Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model

Journal name:
Nature
Volume:
548,
Pages:
592–596
Date published:
DOI:
doi:10.1038/nature23664
Received
Accepted
Published online

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.

At a glance

Figures

  1. Behavioural analysis of monkeys.
    Figure 1: Behavioural analysis of monkeys.

    a, b, Neurological scores (a) and score improvements (b) of the monkeys. Vehicle, monkeys that received control injections of vehicle; PD, monkeys transplanted with cells derived from patients with PD; Healthy, monkeys transplanted with cells derived from healthy individuals. Lines show mean values (n = 3 for vehicle and PD groups, 4 for healthy group). Two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was performed; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 versus vehicle group. c, d, Linear regression analysis of moving time of monkeys analysed with video recordings when the threshold was set to 5,000 (c) or 10,000 (d) pixels per 0.033 seconds. Values are shown relative to each pre-operative value, which was set to 1 (n = 3 for vehicle group, 7 for transplanted group). The P and r2 values and regression equations are 0.0016, 0.33 and y = 0.17x + 0.88 (c, transplanted), 0.44, 0.067 and y = 0.052x + 0.85 (c, vehicle), 0.0007, 0.37 and y = 0.85x − 0.31 (d, transplanted) and 0.18, 0.051 and y = 0.23x + 1.3 (d, vehicle), respectively. All Source Data for the graphs are provided with the online version of the paper.

  2. Growth and survival of iPS cell-derived dopaminergic neuron progenitors in vivo.
    Figure 2: Growth and survival of iPS cell-derived dopaminergic neuron progenitors in vivo.

    a, Representative MRI of a cell-transplanted monkey (number 11, 12 months after transplantation). Arrowheads indicate grafts. b, Graft sizes estimated from MRI. c, Graft sizes at the time of euthanasia. d, Representative haematoxylin–eosin staining of brain tissue from monkey 1. The box in each image outlines the area magnified to the right. Dotted white lines designate the graft. Scale bars, 5 mm, 1 mm, 200 μm and 50 μm from left to right. e, Representative image of double staining for STEM121 and Ki-67+. f, g, Magnified view of the graft (f) and ventricular wall (g, positive control) in e. Scale bars, 2 mm (e), 50 μm (f) and 10 μm (g). h, A representative image obtained by [18F]FLT-PET. Dotted white lines designate the putamen.

  3. Characteristics of surviving TH+ cells.
    Figure 3: Characteristics of surviving TH+ cells.

    a, b, Successive images of tyrosine hydroxylase staining of monkeys 10 (PD group, a) and 3 (vehicle group, b). Scale bars, 5 mm. c, Percentage of area in which TH+ fibres are innervated in caudate nuclei, putamen, and whole striatum of each monkey. d, Magnified view of TH+ neurons in the graft (left) and substantia nigra (right). Scale bar, 50 μm. e, Representative immunostaining images for FOXA2 and tyrosine hydroxylase. Scale bars, 200 μm (left image) and 50 μm (three images to the right). f, The number of TH+ cells and FOXA2+ cells surviving in each monkey. g, h, Representative images of double staining for DAT and G-protein-activated inward rectifier potassium channel 2 (GIRK2, h). Scale bars, 50 μm. i, Time course of MRI and PET of a representative monkey (number 1). Dotted white lines designate the putamen. m, months; w, weeks. j, Ki values obtained from [18F]DOPA-PET. Lines show mean values (n = 3 for vehicle and PD groups, 4 for healthy group). Two-way ANOVA with Dunnett’s multiple comparisons test was performed. *P < 0.05 versus vehicle group. k, Ki values obtained from [18F]DOPA-PET in each monkey. Dotted lines in j and k designate the average Ki value for two intact monkeys. l, Correlation between surviving TH+ cells and Ki values from [18F]DOPA-PET. Two-tailed Pearson’s correlation analysis was performed, and r and P values are shown. A linear regression line is also shown. Data for the healthy group are shown in blue, and the PD group in red.

  4. In vitro analysis of dopaminergic neuron progenitors.
    Extended Data Fig. 1: In vitro analysis of dopaminergic neuron progenitors.

    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.

  5. Behavioural analysis of monkeys.
    Extended Data Fig. 2: Behavioural analysis of monkeys.

    a, Rating scale of PD model monkeys. b, PD scores of each monkey. cf, 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). gj, 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.

  6. Estimation of graft growth.
    Extended Data Fig. 3: Estimation of graft growth.

    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.

  7. Tyrosine hydroxylase histology of monkeys.
    Extended Data Fig. 4: Tyrosine hydroxylase histology of monkeys.

    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).

  8. [18F]DOPA- and [11C]PE2I-PET of monkeys.
    Extended Data Fig. 5: [18F]DOPA- and [11C]PE2I-PET of monkeys.

    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.

  9. Correlation between surviving TH+ cells and functional recovery.
    Extended Data Fig. 6: Correlation between surviving TH+ cells and functional recovery.

    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.

  10. Inflammation in the brains of cell-transplanted monkeys.
    Extended Data Fig. 7: Inflammation in the brains of cell-transplanted monkeys.

    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.

  11. Representative MHC class II staining of each monkey.
    Extended Data Fig. 8: Representative MHC class II staining of each monkey.

    Scale bars, 5 mm.

  12. Gene expression analysis of the transplanted cells.
    Extended Data Fig. 9: Gene expression analysis of the transplanted cells.

    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).

Tables

  1. Blood concentration of FK506 in each monkey (ng ml−1)
    Extended Data Table 1: Blood concentration of FK506 in each monkey (ng ml−1)

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan

    • Tetsuhiro Kikuchi,
    • Asuka Morizane,
    • Daisuke Doi,
    • Hiroaki Magotani &
    • Jun Takahashi
  2. Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe 650-0047, Japan

    • Hirotaka Onoe,
    • Takuya Hayashi,
    • Hiroshi Mizuma &
    • Sayuki Takara
  3. Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

    • Ryosuke Takahashi
  4. Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan

    • Haruhisa Inoue
  5. Department of Biomedical Statistics and Bioinformatics, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

    • Satoshi Morita &
    • Michio Yamamoto
  6. Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan

    • Keisuke Okita &
    • Masato Nakagawa
  7. Wallenberg Neuroscience Center and Lund Stem Cell Center, Lund University, 22184 Lund, Sweden

    • Malin Parmar
  8. Department of Neurosurgery, Clinical Neuroscience, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

    • Jun Takahashi

Contributions

T.K. designed the study, performed the culture, transplantation, MRI study, data analysis and interpretation, and wrote the manuscript. A.M. and D.D. assisted with cell culture, cell sorting, transplantation and the MRI study. H.Ma. generated the PD model monkeys and performed the behavioural analysis. H.O., T.H., H.Mi. and S.T. performed the PET imaging and corresponding analysis and interpretation. R.T., H.I., K.O. and M.N. generated the iPS cells. S.M. and M.Y. performed statistical analyses. M.P. provided fetal mesencephalic samples and discussed the gene expression analyses. J.T. conceived and designed the study, assembled the data, carried out the data analysis and interpretation, wrote the manuscript, and gave final approval of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks R. Barker, A. Björklund and F. Gage for their contribution to the peer review of this work.

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Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: In vitro analysis of dopaminergic neuron progenitors. (438 KB)

    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.

  2. Extended Data Figure 2: Behavioural analysis of monkeys. (607 KB)

    a, Rating scale of PD model monkeys. b, PD scores of each monkey. cf, 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). gj, 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.

  3. Extended Data Figure 3: Estimation of graft growth. (178 KB)

    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.

  4. Extended Data Figure 4: Tyrosine hydroxylase histology of monkeys. (652 KB)

    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).

  5. Extended Data Figure 5: [18F]DOPA- and [11C]PE2I-PET of monkeys. (820 KB)

    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.

  6. Extended Data Figure 6: Correlation between surviving TH+ cells and functional recovery. (93 KB)

    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.

  7. Extended Data Figure 7: Inflammation in the brains of cell-transplanted monkeys. (495 KB)

    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.

  8. Extended Data Figure 8: Representative MHC class II staining of each monkey. (413 KB)

    Scale bars, 5 mm.

  9. Extended Data Figure 9: Gene expression analysis of the transplanted cells. (234 KB)

    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).

Extended Data Tables

  1. Extended Data Table 1: Blood concentration of FK506 in each monkey (ng ml−1) (100 KB)

Supplementary information

Additional data