This study is the first documentation of the isolation and extensive characterization of mesenchymal stem cells from canine adipose tissue. Methods previously used by our group to isolate and differentiate human adipose-derived mesenchymal stem cells (hAD-MSCs) have been modified and optimized for derivation of similar cells from canine adipose tissues. The canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs) showed lower proliferation ability and were refractory to osteogenic and adipogenic differentiation under conditions employed to differentiate hAD-MSCs. The differentiation of cAD-MSCs into osteoblasts and adipocytes was effectively achieved under modified conditions, by using laminin-coated plates and peroxisome proliferative activated receptor, gamma (PPARgamma) ligands, respectively. The formation of micromass was sufficient to induce chondrogenesis, unlike hAD-MSCs, which require transforming growth factor beta (TGF-beta). These cells displayed anchorage-independent growth in soft agar, and their colony-forming efficiency in plastic was comparable with human counterparts. The cAD-MSCs expressed genes associated with pluripotency, while their differentiated progeny expressed appropriate lineage-specific genes. The optimization of growth and differentiation of cAD-MSCs should facilitate future stem cell-based reparative and regenerative studies in dogs. The dog is a promising biomedical model that is suitable for evaluation of novel therapies such as those employing stem cells in experimental and in spontaneous disease settings.

This study is the first documentation of the isolation and extensive characterization of mesenchymal stem cells from canine adipose tissue. Methods previously used by our group to isolate and differentiate human adipose-derived mesenchymal stem cells (hAD-MSCs) have been modified and optimized for derivation of similar cells from canine adipose tissues. The canine adipose tissue-derived mesenchymal stem cells (cAD-MSCs) showed lower proliferation ability and were refractory to osteogenic and adipogenic differentiation under conditions employed to differentiate hAD-MSCs. The differentiation of cAD-MSCs into osteoblasts and adipocytes was effectively achieved under modified conditions, by using laminin-coated plates and peroxisome proliferative activated receptor, gamma (PPARgamma) ligands, respectively. The formation of micromass was sufficient to induce chondrogenesis, unlike hAD-MSCs, which require transforming growth factor beta (TGF-beta). These cells displayed anchorage-independent growth in soft agar, and their colony-forming efficiency in plastic was comparable with human counterparts. The cAD-MSCs expressed genes associated with pluripotency, while their differentiated progeny expressed appropriate lineage-specific genes. The optimization of growth and differentiation of cAD-MSCs should facilitate future stem cell-based reparative and regenerative studies in dogs. The dog is a promising biomedical model that is suitable for evaluation of novel therapies such as those employing stem cells in experimental and in spontaneous disease settings.

An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified MECP2 as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRASG12C-addicted cell line after KRAS down-regulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action.

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Background: Both circulating tumor cell (CTC) and total circulating cell-free DNA (ccfDNA) predict cancer patient prognosis. However, no study has explored the prognostic value of the combined use of CTC and ccfDNA. We aimed to investigate individual and joint effects of CTC and ccfDNA on clinical outcomes of metastatic breast cancer (MBC) patients. Methods: We collected 227 blood samples from 117 MBC patients. CTCs were enumerated using the CellSearch System. ccfDNAs were quantified by quantitative real-time PCR and Qubit fluorometer. The individual and joint effects of CTC and ccfDNA levels on patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models. Results: Compared to patients with < 5 CTCs, patients with ≥ 5 CTCs had a 2.58-fold increased risk of progression and 3.63-fold increased risk of death. High level of ccfDNA was associated with a 2.05-fold increased risk of progression and 3.56-fold increased risk of death. These associations remained significant after adjusting for other important clinical covariates and CTC/ccfDNA levels. CTC and ccfDNA levels had a joint effect on patient outcomes. Compared to patients with low levels of both CTC and ccfDNA, those with high levels of both markers exhibited a > 17-fold increased death risk (P < 0.001). Moreover, longitudinal analysis of 132 samples from 22 patients suggested that the inconsistency between CTC level and outcome in some patients could possibly be explained by ccfDNA level. Conclusions: CTC and total ccfDNA levels were individually and jointly associated with PFS and OS in MBC patients.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-lcysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high selfrenewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm 2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

BackgroundLeptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice.MethodsUsing leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells.ResultsWe report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells.ConclusionsTaken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies.

Somatic cell count (SCC), bacteriological findings and electrical conductivity (EC) were measured in milk from 216 quarters of 54 Murrah crossbred buffaloes for characterization of buffalo milk. Mean SCC in normal, subclinical and clinical mastitic milk was 171, 799 and 6039 ¥ 10 3 /mL, respectively. Coagulase negative Staphylococci (CNS) were the major organisms associated with subclinical mastitis, whereas CNS and coliforms were the predominant organisms in clinical mastitis. EC values were found to be significantly higher in buffalo milk with mammary infection. The best trade-off between sensitivity and specificity for diagnosing mastitis in buffaloes by EC score was found at the cut-off value of 3.7 mS/cm. The direct measures such as SCC, EC, bacteriological findings, and appearance of milk were found to be useful for the evaluation of udder health of buffaloes and categorization of milk.