Surface Coating Effects on Rat Pulp Cells Neurodifferentiation In Vitro

Earlier we developed a three-step protocol for neural differentiation of human dental pulp cells. However, the method is not completely applicable for rat dental pulp stem cells (rDPSC). As cell-to-extracellular matrix attachment plays a role in differentiation, coating might be an important element of protocols. Our aim was to find the parameters for optimal isolation, cultivation and differentiation of rDPSC cultures.

Methods:

Cells were isolated from lower incisors of young rats, and cultivated under standard conditions. Two different digestion mixtures and different media were used for isolation. Neural differentiation was performed as we reported before (Király et al. Neurochem Int. 55(5):323-32, 2009), the 3rd step was divided into two stages. Cells were differentiated on 5 different coatings: fetal calf serum (FCS), poly-L-lysine (PLL), poly-L-lysine/laminin (LYLA), ornithine/laminin (ORLA), laminin (LA), and as control, on non-tissue culture (NTC) surface. Neural marker expression was studied with Q-PCR.

Results:

Isolated cells attached to the surface within 24 hours, displaying similar morphologies in different media, forming cobble-stone patterns. Cell attachment was not surface dependent. During pretreatment cells formed ridges on FCS coating, but spread out on other surfaces. During induction, the proportion of cells with neural morphology increased, but during the first maturation stage it decreased. By the second maturation stage the cells had typically displayed either a large epitheloid or a smaller fibroblast-like morphology. On LYLA, ORLA and PLL surfaces neuronal-like cells were observed. At the end of differentiation the expression of late neural markers such as NFM and NSE increased 6x and 2x, respectively, on LYLA and ORLA surfaces, while N-tub and Nestin decreased compared to NTC.

Conclusions:

Our data suggest that during neuronal transdifferentiation of rDPSC cultures surface coating by combination of poly-L-lysine, laminin and ornithine considerably enhance the manifestation of neuronal phenotype.