To direct human embryonic stem (HES) cells to a dopaminergic neuronal fate, we cocultured HES cells that were exposed to both sonic hedgehog and fibroblast growth factor 8 with telomerase-immortalized human fetal midbrain astrocytes. These astrocytes substantially potentiated dopaminergic neurogenesis by both WA09 and WA01 HES cells, biasing them to the A9 nigrostriatal phenotype. When transplanted into the neostriata of 6-hydroxydopamine–lesioned parkinsonian rats, the dopaminergic implants yielded a significant, substantial and long-lasting restitution of motor function. However, although rich in donor-derived tyrosine hydroxylase–expressing neurons, the grafts exhibited expanding cores of undifferentiated mitotic neuroepithelial cells, which can be tumorigenic. These results show the utility of recreating the cellular environment of the developing human midbrain while driving dopaminergic neurogenesis from HES cells, and they demonstrate the potential of the resultant cells to mediate substantial functional recovery in a model of Parkinson disease. Yet these data also mandate caution in the clinical application of HES cell–derived grafts, given their potential for phenotypic instability and undifferentiated expansion.
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We thank B. Germin, J. Powers and M. Edgar for their neuropathological evaluations and H.M. Keyoung for advice. We are grateful to the late W.K. Rashbaum for his obstetric assistance in providing fetal tissue samples for this research. We are also grateful to Melissa Carpenter and Geron Corp. for providing the H9 cell line and retroviral hTERT used in this study, Curis for sonic hedgehog protein, and G. Corte (National Institute of Cancer Research, Genova, Italy) for the antibody to Otx2. Work in the Goldman laboratory was supported by NINDS and the Michael J. Fox Foundation, and work in the Beal laboratory was supported by the US Department of Defense and the Michael J. Fox Foundation.
The authors declare no competing financial interests.
Dopaminergic neurons may be induced at high efficiency from H1 HES cells. (PDF 131 kb)
Neuronal phenotypes in cultures of differentiated H9 and H1 cells consists of minor populations of GABAergic and serotonergic cells. (PDF 677 kb)
HES-derived dopaminergic neurons are of A9 phenotype and functionally active. (PDF 114 kb)
Animals transplanted with H1-derived cells show functional improvement as well as histological integration and differentiation of xenografted cells. (PDF 340 kb)
Transplanted naive HES cells rapidly generate teratomas. (PDF 207 kb)
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Roy, N., Cleren, C., Singh, S. et al. Functional engraftment of human ES cell–derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12, 1259–1268 (2006). https://doi.org/10.1038/nm1495
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