Peptide-based Pure Synthetic Substrates For The In Vitro Xeno-free And Serum-free Expansion Of NSCs

Background and Objective:The application of neural stem cell has become one of the key research directions in our nation due to its great potentials in biomedical and clinical applications.The safety,standardization,and quality control of cell cultures are of vital importance for stem cell research and clinical translation,which highly favor for serum-free,xeno-free,and chemically defined cell culture media and substrates.In conventional stem cell cultures,whole Extracellular Matrix(ECM)proteins derived from human or animal tissues are often employed.Such ECM protein preparations involve high-cost,time consuming,and labor-intense procedures,and may run the risk of pathogen or immunogen transmissions to the cells in cultures.ECM or ECM-like molecules are not only necessary for in vitro growth of stem cells,but also play significant role in regulating their bio-activities.Therefore,ideal ECM molecules should be developed with defined components as well as functions.Generally the interactions between cells and ECM molecules are induced by receptors on cell surfaces binding to certain sequences on ECM,while integrins represent the most important trans-membrane receptors.The study on integrin function may shed lights on the role of cell-ECM interactions in stem cell biology,as well as the development in new ECM or ECM-like molecules.The efforts to identify specific integrin binding peptides may provide valuable knowledge in understanding the integrin functions in stem cell biology.In this study,a highly bioactive looped peptide on vinyl sulfone functionalized surface will be used to build a coating substrate system suitable for in vitro cultue of human neural stem cells(hNSCs).The integrin-related mechanism for NSC growth will be investigated with a novel integrin binding peptide array technology.With this complete integrin binding peptide array,one can quickly identify the types of integrins on any cell surface and develop pure synthetic substrates to support in vitro growth of a wide range of cells.This novel array will be tested in culturing human induced pluripotent stem cells(iPSCs)and iPSC derived neuroepithelial precursors(NEPs)to evaluate its potential in supporting reprogramming neural stem cell sources.Methods:1.A vinyl sulfone-functionalized surface is developed to immobilize the looped peptides on the surfaces of conventional culturewares.The adhesion,viability,proliferation,and differentiation of hNSCs were evaluated and compared with conventional laminin coated culturewares.2.Integrin binding peptide array was established with the same coating system,NSC adhesion,proliferation and differentiation on various integrin binding peptides were investigeted to explore ECM and integrin-related mechanisms in NSC growth..3.The adhesion and self-renewal of iPSCs and iPSCs derived NEPs on integrin binding peptide array were researched to identify the approporiate peptides for culturing these cells,and to evaluate the potential of our system in reprogramming NSC sources.Results:1.The looped IKVAV peptide on VS functionalized surface supported adhesion,proliferation and differentiation of NSCs and performed equally or better comparing to conventional LN coating.The novel artificial substrate was good for self-renewal and long term passaging of NSCs,even after stored for up to 1 year at room temperature,which indicates highly stable nature of the synthetic peptide coatings.2.NSC adhesion and self-renewal were found on 12 of total 16 integrin binding peptide surfaces.The proliferation rates varied while ?5?1,?V?1,?IIb?3 binding peptide supported faster proliferation than others.The role of integrin binding peptides in hNSCs varied and were protocol dependent.3.iPSCs and iPSC derived NEPs could attach to ?5?1 binding peptide surfaces and maintain fast proliferation.iPSCs could be induced into NEPs on the pure synthetic ?5?1 binding peptide coated surfaces.Conclusions:1.A chemically defined,serum-free,xeno-free,pure synthetic coating system based on looped peptides and VS functionalized polymer linkers was developed for the long-term culture of hNSCs,which will abandon human-or animal-derived ingredients.2.Specific binding of integrins and peptides could affect NSC adhesion,proliferation and differentiation,which turned out to be important mechanism of NSC growth on our pure synthetic substrate.3.Sythetic substrate based on ?5?1 integrin binding peptide could support in vitro growth of iPSCs and iPSCs derived NEPs,thus indicating that our system can also be used in iPSCs derived neural stem cell sources.