Final Activity Report Summary - NEURONAL REPLACEMENT (Neuronal Replacement in the Injured Brain)
Neurological disorders and brain injury cause the loss of neurons and glial cells. Self-repair mechanisms are initiated in the injured brain to promote recovery. The production of new neurons to replace those lost during the injury is among these repair mechanisms. However, the efficiency of neuronal replacement from endogenous sources is extremely limited. Although transplantation of stem cells could be seen as a valuable strategy to complement endogenous neurogenesis, the multipotent characteristics of stem cells could lead to the generation of teratoma, and eventually death. To avoid that problem, one solution could be to use stem cells with limited proliferation capability, yet sufficient differentiation potential, to be tailored to therapeutic needs. The current project proposed to examine whether neuronal precursor cells could serve as sources for neuronal replacement after injury.
The first achievement of our project was to generate molecular tools to isolate neuronal precursors. The promoter sequence of doublecortin, i.e. a gene expressed in neuronal precursors, was cloned and used to control reporter genes, such as the gene encoding for the green fluorescence protein or luciferase gene. Using these reporting systems, we demonstrated the selectivity of the activity of the cloned promoter sequence to neuronal precursors. These cloned promoters served to generate expression vectors to follow cell differentiation processes of mouse embryonic stem cells (ESC) in vitro. They also allowed for screening of transcription factors whose activity was present at this early stage of neuronal differentiation.
We characterised two protocols to promote in vitro ESC differentiation into neuronal precursors. We showed that, while neuronal precursors could represent the main proportion of the ESC population after several days of differentiation, no fully mature neurons could be obtained using these in vitro protocols. However, undifferentiated ESC were not detected, neither was suggested that all cultured cells were engaged in some defined lineage, mainly neural.
Finally, we described the effects of transplantation of neuronal precursors in the injured brain. Pre-differentiated ESC were transplanted into a mouse model of cerebral ischemia and their therapeutic benefit was tested. We observed that transplantation of ESC, pre-differentiated for 8 days in vitro, did not ameliorate functional recovery of ischemic mice. Further analyses were still ongoing, by the time of the project completion, to examine the therapeutic potential of ESC at various stages of differentiation to treat ischemic mice.
This proposal explored new concepts regarding the biology of neuronal precursors and designed new tools to select ESC subpopulations. It finally opened new strategies to neuronal replacement and brain repair following injury.
The first achievement of our project was to generate molecular tools to isolate neuronal precursors. The promoter sequence of doublecortin, i.e. a gene expressed in neuronal precursors, was cloned and used to control reporter genes, such as the gene encoding for the green fluorescence protein or luciferase gene. Using these reporting systems, we demonstrated the selectivity of the activity of the cloned promoter sequence to neuronal precursors. These cloned promoters served to generate expression vectors to follow cell differentiation processes of mouse embryonic stem cells (ESC) in vitro. They also allowed for screening of transcription factors whose activity was present at this early stage of neuronal differentiation.
We characterised two protocols to promote in vitro ESC differentiation into neuronal precursors. We showed that, while neuronal precursors could represent the main proportion of the ESC population after several days of differentiation, no fully mature neurons could be obtained using these in vitro protocols. However, undifferentiated ESC were not detected, neither was suggested that all cultured cells were engaged in some defined lineage, mainly neural.
Finally, we described the effects of transplantation of neuronal precursors in the injured brain. Pre-differentiated ESC were transplanted into a mouse model of cerebral ischemia and their therapeutic benefit was tested. We observed that transplantation of ESC, pre-differentiated for 8 days in vitro, did not ameliorate functional recovery of ischemic mice. Further analyses were still ongoing, by the time of the project completion, to examine the therapeutic potential of ESC at various stages of differentiation to treat ischemic mice.
This proposal explored new concepts regarding the biology of neuronal precursors and designed new tools to select ESC subpopulations. It finally opened new strategies to neuronal replacement and brain repair following injury.