Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis.

Endothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl4 administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl4-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12 h after acute CCl4-intoxication. Tissue levels of ET-1, ETA- and ETB receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl4-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.

Characterization and Differentiation of Circulating Blood Mesenchymal Stem Cells and the Role of Phosphatidylinositol 3-Kinase in Modulating the Adhesion.

Bone marrow mesenchymal stem cells (BM MSCs) can differentiate into multi-lineage tissues. However, obtaining BM MSCs by aspiration is difficult and can be painful; therefore peripheral blood (PB) MSCs might provide an easier alternative for clinical applications. Here, we show that circulating PB MSCs proliferate as efficiently as BM MSCs in the presence of extracellular matrix (ECM) and that differentiation potential into osteoblast in vitro and in vivo. Both BM MSCs and PB MSCs developed into new bone when subcutaneously transplanted into immune-compromised mice using hydroxyapatite/tricalcium phosphate as a carrier. Furthermore, LY294002 and Wortmannin blocked mesenchymal stem cell attachment in a dose-dependent manner, suggesting a role of phosphatidylinositol 3-kinase in MSC attachment. Our data showed that the growth of PB MSCs could be regulated by interaction with the ECM and that these cells could differentiate into osteoblasts, suggesting their potential for clinical applications.

Phenotypic characterization and in vivo localization of human adipose-derived mesenchymal stem cells.

Human adipose-derived mesenchymal stem cells (hADMSCs) are a potential cell source for autologous cell therapy due to their regenerative ability. However, detailed cytological or phenotypic characteristics of these cells are still unclear. Therefore, we determined and compared cell size, morphology, ultrastructure, and immunohistochemical (IHC) expression profiles of isolated hADMSCs and cells located in human adipose tissues. We also characterized the localization of these cells in vivo. Light microscopy examination at low power revealed that hADMSCs acquired a spindle-shaped morphology after four passages. Additionally, high power views showed that these cells had various sizes, nuclear contours, and cytoplasmic textures. To further evaluate cell morphology, transmission electron microscopy was performed. hADMSCs typically had ultrastructural characteristics similar to those of primitive mesenchymal cells including a relatively high nuclear/cytosol ratio, prominent nucleoli, immature cytoplasmic organelles, and numerous filipodia. Some cells contained various numbers of lamellar bodies and lipid droplets. IHC staining demonstrated that PDGFR and CD10 were constitutively expressed in most hADMSCs regardless of passage number but expression levels of α-SMA, CD68, Oct4 and c-kit varied. IHC staining of adipose tissue showed that cells with immunophenotypic characteristics identical to those of hADMSCs were located mainly in the perivascular adventitia not in smooth muscle area. In summary, hADMSCs were found to represent a heterogeneous cell population with primitive mesenchymal cells that were mainly found in the perivascular adventitia. Furthermore, the cell surface markers would be CD10/PDGFR. To obtain defined cell populations for therapeutic purposes, further studies will be required to establish more specific isolation methods.

Comparison of the ultrastructural and immunophenotypic characteristics of human umbilical cord-derived mesenchymal stromal cells and in situ cells in Wharton's jelly.

The umbilical cord contains mucinous connective tissue, called Wharton's jelly. It consists of stromal cells, collagen fibers, and amorphous ground substances composed of proteoglycan. Recently, these stromal cells have been redefined as a new cell therapy source, named human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). However, there are few studies on the ultrastructural features and immune-phenotypic characteristics of isolated hUCMSCs and comparisons with the cells found in original cord tissues. In this study, the authors describe and compare the phenotypic characteristics of hUCMSCs with cells in the umbilical cord in order to know the kinds of cells and ultrastructural changes. Isolated hUCMSCs showed similar ultrastructure with few structural differences from in situ stromal cells, and they are relatively homogenous and well-developed mesenchymal cells that demonstrate a myofibroblastic phenotype.

The establishment of mouse embryonic stem cell cultures on 96-well plates for high-throughput screening.

Embryonic stem (ES) cells can be valuable for monitoring differentiation processes and for improving applications in basic developmental biology. The application of ES cells can be a useful tool for drug discovery and toxicology. Therefore, we suggest the high-throughput screening (HTS) system based on ES cells in this study. Firstly, we optimized the feeder-free condition and seeding cell number which can maintained for at least 7 days without over-confluency. We analyzed the system by cell viability, proliferation activity, RT-PCR and morphologic/immunohistochemical evaluations. The optimal cell seeding number was 30/well that was maintained the typical colonial morphology over 9 d with 1,000 U/ml LIF in the limited space. The cell in optimized condition expressed ALP, SSEA-1, Oct 4 and Nanog and the genetic expressions showed similar to protein expressions. The cell lineage marker expressions showed faint or none. The cell viability and proliferation activity were increased in time-dependent manner in our optimized HTS system. In conclusion, the novel HTS system using ES cells can by useful for developing models for drug discovery as well as toxicological screening in the near future.

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-ß3 and bone morphogenetic protein-6 in a normal healthy impacted third molar.

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-ß3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-ß3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-ß3 and 220% by BMP-6. The synergetic effect of TGF-ß3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-ß3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

The Porcine Aortic Tissue Culture System in vitro for Stem Cell Research.

BACKGROUND AND OBJECTIVES: Due to the shortage of human donors for transplantation, the use of animal organs for xenotransplantation has come into great interest. Xeno-derived vessels and cardiac valves would be possible alternatives for the patient suffering from cardiovascular diseases. Therefore, we established in vitro culture system of a porcine vessel that could be helpful for the research of xenograft and stem cell research. METHODS AND RESULTS: We primarily isolated porcine thoracic aorta, cultured square-shaped pieces up to 17 days and analyzed its morphology and characters. The endothelial cells were primarily isolated from cultured porcine aortic pieces and their morphology, function and character were analyzed in order to confirm them as endothelial cells at day 3, 4, 8, 10 and 17. Even at day 17, the morphology exhibited the intact endothelial layer as well as specifically expressed CD31 and von Willebrand factor. The morphology of primarily isolated cells from cultured tissues was identical as an endothelial cell. By flow cytometry analysis, more than 80% of the isolated cells expressed CD31 and up to 80% took up acetyl low density lipoprotein (ac-LDL) until day 10 of tissue culture period even though it decreased to about 50% at day 17 that means they not only showed typical endothelial cell characters but also functioned properly. CONCLUSIONS: We successfully established and optimized a porcine vascular tissue in vitro culture system that could be a valuable model for in vitro study of xenotransplantation and stem cell research.

Detection and identification of Mycobacterium tuberculosis in joint biopsy specimens by rpoB PCR cloning and sequencing.

Osteoarticular tuberculosis (OAT) is an extrapulmonary tuberculosis and accounts for 1 to 3% of all tuberculosis cases. We used an rpoB PCR-plasmid TA cloning-sequencing method to detect and identify tubercle bacilli in surgical specimens from patients suspected of having OAT. By comparing the similarities of the rpoB sequences determined with those in GenBank, Mycobacterium tuberculosis was detected in 23 of 43 samples. Three of the 23 positive samples had mutations at codon 531, which are commonly observed in rifampin-resistant M. tuberculosis strains. Our results suggest that the rpoB PCR-TA cloning-sequencing method developed, which detects M. tuberculosis and which simultaneously determines its rifampin susceptibility, can also be used efficiently for the diagnosis of OAT.