Physical stimulation by REAC and BMP4/WNT-1 inhibitor synergistically enhance cardiogenic commitment in iPSCs.

It is currently known that pluripotent stem cells can be committed in vitro to the cardiac lineage by the modulation of specific signaling pathways, but it is also well known that, despite the significant increase in cardiomyocyte yield provided by the currently available conditioned media, the resulting cardiogenic commitment remains a highly variable process. Previous studies provided evidence that radio electric fields asymmetrically conveyed through the Radio Electric Asymmetric Conveyer (REAC) technology are able to commit R1 embryonic stem cells and human adipose derived stem cells toward a cardiac phenotype. The present study aimed at investigating whether the effect of physical stimulation by REAC in combination with specific chemical inductors enhance the cardiogenic potential in human induced pluripotent stem cells (iPSCs). The appearance of a cardiac-like phenotype in iPSCs cultured in the presence of a cardiogenic medium, based upon BMP4 and a WNT-inhibitor, was consistently increased by REAC treatment used only during the early fate differentiation for the first 72 hours. REAC-exposed iPSCs exhibited an upregulation in the expression of specific cardiogenic transcripts and morphologically in the number of beating clusters, as compared to cells cultured in the cardiogenic medium alone. Our results indicate that physical modulation of cellular dynamics provided by the REAC offers an affordable strategy to mimic iPSC cardiac-like fates in the presence of a cardiogenic milieu.

Radio Electric Asymmetric Conveyer (REAC) technology to obviate loss of T cell responsiveness under simulated microgravity.

Alterations of the gravitational environment are likely to modify cell behavior. Several studies have proven that T cells are sensitive to gravity alterations and that microgravity conditions may induce immunosuppression and weakened T cell immune response in humans during spaceflights. The aim of this work was to elucidate if a specific treatment of Radio Electric Asymmetric Conveyer (REAC) technology could restore, after mitogenic activation (Con A), a correct expression of cytokine IL2 gene and its receptor IL2R alpha, which are inhibited in T cells under microgravity conditions, as demonstrated in several studies. The results of this study, conducted in microgravity simulated with Random Positioning Machine (RPM), confirm the T cell activation recovery and offer the evidence that REAC technology could contribute to the understanding of T cell growth responsiveness in space, reducing the impact of weightlessness on the immune system experienced by humans in long duration space missions.

REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence.

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30(th) passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

Osteogenesis from Dental Pulp Derived Stem Cells: A Novel Conditioned Medium Including Melatonin within a Mixture of Hyaluronic, Butyric, and Retinoic Acids.

Human dental pulp stem cells (hDPSCs) have shown relevant potential for cell therapy in the orthopedic and odontoiatric fields. The optimization of their osteogenic potential is currently a major challenge. Vascular endothelial growth factor A (VEGF A) has been recently reported to act as a major conductor of osteogenesis in vitro and in vivo. Here, we attempted to prime endogenous VEGF A expression without the need for viral vector mediated gene transfer technologies. We show that hDPSCs exposure to a mixture of hyaluronic, butyric, and retinoic acids (HA + BU + RA) induced the transcription of a gene program of osteogenesis and the acquirement of an osteogenic lineage. Such response was also elicited by cell exposure to melatonin, a pleiotropic agent that recently emerged as a remarkable osteogenic inducer. Interestingly, the commitment to the osteogenic fate was synergistically enhanced by the combinatorial exposure to a conditioned medium containing both melatonin and HA + BU + RA. These in vitro results suggest that in vivo osteogenesis might be improved and further studies are needed.

Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson's disease.

Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson's disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology.

Anti-senescence efficacy of radio-electric asymmetric conveyer technology.

Recent evidence suggests that ageing-related diseases could result in an accelerated loss of self-renewal capability of adult stem cells, normally involved in replacing damaged cellular elements. In previous works, we highlighted that a specific treatment, named tissue optimization-regenerative (TO-RGN), of radio-electric asymmetric conveyer (REAC) technology, influenced gene expression profiles controlling stem cell differentiation and pluripotency of human skin-derived fibroblasts in vitro. The purpose of the present work was to verify whether TO-RGN may also be effective in counteracting the expression of the senescence marker beta-galactosidase and of senescence-associated gene expression patterning, engaged during prolonged culture of human adipose-derived stem cells (hADSCs). Following TO-RGN exposure, we observed a significant downregulation in beta-galactosidase staining and in the expression of the senescence mediator genes p16INK4, ARF, p53, and p21(CIP1). Moreover, differently formed untreated cells, TO-RGN-exposed hADSCs maintained their typical fibroblast-like morphology and exhibited a multilineage potential even at late passages, as shown by the remarkable preservation of commitment to osteogenic, adipogenic, chondrogenic, and vasculogenic fates, both at morphologic and gene expression levels. In conclusion, our study highlights a positive effect of TO-RGN in counteracting degenerative senescence processes in vitro.

Radioelectric asymmetric conveyed fields and human adipose-derived stem cells obtained with a nonenzymatic method and device: a novel approach to multipotency.

Human adipose-derived stem cells (hASCs) have been recently proposed as a suitable tool for regenerative therapies for their simple isolation procedure and high proliferative capability in culture. Although hASCs can be committed into different lineages in vitro, the differentiation is a low-yield and often incomplete process. We have recently developed a novel nonenzymatic method and device, named Lipogems, to obtain a fat tissue derivative highly enriched in pericytes/mesenchymal stem cells by mild mechanical forces from human lipoaspirates. When compared to enzymatically dissociated cells, Lipogems-derived hASCs exhibited enhanced transcription of vasculogenic genes in response to provasculogenic molecules, suggesting that these cells may be amenable for further optimization of their multipotency. Here we exposed Lipogems-derived hASCs to a radioelectric asymmetric conveyer (REAC), an innovative device asymmetrically conveying radioelectric fields, affording both enhanced differentiating profiles in mouse embryonic stem cells and efficient direct multilineage reprogramming in human skin fibroblasts. We show that specific REAC exposure remarkably enhanced the transcription of prodynorphin, GATA-4, Nkx-2.5, VEGF, HGF, vWF, neurogenin-1, and myoD, indicating the commitment toward cardiac, vascular, neuronal, and skeletal muscle lineages, as inferred by the overexpression of a program of targeted marker proteins. REAC exposure also finely tuned the expression of stemness-related genes, including NANOG, SOX-2, and OCT-4. Noteworthy, the REAC-induced responses were fashioned at a significantly higher extent in Lipogems-derived than in enzymatically dissociated hASCs. Therefore, REAC-mediated interplay between radioelectric asymmetrically conveyed fields and Lipogems-derived hASCs appears to involve the generation of an ideal "milieu" to optimize multipotency expression from human adult stem cells in view of potential improvement of future cell therapy efforts.

Amniotic fluid stem cells morph into a cardiovascular lineage: analysis of a chemically induced cardiac and vascular commitment.

Mouse embryonic stem cells were previously observed along with mesenchymal stem cells from different sources, after being treated with a mixed ester of hyaluronan with butyric and retinoic acids, to show a significant increase in the yield of cardiogenic and vascular differentiated elements. The aim of the present study was to determine if stem cells derived from primitive fetal cells present in human amniotic fluid (hAFSCs) and cultured in the presence of a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids show a higher yield of differentiation toward the cardiovascular phenotype as compared with untreated cells. During the differentiation process elicited by exposure to HA + BU + RA, genes controlling pluripotency and plasticity of stem cells, such as Sox2, Nanog, and Oct4, were significantly downregulated at the transcriptional level. At this point, a significant increase in expression of genes controlling the appearance of cardiogenic and vascular lineages in HA + BU + RA-treated cells was observed. The protein expression levels typical of cardiac and vascular phenotypes, evaluated by Western blotting, immunofluorescence, and flow cytometry, were higher in hAFSCs cultured in the presence of HA + BU + RA, as compared with untreated control cells. Appearance of the cardiac phenotype was further inferred by ultrastructural analysis using transmission and scanning electron microscopy. These results demonstrate that a mixture of HA + BU + RA significantly increased the yield of elements committed toward cardiac and vascular phenotypes, confirming what we have previously observed in other cellular types.

Effects of regenerative radioelectric asymmetric conveyer treatment on human normal and osteoarthritic chondrocytes exposed to IL-1ß. A biochemical and morphological study.

PURPOSE: Osteoarthritis (OA) is a degenerative disease characterized by a progressive loss of articular cartilage extracellular matrix and is due to functional impairments occurring in chondrocytes. In previous works, we highlighted that Regenerative Tissue Optimization (TO-RGN) treatment with radioelectric asymmetric conveyer (REAC) technology influenced the gene expression profiles controlling stem cell differentiation and the pluripotency of human skin-derived fibroblasts in vitro. Since interleukin-1 beta signaling has been implicated in the induction and progression of this disease (through metalloproteinase-3 synthesis and nitric oxide production), we investigated whether REAC TO-RGN might influence the biochemical and morphological changes induced by interleukin-1 beta in normal and OA chondrocytes. METHODS: The induction of metalloproteinase-3 and proteoglycan synthesis was evaluated by a solid-phase enzyme-amplified sensitivity immunoassay, and nitric oxide production was evaluated with the Griess method. Ultrastructural features were observed by transmission electron microscopy. RESULTS: REAC TO-RGN treatment decreased nitric oxide and metalloproteinase-3 production in normal and OA chondrocytes, while inducing an increase in proteoglycan synthesis. OA chondrocytes were more affected by REAC TO-RGN treatment than were normal chondrocytes. Ultrastructural changes confirmed that REAC TO-RGN may counteract the negative effects of interleukin-1 beta incubation. CONCLUSION: The results of this in vitro study suggest that REAC TO-RGN treatment may represent a new, promising approach for the management of OA.

Radio electric conveyed fields directly reprogram human dermal skin fibroblasts toward cardiac, neuronal, and skeletal muscle-like lineages.

Somatic cells can be directly reprogrammed to alternative differentiated fates without first becoming stem/progenitor cells. Nevertheless, the initial need for viral-mediated gene delivery renders this strategy unsafe in humans. Here, we provide evidence that exposure of human skin fibroblasts to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields at a radiofrequency of 2.4 GHz, afforded remarkable commitment toward cardiac, neuronal, and skeletal muscle lineages. REAC induced the transcription of tissue-restricted genes, including Mef2c, Tbx5, GATA4, Nkx2.5, and prodynorphin for cardiac reprogramming, as well as myoD, and neurogenin 1 for skeletal myogenesis and neurogenesis, respectively. Conversely, REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 h, followed by a downregulation at later times. The REAC action bypassed a persistent reprogramming toward an induced pluripotent stem cell-like state and involved the transcriptional induction of the NADPH oxidase subunit Nox4. Our results show for the first time the feasibility of using a physical stimulus to afford the expression of pluripotentiality in human adult somatic cells up to the attainment of three major target lineages for regenerative medicine.

Regenerative treatment using a radioelectric asymmetric conveyor as a novel tool in antiaging medicine: an in vitro beta-galactosidase study.

BACKGROUND: Beta-galactosidase is the most widely used biomarker for highlighting the processes of cellular aging, including neurodegeneration. On this basis, we decided to test in vitro whether a set of rescuing/reparative events previously observed by us in subjects treated with radioelectric asymmetric conveyor (REAC) technology may also involve antagonism of a marker of aging-related degenerative processes, as assessed by a reduction in beta-galactosidase at the cellular level. METHODS: Human adipose-derived stem cells were cultured at different passages, ranging from 5 to 20, with or without REAC exposure for 12 hours. The cells were then processed for biochemical beta-galactosidase staining and morphological microscopy analysis. RESULTS: We observed a significant reduction in expression of senescence associated-beta-galactosidase, and a persistence of fibroblast-like morphology typical of human adipose-derived stem cells, even at late passages. CONCLUSION: Our results indicate the ability of REAC technology to counteract in vitro senescence of human adipose-derived stem cells, and prompt the hypothesis that such technology may be exploited to antagonize in vivo senescence of tissue-resident or transplanted stem cells playing an important role in clinical treatment of age-related processes.

Radiofrequency energy loop primes cardiac, neuronal, and skeletal muscle differentiation in mouse embryonic stem cells: a new tool for improving tissue regeneration.

Radiofrequency (RF) waves from Wi-Fi (wireless fidelity) technologies have become ubiquitous, with Internet access spreading into homes, and public areas. The human body harbors multipotent stem cells with various grading of potentiality. Whether stem cells may be affected by Wi-Fi RF energy remains unknown. We exposed mouse embryonic stem (ES) cells to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering Wi-Fi RF of 2.4 GHz with its conveyer electrodes immersed into the culture medium. Cell responses were investigated by real-time PCR, Western blot, and confocal microscopy. Single RF burst duration, radiated power, electric and magnetic fields, specific absorption rate, and current density in culture medium were monitored. REAC stimulation primed transcription of genes involved in cardiac (GATA4, Nkx-2.5, and prodynorphin), skeletal muscle (myoD) and neuronal (neurogenin1) commitment, while downregulating the self renewal/pluripotency-associated genes Sox2, Oct4, and Nanog. REAC exposure enhanced the expression of cardiac, skeletal, and neuronal lineage-restricted marker proteins. The number of spontaneously beating ES-derived myocardial cells was also increased. In conclusion, REAC stimulation provided a "physical milieu" optimizing stem cell expression of pluripotentiality and the attainment of three major target lineages for regenerative medicine, without using chemical agonists or vector-mediated gene delivery.