Impaired In Vitro Neuronal Differentiation Capability Of Mouse APP/PS1?E9 Transgenic Embryonic Stem Cells

Alzheimer's disease(AD),also known as senile disease,is a kind of degenerative disease of the nervous system and the clinical manifestations are insidious onset.Pathological features of AD are characterized mainly by the senile plaques,neurofibrillary tangles and neuronal injury.In recent years,AD incidence is rising,which has seriously affected the life quality of the elders,posing a severe social problem and economic burden.However,the causes of this disease are complex,and detailed pathogenesis is still unclear.There is no effective treatment for this disease.Currently,many researchers are using animal models to investigate the molecular mechanism of AD.As a widely used mouse mode,APP/PS1?E9 transgenic mice produce large amounts of human amyloid precursor protein(APP)and presenilinl protein mutant with deletion of the ninth exon(PS1?E9)in brain.PS1?E9 can accelerate the selective decomposition of APP into harmful A?42,which has an abnormal fibrillary structure and is easy to form aggregation and deposits in the striatum.Therefore,APP/PS1?E9 mice show symptoms of disease and are good animal models to study the pathogenesis of AD.Although AD animal models provide advantages in the study of physiology,pathology and cognition in the development of AD,it is difficult to investigate molecular mechanism of AD disease deeply.Embryonic Stem Cells(ES cells),derived from the inner cell mass(ICM)of blastocyst,have the ability to self-renewal and differentiate into various cell types of three germ layers.ES cells have provided good models for studying cellular differentiation and organ development in vitro.Notably,the formation of embryoid bodies(EBs)can be induced to differentiate into neural cells by retinoic acid,which has become an important mean of investigating the development of nervous system in vitro.Recently,CHIR99021 has been reported to maintain pluripotent state of ES cells efficiently and endow the isolation and propagation of pluripotent cells from many challenging species such as the rat ES cells.Result:In this study,we firstly established ES cells from APP/PS1?E9 transgenic mice.Removal of zona pellucida,the E3.5d blastocysts were cultured in ES culture medium supplemented with GSK3 inhibitor CHIR99021.one week later,nine of total twenty blastocysts showed outgrowth of cell colony.These colonies were picked and trypsynized for further culture.After another week of culture,all cells displayed typical morphology of ES cells,showing the growth of adherent colonies,shiny and clear surrounding boarder,high nuclear/cytoplasm ratio,tightly packed clones and vigorous growth.PCR genotyping analysis revealed that two APP/PSAE9 ES cell lines were established,confirmed by the expression of APP and PS1?E9 expressed in AD ES cells but not in wild-type ES cells.Both WT and AD ES cells showed positive alkaline phosphatase activityand expressed pluripotency factor Oct4.qRT-PCR analysis confirmed that the expression of pluripotency factors Oct4 and Sox2 is comparable.Moreover,MTT assay showed that the proliferation rate of AD ES cells is similar to that of WT ES cells.All these data indicated that transgenic APP and PS1?E9 did not affect the self-renew of mouse ES cells.As AD is a disease of CNS,we asked whether there is any difference in the neural differentiation between WT and AD ES cells.ES cells were differentiated into neural cells used "4d-/4d+RA" method followed by 3 days of adherent culture.Immunofluorescence staining showed that at day 11,Nestin,a marker of neural stem/precursor cells,positive cells can be observed in differentiated cells derived from either WT or AD ES cells.qRT-PCR analysis confirmed that mRNA levels of neural precursor cell marker genes Nestin,Sox l,and Musashi in AD ES cells-derived cells were comparable to those of WT ES cells-derived neural cells.Therefore,the APP/PS1AE9 transgenes and their products doesn't affect the generation of neural precursor cells from ES cells.PS1?E9 selectively accelerates APP decomposition into excessive A?42 which deposits in hippocampus leading to degradation and loss of neurons.So we analyzed whether the similar phenotype can be produced in vitro.After 18 days neural differentiation,both WT and AD ES cell-derived neural cells displayed positive Tuj1,it's at low efficiency.qPCR analysis showed that the expression of mature neural cells associated genes,like neuron-specific marker genes Tuj1,axon-specific marker genes GAP43,dendritic cell-specific marker genes MAP2,astrocytes specific marker genes GFAP,presynaptic vesicles marker Synaptophysin(Syn)and acetylcholinesterase(AchE),were significantly lower in AD ES cell-derived neural cells than WT ES cell-derived cells.Thus,those data suggest that transgenic APP and PS1?E9 in AD ES cells decreased the efficiency of neuronal differentiation.Our research thus presented an in vitro cell model for further AD mechanism study and drug screening for AD therapy.