Establishment of a DsRed-monomer-harboring ICR transgenic mouse model and effects of the transgene on tissue development.

DsRed-monomer is an enhanced red fluorescent protein that may serve as a marker for studies in biotechnology and cell biology. Since the ICR mouse strain is a widely utilized outbred strain for oncology, toxicology, vaccine development and for aging studies, the objective of this study was to produce a DsRed-monomer transgenic mouse by means of pronuclear micro-injection of a vector driven by the cytomegalovirus (CMV) enhancer/chicken beta-actin promoter. Four transgenic mice were successfully produced, one of which expressed the DsRed-monomer protein in every tissue, although at varying levels. High expression levels were observed in the heart, pancreas and muscle. Moreover, amniotic fluid-derived progenitor cells, which also expressed the DsRed-monomer protein, could be collected from the DsRed-monomer- harboring ICR mice. As compared to wild-type mice, a few biochemical and histological dissimilarities were found in the DsRed-monomer transgenic mice, including the presence of intra-cytoplasmic eosinophilic threadlike materials in the acinar cells. Taken together, transgenic mice stably expressing DsRed-monomer can be produced using pronuclear micro-injection; however, expression of the DsRed-monomer gene or its insertion position may lead to minor influences.

Intramuscular Transplantation of Pig Amniotic Fluid-Derived Progenitor Cells Has Therapeutic Potential in a Mouse Model of Myocardial Infarction.

Acute myocardial infarction (MI) is a fatal event that causes a large number of deaths worldwide. MI results in pathological remodeling and decreased cardiac function, which could lead to heart failure and fatal arrhythmia. Cell therapy is a potential strategy to repair the damage through enhanced angiogenesis or by modulation of the inflammatory process via paracrine signaling. Amniotic fluid-derived progenitor cells (AFPCs) have been reported to differentiate into several lineages and can be used without ethical concerns or risk of teratoma formation. Since pigs are anatomically, physiologically, and genetically similar to humans, and pregnant pigs can be an abundant source of AFPCs, we used porcine AFPCs (pAFPCs) as our target cells. Intramyocardial injection of AFPCs has been shown to cure MI in animal models. However, intramuscular transplantation of cells has not been extensively investigated. In this study, we investigated the therapeutic potential of intramuscular injection of pAFPCs on acute MI. MI mice were divided into 1) PBS control, 2) medium cell dose (1 × 10(6) cells per leg; cell-M), and 3) high cell dose (4 × 10(6) cells per leg; cell-H) groups. Cells or PBS were directly injected into the hamstring muscle 20 min after MI surgery. Four weeks after MI surgery, the cell-M and cell-H groups exhibited significantly better ejection fraction, significantly greater wall thickness, smaller infarct scar sizes, and lower LV expansion index compared to the PBS group. Using in vivo imaging, we showed that the hamstring muscles from animals in the cell-M and cell-H groups had RFP-positive signals. In summary, intramuscular injection of porcine AFPCs reduced scar size, reduced pathological remodeling, and preserved heart function after MI.

Generation and characterization of a transgenic pig carrying a DsRed-monomer reporter gene.

BACKGROUND: Pigs are an optimal animal for conducting biomedical research because of their anatomical and physiological resemblance to humans. In contrast to the abundant resources available in the study of mice, few fluorescent protein-harboring porcine models are available for preclinical studies. In this paper, we report the successful generation and characterization of a transgenic DsRed-Monomer porcine model. METHODS: The transgene comprised a CMV enhancer/chicken-beta actin promoter and DsRed monomeric cDNA. Transgenic pigs were produced by using pronuclear microinjection. PCR and Southern blot analyses were applied for identification of the transgene. Histology, blood examinations and computed tomography were performed to study the health conditions. The pig amniotic fluid progenitor/stem cells were also isolated to examine the existence of red fluorescence and differentiation ability. RESULTS: Transgenic pigs were successfully generated and transmitted to offspring at a germ-line transmission rate of 43.59% (17/39). Ubiquitous expression of red fluorescence was detected in the brain, eye, tongue, heart, lung, liver, pancreas, spleen, stomach, small intestine, large intestine, kidney, testis, and muscle; this was confirmed by histology and western blot analyses. In addition, we confirmed the differentiation potential of amniotic fluid progenitor stem cells isolated from the transgenic pig. CONCLUSIONS: This red fluorescent pig can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation.

Therapeutic potential of amniotic-fluid-derived stem cells on liver fibrosis model in mice.

OBJECTIVE: Liver fibrosis results from the wound healing response to chronic liver damage. Advanced liver fibrosis results in cirrhosis and liver failure, and liver transplantation is often the only option for effective therapy; however, the shortage of available donor livers limits this treatment. Thus, new therapies for advanced liver fibrosis are essential. MATERIALS AND METHODS: Amniotic fluid contains an abundance of stem cells, which are derived from all three germ layers of the developing fetus. These cells do not induce teratomas in vivo and do not pose any ethical concerns. To generate liver fibrosis models, male ICR mice were treated with CCl4 via oral gavage for 4 weeks, and the serum levels of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin were higher than in the control group following chemical induction. To assess the potential of amniotic-fluid-derived stem cells (mAFSCs) to ameliorate liver fibrosis in vivo, mAFSCs were isolated from amniotic fluid of 13.5-day-old transgenic mice, which globally express the fluorescent protein, enhanced green fluorescent protein (EGFP), for tracing purposes (EGFP-mAFSCs). Single cells were injected via the mesentery (1 × 10(6) cells/mouse) of transplanted mice with liver fibrosis. RESULTS: Four weeks after EGFP-mAFSC transplantation, the serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and albumin levels of recipient mice in the EGFP-mAFSC-injected group were significantly decreased when compared with mice in the saline-injected group. Additionally, fibrotic tissues were evaluated using Masson's trichrome staining 4 weeks after cell transplantation. Shrinkage of the fibrotic area was observed in the EGFP-mAFSC-injected group. The tissue-repair effects were also confirmed by hydroxyproline content analysis. CONCLUSION: The possible repair mechanism from our data revealed that EGFP-mAFSCs may fuse with the recipient liver cells. Overall, EGFP-mAFSCs can ameliorate liver fibrosis in mice, thus providing insight into the future development of regenerative medicine.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

OBJECTIVES: Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. METHODS: The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. RESULTS: Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. CONCLUSION: In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue.

Ohx is a homeobox-encoding gene preferentially expressed in mature oocytes.

There is abundant evidence to indicate that the homeobox genes are developmentally important. We used the NCBI dbEST databases of early mouse embryos to identify novel homeobox-containing sequence tags. Ohx (oocyte-specific homeobox gene) was one of several genes identified by in silico cloning. The full-length Ohx cDNA was cloned and its genomic organization was characterized. The Ohx gene spans 1.6 kb, encodes three exons and maps to the proximal region of mouse chromosome 7. Reverse transcriptase polymerase chain reaction analyses show that Ohx is preferentially expressed in one- and two-cell embryonic stages, as well as in the ovary. Whole mount in situ hybridization analysis demonstrates that the Ohx mRNA was exclusively localized to the oocytes of the mature ovary. Ohx is one of the few homeobox-encoding genes preferentially expressed during mammalian oogenesis.