Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson’s disease (PD), in which midbrain dopaminergic neurons progressively degenerate1,2. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.
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We thank K. Sekiguchi, J. Toga and E. Yagi for providing recombinant LM511-E8, Y. Ono for anti-CORIN and anti-NURR1 antibodies, H. Doi, A. Mawatari, M. Tsuji, K. Takahashi, M. Goto, Y. Wada, A. Yamazaki, T. Kawasaki, C. Takeda, N. Shibata, S. Kurai, A. Igesaka, T. Mori, R. Zochi, E. Hayashinaka, M. Yamano, T. Ose, M. Ohno and K. Onoe for supporting the PET study, H. Ohmori for an electrophysiological study, S. Nolbrant for discussions about gene expression by the donor cells, and Astellas Pharma Inc. for FK506. We also thank P. Karagiannis for reading of the manuscript, K. Kubota, Y. Ishii, Y. Morita and Y. Katano for technical assistance, K. Nishimura, M. Motono, Y. Ioroi, B. Samata, Y. Koshiba, Y. Nakajima and Y. Miyawaki for taking care of the animals, and S. Tsuji, J. Mitsui and S. Morishita for whole-exome analysis of patients with PD. This study was supported by grants from the Highway Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Network Program for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) and the Program for Intractable Diseases Research using disease-specific iPS cells from AMED (to H.I.). M.P. is a New York Stem Cell Foundation - Robertson Investigator.
The authors declare no competing financial interests.
Reviewer Information Nature thanks R. Barker, A. Björklund and F. Gage for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, The protocol for the induction of dopamine neuron progenitors. Y, Y-27632; BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; AA, ascorbic acid; dbcAMP, dibutyryladenosine cyclic monophosphate. b, Percentages of CORIN+ cells at day 12. Values are mean ± s.d. (n = 4 each for PD group and healthy group). c, Representative images of immunostaining for FOXA2, NURR1, TUJ1, PAX6, and SOX1 at day 26. Scale bar, 50 μm. d, e, Quantification of immunostaining for FOXA2 (d) and NURR1 (e) at day 26. Values are mean ± s.d. (n = 4 each for PD group and healthy group). f, A representative current-clamp recording of the action potentials induced by brief current pulses at day 70 (1231A3). g, A representative chromatogram from HPLC analysis at day 42 (1231A3). DOPAC, 3,4-dihydroxyphenylacetic acid. t-tests were performed in b, d, and e. There was no significant difference between healthy and PD groups.
a, Rating scale of PD model monkeys. b, PD scores of each monkey. c–f, The video analysis system. Schematic view of the video recording system (c), simplified illustration of the special cage for the video recording (d), a photo of the cage with the LED backlight switched on (e), and a representative capture from the video recording (f). g–j, Representative captures of the video analysis when the monkey’s number of movements was quantified as 5,316 (g) and 15,212 (i), and the moving time of each monkey analysed by video recording when the threshold was set to 5,000 (h) or 10,000 (j) pixels per 0.033 seconds. In h and j, values are shown relative to each pre-operative value, which was set to 1. k, l, Improvement of monkey PD scores (k) or fold change in spontaneous movement analysed by video recording (l) after administration of one-shot l-DOPA or transplantation. Horizontal bars designate the mean value. Two-tailed Wilcoxon matched-pairs signed rank-test was performed. *P < 0.05.
a, Estimated maximum volume of the grafts within 95% confidence upper limit analysed by a linear mixed effect model. b, Correlation between the graft volumes calculated from MRI and measured by histological analysis (n = 16). Data were compared using a two-tailed Pearson’s correlation analysis, and r and P values and linear regression lines are shown.
a, Representative tyrosine hydroxylase staining of each monkey. Scale bars, 5 mm. b, Representative magnified view of TH+ cells in each graft. Scale bars, 100 μm (left) and 50 μm (right).
a, Binding potential (BPnd) values of [11C]PE2I-PET. Lines show mean values (n = 3 for vehicle and PD groups, 4 for healthy group). b, BPnd values of [11C]PE2I-PET in each monkey. c, [18F]DOPA- and [11C]PE2I-PET of each monkey. Dotted white lines designate the putamen.
a, b, Correlation between the number of surviving TH+ cells and score improvement (a) and moving time analysed from the video recording (b). c, d, Correlation between tyrosine hydroxylase-innervated area and score improvement (c) and moving time (d). Two-tailed Pearson’s correlation analysis was performed, and r and P values are shown. Data for the healthy group are shown in blue, the PD group in red, and the vehicle group in black.
a, Uptake ratios of [11C]PK11195-PET and S-[11C]KTP-Me-PET in representative monkeys. Dotted white lines designate the putamen. b, c, Ratio of standardized uptake values of [11C]PK11195-PET (b) and S-[11C] KTP-Me-PET (c). d, Representative images of MHC class II, CD45, and monkey IgG staining of monkey number 9. Dotted lines designate the grafted area. Scale bar, 5 mm for the left side of MHC class II, CD45, and monkey IgG, and 50 μm for the right side of MHC class II.
a, Principal component analysis of the transplanted cells (healthy group in blue, PD group in red), two iPS cells (836B3 and 1231A3, black), fetal ventral midbrain tissue (fVM, green), fetal dorsal midbrain tissue (fDM, green), adult whole brain tissue (WB, navy), and adult substantia nigra tissue (SN, navy). b, Gene list obtained from the microarray analysis. c, Quantitative PCR analysis of the transplanted cells. Values are expressed as relative quantity (RQ).
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Kikuchi, T., Morizane, A., Doi, D. et al. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model. Nature 548, 592–596 (2017). https://doi.org/10.1038/nature23664
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