Role of hepatocyte growth factor in the immunomodulation potential of amniotic fluid stem cells

Human amniotic fluid stem cells (hAFSCs) may be useful for regenerative medicine because of their potential to differentiate into all three germ layers and to modulate immune response with different types of secretion molecules. This last issue has not been completely elucidated. The aim of this study was to investigate the secretome profile of the hAFSC, focusing on the role of hepatocyte growth factor (HGF) in immunoregulation through short and long cocultures with human peripheral blood mononuclear cells. We found that HGF produced by hAFSCs exerts a cytoprotective role, inducing an increase in caspase-dependent apoptosis in human immune cells. This study provides evidence supporting the hypothesis that amniotic fluid is an ideal source of stem cells for expansion and banking properties for therapeutic use. hAFSCs not only are less immunogenic but also can secrete immunoregulatory factors that may be useful in autoimmune diseases or allogenic implants. SIGNIFICANCE: New information about the secretome pattern is reported in this paper. Human amniotic fluid stem cells (hAFSCs) possess immunomodulatory properties involving hepatocyte growth factor production. hAFSCs could be used in immunotherapies and might be able to avoid allogenic rejectio

Nuclear Nox4-derived reactive oxygen species in myelodysplastic syndromes

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML

Pigmented Nodular Basal Cell Carcinomas in Differential Diagnosis with Nodular Melanomas: Confocal Microscopy as a Reliable Tool for In Vivo Histologic Diagnosis

Nodular basal cell carcinoma, especially when pigmented, can be in differential diagnosis with nodular melanomas, clinically and dermoscopically. Reflectance confocal microscopy is a relatively new imaging technique that permits to evaluate in vivo skin tumors with a nearly histological resolution. Here, we present four cases of challenging nodular lesions where confocal microscopy was able to clarify the diagnosis

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector\uae (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector\uae profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine

Nuclear Nox4 Interaction with Prelamin A is Associated with Nuclear Redox Control of Stem Cell Aging

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence

NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging

Aims Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. Materials and methods MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age\ua0 65\ua040\ua0years (cases) and with age\ua0 64\ua037\ua0years (controls). Key findings The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. Significance The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2