Functional engraftment of human ES cell–derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes

  • A Corrigendum to this article was published on 01 March 2007

Abstract

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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Figure 1: Dopaminergic neurons can be generated from HES cells.
Figure 2: hTERT-immortalized human midbrain glia potentiate dopaminergic induction by FGF8 + SHH.
Figure 3: 6-OHDA–lesioned rats given dopaminergic grafts exhibited functional benefit.
Figure 4: HES cell–derived progenitors dispersed and differentiated as TH+ neurons in lesioned striatum.
Figure 5: Dopaminergic differentiation by donor cells is associated with host astroglia.
Figure 6: HES cell–derived dopaminergic grafts exhibit persistent undifferentiated expansion.

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Acknowledgements

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.

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Contributions

N.S.R. generated the hMAST-TERT line with S.A.G., conducted the in vivo experiments done in S.A.G.'s laboratory and performed or supervised the in vitro and histological data analysis. C.C. conducted the transplantation and behavior studies with the H9 line, done in M.F.B.'s laboratory. S.K.S. conducted the transplant and behavior studies with the H1 line, done in S.A.G.'s laboratory. L.Y. conducted dopamine analysis by HPLC. M.F.B. supervised C.C. and L.Y. in their in vivo experiments. S.A.G. supervised the overall project, developed the experimental designs, performed the final data analysis and wrote the paper with N.S.R.

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Correspondence to Neeta S Roy or Steven A Goldman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Dopaminergic neurons may be induced at high efficiency from H1 HES cells. (PDF 131 kb)

Supplementary Fig. 2

Neuronal phenotypes in cultures of differentiated H9 and H1 cells consists of minor populations of GABAergic and serotonergic cells. (PDF 677 kb)

Supplementary Fig. 3

HES-derived dopaminergic neurons are of A9 phenotype and functionally active. (PDF 114 kb)

Supplementary Fig. 4

Animals transplanted with H1-derived cells show functional improvement as well as histological integration and differentiation of xenografted cells. (PDF 340 kb)

Supplementary Fig. 5

Transplanted naive HES cells rapidly generate teratomas. (PDF 207 kb)

Supplementary Methods (PDF 87 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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