ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera The ICEMS group in BENEVENTO

Antonella Lisi, Settimio Grimaldi, Livio Giuliani, Enrico DEmilia and Alessandro Giacomello13, ELF induced stem cell differentation ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

4.17Hz 48uT Bo 48nT B Glutaminic acid in pure water Experimental parameters kT zone no kT zone ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

The effect of energy resonance EMF at Calcium ion cyclotron frequency on human cardiac stem cells ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

Regenerative Medicine with Stem Cells Pluripotent stem cells Stem cells are defined as undifferentiated cells capable of self-renewal or propagation as well the capacity to differentiate into specialized cell types under appropriate conditions. Recent studies have also revealed the presence of cardiac stem cells in the heart and some limited regenerative capacity of the mammalian heart during time of stress. Cell transplantation has emerged as a promising new approach to augment the limited regenerative capacity of the injured human heart. ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

Human biopsy specimens were obtained from patients undergoing clinically-indicated percutaneous endomyocardial biopsy and processed. Samples were cut into fragments, washed, partially enzymatically digested, and the single cells discarded. The remaining tissue fragments were cultured as explants on dishes. A lawn of flat cells spreads from the biopsy and covers the bottom of the dish in days. CSps and CDCs CSps were exposed for five days in the a- magnetic room with simultaneous presence of a static Magnetic Field and a ELF-MF, close to the cyclotron frequency corresponding to the charge/mass ratio of Ca++ ion. CSps and CDCs CSps were exposed for five days in the a- magnetic room with simultaneous presence of a static Magnetic Field and a ELF-MF, close to the cyclotron frequency corresponding to the charge/mass ratio of Ca++ ion. (4Hz, 4µTBo, 40nT B) The equipment for electro-magnetic field (solenoid) production is installed in a-magnetic room. This equipment include cellular incubator made to a-magnetic material were temperature regulation (37± 0.1°C) and atmosphere (5% CO2) and humidity were provided and continuously controlled and recorded by a lab view program embedded computer. The main body of the solenoid is a cylinder in PVC 5 mm thick and has a diameter of 33 cm and a height of 3 m. It is made of 3,300 turns of 1 mm diameter copper wire. It is driven from three amplifiers and a signal generator that generated static and alternate current for electromagnetic field production. This equipment is able to produce 0.01Hz to 1 KHz frequency and 10 nT to 1mT of electromagnetic field and induction respectively at 33 mV RMS drove. The detail of instrument was described in patent N° MI 2005A ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

5) Cardiosphere- derived cells 1) Biopsy 2) Explants4) Cardiospheres3) Cardiosphere forming cells 6) Exposure to ELMF 100 mm Explant day mm CSps (4 days after harvest) 200 mm Biopsy fragment day 1 ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera The detail of instrument was described in patent N° MI 2005A000693

Metabolic activity curves by WST-8 analisys Exposed Unexposed WST-8 (tetrazolium salt) is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The figure shows that cells exposed to ELF-MF () have higher metabolic activity then the unexposed cells ( ). The phenomenon is observable both in polylisine and fibronectin growth cells

ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

The effect of ELF steady state exposure at calcium ion cyclotron energy resonance on the short time scale intracellular calcium varations in human cardiac stem cells Graphic analisys Time A.U ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

The effect of ELF exposure at calcium ion cyclotron energy resonance on human cardiac stem cells differentiation factors: 1-Vascular endothelial growth factor (VEGF) 2- Kinase domain receptor (KDR) 3- Troponin I (cTn1) 4-Nkx MHC Cardiac Miosyn heavy chain protein VEGF is the main regulator of blood vessel growth. VEGF is a small, secreted glycoprotein whose production is increased by tissue hypoxia, and which stimulates the growth and invasion of blood vessels into ischemic tissues. VEGF and its major receptor, VEGFR-2 (also known as FlK-1 0r or KDR), have been the focus of efforts to develop pro- and anti- angiogenic therapies Cardiac Troponin I (cTnI) is a protein part of the cardiac muscle structure and is intimately involved in cardiac contractil process Nkx2.5 is a gene controlling muscle hearth growth MHC protein part of the cardiac muscle structure ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera BIOCHEMICAL EFFECT OF Ca 2+ Ion Energy Resonance exposure on CPS

Troponin INkx2.5VEGEF Nkx Unexposed Exposed VEGFcTnINkxVEGFcTnI Increase of mRNA Real Time PCR on cTN I NKx VEGF Fibronectin Polylisine ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera

cTn1 KDR Western Blot cTnI Fibronectin Polylisine Unexp. Exp. Unexp. ICEMS Venice, Dec. 17th VEGA 1 – Palazzo Lybra- Porto Marghera MHC Indirect immunofluorescence The increase in mRNA, demonstrated by RT-PCR was paralleled by an increase in protein expression.

The exposure of CSps to strictly controlled ELF-MF exposure at Calcium Cyclotron Energy Resonance modulates intracellular Calcium transport and cardiac differentiation: Cardiac markers such as VEGF, Troponin I (TnI) or Myosin Heavy Chain (MHC) were up- regulated. We have obtained data showing that exposure of the ex vivo expanded Cardiospheres to ELF-MF modulates their differentiation turning on cardiogenesis and vasculogenesis. Induction of new blood vessels is clinically relevant following stroke or heart attack. These results should increase the reliability and the clinical feasibility of the use of electromagnetic field as cells and stem cells differentiation factor, and in particular in Cardiac Stem Cells employment for cardiac cell therapy.

Regulation of osteoblast differentiation is an important phenomena that must occur to maintain the continuous supply of mature osteoblast, that are needed for bone growth, repair and remodelling; in particular it could have a significant impact on future clinical strategies. The ability of human mesenchymal stem cells (hMSC) to differentiate into osteoblasts was examined through the use of extremely low frequency electromagnetic field (ELF-MF 50Hz 1mT). This study shows that exposure of human MSC to ELF-MF enhanced expression of osteoblast marker differentiation such as Alkaline phosphatase, (AP), Osteocalcin (OCL), and osteopontin (OPN), analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the markers differentiation factors where up regulated, electromagnetic field down regulate osteoprotegerin (OPG) gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice. The exposure of hMSC for 5 days to the field resulted in a change in shape and in plasma membrane morphology and this modification were also accompanied by a rearrangement in actin filaments, as showed by confocal miscroscopy analysis after cells labelling with FITC-phalloidin.

Actin Confocal microscopy assay Control and exposed cells were cultured for 5 days and then fixed in 4% paraformaldehyde. Actin was labeled with FITC-phalloidin using the procedure of Bellomo. The fluorescence was then monitored using a LEICA TCS 4D Confocal Microscope supplemented with an Argon Krypton laser and equipped with 40x 1.00 and 100x 0.6 oil immersion lenses. Mesenchimal cells exposed to the field showed the same actin organization found in cells after treatment with dexamethasone, start to appear a spreading network of actin when in the control cells is present at the periphery, only around the cellular membrane. The differentiating effect of dexamethasone on were potentiate by exposure to the field

Scanning Electron Microscopy (SEM) assay Control and 5 days mesenchymal cells ELF-EMF exposed were washed in phosphate buffer saline (PBS) and fixed with 2.5% glutaraldhyde in 0.1M Millonig's phosphate buffer for 1 hr at 4°C. After three washes in the same buffer, samples were post-fixed in 1% OsO4, dehydrated through a graded acetone series, and critical-point dried with CO2 in a Balzers CPD 030 critical-point drier. Specimens were coated with gold in a Balzers SCD 050 sputter instrument and observed on a Cambridge S240 scanning electron microscope. ELF-MF induce changes in cellular morphology like treatment with dexamethasone. Exposed cells appeared larger and more polygonal and this effect increase in mesenchymal cells treated with dexamethasone and then exposed to ELF- MF

Cell proliferation assay The rates of the cell growth were determined by an immunoistochemical method using an anti-5-bromo-2 -deoxyuridine (BrdU) antibody (Cell Proliferation Kit; Roche Diagnostic GmbH, Penzberg, Germany). The cells were seeded at 3 x 103 cells/cm2 in a 96-well plastic plate and cultured for 4 days in presence or without exposure to magnetic field. Control and exposed cells were harvested and the number of cells per well was counted every 24 h. BrdU (10 µM) was added to cells to each well for the last 2 h in culture. After the cells were fixed for 30 minutes, a solution containing anti-BrdU antibody was added to each well and removed after 30 minutes at 37°C. One hundred micro liters of 2, 2-Azino-di-[3-ethylbenzthiazoline sulfonate] (ATBS) then was added, and incubated for 30 minutes. The absorbencies of the samples were measured in an ELISA reader at 405 nm. The control culture was kept in the same bore for the same exposure time and conditions without ELF-MF exposure. Mesenchymal cells exposed to ELF-MF alone or in synergy by treatmement with dexamethasone showed a decrease to viability at 3, 4 and 5 division respect to control and dexamethasone treatment respectively.

AB

Low-frequency electromagnetic fields promote the expression of differentiation markers in pluripotent human mesenchimal stem cells (hMSC).

CONCLUSION These results demonstrated that exposure to magnetic field can act as a differentiating agent on mesenchymal human cells suggesting a possible use of ELF-MF as support in regenerative medicine.