Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine.

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for "in vitro" stem cell commitment, preparatory to the "in vivo" stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

The trail from quantum electro dynamics to informative medicine.

Several years ago just before Christmas, in a small meeting room at the Institute of Pharmacology at the University of Rome, we had the opportunity to attend a meeting on "The role of QED in medicine" by Emilio Del Giudice and Giuliano Preparata. Before that meeting, we were more oriented towards a mechanistic view of Biochemistry and Medicine, believing that chemical reactions could only take place when a random collision between molecules with a gain in energy takes place. We envisioned water as just a solvent in which was possible to dissolve a solute. After we listened to Giuliano's and Emilio's speech on the "New physics of water", and on "The possible origin of coherence in cell, tissues and the interaction of very weak and low frequency magnetic fields with the ions, systems of the cell", we realized that living organisms are complex electrochemical systems which evolved in a relatively narrow range of well-defined environmental parameters. Environmental natural electro-magnetic fields are an ubiquitous factor in nature. If nature gave certain organisms the ability to receive information about the environment via invisible electromagnetic signals, then there must also the capability to discriminate between significant and meaningless ones. Bearing in mind that electromagnetic fields can be perceived by living organisms by means a resonance effect, we should not be amazed if they can be able to induce different biological effects. The work that we will present in memory of Emilio is based on the hypotheses that an aqueous system a chemical differentiation agent such as retinoic acid (RA) were electronically captured and transferred to the culture medium of Neuroblastoma Cell Line (LAN-5) and the proliferation rate was assessed to assess cell responses to the electromagnetic information transfer through the aqueous system. Like those enfolded in living organisms could play a synergic role in modulating biological functions, generating dissipative structures under appropriate patterns of electromagnetic signals providing basis for storing and retrieving biological activities. An external electro-magnetic stimulus from a source molecule can be stored, translated, and transferred by the aqueous systems to the biological target, selectively driving their endogenous activity and mimicking the effect of a source molecule.

Copper ion fluxes through the floating water bridge under strong electric potential.

We have performed a series of experiments applying high voltage between two electrodes, immersed in two beakers containing bidistilled water in a way similar to experiments conducted by Fuchs and collaborators, which showed that a water bridge can be formed between the two containers. We also observed the formation of water bridge. Moreover, choosing different pairs of electrodes depending on the material they are made up of, we observed that copper ions flow can pass along the bridge if the negative electrode is made up of copper. We show that the direction of the flux not only depends on the applied electrostatic field but on the relative electronegativity of the electrodes too. These results open new perspectives in understanding the properties of water. We suggest a possible explanation of the obtained results.

Nonpulsed sinusoidal electromagnetic fields as a noninvasive strategy in bone repair: the effect on human mesenchymal stem cell osteogenic differentiation.

In vivo control of osteoblast differentiation is an important process needed to maintain the continuous supply of mature osteoblast cells for growth, repair, and remodeling of bones. The regulation of this process has also an important and significant impact on the clinical strategies and future applications of cell therapy. In this article, we studied the effect of nonpulsed sinusoidal electromagnetic field radiation tuned at calcium-ion cyclotron frequency of 50 Hz exposure treatment for bone differentiation of human mesenchymal stem cells (hMSCs) alone or in synergy with dexamethasone, their canonical chemical differentiation agent. Five days of continuous exposure to calcium-ion cyclotron resonance affect hMSC proliferation, morphology, and cytoskeletal actin reorganization. By quantitative real-time polymerase chain reaction, we also observed an increase of osteoblast differentiation marker expression such as Runx2, alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN) together with the osteoprotegerin mRNA modulation. Moreover, in these cells, the increase of the protein expression of OPN and ALP was also demonstrated. These results demonstrate bone commitment of hMSCs through a noninvasive and biocompatible differentiating physical agent treatment and highlight possible applications in new regenerative medicine protocols.

Exposure to extremely low-frequency magnetic field affects biofilm formation by cystic fibrosis pathogens.

AIMS: To evaluate the in vitro effects of extremely low-frequency magnetic field (ELF-MF) on growth and biofilm formation by Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia strains from cystic fibrosis patients. MATERIALS & METHODS: The motion of selected ions (Fe, Ca, Cu, Zn, Mg, K, Na) was stimulated by the ion resonance effect, then influence on growth and biofilm formation/viability was assessed by spectrophotometry or viability count. RESULTS: Generally, exposure to ELF-MF significantly increased bacterial growth and affected both biofilm formation and viability, although with differences with regard to ions and species considered. CONCLUSION: Exposure to ELF-MF represents a possible new approach for treatment of biofilm-associated cystic fibrosis lung infections.

Non ionising radiation as a non chemical strategy in regenerative medicine: Ca(2+)-ICR "In Vitro" effect on neuronal differentiation and tumorigenicity modulation in NT2 cells.

In regenerative medicine finding a new method for cell differentiation without pharmacological treatment or gene modification and minimal cell manipulation is a challenging goal. In this work we reported a neuronal induced differentiation and consequent reduction of tumorigenicity in NT2 human pluripotent embryonal carcinoma cells exposed to an extremely low frequency electromagnetic field (ELF-EMF), matching the cyclotron frequency corresponding to the charge/mass ratio of calcium ion (Ca(2+)-ICR). These cells, capable of differentiating into post-mitotic neurons following treatment with Retinoic Acid (RA), were placed in a solenoid and exposed for 5 weeks to Ca(2+)-ICR. The solenoid was installed in a µ-metal shielded room to avoid the effect of the geomagnetic field and obtained totally controlled and reproducible conditions. Contrast microscopy analysis reveled, in the NT2 exposed cells, an important change in shape and morphology with the outgrowth of neuritic-like structures together with a lower proliferation rate and metabolic activity alike those found in the RA treated cells. A significant up-regulation of early and late neuronal differentiation markers and a significant down-regulation of the transforming growth factor-α (TGF-α) and the fibroblast growth factor-4 (FGF-4) were also observed in the exposed cells. The decreased protein expression of the transforming gene Cripto-1 and the reduced capability of the exposed NT2 cells to form colonies in soft agar supported these last results. In conclusion, our findings demonstrate that the Ca(2+)-ICR frequency is able to induce differentiation and reduction of tumorigenicity in NT2 exposed cells suggesting a new potential therapeutic use in regenerative medicine.

Nonionizing radiation as a noninvasive strategy in regenerative medicine: the effect of Ca(2+)-ICR on mouse skeletal muscle cell growth and differentiation.

Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

Experimental finding on the electromagnetic information transfer of specific molecular signals mediated through the aqueous system on two human cellular models.

BACKGROUND: Recently the authors reported the experimental evidence of the developing concept of Electro Magnetic Information Transfer (EMIT) of specific molecular signals directly and continuously on target cell picking up the molecular signals from the source chemical effector. This was in agreement with the pioneering work of Jaques Benveniste suggesting that the electronic transmission of the 4-phorbol-12-myristate-13-acetate (PMA) signals could be transferred to target neutrophils by an oscillator when coupled to two electromagnetic coils demonstrating the same biologic activity and so mimicking the biologic function of the original chemical active molecule. The present work is the further development of recent research designed to verify the hypotheses that water could record and replay the EMIT from biologic active chemical molecules. METHODS: Retinoic acid, a well-known chemical differentiating agent, was placed at room temperature in the input coil connected to an oscillator (VEGA select 719), while culture medium for human neuroblastoma cell (LAN-5) and NT2/D1 stem teratocarcinoma human cells was placed into the output coil and exposed to signals for 1 hour. At the end the oscillator was switched off and LAN-5 neuroblastoma and NT2/D1 stem teratocarcinoma cells were seeded, respectively, into the medium conditioned as reported into an incubator under controlled conditions. After 5 days of incubations, cells were examined by different strategies such as morphological and biochemical parameters. RESULTS: It was demonstrated that the electromagnetic signals coming from the retinoic acid molecule could be recorded and stored by the aqueous system of the cell culture medium. Cells seeded in the electronically conditioned medium received physical information generating a statistically significant decrease in metabolic activity and changes in phenotypical structure with protrusion typical of differentiated neuronal cells. CONCLUSIONS: These experimental results provide some evidence that water could be tuned in a resonant manner by the EMIT procedure appropriately carried through a carrier frequency provided by the oscillator in a manner that seems related to the chemical structure of the source molecule as, in this case, retinoic acid.

Differentiation of human LAN-5 neuroblastoma cells induced by extremely low frequency electronically transmitted retinoic acid.

BACKGROUND: Jaques Benveniste suggested that electronic transmission of 4-phorbol-12-b-myristate-13-acetate (PMA) activity, carried out using a simple amplifier configured to function as an oscillator when coupled to two electromagnetic coils, demonstrates the same chemical activity as the active molecule. The results obtained suggested that there are associated signals at the PMA molecules that can be transferred to target neutrophils by artificial physical means in a fashion that mimics the original molecules. METHODS: Retinoic acid was placed at room temperature on one coil attached to an oscillator (VEGA select 719), while LAN-5 neuroblastoma cells were placed on another coil and incubated under controlled condition. The oscillator was then turned on for 12 hours a day for 5 days, after which cells were counted and morphology studied by contrast microscopy. RESULTS: The effect of the differentiating agent added to the cell culture by physical means generates a decrease in cell growth, metabolic activity, and the protrusion of a neuritelike structure typical of the differentiated cells. CONCLUSIONS: These preliminary results suggest that retinoic acid molecules emit signals that can be transferred to LAN-5 neuroblastoma cells by artificial physical means in a manner that seems related to the chemical structure of the source molecules.

Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields.

AIMS: Modulation of cardiac stem cell (CSC) differentiation with minimal manipulation is one of the main goals of clinical applicability of cell therapy for heart failure. CSCs, obtained from human myocardial bioptic specimens and grown as cardiospheres (CSps) and cardiosphere-derived cells (CDCs), can engraft and partially regenerate the infarcted myocardium, as previously described. In this paper we assessed the hypothesis that exposure of CSps and CDCs to extremely low-frequency electromagnetic fields (ELF-EMFs), tuned at Ca2+ ion cyclotron energy resonance (Ca2+-ICR), may drive their differentiation towards a cardiac-specific phenotype. METHODS AND RESULTS: A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca2+-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca2+ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca2+ fluorescent probes. CONCLUSIONS: These results suggest that ELF-EMFs tuned at Ca2+-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.

Calcium ion cyclotron resonance (ICR) transfers information to living systems: effects on human epithelial cell differentiation.

The specific aim of the present work concerns the effectiveness of low-frequency electromagnetic fields treatment to modify biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz electromagnetic field, tuned to calcium ion cyclotron resonance (ICR), showed modifications in the cytoskeleton. These modifications were related to different actin distributions as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression, indicating that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that a 7 Hz calcium ICR electromagnetic field may modify cell biochemistry and interfere in the differentiation and cellular adhesion of normal keratinocytes, suggesting the possibility to use ICR electromagnetic therapy for the treatment of undifferentiated diseases.

Ion cyclotron resonance as a tool in regenerative medicine.

The identification of suitable stem cell cultures and differentiating conditions that are free of xenogenic growth supplements is an important step in finding the clinical applicability of cell therapy in two important fields of human medicine: heart failure and bone remodeling, growth and repair. We recently demonstrated the possibility of obtaining cardiac stem cells (CSCs) from human endomyocardial biopsy specimens. CSCs self-assemble into multi-cellular clusters known as cardiospheres (CSps) that engraft and partially regenerate infarcted myocardium. CSps and cardiosphere-derived-cells (CDCs) were exposed for five days in an incubator regulated for temperature, humidity, and CO(2) inside a solenoid system. This system was placed in a magnetically shielded room. The cells were exposed simultaneously to a static magnetic field (MF) and a parallel low-alternating frequency MF, close to the cyclotron frequency corresponding to the charge/mass ratio of the Ca(++) ion. In this exposure condition, CSps and CDCs modulate their differentiation turning on cardiogenesis and turning off vasculogenesis. Cardiac markers such as troponin I (TnI) and myosin heavy chain (MHC) were up-regulated. Conversely, angiogenic markers such as vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) were down-regulated as evidenced by immunocytochemistry. Exposure to the 7 Hz calcium ion cyclotron resonance (ICR) frequency can modulate the cardiogenic vs. angiogenic differentiation process of ex vivo expanded CSCs. This may pave the way for novel approaches in tissue engineering and cell therapy. With regard to bone remodeling, it has been suggested that bone marrow-derived mesenchymal stem cells (MSC) may be considered as a potential therapeutic tool. Using the Ca(++)-dependent specific differentiation potential of the ELF-MF 7 Hz ICR, we show here that exposure of human MSC to these same MF conditions enhanced the expression of osteoblast differentiation markers such as alkaline phosphatase, osteocalcin, and osteopontin, as analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the differentiation marker factors were up regulated, the ICR electromagnetic field down regulated osteoprotegerin gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice.

Action of combined magnetic fields on aqueous solution of glutamic acid: the further development of investigations.

In the present work the results of the known investigation of the influence of combined static (40 microT) and alternating (amplitude of 40 nT) parallel magnetic fields on the current through the aqueous solution of glutamic acid, were successfully replicated. Fourteen experiments were carried out by the application of the combined magnetic fields to the solution placed into a Plexiglas reaction vessel at application of static voltage to golden electrodes placed into the solution. Six experiments were carried out by the application of the combined magnetic fields to the solution placed in a Plexiglas reaction vessel, without electrodes, within an electric field, generated by means of a capacitor at the voltage of 27 mV. The frequency of the alternating field was scanned within the bounds of 1.0 Hz including the cyclotron frequency corresponding to a glutamic acid ion and to the applied static magnetic field. In this study the prominent peaks with half-width of approximately 0.5 Hz and with different heights (till 80 nA) were registered at the alternating magnetic field frequency equal to the cyclotron frequency (4.2 Hz). The general reproducibility of the investigated effects was 70% among the all solutions studied by us and they arose usually after 40-60 min. after preparation of the solution. In some made-up solutions the appearance of instability in the registered current was noted in 30-45 min after the solution preparation. This instability endured for 20-40 min. At the end of such instability period the effects of combined fields action appeared practically every time. The possible mechanisms of revealed effects were discussed on the basis of modern quantum electrodynamics.

Extremely low frequency 7 Hz 100 microT electromagnetic radiation promotes differentiation in the human epithelial cell line HaCaT.

Electromagnetic therapy is a treatment method in which an electromagnetic or magnetic stimulus is used to achieve physiological changes in the body. The specific aim of the present work concerns the effectiveness of low frequency electromagnetic fields to modify the biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz 100 microT electromagnetic field for one hour (twice daily), indicated modification in shape and morphology. These modifications were also associated with different actin distribution as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-Catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-Catenin expression, supporting the conclusion that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that 7 Hz electromagnetic field, may modify cell biochemistry interfering in the differentiation and cellular adhesion of normal keratinocytes.

Extremely low frequency electromagnetic field exposure promotes differentiation of pituitary corticotrope-derived AtT20 D16V cells.

The pituitary corticotrope-derived AtT20 D16V cell line responds to nerve growth factor (NGF) by extending neurite-like processes and differentiating into neurosecretory-like cells. The aim of this work is the study of the effect of extremely low frequency electromagnetic fields (ELF-EMF) at a frequency of 50 Hz on these differentiation activities. To establish whether exposure to the field could influence the molecular biology of the cells, they were exposed to a magnetic flux density of 2 milli-Tesla (mT). Intracellular calcium ([Ca2+]i) and intracellular pH (pHi) were monitored in single exposed AtT20 D16V cells using fluorophores Indo-1 and SNARF for [Ca2+]i and pHi, respectively. Single-cell fluorescence microscopy showed a statistically significant increase in [Ca2+]i followed by a drop in pHi in exposed cells. Both scanning electron microscopy (SEM) and transmission microscopy of exposed AtT20 D16V cells show morphological changes in plasma membrane compared to non-exposed cells; this modification was accompanied by a rearrangement in actin filament distribution and the emergence of properties typical of peptidergic neuronal cells-the appearance of secretory-like granules in the cytosol and the increase of synaptophysin in synaptic vesicles, changes typical of neurosecretory-like cells. Using a monoclonal antibody toward the neurofilament protein NF-200 gave additional evidence that exposed cells were in an early stage of differentiation compared to control. Pre-treatment with 0.3 microM nifedipine, which specifically blocks L-type Ca2+ channels, prevented NF-200 expression in AtT20 D16V exposed cells. The above findings demonstrate that exposure to 50 Hz ELF-EMF is responsible for the premature differentiation in AtT20 D 16 V cells.

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK).

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.