EID3 Regulates The Transdifferentiation Process From Adipose Derived Mesenchymal Stem Cell To Neural Progenitor Like Cells

Background and objective:Neurodegenerative diseases,neural injuries,and other neurological diseases often cause acute and chronic inflammatory reactions.So far,there still remained many problems to be solved in how to repair these damaged and necrotic neural cells.Since neurons which make up most of the cells in the central nervous system are permanent cells,these cells are often unable to be regenerated after injury and necrosis.Endogenous neural stem cells(NSCs)can be activated into neural progenitor cells(NPCs)during the injury process,which can be further differentiated into supporting cells such as astrocytes and other glial cells to participate in inflammatory response,immune regulation and support for the defective areas.However,NSCs only exist in very few areas in adult brains,such as the subventricular zone,etc.For a long time,researchers have long sought to find new sources of cells in order to explore new and viable treatments.There have been many studies on stem cell transplantation.Allogeneic neural stem cell transplantation is gradually eliminated due to obvious ethical problems.Embryonic stem cell transplantation has been controversial due to the potential of tumorigenicity.There have been many breakthroughs in the induction of pluripotency stem cells(iPSCs),but the cell sources are difficult to obtain,therefore it remains in basic research.In recent years,the related research on mesenchymal stem cells(MSCs)has opened a new door for neurological disorders.MSCs are available from many resources,including bone marrow,umbilical cord,placenta,and adipose.Adipose derived mesenchymal stem cell(ADMSCs,ASC)is one of the most preferable sources of MSCs because of their large quantity,easier availability and safety.However,despite many advances in MSCs-related research,the mechanism of its repairment of the nervous system remains unclear.Moreover,the gene modified MSCs still have potential mutational tumorigenicity,thus reducing their safety and feasibility.Transdifferentiation of neural progenitor cell-like cells(NPCLs,NPLCs)from mesenchymal stem cells has raised more attention because NPCLs have similar biological and genetic properties to endogenous neural progenitor cells.At present,there have been many discoveries in adipose derived mesenchymal stem cell transdifferentiation into neural progenitor like cells.Our preliminary work found that EID3(EP300 interacting of differentiation 3)participate in the regulation of umbilical cord mesenchymal stem cells to transdifferentiate into neural precursor-like cells,and can directly interact with DNMT3A(DNA methyltransferase 3 alpha).Therefore,we wondered whether EID3 can regulate the transdifferentiation process of ASCs into NPCL or not,and we also eagered for the specific mechanism.Therefore,in this experiment,it is desirable to achieve the following objective:whether the EID3 has a function of regulating the transdifferentiation of adipose-derived mesenchymal stem cells into neural precursor-like cells.What is the main mechanism by which EID3 regulates this transdifferentiation.Whether the efficiency of transdifferentiation process can be influenced by EID3 needs to be further elucidated.Methods1.Evaluating the markers associated with neural precursor cells after inducing culture of adipose-derived mesenchymal stem cells with a repregramming medium to verify whether adipose-derived mesenchymal stem cells can be transdifferentiated into neural precursor-like cells.2.During the process of transdifferentiation into neural precursor cells,cell cycle changes were identified by flow cytometry.3.Determine whether EID3 can regulate the transdifferentiation process by knocking down and overexpressing EID3 molecules and measuring neural precursor cell-associated markers.4.Immunofluorescence staining technique and western blotting technique were used to determine the expression and distribution of EID3 molecules during transdifferentiation process.5.Whether there is a direct interaction between EID3 and moleculars related to DNA methylation during transdifferentiation by co-immunoprecipitation techniques.6.Whole-genome methylation sequencing of transdifferentiated NPCL,knockdown/overexpressing EID3 and transdifferentiated NPCL and normal adipose-derived mesenchymal stem cells,and observation of differences in whole-genome methylation levels.Results1.After 9-day inducing culture with the induction medium,ASCs showed similar biological traits as neural precursor cells,and the expression level of NPC-specific markers was significantly increased.2.During the process of transdifferentiation,the cell cycle of ASCs showed obvious changes,which were more similar to the cell cycle changes of neural progenitor cells.3.During the process of transdifferentiation of ASCs into NPCLs,the process of transdiferentiation and efficiency can be accelerated or slowed down by knocking down or overexpressing EID3.At the same time,EID3 molecules gradually transfer from the cytoplasm into the nucleus during transdifferentiation,and inhibition of this translocation process can improve the transdifferentiation efficiency.4.During transdifferentiation,the expression of molecules involved in DNA methylation is increased.Moreover,EID3 can interact with TET1,suggesting that EID3 may be involved in the regulation of the biological functions of methylation and hydromethylation molecules.5.EID3 can regulate the level of genome-wide methylation during transdifferentiation.Conclusion1.EID3 can directly affect the process of ASCs transdifferentiation to NPCLs.2.EID3 can directly interact with the DNA(hydro)methylation-related molecules like TET1.3.EID3 can affect the level of DNA methylation during transdifferentiation.