| Microcephaly is a neurodevelopmental disorder whose pathogenesis and neurological injury mechanisms are not fully understood and effective treatment measures are lacking.The most basic clinical manifestation of microcephaly is the delayed growth of cranial circumference,and cranial deformation-induced compressive stress can directly lead to brain tissue compression and neurological impairment,so it is important to investigate the changes of compressive stress regulating neurological damage in microcephaly to deeply understand the mechanism of neurological damage in microcephaly and develop effective treatment methods.Given the limitations of human brain development research in terms of ethics,detection and analysis methods,cerebral organoids are an ideal platform to investigate the changes of compressive stress-regulated neurological injury in microcephaly due to their human-derived characteristics,3D microenvironment and cerebral organoids tissue structure features.Based on this thesis focuses on the relationship between cranial deformation-induced compressive stress and neurological injury changes in microcephaly,and constructs a controlled loading model of hi PSC-derived cerebral organoids for microcephaly by using biomechanics,neuroscience and cellular tissue engineering.The relationship between compressive stress and the growth,contact modulus and stiffness of cerebral organoids was investigated by setting different loading gradients,and the transmission pathways of cerebral organoids to compressive stress.The transcriptome analysis was used to elucidate the molecular mechanisms underlying the regulation of neural damage in microcephaly by compressive stress.We further investigated the therapeutic effects of loading and releasing and growth factors on the neurological recovery of the injured cerebral organoids,and found that loading and releasing mainly regulated the remodeling effect and cell cycle of the compressive stress neurological injury cerebral organoids through cell adhesion molecules and prominent guidance pathway,while growth factors could regulated the compressive stress neurological injury cerebral organoids through PI3K/AKT pathway and protected neural cells from apoptosis.The results suggested that compressive stress-induced microcephaly can be effectively treated by loading re-release and growth factor intervention.The main research contents and conclusions of this paper are as follows:(1)The role of compressive stress in microcephaly nerve injury and the regulatory mechanism were clarified.?The relationship between compressive stress and cerebral organoids growth,contact modulus and hardness was investigated by setting different loading gradients based on photolithography and photocuring techniques to prepare loading devices with material exchange function.With the gradual increase of compressive stress,the cerebral organoids growth was significantly limited,and the cerebral organoids area decreased by 35%at the loading distance of 200μm,while the contact modulus and hardness of the cerebral organoids increased with the increase of compressive stress,and a significant difference was observed at the loading distance of200μm(p<0.05);?The conduction pathways of cerebral organoids to compressive stress were investigated by immunofluorescence.When compressive stress was applied to microcephaly cerebral organoids,mechanical force channel protein Piezo1 was abundantly expressed and increased with compressive stress,and was densely distributed at the edge of the cerebral organoids,while conducting information transmission through cytoskeleton and Wnt pathway;?The most significant loading interval was selected to clarify the pattern of compressive stress response in different brain regions of the cerebral organoids.Statistical analysis of six brain regions of the cerebral organoids revealed that the overall expression of the loaded cerebral organoids decreased by 31.65%,with the largest difference being in the forebrain region,which decreased by 18.81%;?Changes in the expression and distribution of markers characteristic of microcephaly were evaluated,and abnormal forebrain expression,reduced prefrontal cortex,reduced overall level of neural progenitor cell expression,more tilted and vertically oriented spindle bodies,along with reduced proliferative capacity,and reduced expression levels of newborn neurons were found in the loaded cerebral organoids;?The mechanism of neural recovery in the cerebral organoids of the microcephaly during re-release was elucidated.Cell adhesion molecule channels were opened after re-release,the expression of related genes was up-regulated,and the expression of E-cadherin was significantly increased,and the integrity of both the formed rings and ju nctions was enhanced.The key role of cell adhesion molecules in tissue remodeling during the re-release process was confirmed.Further,the Wnt pathway geneβ-catenin and the axon guidance factor SRGAP1 jointly regulated cell mitosis,thereby promoting cell proliferation capacity.(2)Re-release contributes to the neural recovery of microcephaly neurologically injured cerebral organoids caused by compressive stress.Based on the constructed microcephaly compressive stress-injured cerebral organoids model,we investigated the mechanotransduction pathways and neural recovery mechanism during the release process of microcephaly cerebral organoids in 4 d,15 d and 20 d with the confocal 3D imaging,immunofluorescence,transcriptome and RT-PCR techniques.?Re-release facilitates the growth and development of microcephalic compressive stress-injured cerebral organoids.The growth of cerebral organoids at the cellular level was significantly facilitated by re-release,which significantly increased mitotic levels in the forebrain region of microcephalic cerebral organoids,as reflected by cell division,proliferative capacity and apoptosis.The most beneficial effects of re-release on neurogenic non-proliferative divisions in the loaded cerebellum,while for apoptosis,re-release significantly reduced the level of apoptosis,while the growth of the microcephalic cerebellum after re-release was based on the mechanical regulation of cytoskeleton,cell adhesion and compressive stress channels,which leaded to changes in cell proliferation ability and apoptosis level,and increased in the size of microcephaly cerebral organoids;?Re-release facilitates the occurrence of basal progenitor cells in the microcephaly compressive stress-injured cerebral organoids and self-renewal of the dorsal telencephalon.The large cavities and continuous ventricular surface integrity of the released cerebellum were seen in the early neural differentiation stage,while early release significantly promoted the expression of the dorsal telencephalon marker protein PAX6 and tight junction protein,reflecting that early release enhanced the self-renewal ability of the dorsal telencephalon;?Re-release facilitated the expression and differentiation of neural progenitor cells in microcephaly compressive stress-injured cerebral organoids cells.Morphological change of neural progenitor cells similar to those of normal cerebral organoids were observed from loading to 4 d release,and protruded neuroepithelial tissue containing a large number of more mature neurons was observed at 15 d release,with the most significant release at the beginning of neural differentiation for SVZ intermediate progenitor cell expression;?Re-release promoted the generation of newborn neurons and mature neurons in microcephaly compressive stress-injured cerebral organoids.The number of neonatal neurons similar to that of normal cerebral organoids could be achieved at 4 d release,while the number of neonatal neurons was elevated by 13.68%and 3.83%at 15 d and 20 d release,respectively,while for mature neurons,their expression decreased significantly at loading up to 4 d,but the distribution was similar to that of the control group,concentrated in the marginal amplification region;?After re-release,cerebral organoids regulated cell recycling through synaptic guidance and cell adhesion to promoted cell proliferation.The axon guidance factor SLIT1 was upregulated in large amounts after re-release,which activated the upregulation of SRGAP1 gene and caused the upregulation of the downstream cell cycle gene CDC14C,which can effectively regulated cell mitosis,as well as the mechanotransduction through the cell adhesion protein E-cadherin after re-release,thus promoted the expression of intracellular cytoskeleton actin,and the regulation of cell cycle through theβ-catenin gene and the axon guidance factor SRGAP1 together regulated the cell cycle and promoted cell proliferation.(3)Growth factors effectively promoted neural repair in microcephaly compressive stress-injured cerebral organoids.The neural repair mechanism of growth factor in microcephaly compressive stress-injured cerebral organoids was investigated by immunofluorescence staining,3D imaging and RT-PCR.?Growth factors can effectively promoted the growth of microcephaly compressive stress-injured cerebral organoids.The morphological observation of different time points of the cerebral organoids revealed that FGF10 had a significant growth-promoting effect in the pre-induction phase of the cerebral organoids,while EGF growth factor exhibited a significant effect in the middle and late stages of cerebral organoids growth,which was shown by the significant promotion of cell proliferation ability and a significant reduction of apoptosis level by growth factor,and the horizontal direction of radial glial spindle orientation could be observed The percentage of cells at the end of division was34.57%and 40.46%,respectively;?Growth factors can improved the early development of the cerebral organoids.FGF10 and EGF can promote the expression of the ventral marker protein NKX2-1 and the top localized N-cadherin in the ventricular zone of the forebrain,thus improving the basal neural differentiation and forming a ventricular zone with complete apical basal polarity,and a large number of neural progenitor cells were lost in the early loading group of the cerebral organoids,while the percentage of neural progenitor cells in the cerebral organoids with the addition of FGF10 and EGF in the early loading group,a large number of neural progenitor cells were lost,while the percentage of neural progenitor cells in the FGF10 and EGF cerebral organoids were 39.08%and 32.65%,respectively;?Growth factors can effectively promoted the formation of dorsal telencephalic pattern.The dorsal telencephalon was an important region for the occurrence of microcephaly,which was usually caused by the reduction of the neural progenitor cell pool in this region.A significant reduction in PAX6~+expression was observed in microcephaly compressive stress injury cerebral organoids,while FGF10 and EGF can effectively promoted dorsal telencephalic pattern formation by improved radial glial progenitor cell layer and adherent junctional bands;?Growth factors can promoted neuronal growth and maturation.In the forebrain region of microcephaly cerebral organoids,neuronal loss was observed,while the addition of EGF and FGF10 promoted the growth of the forebrain region,as well as the expression of neurons in the forebrain region.The addition of growth factors had a significant improvement on the expression of mature neurons in cortical regions,and DCX newborn neurons started to migrate externally from radial to glial cell regions,while MAP2~+cells were present in more mature neurons,located outside the VZs and at the tissue edges;?FGF10 through PI3K/AKT signaling pathway The PI3K/AKT pathway can be involved in the regulation of basic processes of cell growth and proliferation,and RT-PCR data showed a related upregulation through a large number,suggested that FGF10 may activate the PI3K/AKT signaling pathway after binding to the single-channel transmembrane proteins Trk A and Trk B,thus regulated neurogenesis,promoted the proliferation and differentiation of neuronal cells,and protected neuronal cells from apoptosis. |
