Differentiation And Functional Integration Of ES Cell-Derived Neural Procursor Cells Engrafted Into The Hippocampus Of Aβ_1-40-Injured Rats

Alzheimer's disease (AD) is a progressive neurodegenerative disease that mainly impairs central nervous system and clinically characterized by progressive memory loss, cognitive decline in elderly population. It is the most common form of senile dementia. The main pathological changes are extracellular deposit ofβ-amyloid peptides, intracellular neurofibrillary tangles, synaptic loss, and brain atrophy. The etiology and mechanism of AD are not clear, but the amyloid cascade hypothesis is widely accepted. The hypothesis presumes that gene mutation or other factors upregulate beta-amyloid (Aβ) and disorder its metabolism, and Aβdeposits as fibril aggregates forming senile plaques (SP) and intracellular neurofibrillary tangles (NFTs), which results in the degeneration and necrosis of neurons. Current therapies, such as treatment with acetylcholinesterase inhibitors to enhance cholinergic function, give only partial and temporary alleviation of symptoms, and would not retrieve the neural loss in the cortex and hippocampus of patients at advanced stage. Neural transplantation is regarded as an inspiring strategy for the treatment of AD.Cell replacement is a promising approach for treating neurodegenerative disorders that may overcome some of the existing limitations of traditional pharmaceutical approaches. Such neuroreplacement strategies offer great therapeutic potential for the treatment of neurological diseases such as Parkinson's disease, Huntington's disease, spinal cord trauma, and stroke. In one study the human undifferentiated NSCs were injected into the brain of 6-month-old and 24-month-old rats respectively. Their results demonstrated that human neural stem cells improved cognitive function of aged brain. Wang and colleagues transplanted ES cells-derived neurospheres into mouse model frontal cortex of Meynert nucleus lesion. They found that transplanted neurospheres survived, migrated and differentiated into many choline acetyltransferase-positive neurons and a few serotonin-positive neurons. The working memory error decreased significantly in the mice grafted with neurospheres. Despite the broad experimental application of neuronal transplantation, few studies have addressed the functional integration of single neurons in the host CNS. Therefore, our purpose focus on the differentiation and integration of engrafted NPCs after transplanted into the hippocampus of Aβ1-40-injured rats.Materials and methods:1. Rats model was established by the intrahippocampal injection of aggregatedβ-amyloid (1-40), the learning and memory of AD model rats was measured by the Morris water maze test. The histopathological changes of AD model was observed by Congo Red staining, Nissl staining, Fluoro-Jade B(FJB) staining and GFAP immunohistochemistry.2. Mouse embryonic fibroblast (MEF) were separated from E13.5 pregnant mouse and passaged 3-5 generation, then used as feeder layers treated by mitomycin C for 150 minutes,. An EGFP-expressing derivative of the ES cell line MESPU35 were expanded on feeder layers combinated with leukemia inhibitory factor (LIF). NPCs were generated from ESCs by the modified serum-free methods, and detected by Nestin immunohistochemistry.3. NPCs generated from mouse ESCs in vitro were transplanted into the hippocampus of Aβ1-40-injured rats. And the survive, migration, differentiation and integration of engrafted NPCs were observed, and the improvement of memory dysfunction of Aβ1-40-injured rats was also observed.Results1. The AD model was established by intrahippocampal injection of aggregated beta-amyloid(1-40). Learning and memory of the AD rats were significantly declineded by Morris Water Maze test two weeks post-surgery. The Congo Red-positive plaques were detected around the injection sites of Aβ1-40. FJB and Nissl staining showed the obvious neural degeneration and loss around the injection site. Furthermore, the GFAP-positive astrocyte were over-activated compared with NS-injected control group for four month longer. This model can mimic the cognitive functions impairment and pathology characters of AD well and facilitate the following research on NPCs transplantation.2. Purified mouse embryonic fibroblasts (MEF) were isolated from E13.5 pregnant mouse to prepare feeder layer for ES culture. ES cells can be expanded in vitro without differentiation under the condition of feed layer combinated with leukemia inhibitory factor (LIF). After removal of feeder cells, hLIF and planted on the bacterial culture dishes, embryonic bodies were formed, and then differentiated into NPCs under the serum-free medium supplemented by N2 plus fibronectin, about 93% of which were Nestin-positive cells.3. After intrahippocampal transplantation of NPCs, the cognitive functions of AD rats were assessed by Morris water maze test. It shows learning and memory impairment of AD rats was improved significantly at 4w,8w,12w,16w post-transplantation.4. Engrafted NPCs migrated steadily farther under the guidance of local circumstance as time goes on. The survival rate of engrafted NPCs was 7.32±0.69% and decreased significantly gradually from 4w to 16w. At the same time, the living NPCs mostly differentiated into GFAP-positive astrocytes and some differentiated into Tuj1-positive neurons.5. A few engrafted NPCs expressed the neuronal glutamate transporter protein (EAAT3) and the rate-limiting enzyme for GABA synthesis (GAD67), suggesting efficient differentiation into glutamatergic neurons and GABAergic neurons. Some engrafted NPCs express ionotropic glutamate and GABA receptors using antibodies to the AMPA receptor (GluR1), the GABAA receptor (βchain), and the NMDA receptor (NR1). Confocal immunofluorescence analysis revealed PSD95-positive puncta were typically found in close proximity to the engrafted NPCs surface and numerous synaptophysin-positive, EGFP-negative patches were found in close apposition to the somatic and dendritic membranes of transplanted cells, suggesting that host-derived presynaptic terminals contact incorporated NPCs-derived neurons. Ultrastructural analysis demonstrated the synapse formation between the donor cell-derived neurons and the host neurons. Morris water maze test bombined immunohistology of Fos-expression indicate some engrafted NPCs-derived neurons incorporated into the host brain circuitry and participated the learning and memory process.