Shih Ping Liu

Glycine N-methyltransferase-/- mice develop chronic hepatitis and glycogen storage disease in the liver.

Glycine N‐methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. To establish a Gnmt knockout mouse model, 2 lambda phage clones containing a mouse Gnmt genome were isolated. At 11 weeks of age, the Gnmt−/− mice had hepatomegaly, hypermethioninemia, and significantly higher levels of both serum alanine aminotransferase and hepatic S‐adenosylmethionine. Such phenotypes mimic patients with congenital GNMT deficiencies. A real‐time polymerase chain reaction analysis of 10 genes in the one‐carbon metabolism pathway revealed that 5,10‐methylenetetrahydrofolate reductase, S‐adenosylhomocysteine hydrolase (Ahcy), and formiminotransferase cyclodeaminase (Ftcd) were significantly down‐regulated in Gnmt−/− mice. This report demonstrates that GNMT regulates the expression of both Ftcd and Ahcy genes. Results from pathological examinations indicated that 57.1% (8 of 14) of the Gnmt−/− mice had glycogen storage disease (GSD) in their livers. Focal necrosis was observed in male Gnmt−/− livers, whereas degenerative changes were found in the intermediate zones of female Gnmt−/− livers. In addition, hypoglycemia, increased serum cholesterol, and significantly lower numbers of white blood cells, neutrophils, and monocytes were observed in the Gnmt−/− mice. A real‐time polymerase chain reaction analysis of genes involved in the gluconeogenesis pathways revealed that the following genes were significantly down‐regulated in Gnmt−/− mice: fructose 1,6‐bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose‐6‐phosphate transporter. Conclusion: Because Gnmt−/− mice phenotypes mimic those of patients with GNMT deficiencies and share several characteristics with GSD Ib patients, we suggest that they are useful for studies of the pathogenesis of congenital GNMT deficiencies and the role of GNMT in GSD and liver tumorigenesis. (HEPATOLOGY 2007.)

MicroRNAs Regulation Modulated Self-Renewal and Lineage Differentiation of Stem Cells

Stem cells are unique cells in the ability that can self-renew and differentiate into a wide variety of cell types, suggesting that a specific molecular control network underlies these features. To date, stem cells have been applied to many clinical therapeutic approaches. For example, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the cells responding to ischemia or injury and engage in effective revascularization to repair within impairment regions. Transplantation of MSCs after stroke and hindlimb ischemia results in remarkable recovery through enhancing angiogenesis. MicroRNAs are a novel class of endogenous, small, noncoding RNAs that work via translational inhibition or degradation of their target mRNAs to downregulate gene expression. MicroRNAs have been strongly linked to stem cells, which have a remarkable role in development. In this study, we focused on the microRNA regulation in multiple stem cells. For example, miR-520h was upregulated and miR-129 was downregulated in HSC. MiR-103, 107, 140, 143, 638, and 663 were associated with MSCs while miR-302s and miR-136 were associated with ESCs. In NSCs, miR-92b, let-7, and miR-125 were the critical regulators. This overview of the recent advances in the aspects of molecular control of stem cell biology reveals the importance of microRNAs, which may be helpful for future work.

Characterization of a glycine N-methyltransferase gene knockout mouse model for hepatocellular carcinoma: Implications of the gender disparity in liver cancer susceptibility

Hepatocellular carcinoma (HCC) is the fifth common cancer in the world and it mainly occurs in men. Glycine N‐methyltransferase (GNMT) participates in one‐carbon metabolism and affects DNA methylation by regulating the ratio of S‐adenosylmethionine to S‐adenosylhomocystine. Previously, we described that the expression of GNMT was diminished in human HCC. Here, we showed that 50% (3/6) male and 100% (7/7) female Gnmt−/− mice developed HCC, and their mean ages of HCC development were 17 and 16.5 months, respectively. In addition, 42.9% (3/7) of female Gnmt−/− mice had hemangioma. Wnt reporter assay demonstrated that Gnmt is a negative regulator for canonical Wnt signaling pathway. Beta‐catenin, cyclin D1 and c‐Myc, genes related to Wnt pathway, were upregulated in the liver tissues from both 11 weeks and HCC stage of Gnmt−/− mice. Furthermore, global DNA hypomethylation and aberrant expression of DNA methyltransferases 1 and 3b were found in the early and late stages of HCC development. Hierarchical cluster analysis of 6,023 transcripts from microarray data found that gene expression patterns of HCC tumors from male and female Gnmt−/− mice were distinctively different. Real‐time PCR confirmed that Gadd45a, Pak1, Mapk3 and Dsup3 genes of mitogen‐activated protein kinase (MAPK) pathway were activated in Gnmt−/− mice, especially in the female mice. Therefore, GNMT is a tumor suppressor gene for liver cancer, and it is associated with gender disparity in liver cancer susceptibility.

Decellularization and Recellularization Technologies in Tissue Engineering

Decellularization is the process by which cells are discharged from tissues/organs, but all of the essential cues for cell preservation and homeostasis are retained in a three-dimensional structure of the organ and its extracellular matrix components. During tissue decellularization, maintenance of the native ultrastructure and composition of the extracellular matrix (ECM) is extremely acceptable. For recellularization, the scaffold/matrix is seeded with cells, the final goal being to form a practical organ. In this review, we focus on the biological properties of the ECM that remains when a variety of decellularization methods are used, comparing recellularization technologies, including bioreactor expansion for perfusion-based bioartificial organs, and we discuss cell sources. In the future, decellularization–recellularization procedures may solve the problem of organ assembly on demand.

Differentiation of stem cells: strategies for modifying surface biomaterials.

Stem cells are a natural choice for cellular therapy because of their potential to differentiate into a variety of lineages, their capacity for self-renewal in the repair of damaged organs and tissues in vivo, and their ability to generate tissue constructs in vitro. Determining how to efficiently drive stem cell differentiation to a lineage of choice is critical for the success of cellular therapeutics. Many factors are involved in this process, the extracellular microenvironment playing a significant role in controlling cellular behavior. In recent years, researchers have focused on identifying a variety of biomaterials to provide a microenvironment that is conducive to stem cell growth and differentiation and that ultimately mimics the in vivo situation. Appropriate biomaterials support the cellular attachment, proliferation, and lineage-specific differentiation of stem cells. Tissue engineering approaches have been used to incorporate growth factors and morphogenetic factors—-factors known to induce lineage commitment of stem cells—into cultures with scaffolding materials, including synthetic and naturally derived biomaterials. This review focuses on various strategies that have been used in stem cell expansion and examines modifications of natural and synthetic materials, as well as various culture conditions, for the maintenance and lineage-specific differentiation of embryonic and adult stem cells.

n-butylidenephthalide protects against dopaminergic neuron degeneration and α-synuclein accumulation in Caenorhabditis elegans models of Parkinson's disease.

Background Parkinsons disease (PD) is the second most common degenerative disorder of the central nervous system that impairs motor skills and cognitive function. To date, the disease has no effective therapies. The identification of new drugs that provide benefit in arresting the decline seen in PD patients is the focus of much recent study. However, the lengthy time frame for the progression of neurodegeneration in PD increases both the time and cost of examining potential therapeutic compounds in mammalian models. An alternative is to first evaluate the efficacy of compounds in Caenorhabditis elegans models, which reduces examination time from months to days. n-Butylidenephthalide is the naturally-occurring component derived from the chloroform extract of Angelica sinensis. It has been shown to have anti-tumor and anti-inflammatory properties, but no reports have yet described the effects of n-butylidenephthalide on PD. The aim of this study was to assess the potential for n-butylidenephthalide to improve PD in C. elegans models. Methodology/Principal Findings In the current study, we employed a pharmacological strain that expresses green fluorescent protein specifically in dopaminergic neurons (BZ555) and a transgenic strain that expresses human α-synuclein in muscle cells (OW13) to investigate the antiparkinsonian activities of n-butylidenephthalide. Our results demonstrate that in PD animal models, n-butylidenephthalide significantly attenuates dopaminergic neuron degeneration induced by 6-hydroxydopamine; reduces α-synuclein accumulation; recovers lipid content, food-sensing behavior, and dopamine levels; and prolongs life-span of 6-hydroxydopamine treatment, thus revealing its potential as a possible antiparkinsonian drug. n-Butylidenephthalide may exert its effects by blocking egl-1 expression to inhibit apoptosis pathways and by raising rpn-6 expression to enhance the activity of proteasomes. Conclusions/Significance n-Butylidenephthalide may be one of the effective neuroprotective agents for PD.

Adipose-derived stem cells: Isolation, characterization, and differentiation potential

In mammals, the two main types of adipose tissues, white and brown adipose tissues, exert different physiological functions. White adipose tissue (WAT) is for storing energy, while brown adipose tissue (BAT) is for energy consumption. Adipose-derived stem cells (ADSCs) are abundant in WAT and BAT, have multipotent characteristics, and are easily extracted. ADSCs can be differentiated into several cell lineages, including adipocytes, osteoblasts, chondrocytes (cartilage cells), myocytes, and neuronal cells. Therefore, ADSC could be considered as a strategy for future regenerative medicine and tissue engineering.

Induced Pluripotent Stem (iPS) Cell Research Overview

Stem cells are capable of self-renewal and differentiation into a wide range of cell types with multiple clinical therapeutic applications. The two most important issues associated with embryonic stem (ES) cells are immune rejection and medical ethics. In 2006, induced pluripotent (iPS) cells were generated from somatic cells via the introduction of four transcriptional factors: OCT4, SOX2, c-MYC, and KLF4. Researchers found that iPS cell morphology, proliferation, surface antigens, gene expression, telomerase activity, and the epigenetic status of pluripotent cell-specific genes were similar to the same characteristics in ES cells. iPS cells are capable of overcoming hurdles associated with ES cells due to their generation from mature somatic cells (e.g., fibroblasts). For this reason, iPS cells are considered an increasingly important cell therapy technology. iPS cell production entails the use of retroviruses, lentiviruses, adenoviruses, plasmid transfections, transposons, or recombinant proteins. In this article we discuss the advantages and limitations of each strategy and address issues associated with clinical trials, including the potential for liver tumor formation and low generation efficiency.

Glycine N-methyltransferase affects the metabolism of aflatoxin B1 and blocks its carcinogenic effect.

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B(1) (AFB(1)) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB(1) treatment. Competitive enzyme immunoassays indicated that after AFB(1) treatment, the AFB(1)-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB(1)-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB(1) in the cultural supernatants of cells exposed to AFB(1) showed that the AFM(1) level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB(1). Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB(1). Automated docking experiments showed that AFB(1) binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB(1) interaction is 44.9 microM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB(1), resulting in reduced AFB(1)-DNA adducts formation and cell death.

An overview of concepts for cancer stem cells.

For many years, cancer research has focused on the adult stem cells present in malignant tumors. It is believed that current cancer treatments sometimes fail because they do not target these cells. According to classic models of carcinogenesis, these events can occur in any cell. In contrast, the cancer stem cell (CSC) hypothesis states that the preferential targets of oncogenic transformation are tissue stem cells or early progenitor cells that have acquired the potential for self-renewal. These tumor-initiating cells, or CSCs, in turn, are characterized by their ability to undergo self-renewal, a process that drives tumorigenesis and differentiation, which contributes to the cellular heterogeneity of tumors. Herein, we discuss the definitions and properties of CSCs in the major human cancers.