Paola Lanuti

Proteome analysis of human Wharton's jelly cells during in vitro expansion

BackgroundThe human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Whartons jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.ResultsTo better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.ConclusionsOur work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

Ratiometric-pericam-mt, a novel tool to evaluate intramitochondrial zinc

Zn(2+) can enter mitochondria and promote a plethora of physiological and patho-physiological effects. The issue of measuring changes in intramitochondrial levels is therefore critical. Past studies have employed fluorescent Zn(2+) indicators, like Rhod-2 and RhodZin-3, however, the use of these probes is impaired by their extramitochondrial sequestration. In this study, we show that the ratiometric mitochondria-targeted pericam, RPmt, can be employed to detect changes of intramitochondrial free Zn(2+) ([Zn(2+)](m)) levels. Using RPmt in neuronal and non neuronal cell lines we demonstrate that mitochondria can take up the cation mobilized from the cytosolic pool of protein-bound Zn(2+) and that mitochondrial Zn(2+) sequestration is largely mediated by the activity of the Ca(2+) uniporter.

Aβ1–42 stimulated T cells express P-PKC-δ and P-PKC-ζ in Alzheimer disease

Abstract The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction, and involvement of some PKC isoforms in T-cell activation has been demonstrated. Nevertheless, very little is known about their involvement in the Amyloid β (Aβ)-dependent molecular signals in the T lymphocytes of Alzheimer disease (AD) patients. Therefore, the aim of this study was to investigate the involvement of PKC-α, PKC-δ and PKC-ζ expression and activity in the signaling machinery activated in Aβ-reactive T cells, in adult healthy individuals, elderly healthy subjects, and from patients with AD. The results show that in peripheral T-cells from early AD patients, Aβ1–42 produced a distinct subpopulation highly expressing P-PKC-δ, while in severe AD patients the same treatment induced two distinct P-PKC-δ and P-PKC-ζ T-cell subpopulations. Such subpopulations were not noticeable following CD3/CD28 treatment of the same samples or after treatment of peripheral T cells from healthy adult or elderly subjects with Aβ1–42 or with CD3/CD28. We believe that these findings may be of help in possible attempts to develop further diagnostic strategies useful for the characterization of AD.

A novel flow cytometric approach to distinguish circulating endothelial cells from endothelial microparticles: Relevance for the evaluation of endothelial dysfunction

Circulating endothelial cells (CEC) and endothelial microparticles (EMP) are emerging as markers of endothelial repair and activation/apoptosis. Although significant changes in the number of CEC and EMP in pathological conditions have been reported, their reliable identification and quantification still remain a technical challenge. Here, we present a novel methodology for the identification and quantitation of CEC and EMP based on multicolor flow cytometry. Using a lyse/no wash protocol, we observed that in 50 μl of peripheral blood, the large majority of events expressing an endothelial phenotype (CD45-/CD146+/CD34+) are due to non-nucleated particles (DRAQ5-) carrying mitochondrial activity (MitoTracker+) and, therefore, classified as EMP. We enumerated circulating EMP by single platform absolute count in a lyse/no wash four-color flow-cytometric procedure, which allowed the distinction, within the whole endothelial compartment, of EMP derived from endothelial progenitors (CD45-/CD146+/CD34+/CD117+) and from mature endothelial cells (CD45-/CD146+/CD34+/CD117-). A significant increase in both subsets was observed in patients with diabetes mellitus. Thus, this simple and highly reproducible method may be useful for monitoring endothelial dysfunction in clinical settings.

Enhancement of TRAIL cytotoxicity by AG-490 in human ALL cells is characterized by downregulation of cIAP-1 and cIAP-2 through inhibition of Jak2/Stat3

The ability of death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to selectively kill a variety of cancer cells has been largely described, but one of the major concerns with the treatment is the occurrence of drug resistance and possible toxic side effects. Here, we report that TRAIL induces apoptosis in Jurkat and SUPT1 T cell lines and in human T-ALL blasts but not in healthy subject-derived peripheral blood mononuclear cells. In parallel, the treatment with TRAIL and Tyrphostin (AG-490), a selective Janus kinase 2 inhibitor, produces an evident enhancement of cytotoxicity, characterized by a significant inhibition of Stat3 phosphorylation compared to controls or to TRAIL alone-treated samples, and associated with a dramatic decrease of both cIAP-1 and cIAP-2 mRNA levels. Downregulation of cIAP-1 and cIAP-2 by specific small interference RNAs significantly amplifies TRAIL-reduced cytotoxicity. All together, these findings strongly indicate that cIAP-1 and cIAP-2 downregulation is a fundamental step in the signaling pathways mediating the combinatorial effect of TRAIL and AG-490 on T cell leukemia. These findings may help to open new routes for the development of less toxic pharmacological strategies in the treatment of patients affected by TRAIL-sensitive leukemias.

Gene expression modifications in Wharton's Jelly mesenchymal stem cells promoted by prolonged in vitro culturing.

BackgroundIt has been demonstrated that the umbilical cord matrix, represented by the Wharton’s Jelly (WJ), contains a great number of mesenchymal stem cells (MSCs), characterized by the expression of specific MSCs markers, shared by both human and animal models. The easy access to massive WJ amount makes it an attractive source of MSCs for cell-based therapies. However, as in other stem cell models, a deeper investigation of WJ-derived MSCs (WJ-MSCs) biological properties, probably modulated by their prolonged expansion and fast growth abilities, is required before their use in clinical settings. In this context, in order to analyze specific gene expression modifications occurring in WJ-MSCs, along with their culture prolongation, we investigated the transcriptomic profiles of WJ-MSCs after 4 and 12 passages of in vitro expansion by microarray analysis.ResultsHierarchical clustering analysis of the data set originated from a total of 6 experiments revealed that in vitro expansion of WJ-MSCs up to 12 passages promote selective over-expression of 157 genes and down-regulation of 440 genes compared to the 4th passage. IPA software analysis of the biological functions related to the identified sets of genes disclosed several transcripts related to inflammatory and cell stress response, cell proliferation and maturation, and apoptosis.ConclusionsTaken together, these modifications may lead to an impairment of both cell expansion ability and resistance to apoptosis, two hallmarks of aging cells. In conclusion, results provided by the present study suggest the need to develop novel culture protocols able to preserve stem cell plasticity.

Allele-specific loss and transcription of the miR-15a/16-1 cluster in chronic lymphocytic leukemia

Deregulation of the miR-15a/16-1 cluster has a key role in the pathogenesis of chronic lymphocytic leukemia (CLL), a clinically heterogeneous disease with indolent and aggressive forms. The miR-15a/16-1 locus is located at 13q14, the most frequently deleted region in CLL. Starting from functional investigations of a rare SNP upstream the miR cluster, we identified a novel allele-specific mechanism that exploits a cryptic activator region to recruit the RNA polymerase III for miR-15a/16-1 transcription. This regulation of the miR-15a/16- locus is independent of the DLEU2 host gene, which is often transcribed monoallellically by RPII. We found that normally one allele of miR-15a/16-1 is transcribed by RNAPII, the other one by RNAPIII. In our subset of CLL patients harboring 13q14 deletions, exclusive RNA polymerase III (RPIII)-driven transcription of the miR-15a/16-1 was the consequence of loss of the RPII-regulated allele and correlated with high expression of the poor prognostic marker ZAP70 (P=0.019). Thus, our findings point to a novel biological process, characterized by double allele-specific transcriptional regulation of the miR-15a/16-1 locus by alternative mechanisms. Differential usage of these mechanisms may distinguish at onset aggressive from indolent forms of CLL. This provides a basis for the clinical heterogeneity of the CLL patients carrying 13q14 deletions.

Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation

Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.

Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets

Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.

Expression and phosphorylation of protein kinase C isoforms in Abeta(1-42) activated T lymphocytes from Alzheimers disease.

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction. There is evidence demonstrating altered activity of some PKC isoforms (PKC-α, PKC-δ and PKC-ζ) in the neurons of brains of Alzheimers Disease (AD) sufferers, but little is known about their involvement in the intracellular machinery of amyloid β protein-reactive T lymphocytes in AD. By applying a modified “split-well culture system” for Aβ1–42 reactivity, we carried out flow cytometry analysis and biochemical investigations on the possible involvement of PKC-α, PKC-δ and PKC-ζ in the signalling system activated in Aβ-reactive T cells purified from peripheral blood mononucleate cells (PBMC) from healthy subjects and patients with AD. Flow cytometry analysis of Aβ1–42 activated T lymphocytes in the majority of AD patients highlighted a distinct cellular cluster highly expressing phospho-PKC-δ (P-PKC-δ), while most full-blown AD patients highly expressed two distinct P-PKC-δ and phospho-PKC-ζ (P-PKC-ζ) bright sub-populations. The same investigation performed in freshly purified peripheral T lymphocytes, did not highlight any subpopulation, suggesting that the detection of P-PKC-δ and P-PKC-ζ bright subpopulations is specifically linked to Aβ1–42 activated T lymphocytes. The data presented here, therefore, suggest possible novel hallmarks to discriminate between healthy elderly subjects and beginning or full-blown Alzheimers Disease patients.