In Vitro Monolayer Culture of Dispersed Neural Stem Cells on the E-Cadherin-Based Substrate with Long-Term Stemness Maintenance.

Neural stem cells (NSCs) play an important role in neural tissue engineering because of their capacity of self-renewal and differentiation to multiple cell lineages. The in vitro conventional neurosphere culture protocol has some limitations such as limited nutrition and oxygen penetration and distribution causing the heterogeneity of cells inside, inaccessibility of internal cells, and inhomogeneous cellular morphology and properties. As a result, cultivation as a monolayer is a better way to study NSCs and obtain a homogeneous cell population. The cadherins are a classical family of homophilic cell adhesion molecules mediating cell-cell adhesion. Here, we used a recombinant human E-cadherin mouse IgG Fc chimera protein that self-assembles on a hydrophobic polystyrene surface via hydrophobic interaction to obtain an E-cadherin-coated culture plate (ECP). The rat fetal NSCs were cultured on the ECP and routine tissue culture plate (TCP) from passage 2 to passage 5. NSCs on TCP formed uniform floating neurospheres and grew up over time, while cells on the ECP adhered on the bottom of the plate and exhibited individual cells with scattering morphology, forming intercellular connections between cells. The cell proliferation and differentiation behaviors that were evaluated by Cell Counting Kit-8 assay (CCK-8), immunofluorescence staining, and real-time quantitative polymerase chain reaction showed NSCs could maintain the capacity for self-renewal and ability to differentiate into neurons, oligodendrocytes, and astrocytes after the long-term in vitro cell culture and passaging. Therefore, our study indicated that hE-cad-Fc could provide a homogeneous environment for individual cells in monolayer conditions to maintain the capacity of self-renewal and differentiation by mimicking the cell-cell interaction.