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Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure
Stem cell-based therapy to treat liver diseases is a focus of current research worldwide. So far, most such studies depend on rodent hepatic failure models. The purpose of this study was to isolate mesenchymal stem cells from human placenta (hPMSCs) and determine their therapeutic potential for treating Chinese experimental miniature pigs with acute liver failure (ALF).
hPMSCs were isolated and analyzed for their purity and differentiation potential before being employed as the donor cells for transplantation. ALF models of Chinese experimental miniature pigs were established and divided into four groups: no cell transplantation; hPMSCs transplantation via the jugular vein; X-ray-treated hPMSCs transplantation via the portal vein; and hPMSCs transplantation via the portal vein. The restoration of biological functions of the livers receiving transplantation was assessed via a variety of approaches such as mortality rate determination, serum biochemical analysis, and histological, immunohistochemical, and genetic analysis.
hPMSCs expressed high levels of CD29, CD73, CD13, and CD90, had adipogenic, osteogenic, and hepatic differentiation potential. They improved liver functions
Our data revealed that hPMSCs could not only differentiate into hepatocyte-like cells
Acute liver failure (ALF) is defined as the rapid development of severe acute liver injury with impaired synthetic function and hepatic encephalopathy in the absence of pre-existing liver diseases. The disease carries a high morbidity and mortality. Orthotopic liver transplantation is the most effective therapy for ALF, but it is highly intrusive, irreversible, and limited by a shortage of donor organs, high expense, and the necessity of lifelong immunosuppressive treatments
However, the therapeutic potential of hPMSCs in a large animal model of hepatic injury remains largely unexplored. In the present study, we isolated MSCs from human placentas and characterized their morphology, phenotypic profiles, growth potency, and capacity to differentiate into various lineages. MSCs were transplanted directly into pigs with acute liver failure induced by G-galactosamine (GalN) via two routes: the jugular or portal veins. The intra-portal cell transplantation procedure was guided by B-ultrasonography. After transplantation, their capacity to overcome hepatic injury was investigated.
Isolation and culturing of human placental mesenchymal stem cells (hPMSCs)
Placentas were obtained from donors at the Hangzhou Red Cross Hospital in China after informed consent had been obtained. The study used a protocol approved by the Research Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University.
The placental tissue was washed with preheated phosphate buffered saline (PBS, pH 7.2 ± 0.1, GenomSciences, Hangzhou, China), minced, and digested using 0.1% (w/v) collagenase type IV (Invitrogen, USA) at 37°C for 30 min. Recovered cells and digested cell debris were filtered through a 100-μm cell strainer. Mononuclear cells (MNCs) obtained by lymphocyte isolation (GE Healthcare, Ficoll-Pague™ PLUS, Sweden) were cultured in special medium (MesenCult® Human Basal Medium plus MesenCult® Human Supplement, STEMCELL Technologies Inc., Vancouver, Canada) and adjusted to 2 × 106 cells in T25-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark) maintained in an incubator at 37°C in a humidified atmosphere with 5% (v/v) CO2. Approximately 4 to 6 days later, many colonies had formed. After achieving 60-70% confluence, adherent cells were trypsinized using 0.25% (w/v) trypsin/EDTA (Invitrogen, Carlsbad, CA, USA) and re-plated at a 1:3 dilution. Placenta-derived cells were cultured continuously under the same conditions.
Surface antigen expression
Culture-expanded cells (passages 3 to 6) were washed with PBS-containing 0.5% (w/v) bovine serum albumin (BSA) and their concentration adjusted to 1 × 106 cells/100 μL. Their surface antigen expression was evaluated by incubating with anti-human antibodies against CD90-phycoerythrin (PE), CD13-PE, CD133-PE, CD31- allophycocyanin (APC), CD29-APC, CD73-APC, CD105-APC, CD79B-fluorescein isothiocyanate (FITC), CD34-FITC, CD166-FITC, and CD45-TRI COLOR (PE-Cy5- PC5) (eBioscience, Inc., San Diego, CA, USA). After being labeled with antibodies in the dark at room temperature for 20 min, cells were washed twice with PBS. Two-color flow cytometry was conducted using a Beckman Coulter Cytomics FC 500 MPL (Beckman Coulter, Inc., Los Angeles, CA, USA), and the data were analyzed using MXP software.
Differentiation studies
Adipogenic and osteogenic differentiation
Adherent cells derived from placenta (passages 2 to 6) were plated at a density of 2 × 103/well on six-well multidishes (Nunclon™Δ Surface, Denmark) and cultured in special conditional adipogenic medium (MesenCult® Human Adipogenic Suppl, STEMCELL Technologies, Inc., Canada) as per the manufacturer's protocol. After 4 weeks, adipogenesis was confirmed by oil red O staining (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and lipoprotein lipase (LPL) mRNA detection. Human intraoperative abandoned adipose tissue and hPMSCs cultured in normal growth medium served as controls.
MSCs from placenta (passages 2 to 6) were plated at a density of 2 × 103/well in six- well plates (Nunclon™Δ Surface, Denmark) and expanded in special conditional osteogenic medium (OriCell™ hMSC Osteogenic Differentiation Medium, Cyagen Biosciences, Guangzhou, China) and media was changed twice per week. After 4 weeks, osteogenic differentiation was evaluated by Alizarin Red S staining (Genmed Scientifics Inc., USA, Shanghai, China) and detection of osteopontin (OPN) mRNA. hPMSCs cultured in normal growth medium and immortalized human fetal osteoblastic cells (hFOB 1.19 cell line, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) served as controls.
Hepatic differentiation
Cultured placental cells (1 × 104/mL; passages 2 to 6) were plated in T25-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark) and four-well chamber slides (Nunc Lab-Tek™ Chamber Slide™ System, Denmark) with LG-DMEM (Gibco/Invitrogen, USA) containing 10% (v/v) FBS (Gibco/Invitrogen, USA) for 2 days. Hepatogenic induction was conducted as follows. The medium was changed to IMDM supplemented with 20 ng/mL epidermal growth factor (EGF) and10 ng/mL fibroblast growth factor (FGF) for 2 days. The cells were then treated with IMDM with 20 ng/mL hepatocyte growth factor (HGF), 10 ng/mL FGF, and 0.61 g/L niacin amide and 1% (v/v) insulin-transferrin-selenium (ITS) premix (Sigma-Aldrich, St Louis, MO, USA) for 10 days; the medium was changed every 3 days. Finally, cells were maintained in IMDM containing 20 ng/mL oncostatin M (OSM; St Louis, MO, USA), 1 μmol/L dexamethasone (Dex; Sigma-Aldrich, St Louis, MO, USA), and 1% (v/v) ITS premix for 10 days. For the next few days, cells were cultured with HEPATOZYME-SFM (Gibco/Invitrogen, USA). Cells cultured with low glucose DMEM (LG-DMEM) containing 10% (v/v) FBS served as controls. Albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), and cytokeratin 19 (CK19) levels were determined by immunohistochemistry and RT-PCR; and urea synthesis, low-density lipoprotein (LDL) uptake, glycogen storage, and cytochrome P450(CYP450) enzymatic activity were assayed to evaluate hepatogenic induction on days 7, 14, 21, 28, and 35 after induction.
Cell samples were fixed in 4% (v/v) paraformaldehyde solution at room temperature for 15 min. After being treated with 0.3% (v/v) hydrogen peroxide (H2O2) and blocked in 5% (m/v) BSA, cells were incubated with diluted primary antibody against anti-human CK18 (2 mg/mL, 1:400; Abcam, UK), human AFP (1:200; Abcam, UK), human CK19 (1:400; Abcam, UK), and human ALB (5 mg/mL, 1:500; Abcam, UK), according to the manufacturer's instructions. The primary antibody was detected using horseradish peroxidase-conjugated anti-rabbit IgG (1 mg/mL, 1:1,000; Abcam, UK) antibody followed by incubation with diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs, Burlingame, CA, USA). Cultured human hepatocytes served as positive controls.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA of cells was extracted with the Trizol reagent (Invitrogen, USA). The quantity and purity of RNA were estimated by reading absorbance at 260 and 280 nm. cDNA was synthesized using oligo dT primers with the Improm-II™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The primers for the target products were designed as in Table
Table 1
Primers and conditions used for RT-PCR
Amplicon size (bp)
94°C/30 s; 55°C/40 s; 72°C/60 s
94°C/30 s; 58°C/40 s; 72°C/60 s
94°C/60 s; 64°C/60 s; 72°C/120 s
94°C/30 s; 50°C/40 s; 72°C/60 s
94°C/30 s; 58°C/40 s; 72°C/60 s
94°C/30 s; 56°C/30 s; 72°C/60 s
94°C/30 s; 55°C/45 s; 72°C/60 s
Glycogen storage staining
Primary human hepatocytes were obtained from the Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health, P. R. China.
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were plated in six-well plates and cultured in either LG-DMEM supplemented with 10% (v/v) FBS or HEPATOZYME-SFM (all from Gibco/Invitrogen, USA). Cells were stained with the periodic acid-Schiff (PAS) staining kit according to the manufacturer's protocol (Genmed Scientifics Inc., Shanghai, China).
Low-density lipoprotein (LDL) uptake assay
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) incubated in DMEM containing 10% (v/v) FBS were labeled with DiI-LDL (1,19-dioctadecyl-3,3,39,39-tetramethylllindocarbocyane-low-density lipoprotein, Molecular Probes, Invitrogen, US) at 10 μg/mL for 24 h at 37°C. Cells were harvested, washed with cold PBS, and then stained according to the manufacturer's protocol. Uptake was subsequently evaluated by fluorescence microscopy (Olympus IX81, Japan).
Urea assay
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were cultured for 24 h in HepatoZYME-SFM (serum-free, no phenol red, Gibco®, Invitrogen™, USA) supplemented with 0.9 mmol/L ammonium chloride (NH4Cl, Bio Basic Inc., NY, USA). Supernatants were collected, centrifuged, and urea levels were measured in 96-well plates (Nunclon™Δ Surface, Denmark) at 430 nm by a spectrophotometer (Beckman Coulter® Multimode Detector DTX880, Beckman Coulter, Inc.), according to the instructions of the QuantiChrom™ urea assay kit (DIUR-500, BioAssay Systems, CA, USA).
Determination of cytochrome P450 activity
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were plated in six-well plates and cultured in either LG-DMEM supplemented with 10% (v/v) FBS or HEPATOZYME-SFM (all from Gibco/Invitrogen, USA), both with 10 μM rifampicin for 48 h. The media were then changed to IMDM containing a luminogenic CYP substrate (luciferin-PFBE, 50 μM, 1:40 dilution) for 24 h. Intracellular CYP enzyme activities were assessed by assaying luciferin production, according to the manufacturer's instructions (P450-Glo™ CYP3A4 assay with leciferin-PFBE, Promega, USA). Luciferase activity was expressed as relative luminescence units (RLU).
hPMSCs irradiation
hPMSCs from the placenta (passages 3 to 6) were seeded in T175-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark). After achieving 70-80% confluence, cells were irradiated with 30 Gy at room temperature using six MV X-rays (500 Mu/min, total of 2890 Mu) from a Varian 23EX Linear Accelerator.
Cell-viability assay
The lethal effect of irradiation on hPMSCs was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each group of cells was seeded at a density of 3000 cells per well in 96-well plates (Nunclon™Δ Surface, Denmark). After irradiation for 0, 24, 48, 72, 96, 120, or 144 h, 20 μL of MTT reagent (5 mg/mL, Sigma-Aldrich) was added to each well, and cells were incubated for 4 h at 37°C and 5% (v/v) CO2. The formazan complex was dissolved in 150 μL dimethylsulphoxide (DMSO, Sigma-Aldrich), and absorbance at 490 nm was measured using a spectrophotometer (Beckman Coulter® Multimode Detector DTX880, Beckman Coulter, Inc.). Experiments were performed in triplicate and repeated at least three times. In cell growth curves, the optical density (OD) values were plotted as a function of elapsed time.
Cell-cycle and apoptosis analysis
The effect of irradiation on cell-cycle progression and apoptosis of hPMSCs was investigated by flow cytometry (Beckman Coulter, Inc.). Cell-cycle and apoptosis analyses were carried out at 0 and 6 h and at 3 and 7 days after irradiation treatment. For cell-cycle studies, cells were fixed with pre-cooled absolute ethanol (-20°C) and finally stained using a cell-cycle staining kit (MultiSciences, Hangzhou, China), according to the manufacturer's protocol. The distribution of cell-cycle phases with different DNA contents was determined by flow cytometry and analyzed using multicycle ANOVA analysis software. Cell-apoptosis analysis was carried out by flow cytometry using the Annexin V/PI apoptosis kit (MultiSciences, Hangzhou, China). The percentages of apoptotic cells were analyzed using MXP cytometer software. Experiments were repeated three times.
D-galactosamine-induced acute liver-failure (ALF) model
Male Chinese experimental miniature pigs weighing 10-12 kg were obtained from Beijing Agriculture University (Beijing, China). Animal procedures were approved by the Animal Ethics Committee of Zhejiang University. All pigs were housed in an air-conditioned room with a 12 h dark/light cycle and received humane care for the duration of the study. For catheterization through the external jugular vein, ketalar (0.2 mL/kg) (Fujian Gutian Pharmaceutical Co., Ltd., Fujian, China) was injected intramuscularly to induce sedation. An auricular vein was secured and 2.5 mg/kg/h diprivan (AstraZeneca, Caponago, Italy) administered to achieve continuous anaesthesia. Catheterization was performed as described previously
Experimental groups and cell transplantation
Twenty-four ALF pigs were randomly divided into four groups (
Eighteen hours after GalN administration, hPMSCs (passages 3 to 6; 1.0 × 108 in 5 mL saline) were directly transplanted into the pigs via the jugular venous catheter or the portal vein. For intra-portal cell transplantation, an 18 G PTC needle (Hakko Co., Chikuma-Shi, Japan) with a guide wire was inserted into the left branch of the portal vein inside the liver. The procedure was performed under general anesthesia and guided by color ultrasound (Sequoia 512, Siemens, Germany). By injecting contrast medium SonoVue (Bracco, Milan, Italy), the transplanted cells were seen to be distributed throughout the liver. When the guide wire was withdrawn, blood flow confirmed that the portal vein had been accessed. Then, the hPMSCs preparation was directly and slowly infused into the liver via the portal vein over about 5 min. For jugular vein cell transplantation, the same number and volume of hPMSCs were infused into the jugular vein via a venous catheter. All transplanted pigs were immunosuppressed by daily administration of dexamethasone (Hubei Tianyao Pharmaceutical Co., Ltd, Xiangfan, Hubei) (10 mg/kg/d) during the first week after the transplantation. Blood samples were collected for biochemical analysis at the following time points: pre-and post-GalN administration (18 h), 1 to 7 days after cell infusion, then every 2 weeks from the second week until death. Liver samples from surviving animals were harvested every 2 weeks post-transplantation using an automatic biopsy needle, and the livers of dead animals were directly removed by dissection. These liver tissues were immediately stored at -80°C for molecular detection or fixed in 10% (v/v) formalin for histological and immunohistochemical analysis.
Biochemical analysis
Blood samples were obtained from each pig and centrifuged for 10 min at 1751 ×
Cytokine level measurement
Supernatants of hPMSCs (passages 3 to 6), hPMSCs co-cultured with 1 × 106 lymphocytes from healthy donors for 24 h and serum from model pigs, were collected, centrifuged, and leukemia inhibitory factor (LIF) levels were analyzed using a human LIF platinum ELISA Kit (eBioscience, Inc., San Diego, CA, USA). Tests were performed according to the manufacturer's protocol and every sample was run in duplicate. LIF concentration (pg/mL) was determined by comparison with the standard curve.
hPMSCs (1 × 106) and their supernatants were co-cultured with PBMC (1 × 106) for 24 h. Interferon-γ (IFN-γ) secretion was then assayed by ELISpot, according to the manufacturer's instructions (human IFN-γ ELISpot PRO kit, MABTECH AB, Sweden). The resulting spots were counted and analyzed using a computer-assisted AID ELISPOT Reader System (Immun Spot®, Cellular Technology Ltd.).
Pathology and immunohistochemistry
Pig livers were harvested at the indicated time points after cell transplantation, fixed in 10% (v/v) formalin, and embedded in paraffin. Sections (5-μm thickness) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) for routine histology. For immunohistochemical evaluation, the sections were heated in citrate buffer (0.02 mol/L, pH 5.8) for antigen retrieval. Endogenous peroxidase was prevented by immersion in a 0.3% (v/v) hydrogen peroxide (H2O2) in methanol bath for 15 min. After washing, non-specific binding was blocked with 5% (m/v) bovine serum albumin in PBS. Sections were then incubated with diluted primary antibody against anti-human ALB (5 mg/mL, 1:5,000; Abcam, UK), human AFP (1:250; Abcam, UK), and human CK18 (1 mg/mL, 1:200; Abcam, UK), according to the manufacturer's instructions. Detection of primary antibody was performed using horseradish peroxidase-conjugated secondary antibodies (1 mg/mL, 1:1,000; Abcam, UK). Peroxidase activity was revealed by a 3 to 5 min exposure to diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs). The sections were then washed, counterstained with hematoxylin for one minute, mounted, and observed under a light microscope (TE2000, Nikon, Japan). Normal human tissue obtained under informed consent served as positive controls.
Detection of human
PCR for human
Statistical analysis
Data were presented as the mean ± standard deviation (SD). Differences in serum levels of biochemical parameters were analyzed using a paired-samples
Morphology and phenotype profiles of hPMSCs
Primary adherent monolayer placental cells formed many colonies after 4 to 6 days and exhibited a fibroblastic morphology. They were subcultured every 3 to 4 days (Figure
Figure 1
hPMSCs morphology (4×)
hPMSCs morphology (4×). (A) Three days after isolation from the placenta before changing media; (B) four days after isolation from the placenta after changing media; (C) four weeks after isolation (passage 6).
The immunophenotype of culture-expanded cells (passages 3 to 6) was analyzed by flow cytometry, which revealed that the cells expressed high levels of CD29 (100%), CD73 (95.3%), CD13 (100%), and CD90 (85.2%), moderate levels of CD105 (69.4%) and CD166 (44.1%), but expressed almost no hematopoietic cell markers such as CD45 (0.1%), CD34 (0.5%), and CD133 (0.2%). CD31 and CD79b were expressed in only 0.2 and 0.4% of the population, respectively (Figure
Figure 2
Phenotype profile of adherent cells obtained from placentas
Phenotype profile of adherent cells obtained from placentas. Immunophenotype of hPMSCs determined by flow cytometry at passage three using labeled antibodies specific for the indicated human surface antigens or negative controls.
Adipogenic differentiation potential of hPMSCs
hPMSCs were cultured for 4 weeks in adipogenic medium, and adipogenic differentiation was assessed by oil red O staining and LPL mRNA detection. Differentiated hPMSCs formed intracytoplasmic lipid droplets and could be identified by oil red O staining, whereas hPMSCs cultured in normal growth medium had no lipid droplets and exhibited negative staining. Human adipose tissue (positive control) exhibited large lipid vesicles (Figure
Figure 3
Adipogenic (A-C, H) and osteogenic (D-G, I) differentiation of hPMSCs
Adipogenic (A-C, H) and osteogenic (D-G, I) differentiation of hPMSCs. (A) hPMSCs maintained in normal growth medium (10×); (B) human adipose tissue as a positive control (10×); (C) hPMSCs that had undergone adipogenic differentiation (accumulation of lipid droplets shown by red staining) (20×); (D) hPMSCs maintained in normal growth medium; (E) osteoblast as a positive control(4×); (F, G) hPMSCs that had undergone osteogenic differentiation (bone nodules formed), before and after staining (4×). (H, I) Representative RT-PCR analyses of hPMSCs cultured in adipogenic and osteogenic medium for 28 days. No LPL expression was found in hPMSCs maintained in regular medium (H, lane 2). After adipogenic induction, hPMSCs expressed LPL mRNA (H, lane 1). Human adipose tissue expressed LPL mRNA (H, lane 3). No OPN expression was found in hPMSCs maintained in growth medium (I, lane 2). After osteogenic induction, hPMSCs expressed OPN mRNA (I, lane 1). Osteoblasts expressed OPN mRNA (I, lane 3). LPL, lipoprotein lipase; OPN, osteopontin.
Osteogenic differentiation potential of hPMSCs
hPMSCs were cultured for 4 weeks in osteogenic medium, and osteogenic differentiation was assessed by Alizarin Red S staining and bone-specific gene (OPN) mRNA detection. Differentiated hPMSCs and osteoblast formed bone nodules and stained positive, whereas hPMSCs cultured in normal growth medium showed no positive staining (Figure
Hepatic differentiation potential of hPMSCs
hPMSCs at passage three were cultured in differentiation medium with hepatogenic supplements for 3 weeks. Differentiation efficiency was evaluated at the protein and mRNA levels. Immunocytochemistry revealed the presence of hepatic markers (ALB, AFP, CK18, CK19) in hPMSCs subjected to the hepatogenic differentiation protocol as well as in hepatocytes, but not in undifferentiated cells (Figure
Figure 4
Hepatic differentiation of hPMSCs
Hepatic differentiation of hPMSCs. (A) Immunocytochemistry analysis of hepatically differentiated or undifferentiated hPMSCs. (A, D, G, and J) Staining for the hepatic markers ALB, AFP, CK18, and CK19; a large number of cells had a light-brown coloration. (B, E, H, and K) hPMSCs cultured in growth medium did not display positive immunocytochemical staining. (C, F, I, and L) Efficiency of immunocytochemical staining was tested on cultured hepatocytes as positive controls (10×). (B) RT-PCR analysis of hepatic-specific gene expression in hepatically differentiated or undifferentiated hPMSCs. Lane 1, hPMSCs that had undergone hepatic differentiation; lane 2, hPMSCs cultured in growth medium; and lane 3, human liver tissue as a positive control.
Hepatocyte-like cells differentiated from hPMSCs exhibited characteristics typical of hepatocytes such as LDL uptake, glycogen storage, urea synthesis, and CYP-450 enzymatic activity, which were absent in undifferentiated MSCs (Figure
Figure 5
Functional parameters of hepatocyte-like cells differentiated from hPMSCs
Functional parameters of hepatocyte-like cells differentiated from hPMSCs. (A-D): glycogen storage capacity. (A) Primary human hepatocytes as a positive control (10×); (B) hPMSCs cultured in normal growth medium did not display positive PAS staining (4×); (C, D) hPMSCs cultured in hepatogenic differentiation medium for 28 days were positively stained, revealing cytoplasmic glycogen accumulation (C, 4×; D, 10×). (E-H) LDL uptake. (E) Primary human hepatocytes served as a positive control (10×); (F) hPMSCs maintained in growth medium were stained negatively; (G, H) LDL uptake was detected in hepatocyte-like cells 21 days after differentiation (G, 10×; H, 20×). (I) urea synthesis assay. After hepatogenic differentiation culture for 28 days, urea levels in culture supernatants were significantly increased. (J) CYP-450 enzymatic activities were determined. After hepatogenic differentiation culture for 35 days, CYP-450 enzymatic activity was significantly higher. Data are mean ±
Effect of X-ray irradiation on the viability and cell-cycle progression of hPMSCs
To investigate the effect of X-ray irradiation on the viability of hPMSCs, MTT assays were performed and a cell growth curve generated. No increase in cell numbers was found in hPMSCs treated with X-rays (Figure
Figure 6
Effects of X-ray irradiation on hPMSCs viability
Effects of X-ray irradiation on hPMSCs viability. Cells were treated with X-rays for the times indicated, and their viability was measured by MTT assay. OD indicates optical density at 490 nm.
To assess the effects of X-ray irradiation on cell-cycle progression and apoptosis, flow cytometry was utilized. Compared to normal hPMSCs (G1, 87.4%; S, 6.87%; G2/M, 5.8%), 89.3% of X-ray treated cells (7 days post-treatment) were in G1 phase, 10.7% in S phase, and 0% in G2/M phase (Figure
Figure 7
Effects of X-ray irradiation on cell-cycle progression and apoptosis of hPMSCs
Effects of X-ray irradiation on cell-cycle progression and apoptosis of hPMSCs. Cells were irradiated with X-rays for the times indicated, and cell-cycle progression and apoptosis were analyzed by flow cytometry. (A) Representative cell-cycle profile of hPMSCs. (B) Cytograms of hPMSCs apoptosis assays. (C, D) Quantitative analyses of the FACS data. Data are presented as means ± SD of triplicate experiments; *
Survival of ALF pigs transplanted with hPMSCs
In Group I, all pigs died within 6 days (survival time, 94.7 ± 15.4 h); in Group II, all pigs died within 4 days (survival time, 67 ± 22.5 h) except one (sacrificed at 3 months); in Group III, four pigs died within 6 days (survival time, 75.8 ± 48.0 h) and two others were sacrificed at 3 months; in Group IV, two pigs died after 7 days (survival time, 135 ± 33.9 h) and four were sacrificed at 5 months. In Groups I to IV, 7-day survival rates were 0, 16.7, 33.3, and 66.7%, respectively. The survival of pigs in Group IV was prolonged significantly compared with the other three groups (
Figure 8
Survival curves of the four groups
Survival curves of the four groups. Group I: injured, non-transplanted; Group II: injured, transplanted with normal hPMSCs via the jugular vein; Group III: injured, transplanted with X-ray-treated hPMSCs via the portal vein; Group IV: injured, transplanted with normal hPMSCs via the portal vein. The survival rate of Group IV was prolonged significantly compared with the other three groups (
Restoration of liver functions in ALF pigs transplanted with hPMSCs
Biochemical parameters (ALT, AST, ALP, CHE, TBIL, and TBA) were measured at selected time points to determine the restoration of liver functions in ALF pigs transplanted with hPMSCs (Figure
Figure 9
Biochemical analyses
Biochemical analyses. (A) Alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) alkaline phosphatase (ALP); (D) cholin esterase (CHE); (E) total bile acids (TBA); (F) total bilirubin (TBIL). Group I: injured, non-transplanted; Group II: injured, transplanted with normal hPMSCs via the jugular vein; Group III: injured, transplanted with X-ray-treated hPMSCs via the portal vein; Group IV: injured, transplanted with normal hPMSCs via the portal vein. Data are presented as means ± SD. The dotted line represents serum biochemical results of surviving pigs in Groups II and III.
Cytokine secretion and immunoregulation of transplanted hPMSCs
To determine the immunoregulatory activity of hPMSCs, human LIF and IFN-γ were detected in the sera of model pigs and hPMSCs supernatants. In the four long-term survivors of Group IV, human LIF in the serum peaked on day 4 and then returned to normal (Figure
Figure 10
Levels of human LIF and IFN-γ
Levels of human LIF and IFN-γ. (A) LIF curve in the serum of Group IV pigs; (B) LIF was markedly increased after hPMSCs were co-cultured with PMBCs for 24 h. **
ELISpot assay revealed that hPMSCs themselves did not secret IFN-γ, but they could stimulate PBMCs to secret IFN-γ (Figure
Therefore, hPMSCs may contribute to therapeutic effects in ALF models through immunoregulation.
Histological features of the ALF pig livers transplanted with hPMSCs
Livers from each group were collected at selected time points and H&E staining was performed to examine liver pathology. Galactosamine-induced liver injury in the control group (Group I) was indicated by large areas of inflammation, hepatocyte denaturation, and necrosis, as well as sinusoid congestion and hemorrhage (Figure
Figure 11
H&E staining
H&E staining. (A) Group I (day 4); (B) group II (day 1); (C, D) group III (days 4, 7); (E-L): group IV (days 2, 4, 7, 14, 28, 42, 150, and 155) (10×); (G-J) liver puncture biopsy specimens.
Fate of transplanted hPMSCs in ALF pig livers
To investigate whether hPMSCs are capable of engrafting and undergoing hepatic differentiation
Figure 12
Immunohistochemistry results of transplanted ALF pig liver samples
Immunohistochemistry results of transplanted ALF pig liver samples. (A) No ALB+cells were found in liver puncture biopsy samples 8 weeks after transplantation in Group IV (40×); (B) ALB+ cells were detected 6 weeks after hPMSCs infusion in the specimen harvested using an automatic biopsy needle (40×); (C-H) ALB+cells were observed in three pigs at 5 months (C, E, G, 20×; D, F, H, 40×); (I-P) CK 18+cells were observed in three pigs at 5 months (I, K, M, O, 20×; J, L, N, P, 40×); (Q, S) non-transplanted pig livers served as negative controls for ALB (Q) and CK 18 (S) staining (20×); (R, T) sections of human liver served as positive controls for ALB (R) and CK 18 (T) staining (40×). It is noteworthy that almost all ALB+ or CK18+cells were adjacent to vascular structures.
Similar results were obtained by RT-PCR analysis using human-specific primers. A 154-bp albumin band and a 269-bp CK18 band were detected in sixth-week liver biopsy specimens (in one of four pig samples) as well as three pigs sacrificed at 5 months (in three of four samples). Again, human AFP mRNA was not detectable by RT-PCR in the livers from hPMSCs-transplanted pigs, confirming the results obtained by immunohistochemistry. No expression of human albumin, AFP, or CK18 transcript was detected in non-transplanted pig livers (Figure
Figure 13
Detection of human-specific hepatic markers by RT-PCR (A) and
Detection of human-specific hepatic markers by RT-PCR (A) and
To identify transplanted human cells in recipient pigs, genomic DNA was extracted from recipient livers, and human-specific
According to the International Society for Cellular Therapy (ISCT), MSCs are defined by their expression of CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. Additionally, they usually differentiate to osteoblasts and adipocytes
Comparing with primary human hepatocytes, hepatocyte-like cells derived from hPMSCs showed much lower levels of LDL uptake and CYP-450 enzymatic activity. Since hepatocytes in intact liver are maintained in a three-dimensional environment and supported by surrounding cells such as Kupffer cells and liver endothelial cells, even primary hepatocytes will quickly lose activities during routine two-dimensional
Currently, rodent models, including rat and mouse, are used to evaluate the potential of MSCs from the BM and placenta in repairing liver damage
To verify that the observed effects were caused by transplanted hPMSCs but not the transplantation procedure
The number of MSCs transplanted is a key factor, yet little is known about the optimum dose. Most studies have used doses of 2-10 million MSCs per kg in small-animal experiments
Choosing an appropriate MSC transplantation route is vital for cell survival, induction of cell differentiation, and restoration of liver function. Routine transplantation paths include those through the spleen, liver, belly cavity, and peripheral and portal veins, each of which has disadvantages. MSC transplantation through the spleen is an accepted route, but the transplanted cells usually grow in nodules and hence contribute little to the restoration of liver function. MSC transplantation by hepatic multi-site injection involves the risk of damaging important liver vessels, causing severe complications including hemorrhea, portal hypertension, and so on. MSC transplantation through the belly cavity is simple but also involves the risk of inducing inflammation and fever and causing peritonitis and adhesion in the belly cavity. We compared the effects of transplantation through peripheral (jugular) and portal veins and our data suggested that both transplantation routes were safe, with no portal vein thrombosis. Additionally, hPMSCs were well distributed in the liver after transplantation through the left branch of the portal vein under guidance of B-ultrasonography. The long-term survival rate in the portal vein transplantation group was significantly higher than that in the peripheral vein group (66.7%
To determine whether transplanted MSC-derived cells colonized the pig liver,
Taken together, our results suggest that MSCs from the human placenta could be used as an excellent source of transplanted cells for the repair of liver damage. Portal vein transplantation under B-ultrasonography guidance is safe and effective which could restore liver functions in pigs with liver failure to a substantial extent; however, the underlying mechanism remains to be further clarified.
AFP: alpha-fetoprotein; ALB: albumin; ALF: acute liver failure; ALP: alkaline phosphatise; ALT: alanine aminotransferase; APC: allophycocyanin; AST: aspartate aminotransferase; BSA: bovine serum albumin; CCl4: carbon tetrachloride; CHE: cholin esterase; CK18: cytokeratin 18; CYP450: cytochrome P450; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene diamine tetraacetic acid; EGF: epidermal growth factor; ELISA: enzyme-linked immunosorbent assay; ELISpot: enzyme-linked immunospot; FGF: fibroblast growth factor; FITC: fluorescein isothiocyanate; GalN: D-galactosamine; HE: hematoxylin and eosin; HGF: hepatocyte growth factor; H2O2: hydrogen peroxide; hPMSCs: human placental mesenchymal stem cells; IFN: interferon; IMDM: Iscove's modified Dulbecco's medium; ITS: insulin-transferrin-selenium; LDL: low density lipoprotein; DiI-LDL: 1,19-dioctadecyl-3,3,39,39-tetramethylllindocarbocyane-low-density lipoprotein; LIF: leukemia inhibitory factor; LPL: lipoprotein lipase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MNCs: mononuclear cells; MSCs: mesenchymal stem cells; NH4Cl: ammonium chloride; OD: optical density; OPN: osteopontin; PAS: periodic acid-schiff; PBS: phosphate buffered saline; PBMC: peripheral blood mononuclear cell; PCR: polymerase chain reactions; PE: phycoerythrin; TBA: total bile acid; TBIL: total bilirubin.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors participated in interpretation of the findings. HC and LL were responsible for the conception and design of the study. JL and PZ generated the animal models. JY, JY, QP, and YW carried out cell biology and molecular biology experiments. YL and JL performed pathology tests. XP was responsible for the primary human hepatocytes culture and function analyses. HC, JY, and YW performed the statistical analysis and drafted the manuscript. All authors read and approved the final version of the paper. All authors confirm that the content has not been published elsewhere and does not duplicate their published work.
We would like to thank the staff and the patients of Hangzhou Red Cross Hospital for providing the placentas and Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health of China for providing the primary human hepatocytes. We are grateful to Dr. Senxiang Yan and Dr. Zhongjie Lu for their help with cell irradiation and to Dr. Tianan Jiang, Dr. Qiyu Zhao, and Mr. Linfeng Jing for the performance of cell transplantation and biopsy procedures. This work was financially supported by the Chinese High Tech Research & Development (863) Program (No.: 2011AA020104), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (No.: 81121002) and the Technology Group Project for Infectious Disease Control of Zhejiang Province (No.: 2009R50041).
Fetal and adult liver stem cells for liver regeneration and tissue engineering
Cell-based therapy for liver diseases
Prospects for stem cell transplantation in the treatment of hepatic disease
Cell-based therapies for metabolic liver disease
[Hepatocyte transplantation. From experimentation to therapeutic attempts in man]
Human hepatocyte transplantation overview
Hepatocyte transplantation-biology and application
Mesenchymal stem cells: molecular targets for tissue engineering
Pluripotency of mesenchymal stem cells derived from adult marrow
Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors
Stem cells from human exfoliated deciduous teeth--isolation, long term cultivation and phenotypical analysis
Isolation and characterization of postnatal stem cells from human dental tissues
Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol
Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue
Evaluation of wound healing effect on skin-defect nude mice by using human dermis-derived mesenchymal stem cells
Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta
IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury
Umbilical cord blood mesenchymal stem cells
[Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration]
Stem cells for liver tissue repair: current knowledge and perspectives
Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow
Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion
Effect of human umbilical cord blood-derived mesenchymal stem cells in a cirrhotic rat model
Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure
The umbilical cord: a rich and ethical stem cell source to advance regenerative medicine
Comparison of human placenta- and bone marrow-derived multipotent mesenchymal stem cells
Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells
Mesengenic progenitor cells derived from human placenta
Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation
In vitro differentiation of human placenta-derived multipotent cells into hepatocyte-like cells
Anti-fibrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: potential application to the treatment of hepatic diseases
Evaluation of a bioartificial liver based on a nonwoven fabric bioreactor with porcine hepatocytes in pigs
Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement
Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma
Engraftment potential of human amnion and chorion cells derived from term placenta
Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo
Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse
Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model
Mesenchymal stem cell infusion therapy in a carbon tetrachloride-induced liver fibrosis model affects matrix metalloproteinase expression
Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice
Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats
Changing potency by spontaneous fusion
Stem cell-based therapy in gastroenterology and hepatology
Transplanted bone marrow regenerates liver by cell fusion
Development of liver regenerative therapy using glycoside-modified bone marrow cells
Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat
Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells
Potentials of regenerative medicine for liver disease
Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes
Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha
Mesenchymal stem cells as trophic mediators
Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement
Mesenchymal stromal cells: Tissue repair and immune modulation
Immunosuppressive properties of cloned bone marrow mesenchymal stem cells
A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1
Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated immunosuppression
Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells
A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells
Mesenchymal stem cells can induce long-term acceptance of solid organ allografts in synergy with low-dose mycophenolate
Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model
Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution
A method for distinguishing human and mouse cells in solid tumors using in situ hybridization
Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples
Pre-publication history
The pre-publication history for this paper can be accessed here:


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subset: commoncrawl


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FRIDAY NIGHT COOKIE JAR BASS TOURNAMENT: 7 p.m.-midnight, Morrison Parkway public landing, False River, New Roads. Fee $30/boat. Friday nights through Sept. 15. Call Guy Perkins (225) 293-3402.
17TH ANNUAL SAVE OUR LAKE & COAST FISHING RODEO: New Canal Lighthouse, New Orleans. Fee $40/Open & Kayak divisions, $15/15-and-younger. Weigh-in 2-5 p.m. Friday, Noon-4 p.m. Saturday. Call (504) 836-2205. Website: www.saveourlake.org.
B.A.S.S. COLLEGE WILD CARD TOURNAMENT: Pickwick Lake, Florence, Ala. Website: www.bassmaster.com.
GRAND ISLE FLY FISHING WEEKEND: Grand Isle. Sponsored by South Louisiana and Mississippi fly-fishing clubs. Preregistration required. Call Randy Leonpacher (225) 769-1895. Website: www.rsff.org.
BLUE JAY FISHING RODEO: Weigh-in at John Ryan Stadium, 100 Blue Jay Way, Metairie. Jesuit High, New Orleans. Website: www.jesuitnola.com/events.
CCA SPECKS & SPOTS TOURNAMENT: 6 a.m., Quintana Landing, Cypremort Point. 1-3 p.m. weigh-in. Speckled Trout (2 trout, 10 places), Redfish (2, 16 inches minimum/less than 27 inches, 10 places) & Kayak (one each trout & redfish, 3 places) divisions. Fee $150/boat, $25/kayak. Also Youth Division. $15,000 prize money (based on 100 boats). Captain's meeting 6:30 p.m. Friday, Quintana Landing. Call CCA office (225) 952-9200.
FAMILY FISH FEST: Registration 5:30-10 a.m., in conjunction with CCA Specks & Spots Tournament, Quintana Landing, Cypremort Point. Fishing & other activities including lunch. Fee $10. Little Angler/10-under, Junior Angler/11-16 & Team/adult & youth divisions for boat and shore anglers. Must bring own tackle & transport bags. Live redfish and trout tagged & released. First 50 to register receive rod and reel. First 100 receive "goodie" bag. Preregistration website: www.wlf.la.gov/familyfishfest. Call Danica Williams (504) 628-7282. Email: email@example.com.
THE CAMP FLY FISHING SCHOOL/ADVANCED: 8 a.m.-5 p.m., The Camp, Breaux Bridge. Fee $135 includes lunch, handouts. Casting mechanics. FFF-certified instructors. Call Keith Richard (337) 344-0908. Website: www.thecampflyfishingschool.com.
NRA OUTDOOR CONVENTIONAL PISTOL (1800) MATCH: 9 a.m., Palo Alto Rifle & Pistol Club, 1393 La. 943-North, Donaldsonville. NRA Short Course/25 yards, 180 rounds in .22, centerfire & 45 groupings. NRA rules. Fee $10. Open to public. Call Skip Blanchard (504) 427-0050. Email: firstname.lastname@example.com.
LIVINGSTON PARISH BASSMASTERS HIGH SCHOOL STATE CHAMPIONS FUNDRAISER: 3:30 p.m. weigh-in, Doiron's Landing, Stephensville. Pick-your-partner event. Anglers allowed to fish either side of Atchafalaya Basin levee. Fee $75. Raising fund to defray expenses for B.A.S.S. state high school champions Dillon King & Kody Kelly to participate in National High School Bass Championship. Call John Chaney (225) 413-5142 or Jim Breaux (225) 772-3026.
SOUTH LOUISIANA HIGHPOWER CLUB MATCH: 8:30 a.m., Ascension Parish Sheriff's Range, 9300 St. Landry Rd. Gonzales. NRA 200-yard/50-rounds match course. Fee $12 members, $15 nonmembers, $5 juniors. $15 annual Civilian Marksmanship Program membership. Email: firstname.lastname@example.com.
CAPITAL CITY BASS ELITE TEAMS TOURNAMENT: Bayou Black Marina, Gibson. $30 membership fee. $100/team tournament fee, 100-percent payback. Seventy in 8-tournament series. Call Jesse Brooks (225) 397-2071. Website: www.capitalcitybassla.com.
FIN-ADDICT FLY FISHERS MEETING: 6:30 p.m., Spahr's Seafood Restaurant, 1400 W. Tunnel Blvd, Houma. Call Stephen Robert (985) 850-0082. Website: finaddictflyfishers.blogspot.com.
CANAL BANK BASS TOURNAMENT: 5-8 p.m., Canal Bank Club, Diversion Canal. Wednesdays through Aug. 13th. Membership fee $15/angler. Weekly fee $40/boat. Call Todd Rabalais (225) 413-3634. Email: firstname.lastname@example.com.
JUNE 28 — FLY-TYING AT BASS PRO SHOPS: 9:30 a.m.-noon, Conference Room, Bass Pro, Denham Springs. Call Ron Brooks (225) 271-3100.
JULY 3 — LOUISIANA WILDLIFE & FISHERIES COMMISSION MEETING: 9:30 a.m., Louisiana Room, state Wildlife & Fisheries headquarters, Quail Drive, Baton Rouge.
JULY 3-5 — GOLDEN MEADOW-FOURCHON TARPON RODEO: Moran's Marina, Fourchon.
JULY 5 — PALO ALTO MID-RANGE MATCHES: Palo Alto Rifle & Pistol Club, Donaldsonville. 600-yard, F-Class/Prone match. Monthly event. Call Richard Bordelon (225) 753-7675. Email: email@example.com.
july 12 — Palo Alto Club Championship Match: Head-to-head competition, 3 strings at 600-yard line. Field Precision Rifle, FTR and F-Open categories. Website: www.PaloAltoGunClub.com.
JULY 19 — 4TH ANNUAL CASTING FOR KIDS RODEO: Bobby Lynn's Marina, Leeville. Fees $40 adults, $20 children. Boater & Kayak divisions. Benefits Palliative Care Foundation of Baton Rouge. Call Janet Rhodus (225) 413-4414. Website: www.palliativecarebr.org.
STATEWIDE TOURNAMENT & ANGLERS' RODEO—S.T.A.R.: Through Sept. 1. Sponsored by CCA-Louisiana. Entry fee/CCA membership required. Call CCA-Louisiana (225) 952-9200.
STATE RECREATIONAL RED SNAPPER SEASON: Open in state waters. Daily limit 2 red snapper measuring 16 or more inches long. Anglers required to have fee-free Louisiana Recreational Offshore Landing Permit. Website: www.rolp.wlf.la.gov.
FEDERAL RECREATIONAL Red Snapper Season: Closed in federal waters throughout the Gulf of Mexico.
SPRING INSHORE SHRIMP SEASON: Open until further state notice in all state inside and outside waters.
RECREATIONAL FISHERIES cLOSURES: Gag grouper through Monday, June 30; greater amberjack, through July 31; gray triggerfish, through 2014 in state and federal waters.
Email: firstname.lastname@example.com


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Start 2017 on the Right Path
New Year's resolutions, beloved by some and loathed by others, are an integral part of the New Year's tradition. We wish our relationship to the new year to be one of joy and anticipation, and we ...View Article
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| Taking an Exercise Break
| Exercise breaks happen. Sometimes we schedule a week-long break - for example, at the end of a 12-week training cycle. At other times life intervenes, and our exercise intentions go out the window.
We want exercising to be a lifelong commitment, something we've taken on for the long term. This is distinct from the binge exerciser, who periodically "wants to get in shape", struggles for a month or two, and then quits.
If we're in it for the long haul, we can withstand a break here and there, even a prolonged month-long break if necessary. We've built a strong base. Our bodies are trained. Our muscular functions are optimized for performance. So, while we don't really want to take a break, we're prepared physically to get back in shape quickly.
Bottom line - if you need to take a holiday break from exercising, go ahead. If you're a regular exerciser, you can take a week or two off secure in the knowledge that you won't have lost that much. You'll recover quickly and possibly even recover to a higher level of fitness.
Nutritional Counseling
Spinal Decompression (IDD Therapy)
Physical Therapy


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Remember that Syrian passport found next to the body of one of the Paris attackers? And the Republican leadership which was so quick to claim that it meant a Syrian refugee was one of the attackers?
Turns out, the passport was a fake—it is, in fact, a passport with the same details as another man arrested in a Serbian refugee center. Both individuals claimed to be Ahmad al-Mohammad. Mohammad actually died months earlier fighting in the Syrian army.
A myriad of individuals are desperate to acquire Syrian passports because possession of the document now automatically grants the right to asylum in any country; thus, a potentially dangerous black market for Syrian passports has materialized.
The individuals hankering for these passports are not all involved with militant organizations or members of ISIL—most are Syrian refugees, displaced due to the Syrian civil war. Ewa Moncure, a spokeswoman for the European Union's border agency, told NPR that, "most of those who sought the documents [are] in fact victims of the Syrian Civil War…they are coming from a war-torn country. Probably many had to leave their homes rather quickly. Maybe some didn't have passports, and obtaining a Syrian passport right now—it's probably extremely difficult. These are people who've been on the move, sometimes for several years. These are people who some of their children were born outside Syria."
And it's not just Syrian refugees—according to Kevin Knodell, "several European countries have agreed to take in Syrian refugees, but for millions more — Iraqis, Afghans, Libyans, Eritreans, Yemenese, Somalis and others — are fleeing violence and deprivation in their homelands but are disqualified for programs aimed at Syrians."
The issue is that terrorists working for the Islamic State can take advantage of this same market, as one of the Paris attackers did. According to Sky News, even in February, "Foreign fighters seeking to join Islamic State are using black-market Syrian passports to enter through Turkish border checkpoints," which is clearly a significant problem, as once they enter Europe, it becomes easier to move between countries.
The difficulty lies in reconciling the legitimate need of many refugees to obtain passports so they can seek the asylum they critically need, and the ease this affords terrorists who wish to infiltrate countries accepting refugees.
Banner Image Credit: Twitter, @Reuters


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There is no fingerprint authentication, even worth, there's no option to remember user. I tired of entering my long email address and password each and every time.
This is not an app, just a wrapper of a mobile site. Its slow and not mobile friendly.
Will not provide my personal info just to contect to my car.ugh
Glitchy, slow, and irritating. Not wise to make trying to get paid so difficult. I'll use something else
Seems to only open the mobile site.. lol! This is the 'app' or was this a revert since the app was so poor?
The app is slow and does not allow me to register. Tried several times but in vain.
I agree with JSANCHEZ. Quick install. Once installed I didn't experience any difficulty.
the app is based on a mobile site. too Clichy for regular users, you will be better off using your mobile browser to access this service.
Been trying to log into my account to change the password for 2 weeks and it doesn't allow me to. Tried online and on my phone. Doesn't give a temporary password or anything. Waste of time.
It's exactly like the web page and I have no problems whatsoever. But, all it really needs is to be able to remember my username.
I would give it and your website a zero. Once i login it just keeps prompting me to register my account and then says I'm already registered. I cannot do anything on either.
My mine works except it is slow in exciting commands
It opens then it doesn't let you do anything else. Horrible app. This should be working all my other apps open fine and thats how I pay all my bills. If I dont see improvement gonna uninstall! !!
This app is as good as the customer service number. Both are a disgrace. Shame on Nissan
It opens and that's it..Uninstalling . thanks for nothing.
Worst app ever
App works fine for me. I schedule future payments free of charge (no convenience fee). Plus I've never be able to get through to a live person on their toll free number so it's better than being on hold for 45minutes
This is one of the worst apps I have ever used, it freezes a lot and is impossible to log out.....!! I hope they didn't pay to create this app
let's you log in then it's all blank
It's basically the website in an app. Looks no different.
Stupid thing! It won't let me log on. Every time I put my username and password and press login, it brings me to the registration page! Smh!
I cannot even get the app to let me login in. Horrible app that needs to be redesigned.
Payment features work fine, but the whole app is slow. Which is about the same as the website.
Confusing piece of crap
Does not work
It works for me.
Works perfectly!
It doesnt let me go to the second page after entering my my information.
Freezes everytime i try to register myself. Tried 4 times different days.
I can log in on the website but not on the app. It says "incorrect password".
I see a lot of people complaining about passwords, create one using a computer. Then download this App and login. It works flawless. Just scheduled my payment with it.
The app works great, but it does need a lot of design improvements such as the login screen the screen resolution doesn't seem right and the fonts seem stretched out. Also the top area for lollipop it's black. So I hope it gets better in the future. All the other Nissan apps don't function so great
Good luck choosing a password that works. I have never in my life had such a hard time giving somebody else my money. Wtf Nissan
Gives ridiculous password requirements even when they are correctly met. Shows password validation on 3rd step after password entry was step 1. Frustrating app. By the way even the website didn't work
GUYS I FIGURED OUT THE PASS PROBLEM: It requires a special character. For some reason, it doesn't tell us that you require a special character. But all it requires is an uppercase letter numbers minimum of eight and a special character. Hope I helped some of you.
Won't let me register on the app or online
Has ANYONE ever been able to make a password with the app or online???
Getting through to make a payment ends up with all sort of errors. Calling the dealer, ends up in AVR. But Sale is very good. Very Frustrating, Disappointing. Called Nissan IT, can give a solution but would ask my information to record the call. Nissan Hemet, Ca, after sale customer service not good.
Good job Nissan


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Monday, January 10, 2011
Synthetic Genomics Brings Funding Up To $35.0M
La Jolla-based Synthetic Genomics, a developer of genomics technology for the biofuels industry, has now raised $35M of its ongoing $50M funding round, according to a recent regulatory filing from the company. Synthetic Genomics is backed by ExxonMobil, Draper Fisher Jurvetson, Meteor Group, Biotechonomy LLC and Plenus, S.A., BP plc, and ACGT Sdn Bd. The firm is headed by J. Craig Venter, the inventor of DNA sequencing. The firm is currently focused on using genetics technology to modify microalgae and cyanobacteria to more efficiently convert carbon dioxide into cellular oils and long-chain hydrocarbons, for use in the biofuels and chemicals industry. ExxonMobil and Synthetic Genomics have said that ExxonMobil is investing up to $300M in the firm around their joint efforts. More information »


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An office you won't want to leave. By Paula Robinson
My dream is to have a garden office. I love the idea of leaving home behind and "commuting" down the garden to work … then, at the end of a long, productive day, closing the office door on all things serious. For anyone working from home, a garden office offers a pleasant discipline and routine, and a chance to escape the hustle and bustle of the household.
Converting an existing structure into a garden office - or starting from scratch - is an involved project. The structure needs to be properly insulated, have power, telecommunications, heating (either underfloor or a convection heater with a thermostat), and you may want to connect it to water and waste utilities. This is an ideal opportunity to try eco-building materials (especially insulation), and alternative energy sources (solar panels in particular). Think about whether you need a path from the house to the office, or a decked area outside it.
Be sure to include plenty of windows in the design, as daylight will enhance your office. If the idea of working in nature appeals, consider installing folding and sliding doors that can open up completely in summer. Go a step further and have a structure that blends with its surroundings and that's attractive to wildlife: either grow your own (visit www.livingroofs.org for advice on how to go about it) or buy readymade. Alternative Space offers garden offices with a "living roof", which is also a good insulator in winter and summer. However, if a sleek, contemporary design statement is more your style, have a look at the eco-friendly Qube garden office.
Unlike a home office, the garden office's interior doesn't have to blend with the rest of your home. Instead, it can be a true expression of what you need from a working environment, tailored just for you: conservative, artistic, dramatic, quirky, minimalist, or whatever takes your fancy.
A garden office allows you to rethink your working style and to find a rhythm that's right for you. Not everyone produces their best chained to a desk. If you're like me and prefer to work curled up on a sofa or armchair, I strongly recommend investing in a Tablet PC (the screen rotates 180 degrees and lays flat over the keyboard, allowing you to write comfortably on the screen with the pen provided). Wireless technology is also a good investment for a garden office.
However, working in the same position day after day can be mindnumbing. Movement and change keep the brain alert and ideas flowing, so plan your garden office with distinct areas for different tasks.
This will encourage you to gravitate to other areas as the day progresses - but be sure to include practical work surfaces in each activity corner.
I like the idea of a built-in "work island" - positioned slightly offcentre to allow sufficient space elsewhere in the office for comfortable seating for relaxing and brainstorming. A well designed work island (with a generous-sized work surface and a base containing the maximum practical storage) will give you flexibility: your main position can overlook the garden, with the option of working on the three other sides. Include ample leg room on all four sides. If you want to work standing up, consider building the work island at bar height. Make sure your seating gives adequate back support and includes a foot rest.
If the budget won't stretch to a bespoke work island, your office layout can still be creative and dynamic. Don't let furniture hug the walls, with a large, empty space in the middle. Try positioning an attractive desk (check out the designs on www.shedworking.co.uk) towards the centre of the office with floor-toceiling bookshelves and storage within easy reach on the wall behind you. Floor sockets will be essential for power. Place a sofa immediately in front of the desk, facing out on to the view. Your office will be comfortable and practical for you, but inviting and interesting to visitors, too.
Practical tips
- Planning permission isn't required for a garden office if: its overall height doesn't exceed 4 metres for a pitched roof and 3.5m for a flat roof; the total floor area doesn't exceed 30 sq m; the structure is sited at least 5m from the house; and it's not in a conservation area.
- Think about the placement and size of your office from your neighbours' perspective and speak to them about what you are planning. Most neighbours are accommodating if you keep them informed.
- Don't neglect security: your garden office may need to be alarmed. Also, check carefully with your insurance company: the office's contents must be covered for all eventualities.
- If you'd like to take your garden office with you when you move, discuss this with your supplier as some models can be built accordingly.
- Make sure you have sufficient lighting in the form of good general illumination (preferably on a dimmer switch) and appropriate task lighting.
Useful contacts
Natural insulation & eco building materials Womersleys, www.womersleys.co.uk, 01924 400651
Garden office with 'living' roof Alternative Space, www.alternativespace.com, 07932 340349
Contemporary garden office The Qube, www.theqube.co.uk, 01604 785786; The Garden Escape www.thegardenescape.co.uk, 0870 2427024
Eco-friendly, classic-style garden offices Oakland Garden Offices, www.oaklandoffices.co.uk, 01636 816556
Tablet PC Microsoft, www.microsoft.com
Green office products and recycled paper The Green Stationery Company, www.greenstat.co.uk, 01225 480556


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COLLEGE FOOTBALL; In Nebraska, Hardships Ease on Each Fall Saturday
By MALCOLM MORAN
Published: August 21, 1988
SCOTTSBLUFF, Neb.— Right about 2 o'clock in the morning on those special Saturdays, when the automobile turns southeast onto State Highway 92, Harold and Polly Gentry and Darrel and De Willet, two retired couples who live in nearby Gering, begin another autumn adventure.
For miles and miles, theirs is almost always the only car on the two-lane highway through the middle of the night. They watch out for deer as they pass ranches and leave the darkened sand hills in the Nebraska panhandle.
They turn south onto 385 at Bridgeport, and finally reach Interstate 80 at Sidney. An hour disappears as the car passes from mountain to central time, and soon, about a half-hour before their 8 A.M. breakfast stop in Kearney, a large orange sun appears on the horizon to signal the start of a football Saturday. By then they have joined the others on the interstate, almost all in red, as they point east toward Lincoln, the state capital and emotional focal point.
There is no major league sports franchise to dilute the affection in this state for the University of Nebraska Cornhuskers. It exists from the Iowa line in the east to Wyoming and Colorado to the west. No other major university in the state can divide the passionate loyalties from South Dakota to the north to the Kansas line to the south.
Southern California has to deal with U.C.L.A. Penn State copes with Pittsburgh. Michigan contends with Michigan State. Florida State must recruit against Florida and Miami. Alabama bears the presence of Auburn.
But here, through cattle country, the irrigated fields and the less fortunate farmlands affected by the drought, there is one team, one approved color to wear, and one line that follows the fanfare at the start of the fight song: ''There is no place like Nebraska. . . .''
So many Nebraskans face crucial business decisions and time-consuming tasks each autumn, and yet, with the tickets already sold for the six home games at 73,650-seat Memorial Stadium, the streak of sellouts that began in 1962 will this year reach 161. There is important work to complete, but. . . .
''It's also the time you need college football to help you keep your perspective and get through it,'' said Bill Dicke, a cattle nutrition consultant from McCook. ''You just work harder the rest of the week to make time.''
''Any other day of the week, if something goes wrong, you stay there and fix it,'' said Reed McClymont, whose family has been feeding cattle in Holdrege, in the south central part of the state, for more than a century.
''But in the football season,'' said his brother, Ed, ''it's always, 'It can wait until tomorrow.' ''
The passion has been more powerful than the hardships that periodically exist in agricultural and ranching communities. ''In some places,'' said Tom Osborne, the head coach and a native of Hastings, ''football tickets might be one of the first things to go. But with a lot of people in Nebraska, the football tickets would be one of the last things to go.
''You can do without a lot of other things before you give up football tickets, because you know you may not get another shot at them. People have made a lot of sacrifices to have enough money to see the games and stay overnight.''
It does not seem to matter that the quarterback, a wingback and the kicker are from California, the left outside linebacker is from Houston, the cornerbacks are from Kansas City and Colorado Springs, and the New Jersey pipeline remains important. ''When they're here,'' said Allen Swanson, a rancher from Arthur, ''they're Nebraskans. That's the way it is.''
Generations of followers, up to and including some of the players who will begin the 99th year against Texas A&M in the Kickoff Classic next Saturday night, have memories of listening to the radio as they go about their work on Saturday afternoons. They would wait earnestly, in trucks and barns, grocery stores and hospitals and any room in the house, for the moment that would inspire Lyell Bremser, the voice of the Huskers from 1938 through 1983, to shout his signature exclamation: ''Man, woman and child! Look at those Cornhuskers roll!''
The joy produced by such moments pushes the Gentrys and Willets to make their trip. They are back in the car after the final gun, heading west, listening to the interviews, digesting the game, waiting for other scores, wondering about their bearing on the next week's rankings.
They arrive home at 2 A.M., 24 hours and more than 820 miles removed from their departure. ''It's not nearly as tiring as you'd think,'' Harold Gentry said. ''But when they lose, it's a long trip home.''
There have been few such trips in the past quarter century. Nebraska can break the modern record of 26 consecutive winning seasons that it now shares with Alabama and Penn State. The Huskers have won no fewer than 9 games for 19 years, including a bowl appearance each of those years.
The devotion can be found across the state. Bernice High, an 87-year-old fan in Bertrand, is legally blind. Legally, selectively blind. ''She somehow sees when they're on TV,'' said her son-in-law, Keith (Swede) Fastenau, a farmer.
''She can see interest checks and Nebraska football,'' said her daughter, Miriam (Shorts) Fastenau.


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The Pittsburgh Steelers have lost wide receiver and kickoff return man Quincy Morgan to a fractured right fibula sustained in Sunday's wild-card victory at Cincinnati, an injury that forced the five-year veteran onto injured reserve Tuesday and will require surgery.
To replace Morgan on the roster, Pittsburgh re-signed wide receiver Lee Mays, who had been with the Steelers the past three years but was released early this season.
It is believed that starting cornerback Ike Taylor, who took over the kickoff return chores last weekend after Morgan left the game in the first quarter, will assume those duties for Sunday's divisional-round matchup against the Indianapolis Colts. Morgan also served as the Steelers' No. 4 wide receiver and Sean Morey will probably take that role.
The injury to Morgan was originally diagnosed as a high ankle sprain but further tests revealed the fracture.
Morgan was signed by the Steelers just before the start of the season after he was released by the Dallas Cowboys. The former Kansas State star averaged 25.3 yards on 23 kickoff returns and also had nine receptions for 150 yards and two touchdowns. Morgan will be an unrestricted free agent this spring and, while Pittsburgh would like to retain him, he might test the market.
Taylor has returned kickoffs in the past but he logged just three runbacks this season for an average of only 19.7 yards.
Mays had 11 catches for 154 yards in his three previous season with the Steelers and was used primarily as a No. 4 wideout and on special teams.
Len Pasquarelli is a senior NFL writer for ESPN.com. To check out Len's chat archive, click here.


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Located someone who is attractive to you? Drop them a line or just a wink and wait for their response. With a little luck you'll be on your way to uncover that special someone for you.
It only takes a few clicks


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Ans. A person can send letters, post cards, parcels. International postal services are also offered.
Ans. Postal dept offers various services like post office saving schemes, postal life insurance, mutual funds, recurring deposits account, time deposit, post office monthly investment schemes, public provident fund, kisan vikas patra, national saving schemes etc.
Ans. The maximum weight for a letter is 2kgs, registered parcel can be upto 4kgs & unregistered parcel can be 20kgs.
Ans. the max time for delivery in local & metro cities is 2 days & rest of India is 4-6 working days, whereas for international posts it is 4-10 working days exclusive of time taken in custom clearance.
Ans. A consumer can check the status of his post online on the indiapost.gov.in
Ans. The compensation for delayed post are the speed post charges paid by the consumer. & if the post is lost, theft or totally damaged then the consumer is to be compensated for double the charges paid by him or Rs. 1000/- whichever is less.
Ans. Yes, for Registered letters, Value Payable Letters, Registered Parcels , Value Payable Parcels, subject to the following conditions:
Ans. Postal dept. offers money orders, instant money orders, and international money transfer services to consumers.
Ans. A consumer can send upto Rs. 5000/- by money order,Rrs. 1000-50000/- by instant money order & upto Rs. 50000/- by international money transfer.
Ans. Employees from any govt. or autonomous body are eligible for PLI.
Ans. Recurring Deposit is a special kind of Term Deposit offered by banks in India which help people with regular incomes to deposit a fixed amount every month into their Recurring Deposit account and earn interest at the rate applicable to Fixed Deposits. It is similar to making FDs of a certain amount in monthly installments, for example Rs 1000 every month. This deposit matures on a specific date in the future along with all the deposits made every month.
Ans. E-Payment is a smart option for businesses and organizations to collect their bills or other payments through Post Office network. When businesses require collection of bills and other payments from customers across the country, Post Office offers them a simple and convenient solution in the form of e-Payment.e-Payment is a many-to-one solution which allows collection of money (telephone bills, electricity bills, examination fee, taxes, university fee, school fee etc.) on behalf of any organization. The collection is consolidated electronically using web based software and payment is made centrally through cheque from a specified Post Office of biller's choice.
Ans. Yes such services are transferable from one post office to another post office anywhere in India by giving a written application.
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posted by r .
To extract gold from its ore, the ore is treated with sodium cyanide solution in the presence of oxygen and water.
4 Au(s) + 8 NaCN(aq) + O2(g) + 2 H2O(l) 4 NaAu(CN)2(aq) + 4 NaOH(aq)
Determine the mass of gold that can be extracted if 27.4 g sodium cyanide is used.


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1 dead, hundreds stranded in Adriatic Sea ferry disaster
Updated 7:11 pm, Sunday, December 28, 2014
ATHENS — Hundreds of passengers and crew endured a second night of smoke, frigid temperatures and gale-force winds as they waited to be evacuated from a burning ferry adrift early Monday in rough seas between Italy and Albania. At least one person died and two were injured in the risky rescue operation.
The Italian coast guard said 201 of the 478 people on the ferry, sailing from the Greek port of Patras to Ancona in Italy, had been evacuated by early Monday. Most were airlifted by helicopter to other merchant vessels sailing nearby, though a few were flown to hospitals in southern Italy to be treated for hypothermia.
Latest news videos
"It will be a very difficult night — a night in which we hope we will be able to rescue all on board," Greek Merchant Marine Minister Miltiadis Varvitsiotis said in Athens.
He said 10 merchant ships were in the area assisting rescue efforts, and that those that had already taken on dozens of passengers from the stricken ferry would remain in the area until the operation was over.
The fire broke out before dawn Sunday on a car deck of the Italian-flagged Norman Atlantic, carrying 422 passengers and 56 crew members. Passengers huddled on the vessel's upper decks, pelted by rain and hail and struggling to breathe through the thick smoke, passengers told Greek media by phone.
"We are outside. We are very cold. The ship is full of smoke," passenger Giorgos Stiliaras told Greek Mega TV.
The Italian Coast Guard, which was coordinating the rescue operation, said the flames visible from outside the ship had been extinguished by about 8:30 p.m., about 16 hours after the blaze began. But the ferry was still enveloped in dense smoke, which the coast guard said probably was being fueled by some hotspots inside the ship.
The Greek coast guard said the Greek man who died and his injured wife were found in a lifeboat rescue chute.


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NIKE iD - customize your own Nikes.
Because I'm a designer. And a control freak.
I bought this on sale last week in this Grapeseed color, and I have worn it with everything! The color is really versatile (espec if you LOVE purple..like me), and it's super cozy.
The back of this shirt is so tasteful and looks good in fitness classes or while walking to one. I wore mine (I have it in black) with jeans and flip-flops yesterday! Sooo versatile.
I love the longer version of this too, but the short version here is a great layering piece and shows off your hard work on those upper abs!Tags: fitness, sports, Clothes, gym, bras, activewear, lululemon, sportbras, fitness wearAdded 7 Years Ago from Lululemon
I honestly NEVER thought that I would like these on...but then I tried them and fell head over heels in lust. They're baggy, so I paired them with a tight cropped tank (the Eagle top) and I look fantastic.
I just picked TWO of these up today (purple and grey). They are lighter weight than the normal ones and sooo flattering!
The perfect running/gym shorts. I've never been more comfortable in a pair of running shorts.
I prefer bikini tops that don't tie because I'm so active in the water that it is guaranteed to fall off. This one is all attached and has a colorful print! Plus the shape is ideal for tiny tatas.
ALO (air.land.ocean) makes flowy workout wear and interestingly cut sport bras. This tank is perfect for low-impact workouts (and then drinks afterward!).
These shorts don't ride up and wick away sweat so that you can get through your sun salutations without a wedge. They also make it into my bag for weightlifting sessions, cycle and group fitness.
I love these capris so much that I have them in grey, white and black. The people at the Nike store in Eugene thought that I was insane. They are the perfect length for petites and hide any insecurities in the hip area.
Another great Jersey Shore t-shirt that plays on the phrase "gym tan laundry."
In case you need a constant reminder about what the daily activities should be, gym, tan, laundry.
If you are a runner, you know the importance of wearing reflective clothing. This top looks super cute on, but the real benefit from it is that it has reflective piping so that those cars' headlights can see you.Added 8 Years Ago from Road Runner Sports
These fitness pants have a higher waistband that is super thick and creates the smoothest thighs, belly and butt. They create the illusion of being 2 sizes smaller than you really are (and they're moisture wicking!).
YES! These go in your lucy sportsbra to give you "lift" and shape when working out. Oh hello, I'll take these! They might make my A-cups look like B-cups when I spend hours in the gym everyday. Sportsbra uniboob = NOT sexy.
The interesting band makes the basic black short a little bit better. Now when I wear spandex shorts with a nice shirt because my pants don't fit (hey, chocolate cake happens to the best of us) I will look semi-stylish...
So unusual and still completely appropriate with a black racerback tank! These are my new favorite pant to teach in.
Check out these totally cute shorts by Circular Accessories! I love the silly little birdie print!
A track Jacket with a twist for the ladies: This Pro Mama Track Jacket is BY Mama and it costs........$82.00
My list is turning into an after-the-fact New Year's Resolutions list... I like going to the gym... I HATE getting ready to go to the gym. Finding a pair of matching socks, a cute (but not too cute) outfit, shaving your legs, ... My list is turning into an after-the-fact New Year's Resolutions list... I like going to the gym... I HATE getting ready to go to the gym. Finding a pair of matching socks, a cute (but not too cute) outfit, shaving your legs, a ponytail holder, the right shoes, a water bottle, iPod....... This top is really pretty- I like the double straps and the color... not sure if I would get any street cred when I am trying to lift heavy weights with the guys but perfect for an afterwork Spin Class.Added 10 Years Ago from Fitcouture


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4260 Washtenaw Avenue, Ann Arbor 48108
See more homes for sale in Ann Arbor, MI
This property has been Sold!
07/01/2016 at $212,000
Plenty of space inside this 4-bedroom, 2 and a half bath home that sits on a fenced, double lot! (Parcel #L-12-01-326-027 included) Large living room with hardwood floors and wood-burning fireplace leads to a light-filled 3-season porch with access to backyard and patio. Formal dining room, plus a spacious kitchen with plenty of storage on main level. Upstairs you'll find 4 oversized bedrooms, plus a full bath. Even more living space can be found in the lower level which features a rec room with built-in shelving and a full bath. This colonial is within walking distance of elementary school. It is also close to bus stops, expressway, and plenty of shopping options. Schedule a showing today!
Property Information
- Total Finished SqFt
- 2,264
- Lower Finished SqFt
- 400
- Above Grade SqFt
- 1,864
- Garage Desc.
- Attached, Door Opener
- Water
- Public
- Sewer
- Public
- Year Built
- 1943
- Home Style
- 2 Story
- Summer Taxes
- $2,248
- Winter Taxes
- $1,184
- Association Fee
- $0
Rooms and Land
- MasterBedroom
- 2nd Floor
- Bedroom2
- 2nd Floor
- Bedroom3
- 2nd Floor
- Bedroom4
- 2nd Floor
- Dining
- 1st Floor
- Kitchen
- 1st Floor
- Living
- 1st Floor
- Basement
- Full, Partially Finished
- Cooling
- Central
- Heating
- Forced Air, Gas
- Acreage
- 0.52
- Lot Dimensions
- 80 x 150
- Appliances
- Dishwasher, Disposal, Dryer, Range/Oven, Refrigerator, Washer
- Fireplace Desc.
- One, Wood
- Exterior Materials
- Brick/Aluminum
- Exterior Features
- Cable Available, Patio, Porch
Listing Video for 4260 Washtenaw Avenue, Ann Arbor MI 48108
Mortgage Calculator
Listing Broker
Listing Courtesy of Keller Williams Ann Arbor
Office Address 2144 S. State Street
Listings last updated: . Some properties that appear for sale on this web site may subsequently have been sold and may no longer be available.
Our Michigan real estate agents can answer all of your questions about 4260 Washtenaw Avenue, Ann Arbor MI 48108. Real Estate One, Max Broock Realtors, and J&J Realtors are part of the Real Estate One Family of Companies and dominate the Ann Arbor, Michigan real estate market. To sell or buy a home in Ann Arbor, Michigan, contact our real estate agents as we know the Ann Arbor, Michigan real estate market better than anyone with over 100 years of experience in Ann Arbor, Michigan real estate for sale.
The data relating to real estate for sale on this web site appears in part from the IDX programs of our Multiple Listing Services. Real Estate listings held by brokerage firms other than Real Estate One includes the name and address of the listing broker where available.
IDX information is provided exclusively for consumers personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
Provided through Ann Arbor Association Board of Realtors. © 2017 Ann Arbor Associate Board of Realtors Ltd. shareholders.


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COVER STORY: Foreign Minister launches Paul Gray's new book on Islam
EDITORIAL: Australia and the Timor Gap Treaty
CANBERRA OBSERVED: Latham loses lustre as poll looms
INTERNATIONAL TRADE: Behind the WTO talks
RURAL POLICY: Facing up to the farm income crisis
UNITED STATES: Transsexual case and marriage law
FOREIGN AFFAIRS: Pakistan and the Islamic N-bomb
POPULATION: 'Gender equality' partly to blame for fertility decline, says UN official
DOCUMENTARY: 'My Foetus' prompts abortion re-think
OPINION: US law professor blasts US court on gay marriage
OPINION: Distributism and capitalism
STRAWS IN THE WIND : Part-timers: pros and cons / Family Law changes / Timor's Labor pain
Latham, Iraq and free trade deal (letter)
Fishermen protest in marginal seats (letter)
Ethanol stand challenged (letter)
BOOKS: The Red Millionaire: Willi Münzenberg, by Sean McMeekin
BOOKS: GETTING ON TRACK: A Business Plan for Australia
BOOKS: Just War Against Terror, by Jean Bethke Elshtain
BOOKS: The Mystery of Olga Chekhova, by Anthony Beevor
Join e-newsletter list
COVER STORY The Great Barrier Reef is dying? ... Again?
EDUCATION FUNDING With Gonski reboot, Turnbull taps in to way to lose Catholic vote
EDITORIAL No heads roll despite quarantine foul-ups
CANBERRA OBSERVED McCain, Keating wade into South China Sea
ELECTRICITY Bad science + bad economics = bad policy
COVER STORY 'Safe Schools' and every school's duty of care
CANBERRA OBSERVED Catholic education: not gone but Gonski'd


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Cilic also ranked as among the most effective player in the ATP singles ranking, Cilic has attained great accomplishment in tennis and he's won many singles matches and then ranked (status) among the most effective player on the planet.
Cilic is disciplined and hard working man and goes for training session all day in the tennis court daily. Cilic can also be amusing and interesting man and sporty man, Cilic is tall and his height is all about 6 feet 6 in and is a right handed player with great power, stamina and precision. Cilic played in the Crotia team playing various kinds of matches against different teams and demonstrated that he's got an excellent potential.
Cilic has lots of followers since he's a tall proficient player, hot guy with amazing appearances, Cilic has lots of followers in twitter, Cilic uses twitter for the the majority of his day-to-day work and daily picture are shared in twitter as what he does and how he spend time. Cilic additionally shares he is advancement in the social media like other tennis players and consistently go together with the stream. Cilic accomplishment also seemed many telivision displays with interview, Cilic is renowned through the planet as a awesome tennis player. Cilic always had a love and fondness toward tennis and realized it, Cilic is at present is expecting to be the world champion.


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Classic Movie Night - Casablanca
Classic Movie Night - Casablanca Friday, June 2 Jefferson Theatre $5.00 per person 6:00pm doors 7:30pm show time Tickets are available at the Beaumont Civic Center Box Office (701 Main Street) or at the Jefferson Theatre immediately before the show. Concessions, including beer and wine, will be available for sale. Classic Movie Nights are sponsored by ExxonMobil, iHeart Media, and 2 Row/Del Papa Distributing. For more information call 409.838.3435.
Jefferson Theatre
345 Fannin St, Beaumont, TX


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After this blog post goes live, you will have 12 hours to put any and all Category:OC that is not listed on the User Submissions page on the User Submissions page, or the tag WILL BE DELETED. This is your FINAL WARNING. You have had over a month, if not more.
Following this post going live, any pages newly tagged OC and not added to the Submissions page within 30 minutes will have their tag removed.
Starting in October, failure to properly put THREE pages on the User Submissions page after tagging them as OC will earn you a three-day ban.
I hope this will keep me from having to find 60 pages out of almost 300.


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My partner and I popped into this place for lunch as we were heading to Roncesvalles. They had two menus - one for weekend brunch, the other a lunch menu consisting mostly of sandwiches - plus a bunch of specials. We ended up both ordering specials. I got the Better Mac, a homemade Big Mac style burger, with a side of chilled lima bean soup. The burger was perfectly cooked - just a slight hint of pink, very juicy. The bun was soft, but didn't get soggy. The soup was garnished with crispy garlic slivers and thinly sliced granny smith apples - super delicious! My partner ordered fish tacos - three cornmeal crusted fried basa tacos, with pickled cabbage, crema, and avocado. The fish was so light and crispy. Super good!
For drinks, we got homemade sodas - blueberry and almond & pineapple and sage. They also served us Q water for free in a big pitcher. For $30 (taxes in), it was a fantastic deal! I can't wait to go back.


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Solitaire Engagement Ring
By Dirk Rendel
A Solitaire Engagement Ring Is Really The Only Choice
What is the most popular setting for a diamond? It's the solitaire setting. Don't worry guys and girls, it's not boring at all, pick one of these rings out and you won't be disappointed. Nothing says love like a spectacular rock on a thin band. It's simple and fresh, and really you can't go wrong with a diamond solitaire engagement ring. Seriously, guys if you get this for your significant other, you're going to be scoring some serious points.
Women often are extremely specific in what type of jewelry they wish to wear and even how the stone is to be cut. Thankfully, solitaire engagement rings are an easy choice to please many women. Solitaire rings focus on having just one diamond instead of numerous diamonds.
The round brilliant cut was originally the only preferred cut but today the demand for variety has made other choices available. Solitaire engagement rings are the traditional expression of the one true love you will share for a lifetime. It's traditional, but modern, both contemporary and old fashioned. It will never go out of style. They feature simply a band and one gemstone making solitaire engagement rings simple, yet bold. The statement made by a solitaire setting is about the purity of the diamond and the band it is set on.
Solitaire diamonds can be cut to various shapes and sizes. As we mentioned, the round brilliant was the only preferred earlier cut but today cuts like the princess cut, marquise cut, cushion cut and emerald cut are also available. You can go absolutely hog wild when it comes to picking out the shape of the solitaire diamond. Ever seen a heart shape diamond? How about a pear? You're certainly not limited when it comes to a solitaire engagement ring.
The round brilliant looks best in a four-prong setting, but the newer cuts come in different, new prong settings creating a wide variety of choices from and most are available in yellow or white gold and platinum. Solitaire means a single gemstone or diamond set by itself in a beautiful setting and solitaire engagement rings used to mean colorless diamonds. Today, colored diamond solitaire rings are also becoming popular. A solitaire engagement ring is a gift that will be treasured for a lifetime.
Another important factor is your choice is the size and style of the band. Narrower bands create the appearance of making a smaller diamond seem larger which creates a delicate and beautiful look on smaller fingers. Thicker bands give more options on how you choose to have the diamond set. You have a lot of choices when it comes to the band. Just make sure you measure your significant others finger before you make the plunge. If you're planning to pop the question, don't get caught with a ring that's too small and won't go over the knuckle. Embarrassing!
When looking at the shape of the diamond, in order to lengthen the look of the gem, another way to make it appear larger than it actually is to go with the emerald and marquise cut or oval and pear-shaped stones. Well-cut, fiery round brilliant gems always stand out no matter what size it is. Uncommonly shaped diamonds, like the trillium cut or heart-shape, will also catch the eye because of its rarity. Since larger stones are far less common than smaller stones the cost is higher, but just because you choose a simple solitaire engagement ring setting that doesn't mean you sacrifice exquisiteness and style.
The elegantly understated simplicity of a solitaire engagement ring is ageless and timeless. If you think solitaire engagement rings are for old people, then I encourage you to look again. They're modern and fresh and will never go out of style. Nothing can compare to the classic elegance, charm and simplicity of solitaire rings.
Gold and silver bands are available for a bride to select for her wedding or engagement ring. Platinum and other metals make the choices that a woman has for her amazing solitaire engagement ring even more numerous. You might be surprised at how stunning and beautiful the color of a diamond glows against a white gold band. You need to carefully examine a solitaire engagement ring before purchase. The prongs for the setting need to be strong. Many consumers do not realize that the prongs of the ring are loose until they have the stone fall out and say a few colorful words. The setting for the ring will make a difference! Also, ensure that the band width is set properly for the stone. A small diamond on a narrower band will look much larger. This means that a woman can have a beautiful diamond without having to have an extremely large ring.
The cut of the stone is extremely important to the desired look of a solitaire engagement ring. For example, if a woman wants to have a larger looking stone she can have an emerald or oval cut. A rare cut such as a heart shaped stone will also catch the casual glance from a stranger. It is amazing what some of the unique cuts that are featured on solitaire engagement rings can do to help individuals to make friends.
The larger the stone, the higher the price of the ring will naturally become. This is due to the rarity of larger stones. However, you should strongly consider purchasing even a two or three carat diamond. The color and clarity that these unique diamonds can offer will be brilliant and dazzling for everyone to see on a solitaire engagement ring.


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UK Dating
thank you so much for having this website, its so WICKED man...........spk sn xxxxx... more..
Other UK Dating Group Sites
Kara Tointon and Artem Chigvintsev appear happier than everPosted on: May 27, 2011 09:58:29
Lonely hearts looking for love using online dating websites could be interested to hear that Kara Tointon's love life appears to be going well.
The stunning 27-year-old actress was photographed looking rather happy with her boyfriend and former dance partner, Artem Chigvintsev.
After meeting last year during Tointon's run as a contestant on BBC1 talent show Strictly Come Dancing, the pair have been full of praise for one another.
During an interview with the Daily Mirror earlier this year, Tointon revealed she had not been looking for a relationship when she signed up for the series.
However, she went on to say: "Now I feel really lucky and I am not going to regret anything at all.
"It has just been fantastic. Long may it continue, fingers crossed."
The former EastEnders star was dressed to impress for the after party on her opening night in Pygmalion in London's West End.
She laughed and smiled as she posed for photographs alongside Chigvintsev and her family, including her sister - Inbetweeners star Hannah.
Tired of looking for love? Try online dating!
Related Online Dating News
Hong Kong Phil boss "dreams of working with Jackie Chan"Posted on: April 17, 2014 16:26:26
The musical director of one of Asia rsquo s most well-known orchestras dreams of bringing in some unorthodox talent to perform with his musicians Speaking to the South China Morning...
(read more)
Lee Ryan and Casey Batchelor steam up the CBB housePosted on: January 6, 2014 17:25:01
They may have been shackled together when they entered the house but Lee Ryan and Casey Batchelor have done anything but drift apart since they were set free In fact...
(read more)
The online dating racePosted on: January 6, 2014 16:22:05
New research has revealed how likely single people are to get a message from an online dating site throughout According to MailOnline age matters more to men than women as...
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Sweethearts get engaged after failures on reality TVPosted on: January 3, 2014 19:02:42
When Danielle Cornwall-Nash appeared on ITV s prime time dating show Take Me Out last year she hoped that she would find a new partner and maybe even a new...
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Black Dating (7)
BBW Dating (11)
Lesbian Dating (12)
Military Dating (15)
European Dating (30)
South African Dating (5)
USA Dating (154)