Diapositiva 1

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The ICEMS group in BENEVENTO
ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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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
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ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
Glutaminic acid in pure water
kT zone
no kT zone
4.17Hz 48uT Bo 48nT B
Experimental parameters
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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
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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
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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 14-20 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. (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 0.1C) 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 2005A000693.
ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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The detail of instrument was described in patent
N MI 2005A000693
ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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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

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ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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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
A.U
Time
ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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BIOCHEMICAL EFFECT OF Ca 2 Ion Energy Resonance
exposure on CPS
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-Nkx2.5 5-
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
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ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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The increase in mRNA, demonstrated by RT-PCR was
paralleled by an increase in protein expression.

Indirect immunofluorescence
cTn1
KDR
MHC
ICEMS Venice, Dec. 17th VEGA 1 Palazzo Lybra-
Porto Marghera
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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.
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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.
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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
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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 4C. 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
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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 37C. 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.
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Low-frequency electromagnetic fields promote the
expression of differentiation markers in
pluripotent human mesenchimal stem cells (hMSC).
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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.
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THANK FOR YOUR KINDNESS