ABSTRACT
Title
Adult Mouse Post Mortem Neural Precursors regenerate neuronal tissue and promote functional recovery after transplantation in a spinal cord injury model
Authors
A. Raspa1
Dottorato di Ricerca in Fisiopatologia, Farmacologia e Malattie Metaboliche
1 Laboratories of Pharmacology, Dept. Medicine, Surgery and Dentistry, University of Milan; Milan Italy.
Dottorato di Ricerca in Fisiopatologia, Farmacologia e Malattie Metaboliche
1 Laboratories of Pharmacology, Dept. Medicine, Surgery and Dentistry, University of Milan; Milan Italy.
Abstract
Neural stem cells from the subventricular zone of the forebrain, because of their proliferation and differentiation characteristics, are a good tool for tissue replacement therapies. We recently isolated neural progenitors, capable of surviving a powerful ischemia insult: these cells were named Post Mortem Neural Precursor Cells (PM-NPCs). Differentiation yield mostly neurons (about 30-40%) compared to regular NPCs.PM-NPCs are particularly sensitive to mTOR activity. The average dose of rapamycin normally used to inhibit mTOR is 5 ng/mL but we observed that the effect was significantly evident with a dose 100 fold lower. The higher ERK activation observed in undifferentiated PM-NPCs is also involved in their differentiation process, since the exposure to ERK inhibitor PD98059, downregulates significantly the extent of neuronal differentiation. PM-NPCs but not classical NPCs synthesize EPO that it is known to be active as a signalling molecule promoting stem cell-derived neurogenesis and neuronal differentiation. Blocking the EPO pathway by means of monoclonal antibodies anti-EPO or anti-EPOR markedly inhibits the differentiation of PM-NPCs towards the neuronal phenotype. Differently the exposure of regular NPCs to exogenous EPO increases their differentiation ability close to level of PM-NPCs.
The potential of PM-PCs in terms of replacement therapy was investigated in a mouse model of spinal cord injury. PM-PCs kept from animal 6 hours after death (T6), were administered intravenously within 2 hours after the traumatic injury of the cord. The improvement of animal functional recovery and the transplanted cells fate were studied. 30 days after transplantation animals treated with T6 PM-NPCs show a remarkable improvement of the rate of hind limb function evaluated by Basso Mouse Scale compared with animals treated with placebo. PM-NPCs migrate predominantly at the injury site and differentiate predominantly into neurons, reconstitute a rich axonal and dendritic network and promote a marked axonal regeneration across the injury site of monoaminergic fibers. Moreover the molecular analysis of the lesion site show that PM-NPCs induce a remodulation of inflammatory response and release of neurotrophic factors. Pro-inflammatory cytokines levels significantly decrease after 48 hours from spinal cord injury and PM-NPCs transplantation, while after 7 days we observe a small increase of IL-6 and TNF alpha. In conclusion, we purified a new class of neural precursors able to survive after a powerful ischemic insult (PM-NPCs). Their neuronal differentiation requires the activity of mTOR and MAPK and is prevented by exposure to anti-EPO and anti-EPOR antibodies. Moreover, these cells represent a liable source for cellular therapy in neurodegenerative disorders, specially on spinal cord injury.
To determinate the amount of proliferative cells in T6 SVZ we used BrdU administration (i.p., 50mg/kg body weight), two or twelve hours before animal sacrifice, and subsequently the studies of the regional localization of neurogenesis have been made by immunohistochemical detection together with stem cells’s and precursor cells’s markers.
The potential of PM-PCs in terms of replacement therapy was investigated in a mouse model of spinal cord injury. PM-PCs kept from animal 6 hours after death (T6), were administered intravenously within 2 hours after the traumatic injury of the cord. The improvement of animal functional recovery and the transplanted cells fate were studied. 30 days after transplantation animals treated with T6 PM-NPCs show a remarkable improvement of the rate of hind limb function evaluated by Basso Mouse Scale compared with animals treated with placebo. PM-NPCs migrate predominantly at the injury site and differentiate predominantly into neurons, reconstitute a rich axonal and dendritic network and promote a marked axonal regeneration across the injury site of monoaminergic fibers. Moreover the molecular analysis of the lesion site show that PM-NPCs induce a remodulation of inflammatory response and release of neurotrophic factors. Pro-inflammatory cytokines levels significantly decrease after 48 hours from spinal cord injury and PM-NPCs transplantation, while after 7 days we observe a small increase of IL-6 and TNF alpha. In conclusion, we purified a new class of neural precursors able to survive after a powerful ischemic insult (PM-NPCs). Their neuronal differentiation requires the activity of mTOR and MAPK and is prevented by exposure to anti-EPO and anti-EPOR antibodies. Moreover, these cells represent a liable source for cellular therapy in neurodegenerative disorders, specially on spinal cord injury.
To determinate the amount of proliferative cells in T6 SVZ we used BrdU administration (i.p., 50mg/kg body weight), two or twelve hours before animal sacrifice, and subsequently the studies of the regional localization of neurogenesis have been made by immunohistochemical detection together with stem cells’s and precursor cells’s markers.