Study On The Synergistic Effect Of Glycosaminoglycan Mimics And Photothermal Nanoparticles On Stem Cell Neural Differentiation

The neural differentiation of embryonic stem cells(ESCs)is of great value in the treatment of neurodegenerative diseases,with urgent need for improved methods for the precise and efficient regulation of neurogenic differentiation.Glycosaminoglycan(GAG)-related and retinoic acid(RA)-related signaling pathways,two of the most critical signaling pathways in the neuronal differentiation process of stem cells,have received extensive attention in both basic research and practical applications.However,low bioavailability of signaling molecules is the general problem existing in the above strategies.Therefore,a novel cell differentiation induction platform have been developed in this thesis,in which glycosaminoglycan mimics were used for activating GAG-related signaling pathway and RA were effectively used to promote neuronal differentiation and maturation.Neural differentiation of stem cells with high efficiency and purity were achieved by simultaneously acting on the two related signaling pathways.The detailed research works are introduced as follows(1)Synergistic effect of glycosaminoglycan mimics and RA delivery on stem cell neural differentiation by taking gold nanoparticles(GNP)as a carrier.Firstly,syntheses of PM AG and PSS(containing glyco and sulfonate units respectively)were carried out by reversible addition fragmentation chain transfer(RAFT)polymerization,using 2-(methacrylamido)glucose(MAG)and sodium styrene sulfonate(SS)as monomers respectively.Thiolated polymers were obtained by aminolysis,following by attaching to GNP surface at different ratios.The prepared polymer-modified nanocomposites can improve differentiation efficiency via GNP-membrane interactions to activate signaling pathway,and further promote the differentiation of stem cells into nerve cells under the optional conditions of laser irradiation by transferring more RA bind to its receptors on the nucleus.The results of immunofluorescence staining and quantitative polymerase chain reaction(qPCR)proved that assembly of glycosaminoglycan mimics on the GNP promoted the neural differentiation of ESCs,and when the feed ratio of the PMAG-to-PSS was 1:1,the nanocomposite GNP-M1S1 showed the greatest promotion effect.More importantly,?3-tubulin gene expression of GNP-M1S1 group at a power density of 0.80 W/cm2 and 10 s increased by 320-fold and the percentage of nerve cells in differentiated cells increased to 87%.Those results indicated that synergistic effect of functional units on the surface of nanocomposites and its photothermal effect not only facilitated the differentiation efficiency remarkably,but also noticeably improved the purity of the differentiated nerve cells.(2)Effect of glycosaminoglycan mimics and photothermal gold nanomaterials on stem cell neural differentiation,based on electrospun scaffold.Polycaprolactone(PCL)scaffold loading with glycosaminoglycan mimics and gold nanorods(GNR)was obtained via electro spinning technology,with PMAG and PSS inner and GNR containing shell.High-porosity of the prepared scaffold can support cell growth by simulating in vivo microenvironment.Released polymers from the scaffold fibers can promote neural differentiation by activating GAG-related signaling pathway.Meanwhile,RA entered cells under photothermal effect of GNR,further improving neurogenesis efficiency once bind to receptors on the nucleus.The results showed that the introduction of glycosaminoglycan mimics and GNR in the fibers can both benefit neurogenesis.And the synergism of photothermal effect of GNR can significantly enhance neurogenic differentiation efficiency of PCL@GNR and PCL&P@GNR scaffolds.Especially,PCL&P@GNR scaffold had a strongest promotion of neurogenesis under laser irradiation at a power density of 0.66 W/cm2 and 30 s,with a 250-fold increase in mature neural marker expression.These results indicated that the novel composited scaffold has obvious promotion effect on neuronal differentiation.In summary,based on the significant role of GAG mimics and differentiation-inducing factor RA on ESCs neural differentiation,GNP and electrospun scaffold wer e taken as carriers respectively.Glycosaminoglycan mimics can activate GAG-related signaling pathway and RA can be effectively delivered into cells.Improved neuronal differentiation efficiency and purity were achieved by the synergistic effect of these two pathways.The strategy developed in this work provides a viable alternative for the promotion of stem cell neural differentiation.It may have theoretical significance and practical application value in the cell therapy of neurodegenerative diseases,and may provide guidance for promoting stem cell differentiation into other cell types.