Optimization of RGD-Peptide Nano Scaffolds with Copolymrization Method for Axonla outgrowth in Differentiated hESCe-Neural Stem Cells


Rahjouei A *, Zare N , Kiani S , Shahbazi E , Baharvand H ,

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Objective: Today, 3D scaffolds find more application in culture of various cell types with goal of artificial tissues production. RGD-peptide amphiphil is a kind of these scaffolds from self-assembly peptide family. Previous reported showed a range benefits for this scaffolds, such as induction of cell survival, facility in cell migration and more rate of cell proliferations in compare with 2D surfaces. But, RGD-peptide amphiphil purely is not supportive for neural differentiation. Therefore in this study with attention to previous reports about benefits of laminin surface for neural differentiation, we made a composite nano scaffolds from combination RGD-peptide amphiphil and laminin with copolymerization method for differentiation of “neural stem cells” derived from Human embryonic stem cells to “mature neurons”. Materials and Methods: Cell Culture, Immuno fluorescence staining for marker of mature neurons (MAPII), RT-PCR, Cell viability test (trypan Blue) and quantification of axonal growth with Software (ImageJ). Results: Human embryonic stem cells after 5 days expansion in culture media on Matrigel surface induced to neural stem cells by induction media (1 DMEM/F12: 1 Neurobasal) supplemented with Noggin and N2. After 12 days appeared rosette and neural tube like structure. Differentiation to Neural stem cells confirmed by RT-PCR with expression of specific markers of this stage, such as nestin, sox1, Pax6 in dissociated structure and also with morphologically maturation of these cells in maturation media. Produced neural stem cells were seeded in two groups of prepared scaffolds, pure scaffolds and composite nano scaffolds respectively. Cells in pure nano scaffolds showed more proliferation and cell migration, but in this group after 2 weeks treated with differentiation observed in a circular form with viability around 68%. In composite nano scaffolds, seeded cells after passing same situation had low proliferation and cell migration but, they showed 49 times axonal outgrowth more than cells in pure nano scaffolds group. In this stage maturation of this cells confirmed MAPII immune fluorescence staining. Conclusion: Pure RGD-peptide amphiphile nano scaffolds can support from neural stem cells migration and proliferation and in combination to laminin can support from axonal outgrowth. Therefore, with attention to previous reports in other cell types and our study for neural stem cells we can say that this peptide in combination with other peptide can produce optimize condition in a 3D nano space for differentiation of various cell types.