Haijun Huang

PPARγ and MyoD are differentially regulated by myostatin in adipose-derived stem cells and muscle satellite cells

Myostatin (MSTN) is a secreted protein belonging to the transforming growth factor-β (TGF-β) family that is primarily expressed in skeletal muscle and also functions in adipocyte maturation. Studies have shown that MSTN can inhibit adipogenesis in muscle satellite cells (MSCs) but not in adipose-derived stem cells (ADSCs). However, the mechanism by which MSTN differently regulates adipogenesis in these two cell types remains unknown. Peroxisome proliferator-activated receptor-γ (PPARγ) and myogenic differentiation factor (MyoD) are two key transcription factors in fat and muscle cell development that influence adipogenesis. To investigate whether MSTN differentially regulates PPARγ and MyoD, we analyzed PPARγ and MyoD expression by assessing mRNA, protein and methylation levels in ADSCs and MSCs after treatment with 100 ng/mL MSTN for 0, 24, and 48 h. PPARγ mRNA levels were downregulated after 24 h and upregulated after 48 h of treatment in ADSCs, whereas in MSCs, PPARγ levels were downregulated at both time points. MyoD expression was significantly increased in ADSCs and decreased in MSCs. PPARγ and MyoD protein levels were upregulated in ADSCs and downregulated in MSCs. The CpG methylation levels of the PPARγ and MyoD promoters were decreased in ADSCs and increased in MSCs. Therefore, this study demonstrated that the different regulatory adipogenic roles of MSTN in ADSCs and MSCs act by differentially regulating PPARγ and MyoD expression.

MyoD promotes porcine PPARγ gene expression through an E-box and a MyoD-binding site in the PPARγ promoter region

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in adipogenesis and can be regulated by adipogenesis-related factors. However, little information is available regarding its regulation by myogenic factors. In this study, we found that over-expression of MyoD enhanced porcine adipocyte differentiation and up-regulated PPARγ expression, whereas small interfering RNA against MyoD significantly attenuated porcine adipocyte differentiation and inhibited PPARγ expression. The MyoD-binding sites in the PPARγ promoter region at −412 to −396 and −155 to −150 were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays and chromatin immunoprecipitation further showed that these two regions are MyoD-binding sites, both in vitro and in vivo, indicating that MyoD directly interacts with the porcine PPARγ promoter. Thus, our results demonstrate that an Enhancer box and a binding site for a cooperative co-activator of MyoD are present in the promoter region of porcine PPARγ; furthermore, MyoD up-regulates PPARγ expression and promotes porcine adipocyte differentiation.

Survival and engraftment of mouse embryonic stem cells in the mammary gland

Embryonic stem (ES) cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES cells to improve functional outcome following mammary gland injury. This study investigates the feasibility of implanting mouse ES cells labeled with enhanced green fluorescence protein in the developing mammary glands in order to acquire lineage-committed cells in mammary (mammary gland epithelial cell or luminal cell). Cells implanted in high numbers (5 × 106 cells per mammary gland) survived in the majority of the mice and nearly 38.4% of the surviving cells were CK18+ at 15th week following the transplantation. Our results may provide a technique instrument on advanced therapy of breast diseases and the mammary regeneration after breast ablated partly.

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine β-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.

Immune-Tolerizing Procedure for Preparation of Monoclonal Antibodies Against Glycoprotein E of Pseudorabies Virus

The glycoprotein E (gE) of pseudorabies virus (PRV) is known to be an important marker protein in the control and eradication of Aujeszkys disease. In this study, BALB/c mice were immunized with gE-deleted PRV as tolerogen and with wild-type PRV as immunogen. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. Two hybridoma cell lines that could stably secrete the monoclonal antibody (MAb) against gE were achieved by using indirect ELISA screening and subcloning three times; they were named 1D2 and 2B2. Indirect immunofluorescence assay (IFA) revealed that the MAbs were specifically against gE of PRV. MAbs 1D2 and 2B2 were subgroup IgG1. The MAbs obtained in this study provide useful tools for the development of differential diagnostic methods for PRV.

Mammary Regeneration Using Embryonic Stem Cell Engraftment

Embryonic stem (ES) cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES cells to improve functional outcome following mammary gland injury. This study investigates the feasibility of implanting mouse ES cells labeled with enhanced green fluorescence protein in the developing mammary glands in order to acquire lineage-committed cells in mammary (mammary gland epithelial cell or luminal cell). Cells implanted in high numbers (5 × 106 cells per mammary gland) survived in the majority of the mice and nearly 38.4% of the surviving cells were CK18+ at 15th week following the transplantation. These results maybe provide a technique instrument on advanced therapy of breast diseases and the mammary regeneration after breast ablated partly.

Recombinant PBD-1 (porcine beta-defensin 1) expressed in the milk by transplanting transgenic mES-like-derived cells into mouse mammary gland.

ES (embryonic stem)‐derived cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES‐derived cells to improve functional outcome following partially damaged breast and also the modification of mammary tissue to produce costly proteins. This study investigates the feasibility of implanting mES‐dK (mouse ES‐derived keratinocytes‐like) cells stably transfected with a mammary gland special expression vector for the PBD‐1 (porcine beta‐defensin 1) in developing mammary glands. Our aim was to assess the ability of cell grafting to improve functional outcome following partial damage of the breast, also on the breast modification mammary tissue in mice for the production of PBD‐1 protein secreted in the milk. Our results showed that the ratios of the surviving cells labelled with the myoepithelial or luminal cell markers, EMA (epithelial membrane antigen) and CALLA, were 41.7±15.2% and 28.4±9.6%, respectively, which revealed that transplanted mES‐dK cells survived, integrated in vivo and differentiated into myoepithelial or luminal cells. In addition, Western blot analysis showed that 37.5% (3 out of 8) female transplanted mice had PBD‐1 expression in their milk and reached 0.4998, 0.5229 and 0.5195 μg/ml, respectively.