OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to dedifferentiated fat (DFAT) cells. DFAT cells have many advantages compared with adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs). For example, DFAT cells are homogeneous and could be obtained from donors regardless of their age. Furthermore, DFAT cells also have the same multi-lineage potentials and low immunogenicity as ASCs. As an excellent source of seed cells for tissue engineering and stem cell transplantation, DFAT cells have better prospects in the treatment of many clinical diseases, such as bone defects, neurological diseases, ischemic heart disease and kidney disease. It is necessary to make more intensive studies of DFAT cells. This article summarizes progresses in the immunological characteristics, differentiation ability and potential clinical applications of DFAT cells.
Adipocytes ; cytology ; Cell Dedifferentiation ; Cell Differentiation ; Cells, Cultured ; Humans ; Stem Cell Transplantation ; Tissue Engineering

BACKGROUNDIn vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering.

METHODSBovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining.

RESULTSChondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium.

CONCLUSIONThese findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

Animals ; Cartilage, Articular ; cytology ; Cattle ; Cell Dedifferentiation ; Cells, Cultured ; Chondrocytes ; cytology ; physiology ; Culture Media, Serum-Free ; Fibronectins ; pharmacology ; Real-Time Polymerase Chain Reaction ; SOX9 Transcription Factor ; genetics

Recent studies have confirmed that diverse adult tissue cells can be reprogrammed and induced to pluripotency, that is so-called induced pluripotent stem cells (iPS cells). But most of these dedifferentiated processes are induced by gene delivery with retroviral vectors. Some of the delivered genes are cancer causing. So, in current situation, these adult-derived embryonic stem-like cells cannot be used in clinical therapy to cure human diseases. Recently some articles that were published in the authoritative journals are receiving attentions. They show that, in mice and human, spermatogonial stem cells (SSCs) can be used for generating pluripotent stem cells without the exogenous genes and retroviruses, and they can also be used for autologous transplantation without ethical problems. These findings suggest that human SSCs may have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. In this review, we describe and compare the methods that have been used to isolate, purificate and culture SSCs. We also describe the recent results in which SSCs can be transformed into pluripotent stem cells, and the pluripotent stem cells have potential applications in regenerative medicine and genetic medicine.
Cell Culture Techniques ; methods ; Cell Dedifferentiation ; physiology ; Cells, Cultured ; Humans ; Male ; Pluripotent Stem Cells ; cytology ; Spermatogonia ; cytology ; Stem Cells ; cytology

We describe a 69-year-old woman who presented with a dedifferentiated extraskeletal myxoid chondrosarcoma arising in the left masticator space. Computed tomography and magnetic resonance imaging revealed a 5 cm sized mass in the left masticator space. Histologically, the tumor consisted of two distinct areas. The less cellular area was a low-grade extraskeletal myxoid chondrosarcoma, composed of strands or cords of uniform spindle cells and abundant myxoid stroma. The more cellular, dedifferentiated area corresponded to a high grade myxofibrosarcoma, consisting of anaplastic tumor cells in myxoid stroma and geographic necrosis. The tumor cells of the former area were positive for S-100 protein, microtubule-associated protein-2 (MAP-2) and class III beta-tubulin, but negative for cytokeratin, smooth muscle actin, and desmin. The tumor cells in the latter, pleomorphic area showed MAP-2 and beta-tubulin immunoreactivity with a high Ki-67 labeling index. Based on its histologic and immunohistochemical features, the tumor was considered a dedifferentiated extraskeletal myxoid chondrosarcoma.
Actins ; Aged ; Cell Dedifferentiation ; Chondrosarcoma ; Desmin ; Female ; Humans ; Keratins ; Magnetic Resonance Imaging ; Muscle, Smooth ; Necrosis ; S100 Proteins ; Tubulin

We describe a 69-year-old woman who presented with a dedifferentiated extraskeletal myxoid chondrosarcoma arising in the left masticator space. Computed tomography and magnetic resonance imaging revealed a 5 cm sized mass in the left masticator space. Histologically, the tumor consisted of two distinct areas. The less cellular area was a low-grade extraskeletal myxoid chondrosarcoma, composed of strands or cords of uniform spindle cells and abundant myxoid stroma. The more cellular, dedifferentiated area corresponded to a high grade myxofibrosarcoma, consisting of anaplastic tumor cells in myxoid stroma and geographic necrosis. The tumor cells of the former area were positive for S-100 protein, microtubule-associated protein-2 (MAP-2) and class III beta-tubulin, but negative for cytokeratin, smooth muscle actin, and desmin. The tumor cells in the latter, pleomorphic area showed MAP-2 and beta-tubulin immunoreactivity with a high Ki-67 labeling index. Based on its histologic and immunohistochemical features, the tumor was considered a dedifferentiated extraskeletal myxoid chondrosarcoma.
Actins ; Aged ; Cell Dedifferentiation ; Chondrosarcoma ; Desmin ; Female ; Humans ; Keratins ; Magnetic Resonance Imaging ; Muscle, Smooth ; Necrosis ; S100 Proteins ; Tubulin

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics

Cell reprogramming is a progress in which the memory of a mature cell is erased and then the cell develops novel phenotype and function; ultimately, the fate of the cell changes. Cell reprogramming usually occurs at genes expression levels that no genomic DNA sequence change will be involved. By changing the programs of the genetic expressions of cells in terms of space and time, cell reprogramming alters the differentiation of cells and thus produces the required cells. Further research on cells reprogramming will elucidate the mechanisms that govern the cell development, and thus provides more information of the sources of seed cells used for regeneration medicine. More cells differentiated from many terminally differentiated cells will be obtained, which is extremely important for the understanding of molecular differentiation and for the development of cell replacement therapy. This article summarizes the classification, influencing factors, approaches and latest advances of cells reprogramming.
Animals ; Cell Dedifferentiation ; genetics ; Cell Differentiation ; genetics ; Cellular Reprogramming ; Gene Expression ; Humans ; Nuclear Transfer Techniques

OBJECTIVETo identify the differences in the expression of epithelial or mesenchymal standard proteins between prostate cancer cell lines and tumors, and to investigate the relationship between the process of the prostate cancer cell line forming subcutaneous tumors and epithelial-mesenchymal transition (EMT) by comparing the characteristics of different prostate cell lines forming subcutaneous tumors in SCID mice.

METHODSWe constructed prostate cancer models in male SCID mice by subcutaneous injection of 4 human prostate cancer cell lines DU145, Tsu, PC3 and LNCaP, and compared the characteristics of tumor formation. We used Western blot to detect the expressions of E-cadherin and Vimentin in the cancer cell lines and subcutaneous tumors, observed their differences before and after tumor formation, and explore the relationship between EMT and tumor formation.

RESULTSThe EMT positive cells DU145 and Tsu showed a higher rate and speed of tumor formation than the EMT negative ones PC3 and LNCaP. The expression of E-cadherin was down-regulated in DU145, up-regulated in Tsu, and absent in PC3 and LNCaP.

CONCLUSIONEMT positive cells have a stronger ability of forming tumors than EMT negative cells, and mesenchymal-epithelial transition does exist in subcutaneous tumor formation.

Animals ; Cadherins ; metabolism ; Cell Dedifferentiation ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Epithelial-Mesenchymal Transition ; Humans ; Male ; Mice ; Mice, SCID ; Prostatic Neoplasms ; metabolism ; pathology ; Subcutaneous Tissue ; pathology ; Vimentin ; metabolism