Gianfranco Pigliaru

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.

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.

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.

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.

Stem cell senescence: effects of REAC technology on telomerase-independent and telomerase-dependent pathways

Decline in the gene expression of senescence repressor Bmi1, and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated β-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression.

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.

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 p21CIP1. 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.

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.

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.

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 30th 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.