Human mesenchymal stem cells (hMSCs) constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes, or chondrocytes. So far, the most common source of MSCs has been the bone marrow (BM); however BM-MSC harvesting and processing exhibits major drawbacks and limitations. Thus, identification and characterization of alternative sources of MSCs are of great importance. In the present study, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF). We documented that these cells are of embryonic origin, can differentiate under appropriate conditions into cell types derived from all three germ layers, and express the pluripotency marker Oct-4, the human Nanog protein, and the stage-specific embryonic antigen-4 (SSEA-4). Furthermore, we systematically tested the immunophenotype of cultured MSCs by flow cytometry analysis using a wide variety of markers. Direct comparison of this phenotype to the one derived from cultured BM-MSCs demonstrated that cultured MSCs from both sources exhibit similar expression patterns. Using the two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach, we have generated for the first time the protein map of cultured AF-MSCs by identifying 261 proteins, and we compared it directly to that of cultured BM-MSCs. The functional pattern of the identified proteins from both sources was similar. However, cultured AF-MSCs displayed a number of unique proteins related to proliferation and primitive phenotype, which may confer to the distinct features of the two types. Considering the easy access to this new cell source and the yield of expanded MSCs for stem cell research, AF may provide an excellent source of MSCs both for basic research and for potential therapeutic applications.

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.

Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver.

Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted Bladder cancer (BC) 1 is the second in incidence and mortality cancer of the genitourinary system (1) and estimated to be the ninth most common malignancy (2). It is associated with a high recurrence rate underscoring the need for continuous surveillance following initial treatment. Cystoscopy still remains the gold standard for diagnosis and follow-up monitoring of bladder cancer. However, it is an invasive and unpleasant procedure, rendering particularly the regular surveillance program (e.g. cystoscopy every three months for the first year following initial diagnosis) not well accepted by the patients (3, 4). Urine Cytology is a noninvasive current detection tool for BC, suffering however from suboptimal sensitivity, especially for low grade tumors and being subjected to interobserver variability (5). The invasive nature of cystoscopy and the low effectiveness of cytology have prompted the search for novel and better ways to diagnose the disease with special emphasis on the early detection of disease recurrences and/or progression.Urine is regularly used in clinical practice and yields a wealth of information about the state of an individual's health. Because it can be collected in a noninvasive way it is more accessible than plasma or serum. In addition, there is no need for trained personnel for urine collection. Urine contains cells and cellular debris, inorganic ions (K ϩ , Na ϩ , Cl Ϫ , and Ca ϩ2 ), organic molecules (urea, uric acid, and creatinine) and proteins. If renal function is normal, urinary protein content is less

Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNβ) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNβ, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNβ-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.

The miRNA profile of mesenchymal stem cells (MSCs) derived from amniotic fluid, bone marrow (BM), and umbilical cord blood was analyzed. Initially, 67 different miRNAs were identified that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR‐21 was expressed at higher levels in RS‐AF‐MSCs and BM‐MSCs compared with SS‐AF‐MSCs. Findings suggest that miR‐21 might function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

Secreted proteins play a key role in cell signaling, communication, and migration. We recently described the development of an aggressive variant (T24M) of the bladder cancer cell line T24. Using this cell line model, the objective of our work was the identification of secreted proteins involved in the acquisition of the aggressive phenotype. Using in vitro assays, we demonstrate that conditioned media of the T24M cells promote motility of the parental less aggressive T24 cells. Proteomic analysis of cell culture conditioned media by the use of 2-dimensional gel electrophoresis coupled to MALDI TOF MS and LC-MS approaches resulted in enrichment and detection of multiple classical extracellular and secreted proteins such as fibronectin, cystatin, fibrillin, fibulin, interleukin 6, etc. Comparison of the secretome of the T24 and T24M cells indicated differences in proteins with potential involvement in the mechanisms of cell aggressiveness including SPARC, tPA, and clusterin. These findings were further confirmed by Western blot analysis. In the case of SPARC, further studies involving transwell assays indicated that blockage of the protein in the presence of SPARC-specific Abs results in decreased cell motility. Collectively, our study provides a 2DE-based comprehensive analysis of bladder cancer cell secretome. The results indicate various secreted proteins with potential involvement in bladder cancer cell aggressiveness and more specifically provide initial evidence for special role of SPARC in bladder cancer cell motility and invasiveness.

Cell line models aid in understanding cancer aggressiveness. The aim of this study was the establishment of a metastatic variant (T24M) of the T24 bladder cancer cell line and its initial characterization at chromosomal and proteomic levels. T24M were spontaneously developed in mice from T24 cells, following cycles of subcutaneous injections and culture in vitro. Transwell migration assays and injections in mice revealed increased migration and tumorigenic properties of T24M compared to the T24 cells. Cytogenetic analysis demonstrated that T24M retained several karyotypic characteristics of the parental cells and also acquired novel chromosomal aberrations related to aggressive bladder cancer. Proteomic analysis of the T24 and T24M cells by 2-DE and MS led to the generation of their 2-DE proteomic map and revealed differences in multiple proteins. These include proteases of the lysosomal and proteasome degradation pathways, mitochondrial and cytoskeletal proteins. The 2-DE findings were confirmed by immunoblotting of cell lysates and immunohistochemistry of bladder cancer tissue sections for cathepsin D and activity assays for proteasome. Collectively, our results suggest that the T24M cells reflect many known chromosomal and proteomic aberrations encountered in aggressive bladder cancers but also provide access to novel findings with potentially clinical applications.