download report - Istituto Pasteur

A. Giacomello - Biology and physiology of adult cardiac stem/progenitor cells
5 months the reconstituted mice with transgenic BM
were subjected to surgical LAD ligation. After 3
weeks hearts were excised and plated as primary
explants, and CSps were isolated. CSps from transplanted,
infarcted mice were GFP + , indicating that
GFP + BM-derived cells had been mobilized and had
migrated to the infarcted heart. Much less CSps were
obtained from transplanted, non infarcted mice, and
they were GFP - . To assess the biology and commitment
of kit/GFP + CSps, 1x10 5 CSp-derived cells
(CDCs) were acutely injected into the infarct border
zone of wild-type mice. Three weeks later GFP +
cells had survived and engrafted in the infarct area,
and most of them had differentiated into cardiomyocytes
and vessels. Our data show that, under local
CSC depletion and tissue damage, BM cells can
migrate to the heart and apparently adopt the phenotype
and functional characteristics of the endogenous
CSC pool.
The “paracrine hypothesis” for CSps and CDCs
Many recent pre-clinical and clinical studies, observing
beneficial effects after cardiac cell therapy without
direct massive cardiac regeneration, brought
along the hypothesis that other mechanisms could be
involved. Transplanted cells might produce beneficial
humoral factors, promoting endogenous cardiomyocytes
survival and angiogenesis. Therefore
we investigated the paracrine abilities of CSps and
CDCs. Both stages are able of secreting in vitro
VEGF and HGF in significant amounts, compared to
a control cell line of dermal fibroblasts. CSps also
secrete IGF1, that is not detectable anymore after
this stage, despite many different culture conditions
tested. However both CSps and CDCs express the
corresponding mRNA for VEGF, HGF and IGF1,
and also their receptors. Since so far an in vivo functional
improvement has been demonstrated only for
CDC injection, we assessed whether CDC-conditioned
media (CM) has paracrine effects in vitro in
ischemic-like conditions, that is serum and glucose
54
starvation. CDC-CM was able to significantly reduce
the percentage of apoptotic neonatal rat ventricular
myocytes after 72 hours of hypoxic culture, compared
to fibroblast-CM and control basal media.
Furthermore CDC-CM was able to recover
HUVECs ability to form complex tube networks in
an in vitro angiogenesis assay; this ability was lost in
the basal media and in the fibroblast-CM. The preincubation
with neutralizing antibodies for VEGF
and/or HGF partially but significantly reduced both
the anti-apoptotic and the pro-angiogenic effects,
suggesting that at least in part these beneficial effects
depend on the secreted growth factors. CDCs are
also able to secrete VEGF, HGF and IGF1 in vivo, as
detected by RT-PCR and WB in the same SCID
infarction model where they have been previously
demonstrated to mediate improvement in LV function.
Thus, CSps and CDCs are able to secrete significant
amounts of pro-survival and pro-angiogenic
growth factors. This could be a key mechanism,
together with their spontaneous commitment to the
cardiac lineage, contributing to the beneficial effects
observed in cardiac cell therapy settings.
Selected Publications
Barile L, Chimenti I, Gaetani R, Forte E, Miraldi F,
Frati G, Messina E, Giacomello A. Cardiac stem
cells: isolation, expansion and experimental use for
myocardial regeneration. Nat Clin Pract Cardiovasc
Med. 2007, 4 Suppl 1:S9-S14.
Barile L, Messina E, Giacomello A, Marbán E.
Endogenous cardiac stem cells. Prog Cardiovasc Dis.
2007, 50:31-48.
Smith RR, Barile L, Cho HC, Leppo MK, Hare JM,
Messina E, Giacomello A, Abraham MR, Marbán E.
Regenerative potential of cardiosphere-derived cells
expanded from percutaneous endomyocardial biopsy
specimens. Circulation 2007, 115:896-908.