Integration And Treatment Research Of Engrafted Astrocytes In The Neural Network Of Aging And Alzheimer Mice

Objective:Aging inducesmorphological and functional remodelling of astrocytes,affecting the normal activity of the central nervous system.In addition,studies have shown that the Alzheimer’s disease activates astrocytes,leading to disruption of astrocytic Ca²⁺activity,disturbance of the central nervous system,disruption of the blood-brain barrier and so on that drive the progression of the Alzheimer’s disease.At present,we do not know whether the brain microenvironment can be improved by transplanting healthy astrocytes to maintain normal brain activity in the ageing and Alzheimer’s lesions.Methods:（1）Through gene editing techniques of the Cre-loxp system,we constructed transgenic miceof ALDH1/1-Cre ERT2-GCa MP5G-td Tomato and ALDH1/1-Cre ERT2-GCa MP6f.Embryonic neural stem cells were induced and cultured todifferentiate into glial progenitorcells and astrocytes in vitro.（2）In vitro,the Ca²⁺activity of embryonic neural stem cell-derived astrocytes was obsevered withconfocal calcium imaging.（3）The morphology and expression of functional proteins（CX30,D-serine,AQP4）of engrafted astrocytes in the brain of aging and Alzheimer’s disease（AD）mice were examined by confocal 3D imaging.（4）The escape latency of engrafted glial progenitor cells mice were tested using a footshock escape latency assay.（5）We used novel object recognition and an eight-arm maze tests to examine the spatial learning memory of engrafted glial progenitor cells AD mice.（6）A triple reporter Luciferase-m Ruby2-GCa MP6f gene human iPSC cell line was induced to differentiate into glial progenitor cells and astrocytes.（7）Ca²⁺transientof triple reporter gene human iPSC-derived astrocytes was beenrecording by confocal Ca²⁺imaging.（8）Observation of the survival of transplanted human iPSC-derived glial progenitor cells in the mouse brain by histology.（9）The survival of triple reporter gene human iPSC-derived astrocytes in the mouse brain was observed by small animal live imaging.Results:（1）ATP andthapsigargincouldinduce Ca²⁺signals of embryonic neural stem cell-derived astrocytes.（2）Engrafted astrocytes were able to migrate within the cerebral cortex and maintain stable morphological structure for at least 12 months.（3）Engrafed astrocytesexpressed more AQP4,CX30,and D-Serinethan aged astrocytes.（4）Aged mice transplanted with astrocytes had significantly less escape latency to footshock responses than aged control mice.（5）In the brain of Alzheimer’s mice,transplanted astrocytes have a healthier morphological structure and a more stable cellular territory than host astrocytes.（6）In the brain of Alzheimer’s mice,the engrafted astrocytes were able to produce a corresponding Ca²⁺activity response to footshock.（7）In the predrue of the eight-arm maze training,AD mice engrafted with glial progenitor cells were able to find the target arm faster and more accurately than AD mice.（8）In vitro,a triple reporter Luciferase-m Ruby2-GCa MP6f gene human iPSC cell linewas capable of being induced into astrocyte,which produced Ca²⁺transients.（9）Multimodal methods（histomorphology,small animal live imaging）were able to examine the survival of human iPSC-derived astrocytes in the mice brain.Conclusions:（1）Embryonic neural stem cell-derived glial pregenitor cells were able to integrate into the neural network of hostmice and maintained a long-term excellentmorphological structure.Engrafted astrocytespromoted the response to nociception in aged mice by enhancing the connection with the host neural network,releasing normal glial transmitters and maintaining the polarity of AQP4.（2）Embryonic neural stem cell-derived astrocytes maintained long-term"Stem cell properties"and integrated into the neural network of AD mice at different stages of the disease.Engrafted astrocytes exhibited a relatively healthly morphological structure to perform normal physiological functions,improving the spatial learning and memory ability of AD mice.（3）A human iPSC cell line expressing a triple gene（Luciferase-m Ruby2-GCa MP6f）was able to induce differentiation into astrocytes in vitro with multimodal detection function.The human iPSC cells-derived glial pregenitor cells were able to survive for a long time in the brain of immunodeficient mice...