Effects And Regulatory Mechanisms Of Matrix Stiffness On Adult Hippocampal Neural Stem Cells Characteristics

Injury to the central nervous system(CNS)can lead to inflammation and neuronal death.This can result in motor and sensory dysfunction,cognitive impairment,disability,and even death,greatly affecting the quality of life of patients and causing a significant economic burden to families and society.After CNS injury in the adults,the ability of neurons to regenerate is extremely limited,and mature neurons cannot further proliferate to replace damaged cells.Therefore,CNS injury repair has always been a global challenge in the field of regenerative medicine.Neural stem cells(NSCs)are a type of multipotent cells that exist in the nervous system,with the ability to differentiate into neurons,astrocytes,and oligodendrocytes.They play an important role in CNS injury.Research has shown that transplantation of stem cells can be used to treat CNS injuries by rebuilding damaged neural circuits and functions.Compared to other types of stem cells(mesenchymal stem cells and embryonic stem cells),NSCs are already neural lineage cells and do not require induction,which reduces the risk of incomplete differentiation and tumorigenesis.However,the effectiveness of NSC transplantation alone for treating CNS injury is limited due to the unfavorable microenvironment for cell survival in pathological conditions,where tissue cannot provide sufficient oxygen,nutrients,growth factors,and cytokines.In addition,oxidative stress and immune rejection reactions in the injured site also impede the survival and differentiation of transplanted cells.Hydrogels are 3D structured biomaterials with good biocompatibility,biodegradability and absorbability,and their adjustable physicochemical properties make them widely used in tissue engineering and other biomedical fields.They provide a good microenvironment for cells to obtain nutrients and grow.In addition,hydrogels are a powerful tool for exploring the mechanisms by which the physical properties of the extracellular matrix(ECM)regulate cell behavior.Increasingly,research has shown that the stiffness,pore size,elasticity,and other physical properties of the ECM can regulate cell behavior.This study used a hydrogel constructed based on hyaluronic acid(HA)to simulate the microenvironment for the survival of NSCs,and explored the effect of substrate stiffness on the behavior of adult NSCs in the mouse hippocampus.As pure HA does not have an adhesive effect on NSCs,we modified the HA hydrogel by connecting it with laminin,an extracellular matrix component,to provide adhesive properties.At the same time,by adjusting the concentration of adipic dihydrazide(ADH)added,we generated three different stiffness(Soft,Medium,Stiff)HA-laminin hydrogels.We tested the mechanical properties,swelling,and degradation of these three HA-laminin hydrogels,and the results showed that the compressive modulus of the constructed HA-laminin hydrogel was between 0.1 k Pa and 2.5 k Pa.The results of the biocompatibility experiment show that the HA-laminin hydrogel has no cytotoxicity to adult hippocampal NSCs,and the adult hippocampal NSCs can adhere to the hydrogel,grow,and maintain their stemness.Through the Ed U labeling experiment,we found that adult NSCs in the mouse hippocampus have the strongest proliferation ability on soft HA-laminin hydrogel(0.2k Pa).As the stiffness of the HA-laminin hydrogel increases,the proliferation ability of adult hippocampal NSCs gradually decreases.Under conditions of known hydrogel stiffness range,we also evaluated the proliferation of adult hippocampal NSCs on regular cell culture plastic plates.The study found that the proliferation rate of adult hippocampal NSCs on regular plastic plates was the lowest.Taken together,the above experimental results indicate that the stiffer the matrix material used for cell culture,the slower the proliferation of adult hippocampal NSCs.At the same time,the change in the subcellular localization of YAP(Yes-associated protein,YAP)protein also reflects the response of NSCs to matrix stiffness changes.When the matrix is soft,YAP is mainly localized in the cytoplasm.When the matrix is stiff,YAP is mainly localized in the cell nucleus.Through transcriptome sequencing and bioinformatics analysis of adult hippocampal NSCs cultured on different stiffness materials,we found that the proliferation rate of adult hippocampal NSCs cultured on different stiffness hydrogels were regulated by the PI3K/Akt signaling pathway.We detected the phosphorylation level of AKT,a key downstream protein of the PI3K/Akt signaling pathway,and found that the level of AKT protein phosphorylation gradually decreased with increasing substrate stiffness.Furthermore,we verified changes in the expression of PI3K/Akt signaling pathway-related genes,including Flt1 and Cyclin D1,through RT-q PCR experiments,further confirming that the PI3K/Akt signaling pathway is significantly downregulated when adult hippocampal NSCs grow on a stiffer substrate.Therefore,we speculate that substrate stiffness can regulate the proliferation of adult NSCs in the mouse hippocampus through the PI3K/Akt signaling pathway.In the process of exploring the impact of matrix stiffness on the differentiation potential of adult NSCs in the mouse hippocampus,we found that in soft HA-laminin hydrogel,adult hippocampal NSCs differentiated into neurons and astrocytes in a ratio of 70% and 30%,respectively.In medium HA-laminin hydrogel,the ratio shifted to 40%neurons and 60% astrocytes upon differentiation;in stiff HA-laminin hydrogels,NSCs predominantly differentiated into astrocytes(70%),with a very low proportion differentiating into neurons(30%).These preliminary results indicate that soft hydrogels are more favorable for promoting neurogenesis in adult hippocampal NSCs.Additionally,we also performed transcriptome sequencing on cells differentiated on different stiffness substrates to further confirm the differentiation potential of NSCs based on gene expression.In summary,this project prepared a cross-linked hydrogel(HA-laminin)with laminin as the adhesive protein to promote NSC adhesion.At the same time,by changing the ratio of ADH incorporation,the stiffness of the hydrogel was adjusted,resulting in the production of three types of hydrogel-laminin materials with high,medium,and low stiffness.These materials,combined with plastic culture dishes,were used to investigate the regulatory effects of material stiffness on the proliferation and differentiation of NSCs.Research data indicates that adult hippocampal NSCs have a higher proliferation rate on softer hydrogel materials(0.2 k Pa)compared to harder materials.This is accompanied by significant upregulation of the PI3K-Akt,focal adhesion,HIF-1 signaling pathways,and cell cycle-related genes.Additionally,softer hydrogel materials(0.2 k Pa)can further promote the differentiation of adult hippocampal NSCs into neurons.These findings will provide insights into promoting the survival,proliferation,and controlled differentiation of transplanted neural stem cells in damaged areas.