The Research Of The Effect Of Heparin-Mimicking Polymer On The Differentiation Of Stem Cells

Stem cell therapy is to purify,isolate,culture,and conduct directional induction and differentiation of stem cells in vitro to new cells,tissues,organs,etc.,and transplant them into the body through transplantation technology to replace normal,damaged or dead cells to restore body function.In the process of cell therapy,efficient and targeted differentiation of stem cells is very important.It is of great significance for regenerative medicine to fully understand and make full use of the factors affecting stem cell differentiation.Heparin/heparan sulfate(HP/HS),as the most complex GAGs molecule in stem cell niche,can specifically induce embryonic stem cells and mesenchymal stem cells to differentiate into neural cells and osteoblasts respectively through the interaction of a large number of negatively charged carboxylic and sulfonic groups with soluble signal factors or cell membrane proteins.We synthesized heparin-like polymers through group recombination strategies,simulated the niche microenvironment of stem cells,and designed an artificial stem cell niche microenvironment platform to explore its impact on the targeted differentiation of stem cells.The specific research content is as follows:First,we designed the artificial stem cell niche platform from the perspective of macroscopical 3D.We used 3-sulfopropyl acrylate potassium salt(SPA),2-Methacrylamido glucopyranose(MAG)and N-isopropylacrylamide(NIPAAm)as monomer units to prepare the thermosensitive porous hydrogel PNMS by RAFT polymerization,which was used to encapsulate bone marrow mesenchymal stem cells(BMSCs),and to explore the effect of the platform on the differentiation of BMSCs into osteoblasts.The chemical structure and surface morphology of PNMS hydrogels were characterized by FTIR and SEM respectively.The results showed that the porous heparin-like glycopolymer hydrogel network was successfully synthesized.The pore sizes of PNIPAAm,PNMS1%and PNMS4%gel were in the range of 109.64±7.37?161.94±11.84 um.The gel of PNMS7%and PNMS10%showed macroporous layered structure.When the temperature rises to 37?,each group of gel shows different degrees of temperature sensitive deformation behavior such as more compact structure and smaller pore size.Next,the swelling rate and swelling property of thermosensitive PNMS hydrogel were tested by weighing method.After CCK-8 characterization of porous biohydrogel with good biocompatibility between BMSCs and L929 cells,the osteogenic marker gene was detected at molecular level to explore the effect of the functional platform on the differentiation of BMSCs into osteoblasts.The results showed that after 14 days of osteogenic induction,PNMS4%,PNMS7%and PNMS 10%hydrogel could promote the differentiation of BMSC into osteoblasts compared with the PNIPAAm control group.Among them,PNMS7%showed the best osteogenic differentiation potential,and its mRNA expression of ALP,RUNX2 and BGP was about 10,17 and 22 times higher than that of PNIPAAm.PNMS7%thermosensitive heparin-like glycopolymer hydrogel provides beneficial stem cell niche platform for osteogenic differentiation of BMSC cells.In order to get closer to the microenvironment of stem cell niche from the perspective of micro 3D,we modified the heparin-like glycopolymer PMS synthesized by group recombination strategy and recombinant soluble growth factor FGF2 on the surface of gold nanoparticles with good biocompatibility to prepare the ternary complex of AuNP-PMS/FGF2.The bioactivity,stability and biocompatibility of the composite are investigated in this study comprehensively.The results showed that the AuNP-PMS/FGF2 composite could maintain a long-term stability at room temperature for at least 8 days,and greatly promote the neural differentiation of mESCs.Compared with the other materials,the AuNP-PMS/FGF2 composite could significantly stimulate the expression of the specific neural differentiation markers(nestin and ?3-tubulin),while obviously down-regulate the mRNA production of marker of pluripotency(Oct-4),mesoderm lineages(Flkl expression)and endoderm lineages(Sox17 expression)in mESCs.Moreover,thepromotion effect of the composite on neuronal maturation marker ?3-tubulin expression achieved maximally at the low concentration of FGF2(4 ng/mL),which suggested the high efficiency of AuNP-PMS/FGF2 composite in neural differentiation of mESCs.Meanwhile,both mESCs and L929 cells showed desirable growth during the incubation with AuNP-PMS/FGF2 composite.The AuNP-PMS/FGF2 system presents a new way to achieve HS/FGF2 complex nanomimics efficiently for the neural differentiation of mESCs.To sum up,the synthetic heparin-like glycopolymer has the biological activity of natural heparin/heparin sulfate(HP/HS),and can specifically and efficiently promote the directional differentiation of stem cells in different artificial stem cell niche ecological platforms.These research results provide a good design idea for the construction of artificial stem cell niche based on heparin-like glycopolymer.