Collection of neural inducing factors from PA6 cells using heparin solution and their immobilization on plastic culture dishes for the induction of neurons from embryonic stem cells

Embryonic stem (ES) cells have the ability to replicate themselves and differentiate into various mature cells. Recently, dopaminergic neurons were efficiently induced from ES cells using mouse stromal cells (PA6 cells) as a feeder cell layer. This simple procedure seems to be very efficient to obtain dopamine-releasing cells for future clinical cell transplantation treatment of Parkinsonʹs disease. In this study, we prepared stock solutions containing neural inducing factors (NIFs) by washing PA6 cells with phosphate-buffered saline containing heparin. ES cells grew successfully in culture media supplemented with 33 v/v% NIFs stock solution, and the rate of neural differentiation of ES cell progeny increased with increasing heparin concentration in the culture media. In addition, NIFs-immobilized surfaces were prepared by exposing polyethyleneimine-modified surfaces to NIFs stock solutions. The NIFs-immobilized culture dish effectively supported cell growth as the culture medium supplemented with NIFs stock did, but its induction effect to dopaminergic neurons from ES cells was much smaller than free NIFs. NIFs stock solutions have two different activities. One can stimulate cell growth and the other induces differentiation of ES cells to the neural fate when heparin existed. The former factors were effectively immobilized on the culture dish, but those that induce differentiation may not be. Further optimization is required.