos and are capable to differentiate to multiple tissues. Beside the enormous important application of in vivo tissue engineering in human to develop new therapeutic approaches through regenerating new tissues, this model offers an accessible system for study of organ development and a valuable tool for personalised drug screening and a novel approach for personalised stem cell-based tissue regeneration.

Acute kidney injury (AKI) is a common syndrome featuring the rapid loss of the kidney's excretory function, and presents a common and important diagnostic and therapeutic challenge for clinicians. There are no effective approaches to prevent and treat AKI. During early stage, most ischaemic AKI is reversible, so further research is essential for early intervention in order to prevent and reduce AKI. Cyr61 has been shown to be expressed in renal tubular cells in hypoxic-ischaemic kidney injury, whilst there is no expression in normal tubular cells. In the present study, we have established a stably Cyr61 expressed tubular cell line Cyr61-HK-2 based on HK-2 through recombinant Cyr61-lentivirus, and found that Cyr61 expression promotes tubular epithelial cell proliferation, while the cell apoptosis is inhibited. Further study demonstrated that Cyr61 expression led to BAD phosphorylation, which protects tubular cells from apoptosis, meanwhile Cyr61 activates the Akt and ERK signalling pathways, which are essential for cell proliferation. Altogether, our data showed the Cyr61 expression promotes cell proliferation, while dampening cell apoptosis under hypoxia, potentially suggesting a novel therapeutic approach for AKI.

Worldwide, the ‘Nemo’ ocellaris clownfish (Amphiprion ocellaris, Cuvier 1830) is one of the top three most exported ornamental fishes. It also served as a subject in various fields of study except for cell culture. This first report described a simple explant method for culturing cells from the vertebra of ocellaris clownfish. The fish was first anesthetised with iced cold water and decapitated. The body trunk was disinfected in isopropanol and washed in sterile PBS. The vertebra was aseptically excised, washed two times in PBS and minced in the dissection solution (PBS containing 250 IU/mL penicillin, 250 µg/mL streptomycin, 50 µg/mL gentamycin and 2.5 µg/mL amphotericin-B). Then, the vertebral biopsies were washed three more times in PBS before being seeded in 25 cm² culture flasks containing 1.5 mL of RPMI-1640 supplemented with 20% FBS. A small amount of CO₂ was injected into the flask before it was tightly capped and incubated at 28°C in the regular incubator. When the monolayer reached 40–50% confluence, the vertebral biopsies were dislodged together with the medium to initiate a new primary culture. The cell monolayer was subcultured with short, cold 0.05% trypsin. The Nemo cell line was grown in the medium containing 15% FBS. The cell line at passage 4 had the population doubling time of 39.6 h and the cell line at passage 5 could be cryopreserved with 80% viability. This simple and reliable explant method has been applied successfully to culture cells of both marine and freshwater fishes for the prometaphase chromosome preparation.

We compared airway epithelial cell models relevant for cystic fibrosis (CF): 16HBE cells with endogenous wild-type cystic fibrosis transmembrane conductance regulator (CFTR), CFBE cells with mutated ΔF508-CFTR, corrected CFBE cells overexpressing CFTR, CFSME (CF submucosal) and Calu-3 (non-CF submucosal) cells with respect to the epithelial sodium channel (ENaC), inducible NO synthase (iNOS) and mucins (MUC) (studied by quantitative Real-Time-Polymerase Chain Reaction, qRT-PCR and Western blot), and wound healing.

CFBE cells had significantly more expression of β- and γ-ENaC mRNA and of β-ENaC protein than 16HBE cells. Compared to corrected CFBE cells, CFBE cells had increased mRNA expression of all ENaC subunits and β-ENaC protein. For ENaC, the CFSME/Calu-3 mRNA ratio was very low and contradictory to the ENaC upregulation in CF cells. CFBE cells showed decreased expression of iNOS at both mRNA and protein levels compared to 16HBE cells and only at the mRNA level compared to corrected CFBE cells. CFSME cells showed expression of iNOS whereas Calu-3 cells did not. Higher expression of MUC2 and MUC5B was found in corrected CFBE cells compared to CFBE cells. Wound healing in CFBE cells was delayed compared to corrected CFBE cells, but not to 16HBE cells, and in CFSME cells compared to Calu-3 cells.

Our data suggest CFSME as an inappropriate CF cell model for Calu-3 cells, and provide partial support for the theory that the differences (in ENaC, iNOS and wound healing) between these cell lines are associated to the presence of CFTR in the bronchial airway epithelial cells.

Although induced pluripotent stem cells (iPSCs) are a potential source for transplantation therapy, malignant transformation (tumourigenesis) remains a major concern in their safe clinical application. iPSCs are considered more tumourigenic than embryonic stem cells (ESCs) because of genetic and epigenetic manipulations. We generated 22 human iPSC lines from normal human fibroblasts and injected three of these cell lines into SCID mice, and produced three tumours, all of which were identified as teratomas with at least two germ layers. Using cells cultured from them, RT-PCR showed that the cells expressed undifferentiated cell markers, including OCT4 and NANOG. This suggests that some undifferentiated cells remain in the teratoma during its formation. We also found emergence of cells expressing undifferentiated cell markers from teratoma-derived cells during culturing with the ESC medium. Immunocytochemical analyses showed that NANOG-, OCT4- and SSEA4-positive cells appeared and increased with time in culture. These data indicate that iPSC-like undifferentiated cells can emerge from differentiated cells under certain condition and they may present a potential risk of tumourigenesis, as do residual iPSCs.

We recently presented evidence showing that cementum protein 1 (CEMP1) promotes periodontal ligament (PDL) cell migration, proliferation, expression of bone, and cementum-matrix proteins and mineralisation. In other words, it induces PDL precursor cells commitment toward a cementoblast-like cells phenotype. The intracellular signalling pathways involved in cementoblast differentiation and mineralisation have not been well characterised. JNK and p38 protein kinases (MAPKs) are intracellular signalling pathways and key mediators of cellular processes such as proliferation and differentiation. Since signalling pathways involving MAPKs have been associated with osteoblastic phenotype, in this study we investigated the effect of hrCEMP1 and mineralising media containing β-glycerophosphate and ascorbic acid on the activation of p38-MAPK and JNK–MAPK in cementoblast-like cells. Our results show that mineralising media and hrCEMP1 induced phosphorylation of p38 and JNK kinases. Mineralising media containing hrCEMP1 increased the activation of p38-MAPK and its translocation to the cell nucleus; increased phosphorylation of JNK–MAPK and induced the phosphorylation of the protein C-JUN. We also demonstrate that hrCEMP1 regulates the expression of BSP, OCN, and ALP specific activity. We found that hrCEMP1 and mineralising media promote nodule formation. These findings give an insight into the signalling pathways activated by hrCEMP1 and suggest likely components of the mechanisms that regulate the formation and regeneration of cementum and surrounding connective tissues.

Interferon Stimulated Gene 12a, ISG12a, is a member of a family of small intracellular non-secreted proteins (10–20 kDa), mainly induced by type I IFNs and slightly induced by type II IFN. It has been shown that full length ISG12 (ISG12a) is overexpressed in breast cancer, yet the biological function of ISG12 is largely unknown. Here we show that transient transfection of ISG12a into various mammalian cell lines leads to accumulation of cells initially in the G1 phase of the cell cycle, followed by accumulation of cells in the sub G1 phase, and that cells transfected with ISG12a undergo morphological changes, such as rounding up and detachment from the plate, that are characteristic of apoptosis. Induction of apoptosis by ISG12a was confirmed by Annexin V binding assays and by TUNEL assays. Using general and specific caspase inhibitors, we also showed that ISG12a-induced apoptosis is a caspase dependent, but does not involve p53. Elevation in endogenous ISG12a levels following induction of apoptosis with reagents that induce apoptosis in the intrinsic apoptotic pathway, and reduction in ISG12a-induced apoptosis following co-transfection with Bcl-2, indicated that ISG12a induced apoptosis in the intrinsic apoptotic pathway. Our results suggest a role for the ISG12a gene as a novel pro-apoptotic gene.