download report - Istituto Pasteur

P a r t i c i p a n t s :
Fabio Miraldi, Giacomo Frati, professors; Elisa Messina,
medical manager; Lucio Barile, post-doc fellow; Roberto
Gaetani, Isotta Chimenti, Elvira Forte, PhD students;
Vittoria Ionta, Francesco Angelini, Silvia Turi, undergraduate
students.
C o l l a b o r a t i o n s :
Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma (Dr.
Settimio Grimaldi); Istituto di Tecnologie Biomediche, CNR, Pisa
(Dr. Cristina Magli); Dipartimento di Biotecnologie e Bioscienze,
Università di Milano-Bicocca (Prof. Sergio Ottolenghi); School of
Medicine, Division of Cardiology, Johns Hopkins University,
Baltimore, USA (Prof. Eduardo Marbán).
Report of activity
Aim
The project aims at characterizing the cell biology
and physiology of cardiac stem cells (CSCs) and cardiospheres
(CSps), with particular focus on optimizing
their utility for cardiac cell therapy clinical translation.
The aims are grouped into three categories, as
follows: 1)to improve and optimize cell culture methods
and conditions for CSCs, that is to develop culture
conditions that favor CSC expansion and differentiation
towards the cardiac lineage in vitro; 2) to
verify the efficacy of CSCs in infarct repair in vivo
using animal models; 3) to assess the cell biology of
CSCs and CSps, that is: a) to determine the origin of
human CSCs under physiologic and pathologic conditions;
b) to characterize the physiological properties
of CSCs at various stages of differentiation, in
terms of commitment and potency.
Effects of electromagnetic fields exposure
of CSCs
One of the major limitations to cardiac cell therapy
applicability is the low specific cardiomyogenic
Principal investigator: Alessandro Giacomello
Professor of Pathology
Dipartimento di Medicina Sperimentale
Tel/Fax: (+39) 06 4461481
alessandro.giacomello@uniroma1.it
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Molecular genetics of eukaryotes - AREA 3
Biology and physiology of adult cardiac stem/progenitor cells
with a view to optimizing their utility for autologous
cell transplantation
potential of the candidate cells. Electromagnetic
fields (EMFs) are known to interfere with many
cellular functions, such as proliferations and differentiation,
most likely by altering membrane currents,
in particular through Ca 2+ channels.
Recently, extremely low frequency (ELF) EMFs
have been shown to drive cardiac specific differentiation
in embryonic stem cells. Therefore, we
exposed up to 5 days CSps and CDCs to ELF-
EMFs, tuned at the resonance frequency of the
Ca 2+ ion, inside an a-magnetic room, in order to
guarantee full reproducibility. Exposed CSCs displayed
increased metabolic activity and higher proliferation
rates, compared to unexposed controls.
Transcriptional and translational analysis after 5
days of ELF-EMF exposure revealed increased
expression of cardiac markers, such as Nkx2.5,
TnI, MHC and Cx43, while markers of vascular
lineage, like KDR and SMA, were down-regulated.
Exposure to a control frequency, far from the resonance
of biologically relevant ions, did not elicit
any detectable effect. Chronically exposed cells displayed
marked intracellular Ca 2+ accumulation, as
assessed by Orange-Green fluorescence intensity.
Furthermore compartmentalized analysis of Rhod-
2 fluorescence allowed the detection of Ca 2+ fluxes
among intracellular storage compartments in
exposed CDCs, while no effect was detectable in
unexposed cells or in cells exposed to the control
frequency. In conclusion, the modulation of cell
proliferation and specific cardiac differentiation
elicited by our system through ELF-EMFs could
represent an effective and safe biotechnological tool
to improve their cardiac regenerative potential.
The origin of CSCs
In order to investigate whether bone marrow (BM)derived
cells can contribute to the endogenous c-kit +
CSC pool, we transplanted BM cells from transgenic
mice, expressing GFP under the c-kit promoter, in
wild-type lethally-irradiated syngeneic mice. After 4-