Selection of embryonic stem cell derived eGFP+ dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations

¹ Udall Parkinson's Disease Research Center for Excellence, McLean Hospital/Harvard Medical School, Belmont, Massachusetts; Molecular Neurobiology Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts; Neuroregeneration Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts
² Udall Parkinson's Disease Research Center for Excellence, McLean Hospital/Harvard Medical School, Belmont, Massachusetts; Neuroregeneration Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts
³ Udall Parkinson's Disease Research Center for Excellence, McLean Hospital/Harvard Medical School, Belmont, Massachusetts; Molecular Neurobiology Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts

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

	Abstract

Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson's disease models, but can generate teratomas. Purification of dopamine neurons derived from ES cells by fluorescence activated cell sorting (FACS) could provide a functional cell population for transplantation, while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive eGFP expression in mES cells. First, we evaluated 2.5 kb and 9 kb TH promoter fragments and showed that clones generated using the 9 kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9 kb TH clone with highest eGFP/TH overlap for further differentiation, FACS and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were SSEA-1+ and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1 we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.

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