Cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) (both of which have two isoforms) can form fiber-like subcellular structures termed ‘cytoophidia’ under certain circumstances in mammalian cells. Although it has been shown that filamentation of CTPS downregulates its activity by disturbing conformational changes, the activity of IMPDH within cytoophidia is still unclear. Most previous IMPDH cytoophidium studies were performed under conditions involving inhibitors that impair GTP synthesis. Here, we show that IMPDH forms cytoophidia without inhibition of GTP synthesis. First, we find that an elevated intracellular CTP concentration or treatment with 3′-deazauridine, a CTPS inhibitor, promotes IMPDH cytoophidium formation and increases the intracellular GTP pool size. Moreover, restriction of cell growth triggers the disassembly of IMPDH cytoophidia, implying that their presence is correlated with active cell metabolism. Finally, we show that the presence of IMPDH cytoophidia in mouse pancreatic islet cells might correlate with nutrient uptake in the animal. Collectively, our findings reveal that formation of IMPDH cytoophidia reflects upregulation of purine nucleotide synthesis, suggesting that the IMPDH cytoophidium plays a role distinct from that of the CTPS cytoophidium in controlling intracellular nucleotide homeostasis.

Chemotherapy (CTx)-induced premature ovarian failure (POF) in woman remains clinically irreversible. Amniotic fluid stem cells (AFSCs) have shown the potential to treat CTx-induced POF; however, the underlying mechanism is unclear. Here we demonstrate that AFSC-derived exosomes recapitulate the anti-apoptotic effect of AFSCs on CTx-damaged granulosa cells (GCs), which are vital for the growth of ovarian follicles. AFSC-derived exosomes prevent ovarian follicular atresia in CTx-treated mice via the delivery of microRNAs in which both miR-146a and miR-10a are highly enriched and their potential target genes are critical to apoptosis. The down-regulation of these two miRNAs in AFSC-derived exosomes attenuates the anti-apoptotic effect on CTx-damaged GCs in vitro. Further, the administration of these miRNAs recapitulates the effects both in vitro and in vivo, in which miR-10a contributes a dominant influence. Our findings illustrate that miR-10a has potential as a novel therapeutic agent for the treatment of POF.

Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

BACKGROUND AND PURPOSE Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP‐4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo. EXPERIMENTAL APPROACH Sitagliptin were administered daily to C57CL/B6 mice and eGFP transgenic mouse bone marrow‐transplanted ICR mice that had undergone hindlimb ischaemic surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and circulating levels of endothelial progenitor cells (EPCs) respectively. Cell surface markers of EPCs and endothelial NOS (eNOS) in vessels were studied. KEY RESULTS Sitagliptin elevated plasma glucagon‐like peptide‐1 (GLP‐1) levels in mice subjected to ischaemia, decreased plasma dipeptidyl peptidase‐4 (DPP‐4) concentration, and augmented ischaemia‐induced increases in stromal cell‐derived factor‐1 (SDF‐1) in a dose‐dependent manner. Blood flow in the ischaemic limb was significantly improved in mice treated with sitagliptin. Circulating levels of EPCs were also increased after sitagliptin treatment. Sitagliptin also enhanced the expression of CD 34 and eNOS in ischaemic muscle. In addition, sitagliptin promoted EPC mobilization and homing to ischaemic tissue in eGFP transgenic mouse bone marrow‐transplanted ICR mice. CONCLUSION AND IMPLICATIONS Circulating EPC levels and neovasculogenesis were augmented by the DPP‐4 inhibitor, sitagliptin and this effect was dependent on an eNOS‐related pathway in a mouse model of hindlimb ischaemia. The results indicate that oral administration of sitagliptin has therapeutic potential as an inducer of vasculogenesis.

Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the gelatin molecules were added to the thermosensitive chitosan/beta-glycerol phosphate (C/GP) disodium salt hydrogels to form chitosan/gelatin/beta-glycerol phosphate (C/G/GP) disodium salt hydrogels which were applied as a cell carrier for nucleus pulposus (NP) regeneration. The gelation temperature, gelation time, and gel strength of the C/G/GP hydrogels were analyzed by the rheometer. NP cells were then harvested from the intervertebral discs of the adult New Zealand white rabbits and cultured in monolayer or in C/G/GP hydrogel, respectively. The cell viability, material-mediated cytotoxicity, cell proliferation, production of sulfated glycosaminoglycans, anabolic/catabolic gene expressions, and extracellular matrix-related gene expressions of the NP cells were demonstrated. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1-33.8 degrees C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. Among those, C/GP with 1% gelatin addition showed the most promising gelation time and gel strength and were utilized in the later experiments. From the results of cell activity, cytotoxicity, and cell proliferation assays, NP cells cultured in C/G/GP hydrogel had normal cell viability and cell proliferation that indicated the hydrogel was noncytotoxicity. The amounts of sulfated glycosaminoglycans of NP cells cultured in C/G/GP hydrogels were significantly higher than monolayer cultured. Considering the extracellular matrix-related gene expression, type II collagen and aggrecan of NP cells cultured in the hydrogels greatly increased than those in monolayer culture. On the contrary, the unfavorable gene expression, such as that of type I collagen, was decreased significantly. The results reveal that gelatin added into C/GP hydrogel significantly shortened the gelation time and improved the gel strength without influencing the biocompatibility. NP cells cultured in the C/G/GP hydrogel also displayed better gene expressions when compared with the monolayer culture. This study indicates that using chitosan/gelatin hydrogel for NP cell culture is feasible and may apply in minimal invasive intervertebral disc surgery in the future.

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