Vasiliki Bitsika

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure.

Background There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. Methods AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. Results Both HPL cells and AF-MSCs were incorporated into CCl4-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. Conclusions 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.

Profilin 1 is a Potential Biomarker for Bladder Cancer Aggressiveness

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 proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified proteins, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with BC such as MMP9, fibrinogen forms, and clusterin. In agreement to the immobilized metal affinity chromatography results, aminopeptidase N, profilin 1, and myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis, profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of profilin 1 was investigated in the T24 BC cells where blockage of the protein by the use of antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary proteins and as one main result of this analysis reveals the association of profilin 1 with BC paving the way for its further investigation in BC stratification.

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

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.

A comparison between MALDI-MS and CE-MS data for biomarker assessment in chronic kidney diseases

Non-invasive detection of diseases, based on urinary proteomics, is becoming an increasingly important area of research, especially in the area of chronic kidney disease (CKD). Different platforms have been used in independent studies, mostly capillary-electrophoresis coupled ESI-MS (CE-MS), liquid chromatography coupled mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have compared the performance of CE-MS with MALDI-MS in detecting CKD, based on a cohort of 137 urine samples (62 cases and 75 controls). Data cross-talk between the two platforms was established for the comparison of detected biomarkers. The results demonstrate superior performance of the CE-MS approach in terms of peptide resolution and obtained disease prediction accuracy rates. However, the data also demonstrate the ability of the MALDI-MS approach to separate CKD patients from controls, at slightly reduced accuracy, but expected reduced cost and time. As a consequence, a practical approach can be foreseen where MALDI-MS is employed as an inexpensive, fast, and robust screening tool to detect probable CKD. In a second step, high resolution CE-MS could be used in those patients only that scored negative for CKD in the MALDI-MS analysis, reducing costs and time of such a program.

Sox2 Suppression by miR‐21 Governs Human Mesenchymal Stem Cell Properties

MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle‐shaped (SS) and round‐shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs 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. We further demonstrated for the first time a direct interaction between miR‐21 and the pluripotency marker Sox2. The induction of miR‐21 strongly inhibited Sox2 expression in SS‐AF‐MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR‐21 inhibition in RS‐AF‐MSCs and BM‐MSCs, which led to an enhanced proliferation rate. Finally, miR‐21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS‐AF‐MSCs. Therefore, these findings suggest that miR‐21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

Capillary zone electrophoresis on-line coupled to mass spectrometry: A perspective application for clinical proteomics

Clinical proteomics, a rapidly growing field, intends to use specific diagnostic proteomic/peptidomic markers for initial diagnosis or prognosis of the progression of various diseases. Analyses of disease‐associated markers in defined biological samples can provide valuable molecular diagnostic information for these diseases. This approach relies on sensitive and highly standardized modern analytical techniques. In the recent years, one of these technologies, CZE online coupled to MS (CZE‐MS), has been increasingly used for the detection of peptide biomarkers (<20 kDa) in body fluids such as urine. This review presents the most relevant urinary proteomic studies addressing the application of CZE‐MS in clinically relevant biomarker research between the years 2006 and 2014.

Human Amniotic Fluid-Derived Mesenchymal Stem Cells As Therapeutic Vehicles: A Novel Approach For the Treatment of Bladder Cancer

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.

Analysis of secreted proteins for the study of bladder cancer cell aggressiveness.

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.

Chromosomal and proteome analysis of a new T24‐based cell line model for aggressive bladder cancer

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.

Improving peptide relative quantification in MALDI-TOF MS for biomarker assessment.

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).