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 ß-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

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

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 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

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

The Formation of Future Teacher’s Preparation to Realization of Innovative Educational Activity

У статті розглядаються проблеми готовності майбутнього педагога до інноваційної діяльності. Визначається сутність поняття «інноваційна педагогічна діяльність». Висвітлено основні етапи здійснення інноваційної діяльності. Обґрунтовано зміст структурних елементів інноваційної діяльності, на які опирається майбутній учитель у своїй роботі. Охарактеризовано 5 категорій учителів та описано їхнє ставлення до нововведень. Пояснюється прагнення майбутнього освітянина до самопізнання, самовизначення й осмислення ним свого духовного світу, творчого ставлення до самого себе, прагнення до виявлення та розвитку своїх особистих можливостей.The problems of future teacher’s preparation for the innovation activity are examined. The essence of the idea of «innovation pedagogical activity» is determined. The factors which influence on the development of innovation processes on the modern stage are described. The maintenance of structural elements of future teacher’s innovation pedagogical activity is settled. The basic stages of preparing for the innovation activity are selected. The functional components of future teacher’s innovation activity, which help him to plan his work better, to realize innovations of the pedagogical process, also to correct and estimate results of his innovation activity are described. Five categories of the teachers are characterized, their attitude toward the realization of innovation technologies to the pedagogical process are described. The future teacher’s aspiration to self-recognizing, self-determination and grasping the idea of his spiritual world, own actions, a role and a place in his professional activity, creative attitude to himself in the process of active influence at outer and internal world, aspiring to the exposure and development of his personal possibilities is explained. The role of creative situation in the process of future teacher’s training, the claim of which is possible only in default of internal barriers to creative displays, the organizing of future teacher’s creative work of subconsciousness, searching of new relations, the development of imagination and fantasy, the development of professional sensitiveness, searching of sense in creative activity is described and characterized. The meaning of such directions of future teacher’s development qualities, such as emotional thinking, formulating new types of communication and communicative abilities, the development of internal abilities of a dialogue is generalized. The main conditions of the establishment of innovation technologies into the teacher’s work are characterized. The establishment of innovation technologies into the future teacher’s pedagogical activity, which gives him an opportunity to master the material better, reduces time on the decision of standard tasks, stimulates the positive attitude to the training disciplines, rises the level of informational culture and creates the conditions for the complete future teacher’s opening as a personality is determined

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

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

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

IL2Rα protein expression by Flow cytometric analysis.

<p>(A) IL2Rα gene expression at different time (2-4-12h) of cells pre-treated with REAC before exposition to RPM simulated microgravity (REAC+RPM) for 2-4-12h in comparison to no-REAC-treated cells before exposition to RPM simulated microgravity (noREAC+RPM); (B) REAC pre-treated cells subsequently kept for 2-4-12h at 1<i>g</i> gravity conditions (REAC+GC), compared to 1<i>g</i> gravity conditions not REAC pre-treated (noREAC+GC). (C) IL2Rα cell expression percentage at different time (2-4-12h) of REAC post-treatment after exposition to RPM simulated microgravity (RPM+REAC) for 2-4-12h as compared to REAC untreated cells after exposition to RPM simulated microgravity (RPM+noREAC), (D) REAC post-treated cells after exposure to 1<i>g</i> gravity conditions (GC+REAC) compared to 1<i>g</i> gravity conditions without REAC post-treated (GC+noREAC). Data are expressed as mean ± S.D. (n = 3, *p<0.01; **p<0.001; ***p<0.0001) of independent triplicate samples for each treatment.</p