Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF

Tyrosine hydroxylase (TH)-positive neurons were generated from human embryonic stem (hES) cells by coculturing on astrocytes or PA6 stromal cells. After 3 to 4 weeks in culture, TH-positive cells with neuronal morphology developed. Coculture with astrocytes from the embryonic striatum produced a larger number of TH-positive cells than did coculture with astrocytes from embryonic mesencephalon (329 +/- 149 versus 33 +/- 16 TH-positive cells per well, p < .05). In other experiments using PA6 cells as a substrate, glial-derived neurotrophic factor (GDNF) was added to the media of differentiating hES cells, and this led to a doubling of the number of TH-positive cells (PA6: 443 +/- 105 TH-positive cells per well versus PA6 + GDNF: 934 +/- 136, p < .05). We conclude that substrates of striatal astrocytes and PA6 cells can promote differentiation of human embryonic stem cells to a TH-positive phenotype and that GDNF can increase the number of cells expressing that phenotype.