Stromal cell-derived inducing activity, Nurr1 and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells

¹ Molecular Neurobiology Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts
² Neuroregeneration Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts
³ Department of Physiology, Yonsei University College of Medicine, Seoul, Korea
⁴ Departments of Neurology and Pharmacology, Physiology, & Neurobiology, The University of Chicago, Chicago, Illinois

^* To whom correspondence should be addressed. E-mail: kskim{at}mclean.harvard.edu.

	Abstract

To induce differentiation of embryonic stem (ES) cells into specialized cell-types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ES cells by overexpressing the transcription factor Nurr1 and co-culturing with PA6 stromal cells. Nurr1-expressing ES cells (N2 and N5) differentiated into higher number of neurons (2-fold) than the naïve ES cells (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)-positive neurons than D3 (<30%) and even greater proportion of TH-positive neurons (90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8 and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K⁺ channel 2) and produce and release a higher level of dopamine, compared to D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH⁺ neurons after implantation into mouse striatum. Taken together, our results show that combination of genetic manipulation(s) and in vitro cell differentiation conditions offer a reliable and effective induction of DA neurons from ES cells and may pave the way for future cell transplantation therapy in Parkinson's disease.