Shinn-Chih Wu

Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma

An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.

Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells

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.

Recombinant porcine lactoferrin expressed in the milk of transgenic mice protects neonatal mice from a lethal challenge with enterovirus type 71

The human Enterovirus genus of the piconavirus family causes most of the febrile illnesses that affect children during the summer season in Taiwan. Enterovirus type 71 (EV71) plays a key role in patients with hand-foot-and-mouth disease (HFMD) combined with severe paralysis or encephalitis. It is important to find a method for preventing infection with EV71 since there is no antiviral agent or vaccine for humans. In this study, we developed a transgenic mouse model for demonstrating the protective effects of recombinant lactoferrin (LF) against EV71 infection. Transgenic mice carrying alpha-lactalbumin-porcine lactoferrin (alphaLA-pLF) and BALB/c wild-type mice were subjected to EV71 inoculation. First, we analyzed the expression efficiencies of recombinant pLF (rpLF) in hemizygous and homozygous transgenic mice. Following EV71 inoculation on the 4th day of life, pups ingesting transgenic milk showed the significantly higher survival rate and heavier body weight compared to wild-type mice. RT-PCR analysis for EV71 viral RNA showed that the recombinant pLF had a blocking effect on EV71 infection. Our data suggest that oral intake of pLF-enriched milk exhibited the ability to prevent infection with EV71. The study also provides an animal model for validating the protective effects of pLF.

Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine alpha-lactalbumin regulation sequences

Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manipulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine α-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The αLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3β/β-Catenin signaling in mice

Adiponectin and its receptors are expressed in bone marrow-derived osteoblasts. Previous studies in vivo and in vitro have produced controversial results. The purpose of this study was to use porcine adiponectin receptor 1 transgenic mice (pAdipoR1) as a model to evaluate the role of AdipoR1 on bone physiology at different ages. pAdipoR1 transgenic mice had higher bone mineral density than wild-type mice in both genders at 56 weeks of age. The bone volume and trabecular number, measured by micro-computed tomography (μCT) was significantly greater in transgenic than in wild-type female mice at both 8 and 56 weeks of age. ELISA analysis revealed that both serum osteocalcin and osteoprotegerin (OPG) were significantly increased in 8-week old pAdipoR1 transgenic mice of both genders. Furthermore, serum OPG was elevated at 32 and 56 weeks of age in female and male pAdipoR1 transgenic mice. Serum TRAP5b concentration was reduced in 8 and 56 weeks old male pAdipoR1 mice compared with wild-type male mice. Knock-down of AdipoR1 significantly decreased gene expression of osteocalcin, OPG, alkaline phosphatase and msh homeobox 2 and the mineralization in MC3T3-E1 cells and mesenchymal stem cells. In addition, pathscan analysis and real-time PCR analysis suggest AdipoR1 regulates osteoblast differentiation through GSK-3 β and β-Catenin signaling. Consequently, the lack of AdipoR1 impaired osteoblast differentiation and bone formation. We conclude that AdipoR1 is a critical factor for the osteoblast differentiation and bone homeostasis.

Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

Background While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. Methodology/Principal Findings In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. Conclusions/Significances These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities.

Amniotic Fluid Stem Cells Prevent Follicle Atresia and Rescue Fertility of Mice with Premature Ovarian Failure Induced by Chemotherapy

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.

Docosahexaenoic acid regulates adipogenic genes in myoblasts via porcine peroxisome proliferator-activated receptor γ

The nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) triggers adipocyte differentiation by regulating lipogenic genes. A ligand for PPARgamma is necessary to activate PPARgamma function. Fatty acids are potential ligands for PPARgamma activation. The current experiment was designed to determine the potential for individual fatty acids to activate porcine PPARgamma ectopically expressed in myoblasts. The expression of adipocyte fatty acid binding protein (aP2) and adiponectin in myoblasts stably expressing porcine PPARgamma was increased when docosahexaenoic acid (DHA) was added to the adipogenic medium. The response was positively related to DHA concentration and suggests that DHA may bind to and activate porcine PPARgamma, leading to increased expression of aP2 and adiponectin. The conditioned media collected from myoblasts expressing PPARgamma between d 3 and 6 or between d 6 and 9, but not DHA itself, activated the aP2 gene promoter-driven luciferase activity. These results suggest that a metabolite of DHA is the ligand binding to and activating porcine PPARgamma. The metabolite and pathway for its production are currently unknown.

The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes

Peroxisome-proliferator-activated receptor γ (PPARγ) plays a critical role in regulation of adipocyte differentiation and insulin sensitivity. To become functional, PPARγ must be activated by binding an appropriate ligand. Polyunsaturated fatty acids (PUFA) are potential ligands for PPARγ. The current experiment was designed to determine the potential for PUFA, particularly eicosapentaenoic acid and docosahexaenoic acid, to activate the function of porcine PPARγ in vivo. Transgenic mice, expressing porcine PPARγ in skeletal muscle were generated and fed with a high-saturated fat (beef tallow) or high-unsaturated fat (fish oil) diet for 4 months. When transgenic mice were fed a fish oil supplemented diet, the expression of adipogenic and glucose uptake genes was increased, leading to reduced plasma glucose concentration. The PPARγ transgene increased the expression of Glut4 in the muscle. This result suggests that there was increased glucose utilization and, therefore, a reduced blood glucose concentration in the transgenic mice. Also, the plasma adiponectin was elevated by fish oil treatment, suggesting a role of adiponectin in mediating the PUFA effect. These results suggest that PUFA may serve as a natural regulator of glucose uptake in vivo and these effects are mainly through PPARγ function.

Differential differences in methylation status of putative imprinted genes among cloned swine genomes.

DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.