The evaluation of the effect of PCLF(poly- caprolactone fumarate) three- dimentional scaffold on the behavior of neural stem cells derived from the sub-ventricular zone in the adult mice

Abstract

In the current study, by using multifarious approaches, we aimed to evaluate the effects of three-dimensional PCLF scaffolds on the behaviors of neurons derived from sub-ventricular regions in mature mice. Materials and Methods: In this study, the neural stem cells were isolated from SVZ sub-ventricular areas of adult mice at the age of 2 months. The cells were cultured in three distinct conditions: Control cells (Normally cultured in standard Petri Dishes), the cells cultured in plates covered by laminin, and those cultured over scaffold. In order to characterize the cells in terms of stemness and neuronal identity, the flow cytometry and immunocytochemistry were performed to detect the CD133 and Nestin markers, respectively. The cells were then grown over PCLF scaffold and their adhesion and cell-scaffold connections were assessed using electron microscopy. The MTT test and flow cytometry for Ki67 marker were conducted, respectively, to analyze the cell viability and proliferation following different time intervals (days 1, 2, 3, 7, 14, and 21). Ultimately, the neural stem cell differentiation toward mature neurons and glial cells was assessed using the Map2 and GFAP markers, respectively. Results: Our results confirmed that 80-90% of the cells isolated from sub-ventricular areas in adult mice were neural stem cells due to the high expression of CD133 and Nestin markers. Scanning electron microscopy revealed that the cells well interacted with the scaffold, which showed also a proper cell to cell adhesion over the scaffold. In the MTT analysis, we demonstrated that the cells covered with laminin, and particularly those cultured over the scaffold have greater proliferation rate compared to control group on days 7, 14, and 21 of growth. This was shown to be significantly different between the groups, suggesting that the scaffold could more importantly influence the cell activities following day 7, which were also verified by the Ki67 results. Interestingly, the marker analysis indicated that the cells grown over the scaffold showed a greater expression of Map2 and lower expression of GFAP while compared to the control group, confirming that higher percentage of the cells differentiate into mature neurons rather than astrocytes. These results propose that the PCLF scaffolds can play an influential role in the in vitro differentiation of neural stem cells toward mature neurons.