STEM CELLS
Characterization of the integration, diffusion, survival, differentiation and clinical
outcomes of adult neural stem cells transplanted in the wobbler mouse, a murine
model of motoneuron disease.
Paolo Bigini1, Arianna Ottolina3, Jeff W. M. Bulte2, Luca Saraceno1, Sara Barbera1,
Chiara Rigon1, Linda Chabane4, Vito Lorusso4, Eugenio Parati3 and Tiziana
Mennini1.
1Istituto di Ricerche Farmacologiche “MarioNegri”, Via Eritrea 62 20157 Milano, Italy;
2Johns Hopkins University, School of Medicine, Baltimore, USA; 3Istituto Nazionale
Neurologico”Carlo Besta”, Milano, Italy; 4Centro Ricerche Bracco,Milano, Italy.
Tel:+390239014401; Fax and email:+39023546277 bigini@marionegri.it
Keywords: Amyotrophic Lateral Sclerosis (ALS); Neural Stem Cells (NSCs); wobbler
mice; Magnetic Resonance Imaging (MRI)
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder
selectively affecting upper and lower motoneurons and leading to muscle atrophy,
paralysis and death. The lack of pharmacological strategies for ALS have prompted
scientific community to explore new therapeutic approaches. Stem cells therapy may be a
reliable alternative for the care of different neurological disorders. However, their
interaction with the host tissues is far from defined.
For this reason animal models for human diseases can improve the knowledge about the
homing of neural stem cells and their actual effectiveness for the clinical use.
In our study we investigated the fate of adult murine undifferentiated neural stem cells
(NSCs) transplanted in the brain of early symptomatic wobbler mice. Before
administration NSCs (P8), collected from the sub ventricular zone (SVZ) of 4-week-old
healthy mice, were incubated both with the fluorescent dye Hoechst 33258 and with super
paramagnetic iron oxide particles (Feridex, Berlex Imaging, Wayne, NJ, USA. No
difference in term of cell growth, differentiation and survival were found by comparing
these cells to untreated NSCs.
Wobbler mice received 5 microliter of PBS containing 500,000 NSCs, by bilateral
administration in lateral ventricles, or the same volume of PBS alone. The labeled NSCs
are clearly detected by MR Imaging in the lateral ventricles, in the 3rd and also in the 4th
cerebral ventricle, 24h, 48h and 2 weeks post transplantation.
From 7 to 11 weeks after NSCs administration mice were intracardially perfused, spinal
cords and brains were collected for neuropathological estimations and for detection of
33258 Hoechst positive cells
A high number of 33258 Hoechst positive nuclei were found in the lateral ventricles and
the 3rd ventricle of mice after 7-9 weeks from transplantation, mainly disposed as clusters
or distributed along the surface from ventricles and parenchyma and packed in the
ependymal layer. No 33258 Hoechst positive cells were found in the brain of mice at later
times. Further experiments are ongoing to detect the presence of iron oxide particles, to
determine whether the lack of fluorescent nuclei can be due to the decay of dye or to the
actual loss of cells.
Immunohystochemical experiments revealed no colocalization between 33258 Hoechst
positive nuclei and early markers of neural differentiation (GFAP, nestin and beta tubuline
3), but few 33258 Hoechst cells migrated in the region close to the 3rd ventricle were
positive for a late marker of neuronal differentiation, the NeuN.
Although no 33258 Hoechst positive cells were found in the spinal cord of treated mice,
the NSCs transplantation produced a significant reduction in motoneurons loss compared
to untreated wobbler mice.
In conclusion our study indicates that adult NSC transplanted in the lateral ventricles
diffuse in the ventricular system, survive for at least 7-9 weeks and produce a slight,
although significant, neuropathological improvement in wobbler mice