Investigations Involved GFP+ Naive Mouse Embryonic Stem Cell Transplantation In The Rat Brain

Objective: (1) Totipotent and regionally non-specified ES (embryonic stem, ES) cells provide a powerful tool to understand mechanisms controlling stem cell differentiation in different regions of the adult brain. As the capacity of naive primary ES cells to develop in the adult brain are yet largely unknown, we grafted small amounts of undifferentiated mouse ES (mES) cells in the adult rat brains to explore the survival and differentiation of naive mES cells in different rat brain regions.(2) An alteration of the local environment could also be expected during pathological conditions of the brain. And the specific environment of the cerebral hemorrhage brain would influence the ES cells'behaviour. It remains yet to be determined the influence of the recipient intracerebral hemorrhage rat brain on the commitment of undifferentiated ES cells. We grafted undifferentiated mouse ES cells in the adult intracerebral hemorrhage rat brain to explore the survival and differentiation of mES cells in it.(3)At the same time some problems to be faced with in transplantation exploratory development of ES cells and other stem cells would be exposed to us. These made stage for further investigations in stem cell graft researches.Methods: After marked with green fluorescent protein (GFP), the mouse embryonic stem cells were screened by G418 for 1 month to make them express GFP stably.(1)Then we transplanted the GFP positive mES cells into the hippocampus,septal area,cortex and caudate nucleus in rat brains respectively. Then the rats sacrificed 5 days,14days,and 28 days later. We identified the phenotype of the cells by using nestin,GFAP(glial fibrillary acidic protein) and NeuN(neuronal nuclei protein) antibodies. The mouse embryonic stem cells'living and differentiation were observed by fluorescence stereomicroscopy.(2)The rats intracerebral hemorrhage models were made and the GFP positive mouse embryonic stem cells were transplanted into the opposite lateral ventricle 3 days later. The rats sacrificed 1 month later. The mouse embryonic stem cells living,and differentiation were observed by fluorescence stereomicroscopyResults: (1) We discovered that ES cells undergo gradual migration in recipient rat brain tissues. The survival rate of transplantation cells and their progeny is the highest in hippocampus and the lowest in septal area in all regions (p<0.01). The transplantation cell numbers may also influence the graft survival status. When implanting high cell numbers the grafts exhibited complex tissue types. The grafts are poorly conform to the host and destructed the tissue severely. And we could conclude transplants of low cell numbers led to poor survivorship because of immune reactions. The volume ratio between grafts and recipient organs could also contribute to the growth of the grafts which we couldn't neglect when doing transplantation. We have to use different transplantation protocols to treat various diseases correlated to different sites. The grafted ES cells could differentiate into nestin-ir(immune reactive)neural stem cells. And the efficiency of mES cells differentiation into neural stem cells is also the highest in hippocampus and the lowest in septal area in all regions (p<0.01). mES cells differentiated into end cells such as neurons and glial cells at every transplantation sites in recipient brains. Especially at hippocampus the ES cells differentiated into neurons in large amounts.(2) In the rats intracerebral hemorrhage models the naive mouse embryonic stem cells transplanted into the rat lateral cerebral ventricle could penetrate the cerebral ventricle wall and migrate to the focus of infection. Hemorrhage increases the entry of embryonic stem cells from the ventricles (p<0.05). And the mES cells differentiated to neural cells. Because the sites on the ventricular wall at which these mES cell clusters attach and at which they enter the brain parenchyma were not uniform and they migrated beyond the SVZ (subventriclar zone) and towards the focus of infection the cells differentiation status was very complicated. The control group showed more nestin-positive cells established from transplanted mES cells, especially in hippocampus. On the contrary the stroke group showed very few neuronal markers positive cells with apparently higher abundance of activated astroglia derived from the host around them. The mES derived cells migrated to the focus differentiated into nestin-ir cells contained large areas immunoreactive for NeuN and GFAP. That is to say the mES cells could differentiate into end cells such as neurons and glial cells.Conclusions: (1) These results demonstrate that only some brain areas permitted survival of mES cells and its'progeny and formed instructive environments for neuronal differentiation and development of mES cells. Thus, region-specific presence of microenvironmental cues and their environmental fields in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders and the potentiality of regulate the microenvironment within the implant site when doing transplantation treatments.(2) Hemorrhage increases the entry of embryonic stem cells from the ventricles and decreases the efficiency of the ES cells differentiation into neural cells.(3) The naive mouse embryonic stem cells could survive in brains no matter of hemorrhage or the control rat. They could penetrate the cerebral ventricle wall. At the same time these cells have the potential to differentiate into end neural cells including neurons and glial cells. They pocess the potential to cure cerebral apoplexy and degenerative diseases.