Involvement of caspace-3 in the cleavage of terminal transferase.

To investigate the in vivo role of caspase-3 in Terminal Transferase metabolism DMSO-treated RPMI-8402, a human pre-T cell line was used. In DMSO treated samples3H-dGTP incorporation and TdT phosphorylation occurs after 4 hours of treatment. After 8 hours cells undergo TdT proteolysis in addition to its inactivation. The cleavage of TdT into 32- and 58-KDa proteolytic fragments occurred simultaneously with the activation of Caspase-3, but preceded changes associated with the apoptotic process described after 48 hours of treatment. The Caspase-3 peptide inhibitor V, used as a specific inhibitor, prevented TdT proteolysis prolonging its activity and rescued cells from apoptosis. Our experiments suggest that TdT is a nuclear substrate for Caspase-3, the main apoptotic effector protease in many cell types, and that the cleavage of TdT represents a primary step in a signal cascade leading to pre-T cell apoptosis

Biofabrication and Bone Tissue Regeneration: Cell Source, Approaches, and Challenges

The growing occurrence of bone disorders and the increase in aging population have resulted in the need for more effective therapies to meet this request. Bone tissue engineering strategies, by combining biomaterials, cells, and signaling factors, are seen as alternatives to conventional bone grafts for repairing or rebuilding bone defects. Indeed, skeletal tissue engineering has not yet achieved full translation into clinical practice because of several challenges. Bone biofabrication by additive manufacturing techniques may represent a possible solution, with its intrinsic capability for accuracy, reproducibility, and customization of scaffolds as well as cell and signaling molecule delivery. This review examines the existing research in bone biofabrication and the appropriate cells and factors selection for successful bone regeneration as well as limitations affecting these approaches. Challenges that need to be tackled with the highest priority are the obtainment of appropriate vascularized scaffolds with an accurate spatiotemporal biochemical and mechanical stimuli release, in order to improve osseointegration as well as osteogenesis

mRNAs and miRNAs profiling of Mesenchymal Stem Cells derived from amniotic fluid and skin

Mesenchymal Stem Cells (MSCs) may be isolated from different adult sources and even if the minimal criteria for defining MSCs have been reported, the scientific question about the potential distinctions among MSCs derived from different sources is still opened. In particular, it is debated if MSCs of different origin have the same grade of stemness or if the source affects their undifferentiated status. Here we report not only the isolation and the traditional characterization of MSCs derived from amniotic fluid (AF-MSCs) [1] and skin (S-MSCs) [2], but also a molecular characterization based on mRNAs and miRNAs profiling. Our results show that even if both AF- and S-MSCs are regulated by the same pathways (such as Wnt, MAPK and TGF-β), there is a fine and different control of them as suggested by altered levels of expression of some member of these pathways. In conclusion, it will be necessary to improve the knowledge about the role of each dysregulated miRs/gene because, actually, these differences may strengthen the question about the importance of tissue origin. This work was supported by grant FIRB-RBAP10MLK7_003 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Ital

MSCs and inflammation: not only a guardian role

  The literature on the relation between mesenchymal stem cells (MSCs) and inflammation is continuing to expand at a rapid rate with over 600 entries in PubMed under “MSCs and inflammation” starting from 2002. Inflammation is an essential part of the malignant microenvironment. Chemokines, leukocyte infiltration and cytokines are crucial elements, which contribute to cancer-related inflammation. Attracted by chemokines, MSCs are recruited at injury sites. After exposure to inflammatory factors in the local microenvironment, MSCs secrete several cytokines and vascular endothelial growth factor, which promote immunosuppression, angiogenesis and tumor growth. Here we compare by RT-PCR the expression of selected genes, related to inflammation, on MSCs derived from control (C-MSCs) and inflamed tissues (I-MSCs). First of all, an immunohistochemistry using anti-CD43 antibody was performed to better test the status of inflammation at the moment of tissues’ collection. CD43 is known as marker of inflammation, since it is expressed by most T cells, activated B cells, basophils, macrophages, monocytes and NK cells. Its expression was absent in “control” tissues, while it was strong in the “inflamed”. Subsequently, RNA was extracted, retro-transcribed and used for quantitative PCR. The genes were selected according to their role in inflammation: IL6 and IL8 (known as pro-inflammatory interleukins), TNFα (involved in systemic inflammation), CXCL2 (secreted by monocytes and macrophages and is chemotactic for polymorphonuclear leukocytes), CCL20 (strongly chemotactic for lymphocytes, its expression is induced by inflammatory cytokines), IFNγ (an important activator of macrophages) and TGFβ1 (promoting immunosuppression). Quantification of mRNA expression was calculated with the 2−ΔΔCt method, where ΔCt = Ct (gene of interest) − Ct (control gene) and Δ(ΔCt) = ΔCt (I-MSCs) − ΔCt (C-MSCs). The results revealed that the expression of all tested genes was higher in MSCs derived from inflamed tissues than in MSCs from control tissues (expressed as 1). This study underlines how MSCs are not inert guardians on inflammation, but as they play an active role

Isolation and characterization of Mesenchymal Stem Cells from pituitary tumours

In the past few years the introduction of the cancer stem cells (CSCs) notion opened new perspectives for the diagnosis and cure of solid tumors. According to this theory, CSCs originate from mutated stem cells, maintaining the self-renewal and differentiative abilities. Therefore, the development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients. Actually, no informations are available about stem cells and cancer stem cells on pituitary tumours. This work depicts some essential features of stem cells isolated from pituitary adenomas. Six tumour biopsies (3: GH-secreting; 3: non secreting) were collected and cultured with a specific culture medium. Cell growth and morphology were monitored and cells were subjected to analyses for stemness determination (immunophenotype, gene expression and differentiative potential) [1, 2] and GH secretion. Cells showed a stem-like immunophenotype, the expression of Oct-4, Sox-2, Nanog and Klf-4 and the ability to differentiate towards osteogenic, chondrogenic and adipogenic lineages. The hormone secretion ended after two weeks culturing. Even if further studies are needed for the fully comprehension of the specific nature of these cells and on their role on tumour onset and maintenance, this study opens to the possibility of isolation of stem cells from pituitary tumour, allowing a molecular targeting of it. This work was supported by grant FIRB-RBAP10MLK7_003 from Ministero dell’Istruzione, dell’Università e della Ricerca, Rome, Ital

The inflamed microenvironment: role on MSCs immunobiology and cancer

Inflammation and cancer are an inseparable binomial. The majority of cancers are triggered by somatic mutations and environmental factors with a common element: inflammation. Inflammation creates a microenvironment in which neoplastic cells can profit from the trophic factors secreted by inflammatory cells, useful to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to microenvironment creation by a strong paracrine effect. The linkage between MSCs and inflammation is bidirectional: the inflamed microenvironment affects the complex MSCs immunobiology, but also MSCs can sustain inflammation. Here, we tried to clarify the influence of inflammation on the immunobiology of MSCs and deepen the paracrine effect of MSCs on tumor growth. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). The contralateral part of the same patient, not inflamed, was used as control (C-MSCs). A panel of selected cytokines were analyzed by Real-Time PCR and ELISA. The cytokines expression was different in I-MSCs compared to C-MSCs, revealing that inflammation affects MSCs immunobiology. Then, C- and I-MSCs were indirectly co-cultured with MCF7 cells from breast adenocarcinoma. New analyses on proliferation rate and cytokines expression were performed. C- and I-MSCs gave almost the same results. The over-secretion of all the cytokines referred to the Th1 pathway and the decrease of those belonging to the Th2 pathway revealed the absence of a switch from Th1 to Th2 important to induce a chronic inflammation. The levels of TGF-β and G-CSF linked to the skill to damage the antigen-presenting cell function were decreased. In conclusion, even if MCF-7 proliferation increased after co-culture with I-MSCs, MSCs-derived paracrine effect does not sustain breast adenocarcinoma. These results absolve the breast implants from the insult to enhance adenocarcinoma onset

The role of the mesenchymal stem cells on breast cancer: friends or foes?

Mesenchymal stem cells (MSCs) have been the subject of an increased interest. Because of their ability to give rise to bone, cartilage, fat and muscle, their role in regenerative medicine has been extensively studied and the fact that they can be recruited at sites of inflammation and tissue repair has prompted their potential use as tissue regenerative cells. Contextually there has been a growing interest in the role of MSCs in cancer progression. The nature of the relationship between MSCs and tumor cells appears dual, with effects pro- as well as anti-tumorigenic. This paradox depends on the source and the degree of differentiation of MSCs and the tumor model used. Moreover, with the large range of cytokines and growth factors they produce, MSCs exert regulatory function on apoptosis, angiogenesis and an immunomodulatory role. Here we evaluate the interaction between MSCs derived from the periprosthetic capsule of mastectomyzed women and the breast cancer cell line MCF-7. Capsular tissue around breast implants is a normal inflammatory reaction versus a foreign body and it is rich of MSCs. To asses how MSCs interact with tumor cells, MCF-7 cells were incubated with medium previously conditioned by MSCs or directly co-coltured with MSCs; subsequently, we evaluated the proliferation and the expression of genes implicated in different pathways (angiogenesis, proliferation, anti-apoptosis, EMT transition). Our results showed that MCF-7 cells cultured together MSCs or using their conditioned medium have a more elevated proliferation rate but tumour cells seem less aggressive, like attested by a reduction of the expression of selected genes. The understanding of the mechanisms that control the interaction between MSCs and tumor cells is still at an early stage but recent literature confirm that MSCs and their progeny are not innocent bystanders in the tumor microenvironment. The study of these interactions is a critical area of future investigation that is needed to better define their role in cancer progression and their potential as therapeutic agents or targets

Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma

BAP1 is a gene situated on chromosome 3p in a region that is deleted in over 90% of Renal Cell Carcinomas (RCCs) (1,2). In the present study we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy and we assessed the prognostic value of their expression in terms of patients survival at long-term follow-up. 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis and patient specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BPA-1 in tumoral tissue. Median follow-up was 196.18 months (range 5 to 274); median survival was 125.34 months (range 5 to 274 months). We found that nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1 negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an impact on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant and/or metastatic setting

IGF1 and IGF1/VEGF cross-talk on human mesenchymal stromal cells (hMSCs): the role of stem cell sources in bone healing

Repair of skeletal defects remains a considerable biomedical problem. One of the major obstacles of the different tested strategies still remains the vascularization of engineered scaffolds. To this aim we have examined the ability of IGF1, alone or in association with VEGF, to modulate Periosteum Derived Progenitor Cells (PDPCs) (Ferretti et al., 2012) and Skin-Mesenchymal Stromal Cells (S-MSCs) (Orciani and Di Primio, 2013) osteoblastic or endothelial commitment. A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signalling pathways were investigated. Our results showed a different commitment of PDPCs and S-MSCs under growth factor stimulation: the former are induced towards an osteoblastic differentiation, whilst the latter seem to be brought to an endothelial phenotype. This commitment is also related to a diverse MAPK or PI3K/AKT signalling pathway activation. Our results open intriguing perspective for the development of innovative bone tissue engineering approaches based on associated angiogenesis and osteogenesis. Further investigations are however necessary to gain insight on the real cross-talk between proliferation and differentiation in adult stem cells. The work was supported by Italian FIRB (RBAP10MLK7) and PRIN (2010J8RYS7) project grants