The Study On The Stimulation Effect Of Glycosaminoglycan-mimicking Polymers On The Neural Differentiation Of The Embryonic Stem Cells

Heparin/heparan sulfate(HP/HS)is one kind of glycosaminoglycan(GAG),a natural polysaccharide with linear chain,with complexed structures and various bioactivities.A variety of bioactive molecules,such as growth factors,cytokines,chemokines,morphogen and enzymes are combined with HP/HS in the format of the free molecular chain and lipopolysaccharide and proteoglycan to modulate vital activities.The heparin-mimicking polymers containing sulfonate groups possess similar bio-functions to HP/HS.Furthermore,comparing with HP/HS,heparin-mimicking polymers with sulfonate groups promoted the neural differentiation of embryonic stem cells(ESCs)with more specific and higher efficiency.The structure,degree of polymerization and concentration of heparin-mimicking polymers are important factors which impact its ability on binding and releasing bioactive molecules;meanwhile,sulfonate groups play a key role in the function of heparin-mimicking polymers;therefore,studying the relationship between sulfonate groups' structure in heparin-mimicking polymer and its function has great significance.Three different heparin-mimicking polymers were synthesized through reversible addition-fragmentation chain transfer(RAFT)polymerization with different structure of sulfonate group monomers to study the influence of various structures of heparin-mimicking polymers on the neural differentiation of ESCs.The research contents as follows:1.Sodium 4-styrenesulfonate(SS),3-sulfopropyl acrylate potassium salt(SPA),and 2-acrylamido-2-methyl-1-propanesulfonic acid(AMPS)were chose as monomers with sulfonate groups.2-(methacrylamide)glucose(MAG)as glyco monomer were polymerized with sulfonated monomers to fabricate heparin-mimicking polymers named P(MAG-co-SS),P(MAG-co-SPA)and P(MAG-co-AMPS)by RAFT polymerization ? respectively.Tissue cells were cultured with medium containing heparin-mimicking polymers in order to study the polymer structure influence on cells' growth and differentiation.It showed that,all these three polymers had great cytocompability and were good at supporting the proliferation of ESCs.Due to their similar structure to heparin,heparin-mimicking polymers can promote neural differentiation of ESCs as well.Moreover,heparin-mimicking polymers presented better supports than natural heparin: relative m RNA level of ?3-tubulin,the mark gene of mature neuron of ESCs were 2.40,4.82,2.52 folds,respectively,comparing with that from ESCs response to heparin after treated with P(MAG-co-SS),P(MAG-co-SPA)and P(MAG-co-AMPS)for 18 days.In addition,P(MAG-co-SPA)had the highest affinity to bind with fibroblast growth factor(FGF)and fibroblast growth factor receptor(FGFR)on cells membrane and showed the best promotion to accelerate ESCs neural differentiation.It is suggested that different structure of sulfonated unit in heparin-mimicking polymer is an important factor to promote ESCs neural differentiation difference.2.P(MAG-co-SPA)with different molecular weight were synthetised by RAFT polymerization.After treated with different molecular weight P(MAG-co-SPA),from 6 to 20 KDa,it showed that P(MAG-co-SPA)at 9 KDa had the best neural differentiation promoting effect.When the 0 to 10 ?g/m L of 9 KDa P(MAG-co-SPA)were added in the ESCs' culture medium,the percentage of neuron orientation differentiation increased first and then decreased as the increase of polymer concentration;8 ?g/m L P(MAG-co-SPA)was the optimal concentration to promote ESCs' neural differentiation.These results indicated that the difference in molecular weight and concentration of P(MAG-co-SPA)had an effect on promoting the neural differentiation of ESCs,and performed best in a certain range.In a word,the structure of sulfonate units in heparin-mimicking polymers can affect its promotion on neural differentiation of ESCs;heparin-mimicking polymers with different structure sulfonate units bond to FGF and FGFR in different affinities which causes different degree of the neural differentiation of ESCs.Compared with P(MAG-co-SS)and P(MAG-co-AMPS),P(MAG-co-SPA)performed the best in the promotion on neural differentiation of ESCs.Meanwhile,the variation in its molecular weight and concentration made different effects on neural differentiation of ESCs.Based on our results,it is established a solid foundation to design and prepare heparin-mimicking polymers which can efficiently promote neural differentiation of ESCs.