Functionalized polypeptide hydrogel matrix for ex vivo modulation of proliferation and differentiation of neural stem cells

Abstract

Objective In vitro model with three dimensional cell culture provides the appealing biomimetic platform to probe the biological characteristics of multiple stem cells,which serves as an important in vitro tools to investigate regulating factors controlling the proliferation and differentiation of neural stem cells (NSCs).The present study aims to reconstruct an integrated poly-peptide hydrogel made extracellular matrix (ECM) enhanced with cyclo-RGD molecular [c(RGDfK)] for the exploration of NSCs bio-characteristics.Methods Spinal cords from one-d-old SD rats were collected and spinal-derived NSCs were induced in the complete medium; immunofluorescence staining was employed to observe the NSCs morphology and identify NSCs.Three hydrogel including type Ⅰ collagen,self-assembly poly-peptide nanofiber hydrogel (SAPNH) of RADA16 and RADA16-c(RGDfK) were employed to serve as culturing ECM of spinal-derived NSCs to mimic the ex vivo 3-D culturing.With the theological analysis,cyto-morphological observation was performed,NSCs proliferation was observed by MTT assay,and cell immunochemistty and confocal microscopy were employed to detect the NSCs differentiation.Results SAPNH born appropriate elastic module conducive to the cellular adhesion and proliferation of neural cells (RADA16 and RADA16-c (RGDfK)=(0.42±0.07) kPa and (0.47 ±0.09) kPa,without significant difference (P＞0.05); however,the elastic module in the type Ⅰ collagen group was (0.87±0.12) kPa,which was significantly stronger than the two groups (P＜0.05).Uniform distribution of neuron-shape cells was noted in the extracellular matrix ofRADA16-c (RGDfK) cells,with almost the diameter of cell sphere (200-300 μm); cells in the RADA16 and RADA16-c (RGDfK) had better growth characteristics than the other two groups; RADA16-c(RGDfK) had significantly better cellular adhesion and proliferation of neural cells as compared with RADA16 and collagen groups (P＜0.05).A large number of neural precursor cells differentiated into neurogliocytes was noted in the RADA16 group and RADA16-c (RGDfK) group,with low proportion of neuronal differentiation (17.6％ ±3.1％ and 19.0％±3.6％,respectively); this proportion of neuronal differentiation was significantly higher than that in the type Ⅰ collagen group (10.6％±2.3％,P＜0.05).Conclusions The functionalized SAPNH enhanced with c(RGDfK) presents the excellent biocompatibility and promotes the adhesion and proliferation of spinal NSCs.Serving as the engineered cellular vector,functionalized SAPNH has laid a solid foundation for the studies of neural regeneration and repair with novel neuro-engineering techniques in the subsequent researches. Key words: Self-assembly poly-peptide hydrogel; Extracellular matrix; Three dimensional cell culture; Neural stem cell