Induction dopamine releasing cells from mouse embryonic stem cells and their long‐term culture

Abstract

Cell transplantation therapy using dopaminergic neurons derived from embryonic stem (ES) cells for the treatment of Parkinson's disease has been proposed as one of the major applications for stem cell-based therapy. However, the low collection efficiency of neurons from a culture dish and the rejection of cells after transplantation are expected to limit their future clinical applications. To overcome these problems, we examined the induction of neurogenesis of ES cells under free-floating conditions and microencapsulation of the obtained cell aggregates into an agarose hydrogel. Cell aggregates from ES cells were cultured in various media under the free-floating condition. Immunohistochemical staining for tyrosine hydroxylase (TH) and RT-PCR analyses for TH and Nurr1 showed that dopaminergic neurons were induced in ES cell aggregates cultured in a 1:2 mixture of conditioned medium of PA6 stromal cells and Glasgow minimum essential medium (GMEM) after 16 days in culture. The cell aggregates could be collected and were encased within agarose microcapsules without loss of dopaminergic neurons. The cell aggregates with/without microencapsulation were maintained in CM/GMEM for an additional period. KCl stimulation assays were done at day 23, 30, 37, 44, 51, and 58 to examine dopamine release. Dopamine release abilities were well maintained during 58 days of observation. Amounts of dopamine release from encapsulated cell aggregates were slightly higher than those of unencapsulated cell aggregates from day 16 to 58. Although efficacy for immunoisolation of the agarose microcapsules still remains for future in vivo studies, microencapsulation did not adversely affect viability and functions of the dopamine releasing ES cell progeny.