Differentiation Of IPSCs From Patients With Alzheimer's Disease Into Neural Stem Cells And Neurons And The Effect Of Energy Metabolism During The Differentiation Process

Alzheimer's Disease(AD)is one of the neurodegenerative diseases with the highest incidence.So far,there has been no ideal treatment in clinical practice,and anti-AD drugs that have entered clinical trials mostly end in failure.The possible reasons are as follows:(1)the early diagnosis of AD is difficult and the intervention time is too late.(2)Commonly used cells or animal models cannot reflect the characteristics of human AD.Induced pluripotent stem cells(iPSCs)are generated by the reprogramming of somatic cells.iPSCs from patients obtain disease-related genetic and epigenetic information,and their differentiation process in vitro can reflect the occurrence and development of diseases to a certain extent,which is considered as an ideal tool for the modeling of a variety of disease including AD.There are two types of AD:Familial AD(FAD)and Sporadic AD(SAD).The etiology of FAD is single,and the obtained iPSCs phenotype is consistent,but it is difficult to reflect the characteristics of SAD.Due to the complex etiology of SAD,the obtained phenotypes of iPSCs also have great heterogeneity.Therefore,the study on differentiation of iPSCs from SAD patients into neural stem cells(NSCs)and neurons is of great significance for us to understand the occurrence and development of AD and to carry out targeted drug screening.Based on this,peripheral blood mononuclear cells(PBMCs)from SAD patients were reprogrammed into iPSCs,and the possible cellular phenotypes and functions differences were observed during the differentiation of iPSCs into NSCs and neurons.Through the above research,we aim to establish a model that can reflect the early stage of AD and provide an important tool for the study of the occurrence and development of AD.Based on the phenomenon of abnormal energy metabolism in the early stage of AD,the study on the changes of energy metabolism and regulation mechanism of iPSCs in the differentiation process is carried out,in order to provide experimental basis for early diagnosis and intervention of AD.Part ?.Studies of phenotype and function of iPSCs derived from SAD patients during the differentiation into NSCs and neuronsPBMCs form Young control(YC),Cognitive normal control(CNC)and SAD were reprogrammed into iPSCs,and the differences in cellular phenotypes and functions were observed during the differentiation of iPSCs into neurons.Subsequently,we treated iPSCs-derived neural cells with exogenous stimulus factors(H₂0₂ and cortisol)closely related to the occurrence of AD,and observed their effects on cellular phenotypes and functions.The main results are as follows:1.Phenotypic study of iPSCsThe expression of pluripotency markers,the proportion of EdU-positive cells and embryoid body differentiation of iPSCs derived from SAD were not significantly different from those of YC and CNC,suggesting that there were no significant differences in the reprogramming efficiency,proliferation ability and multidirection differentiation potential of iPSCs among three groups.2.Phenotypic study of iPSCs-NSCsThe ratio of EdU-positive cells of SAD iPSCs-NSCs was lower than that of YC and CNC.During the passage of NSCs,the proportion of S100b-positive cells in some SAD iPSCs-NSCs was significantly increased,while in some SAD iPSCs-NSCs,the proportion of DCX-positive cells was significantly increased,which suggested that SAD iPSCs-NSCs showed obvious heterogeneity.Its dryness was difficult to maintain,and some SAD patients iPSCs-NSCs were easy to differentiate into glial cells,and some were easy to differentiate into neurons.H₂0₂ and cortisol had no significant effect on the viability of NSCs in each group,suggesting that the all three iPSCs-NSCs were not sensitive to exogenous stimulation of H₂0₂ and cortisol.3.Phenotypic study of neurons derived from iPSCsDuring neuronal differentiation,the proportion of DCX positive cells,neurite length and neurite branch points in SAD were significantly higher than those of YC and CNC.The patch clamp results showed that the K+current,Na+current,the number of evoked action potentials and the maximum peak value of SAD iPSCs-neurons were significantly higher than those of YC and CNC.The above results suggested that SAD iPSCs-neurons differentiated faster and had stronger electrophysiological activity and more mature than YC and CNC.H₂0₂ and high concentrations of cortisol can significantly reduced the viability of SAD iPSCs-neurons,neurite length,and neurite branch points,suggesting that SAD iPSCs-neurons were more sensitive to the exogenous stimulating factors of H₂0₂ and cortisol,and were more susceptible to be damaged.Part ?.Studies of the role of energy metabolism in the differentiation of iPSCs into NSCs and neuronsThe results of Part I of this study showed that the stemness of SAD iPSCs-NSCs was difficult to maintain,and in the differentiation of neurons,SAD iPSCs-NSCs differentiated faster and mature accelerated,which was consistent with the pathological phenotype of the increased neurogenesis and excessive consumption of neural stem cell pool in early AD.While abnormal glucose metabolism in the brain was discovered in the early stage of AD,and energy metabolism was also an important regulator of stem cell stemness maintenance and differentiation.Therefore,in this part,we first observed the changes in the energy metabolism phenotype of iPSCs during the differentiation process,and try to explain the possible reasons of accelerated differentiation of SAD iPSCs-NSCs from the perspective of energy metabolism.Next,we inhibited and promoted energy metabolism to observe its influence on the phenotype and function of SAD iPSCs-neural cells,which may provide an important basis for early diagnosis and intervention of AD. The main results are as follows:1.Studies of energy metabolism during the differentiation of iPSCs into NSCs and neuronsExtracellular acidification rate(ECAR)and Oxygen consumption rate(OCR)of SAD iPSCs-NSCs were significantly lower than that of YC and CNC,while in the stage of iPSCs and iPSCs-neurons,there were no significantly differences of ECAR and OCR among three groups.The results indicated that only at the stage of NSCs,the energy metabolism level of SAD iPSCs-NSCs was significantly lower than that of YC and CNC.Expression and activity of triose-phosphate isomerase(TPI)and activity of hexokinase(HK)in SAD iPSCs-NSCs were significantly lower than that of YC and CNC.Pearson correlation analysis showed that the expression of TPI and the activities of HK and TPI were positively correlated with the level of energy metabolism of iPSCs-NSCs.The above results suggested that the decreased energy metabolism of NSCs from SAD may be related to the decreased expression and activity of enzymes involved in energy metabolism.2.The effects of controlling energy metabolism on the proliferation and differentiation of iPSCs-NSCs from SAD patients2-deoxy-D-glucose(2-DG),a hexokinase inhibitor,can significantly inhibited the viability and cell growth of iPSCs-NSCs in each group,increased the expression of DCX and ASCL1 genes,increased the proportion of DCX positive cells in each group,and decreased the proportion of Nestin positive cells.Fructose-6-phosphate(F6P),an intermediate product of glucose metabolism,had no significant effect on the viability,growth,ratio of DCX-positive cells and Nestin-positive cells of iPSCs-NSCs in each group,but it could significantly reduce SAD DCX gene expression.F3SM,a new type of PPAR?/? agonist,can increase the ECAR levels in each group,promote the viability and growth in each group,and reduce the proportion of DCX-positive cells in SAD group.The above results indicated that the rapid differentiation of SAD iPSCs-NSCs into neurons may be related to the decline in energy metabolism and promoting energy metabolism can increase the proliferation of iPSCs-derived NSCs,and inhibit the differentiation of SAD iPSCs-derived NSCs into neurons to a certain extent.In summary,this study mainly draws the following conclusions:(1)iPSCs-NSCs derived from SAD exhibit obvious heterogeneity.During neuronal differentiation,SAD iPSCs-neurons differentiated more faster,and more mature,which may reflect the phenotype of excessively increased neurogenesis in the early stage of AD.SAD iPSCs-neurons are more sensitive to the exogenous stimulating factors of H₂0₂ and cortisol,and are more susceptible to be damaged.The above results suggest that iPSCs derived from SAD and their differentiation of NSCs and neurons can be severed as ideal models to simulate the the occurrence and development of AD,especially the early process.(2)The rapid differentiation of SAD iPSCs-NSCs into neurons may be related to the decrease in energy metabolism caused by decreased expression and activity of enzymes involved in energy metabolism,suggesting that abnormal changes in energy metabolism may be an important basis for early diagnosis of AD.Promoting energy metabolism can inhibit the rapid differentiation of SAD iPSCs-NSCs into neurons to a certain extent,suggesting that regulation of energy metabolism may be served as an early intervention for AD.