Winston Teng-Kui Cheng

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.

Isolation of therapeutically functional mouse bone marrow mesenchymal stem cells within 3 h by an effective single-step plastic-adherent method

Objectives:  Isolation of mouse mesenchymal stem cells (mMSCs), by the approach of plastic adherence, has been difficult due to persistent contamination by haematopoietic cells (HCs); we have observed that this contamination was due to engagement between HCs and mMSCs. The HCs can be lifted together with the mMSCs despite their insensitivity to trypsin digestion. Herein, we provide a single‐step procedure to rapidly segregate mMSCs from HC contaminants using transient lower‐density plastic adherence (tLDA).

Seasonal effect on sperm messenger RNA profile of domestic swine (Sus Scrofa)

Seasonal infertility is a well-known problem in the modern swine (Sus scrofa) industry. The molecular mechanisms responsible for thermal effects on spermatogenesis are, however, just beginning to be elucidated. The existence of specific messenger RNA (mRNA) remnants contained within freshly ejaculated sperm has been identified in several species. Investigators have obtained differential RNA profiles of infertile men compared with fertile individuals; however, there are limited to the probes, which are mostly derived from nucleic acids of testicular tissues of either human or mice. The objective of this study was to investigate mRNA remnants from ejaculated sperm of the domestic swine and uncover important clues regarding the molecular regulation of spermatogenesis under environmental thermo-impacts. We utilized the remnant mRNA collected from swine ejaculated sperm as the target source to detect the global gene expression in summer and in winter by swine sperm-specific oligonucleotide microarray. Sixty-seven transcripts were differentially expressed with statistical differences between seasons of sperm samples collected, including forty-nine in winter (49/67) and eighteen in summer (18/67). There were only 33 of these transcripts that could be annotated to gene ontology hierarchy with the database of Homo sapiens and their functions mostly were involved in variety of metabolic processes. Moreover, these studies also confirmed that significant differences of gene expression profiles were found in swine sperm when comparisons were made between ejaculates collected during the winter and the summer season under the subtropical area such as Taiwan. Even though most of the genes found in our experiments are still poorly understood in terms of their true functions in spermatogenesis, bioinformatics analysis suggested that they are involved in a broad spectrum of biochemical processes including gamete generation. These concordant profiles should permit the development of a non-invasive testing protocol to assess the functional capacity of sperm as well as a new molecular selection scheme for fine breeding swine.

Toward an ideal animal model to trace donor cell fates after stem cell therapy: Production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

Porcine peroxisome proliferator-activated receptor δ mediates the lipolytic effects of dietary fish oil to reduce body fat deposition

Peroxisome proliferator-activated receptor delta promotes fatty acid catabolism and energy expenditure in skeletal muscle and adipose tissues. A ligand for PPARdelta is required to activate PPARdelta function. Polyunsaturated fatty acids are potential ligands for PPARdelta activation. The current experiment was designed to determine the potential for PUFA, particularly from dietary fish oil, to activate porcine PPARdelta in vivo. Transgenic mice were generated to overexpress porcine PPARdelta in the adipose tissue. Mice were fed a high-saturated fat (13% beef tallow), or high-unsaturated fat (13% fish oil) diet, or a diet containing 4 mg/kg of a PPARdelta ligand (L165041) for 4 mo. Compared with beef tallow feeding, fish oil feeding reduced fat mass and decreased (P < 0.05) plasma triacylglycerol and FFA concentrations in the transgenic mice. Adipose tissue expression of genes involved in adipogenesis (i.e., lipoprotein lipase and adipocyte fatty acid-binding protein) was decreased in transgenic mice fed fish oil or the PPARdelta ligand. In the same mice, expression of the lipolytic gene, hormone-sensitive lipase was increased (P < 0.05). Fish oil feeding also stimulated expression of genes participating in fatty acid oxidation in the liver of transgenic mice compared with wild-type mice. Overall, these results indicate that PUFA may serve as natural and effective regulators of lipid catabolism in vivo and many of these effects may be generated from activation of PPARdelta.

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.

Characterization of hTERT-Immortalized Caprine Mammary Epithelial Cells

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and β-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock.

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 × 106 cells per leg; cell-M), and 3) high cell dose (4 × 106 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.

Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

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

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.