Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Co-transplantation of autologous Treg cells in a cell therapy for Parkinson’s disease

Nature volume 619, pages 606–615 (2023)Cite this article

Subjects

Abstract

The specific loss of midbrain dopamine neurons (mDANs) causes major motor dysfunction in Parkinson’s disease, which makes cell replacement a promising therapeutic approach1,2,3,4. However, poor survival of grafted mDANs remains an obstacle to successful clinical outcomes5,6,7,8. Here we show that the surgical procedure itself (referred to here as ‘needle trauma’) triggers a profound host response that is characterized by acute neuroinflammation, robust infiltration of peripheral immune cells and brain cell death. When midbrain dopamine (mDA) cells derived from human induced pluripotent stem (iPS) cells were transplanted into the rodent striatum, less than 10% of implanted tyrosine hydroxylase (TH)+ mDANs survived at two weeks after transplantation. By contrast, TH− grafted cells mostly survived. Notably, transplantation of autologous regulatory T (Treg) cells greatly modified the response to needle trauma, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, intra-striatal co-transplantation of Treg cells and human-iPS-cell-derived mDA cells significantly protected grafted mDANs from needle-trauma-associated death and improved therapeutic outcomes in rodent models of Parkinson’s disease with 6-hydroxydopamine lesions. Co-transplantation with Treg cells also suppressed the undesirable proliferation of TH− grafted cells, resulting in more compact grafts with a higher proportion and higher absolute numbers of TH+ neurons. Together, these data emphasize the importance of the initial inflammatory response to surgical injury in the differential survival of cellular components of the graft, and suggest that co-transplanting autologous Treg cells effectively reduces the needle-trauma-induced death of mDANs, providing a potential strategy to achieve better clinical outcomes for cell therapy in Parkinson’s disease.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Influence of needle-trauma-induced neuroinflammation on transplanted cells.
Fig. 2: Pro-inflammatory-cytokine-induced immunogenicity in transplanted C4-mDAPs.
Fig. 3: Pro-inflammatory-cytokine-induced cell death of C4-mDAPs.
Fig. 4: Effects of Treg co-transplantation with C4-mDAPs in a xenogeneic model of PD.
Fig. 5: In vivo effects of Treg co-transplantation with C4-mDAPs.

Data availability

All data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Parmar, M., Grealish, S. & Henchcliffe, C. The future of stem cell therapies for Parkinson disease. Nat. Rev. Neurosci. 21, 103–115 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Sonntag, K. C. et al. Pluripotent stem cell-based therapy for Parkinson’s disease: current status and future prospects. Prog. Neurobiol. 168, 1–20 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Barker, R. A., Drouin-Ouellet, J. & Parmar, M. Cell-based therapies for Parkinson disease—past insights and future potential. Nat. Rev. Neurol. 11, 492–503 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Lindvall, O. Clinical translation of stem cell transplantation in Parkinson’s disease. J. Intern. Med. 279, 30–40 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Brundin, P. et al. Improving the survival of grafted dopaminergic neurons: a review over current approaches. Cell Transplant. 9, 179–195 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Wenker, S. D. & Pitossi, F. J. Cell therapy for Parkinson’s disease is coming of age: current challenges and future prospects with a focus on immunomodulation. Gene Ther. 27, 6–14 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Barker, R. A., Dunnett, S. B., Faissner, A. & Fawcett, J. W. The time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesencephalon to the striatum. Exp. Neurol. 141, 79–93 (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Emgard, M., Karlsson, J., Hansson, O. & Brundin, P. Patterns of cell death and dopaminergic neuron survival in intrastriatal nigral grafts. Exp. Neurol. 160, 279–288 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Tao, Y. et al. Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys. Nat. Med. 27, 632–639 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hallett, P. J. et al. Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson’s disease. Cell Stem Cell 16, 269–274 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Emborg, M. E. et al. Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain. Cell Rep. 3, 646–650 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Morizane, A. et al. MHC matching improves engraftment of iPSC-derived neurons in non-human primates. Nat. Commun. 8, 385 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  13. Schweitzer, J. S. et al. Personalized iPSC-derived dopamine progenitor cells for Parkinson’s disease. N. Engl. J. Med. 382, 1926–1932 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hiller, B. M. et al. Optimizing maturity and dose of iPSC-derived dopamine progenitor cell therapy for Parkinson’s disease. NPJ Regen. Med. 7, 24 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Piao, J. et al. Preclinical efficacy and safety of a human embryonic stem cell-derived midbrain dopamine progenitor product, MSK-DA01. Cell Stem Cell 28, 217–229 (2021).

    Article  MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  16. Josefowicz, S. Z., Lu, L. F. & Rudensky, A. Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Wood, K. J. & Sakaguchi, S. Regulatory T cells in transplantation tolerance. Nat. Rev. Immunol. 3, 199–210 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Song, B. et al. Human autologous iPSC-derived dopaminergic progenitors restore motor function in Parkinson’s disease models. J. Clin. Invest. 130, 904–920 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kim, J. et al. Spotting-based differentiation of functional dopaminergic progenitors from human pluripotent stem cells. Nat. Protoc. 17, 890–909 (2022).

    Article  MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kriks, S. et al. Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Doi, D. et al. Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson’s disease. Nat. Commun. 11, 3369 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Alam, A. et al. Cellular infiltration in traumatic brain injury. J. Neuroinflammation 17, 328 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Karve, I. P., Taylor, J. M. & Crack, P. J. The contribution of astrocytes and microglia to traumatic brain injury. Br. J. Pharmacol. 173, 692–702 (2016).

    Article  CAS  PubMed  Google Scholar 

  24. Duggleby, R., Danby, R. D., Madrigal, J. A. & Saudemont, A. Clinical grade regulatory CD4+ T cells (Tregs): moving toward cellular-based immunomodulatory therapies. Front. Immunol. 9, 252 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Romano, M., Fanelli, G., Albany, C. J., Giganti, G. & Lombardi, G. Past, present, and future of regulatory T cell therapy in transplantation and autoimmunity. Front. Immunol. 10, 43 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ozaki, M. et al. Evaluation of the immunogenicity of human iPS cell-derived neural stem/progenitor cells in vitro. Stem Cell Res. 19, 128–138 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Yamasaki, S. et al. Low immunogenicity and immunosuppressive properties of human ESC- and iPSC-derived retinas. Stem Cell Rep. 16, 851–867 (2021).

    Article  CAS  Google Scholar 

  28. Zhao, T., Zhang, Z. N., Rong, Z. & Xu, Y. Immunogenicity of induced pluripotent stem cells. Nature 474, 212–215 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Zhao, T. et al. Humanized mice reveal differential immunogenicity of cells derived from autologous induced pluripotent stem cells. Cell Stem Cell 17, 353–359 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Falk, S. et al. Brain area-specific effect of TGF-β signaling on Wnt-dependent neural stem cell expansion. Cell Stem Cell 2, 472–483 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Izsak, J. et al. TGF-β1 suppresses proliferation and induces differentiation in human iPSC neural in vitro models. Front. Cell Dev. Biol. 8, 571332 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Tiklova, K. et al. Single cell transcriptomics identifies stem cell-derived graft composition in a model of Parkinson’s disease. Nat. Commun. 11, 2434 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Xu, P. et al. Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson’s disease model. J. Clin. Invest. 132, e156768 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hess, N. J., Brown, M. E. & Capitini, C. M. GVHD pathogenesis, prevention and treatment: lessons from humanized mouse transplant models. Front. Immunol. 12, 723544 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Becker, C. et al. Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells. Blood 114, 1263–1269 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Finger, S. & Dunnett, S. B. Nimodipine enhances growth and vascularization of neural grafts. Exp. Neurol. 104, 1–9 (1989).

    Article  CAS  PubMed  Google Scholar 

  37. Nakao, N., Frodl, E. M., Duan, W. M., Widner, H. & Brundin, P. Lazaroids improve the survival of grafted rat embryonic dopamine neurons. Proc. Natl Acad. Sci. USA 91, 12408–12412 (1994).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Schierle, G. S. et al. Caspase inhibition reduces apoptosis and increases survival of nigral transplants. Nat. Med. 5, 97–100 (1999).

    Article  CAS  PubMed  Google Scholar 

  39. Sinclair, S. R. et al. GDNF enhances dopaminergic cell survival and fibre outgrowth in embryonic nigral grafts. Neuroreport 7, 2547–2552 (1996).

    Article  CAS  PubMed  Google Scholar 

  40. Takayama, H. et al. Basic fibroblast growth factor increases dopaminergic graft survival and function in a rat model of Parkinson’s disease. Nat. Med. 1, 53–58 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. McKee, C. A. & Lukens, J. R. Emerging roles for the immune system in traumatic brain injury. Front. Immunol. 7, 556 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Bai, R. et al. Long-term kinetics of immunologic components and neurological deficits in rats following repetitive mild traumatic brain injury. Med. Sci. Monit. 23, 1707–1718 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chung, S. et al. Neural precursors derived from embryonic stem cells, but not those from fetal ventral mesencephalon, maintain the potential to differentiate into dopaminergic neurons after expansion in vitro. Stem Cells 24, 1583–1593 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Chung, S. et al. ES cell-derived renewable and functional midbrain dopaminergic progenitors. Proc. Natl Acad. Sci. USA 108, 9703–9708 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Caplan, H. W. et al. Combination therapy with Treg and mesenchymal stromal cells enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy. Stem Cells 39, 358–370 (2021).

    Article  CAS  PubMed  Google Scholar 

  46. Krieglstein, K., Suter-Crazzolara, C., Fischer, W. H. & Unsicker, K. TGF-β superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J. 14, 736–742 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Luo, S. X. et al. TGF-β signaling in dopaminergic neurons regulates dendritic growth, excitatory-inhibitory synaptic balance, and reversal learning. Cell Rep. 17, 3233–3245 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhou, X., Zoller, T., Krieglstein, K. & Spittau, B. TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity. J. Neurochem. 134, 125–134 (2015).

    Article  CAS  PubMed  Google Scholar 

  49. Huang, Y., Liu, Z., Cao, B. B., Qiu, Y. H. & Peng, Y. P. Treg cells protect dopaminergic neurons against MPP+ neurotoxicity via CD47–SIRPA interaction. Cell. Physiol. Biochem. 41, 1240–1254 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Huang, Y., Liu, Z., Cao, B. B., Qiu, Y. H. & Peng, Y. P. Treg cells attenuate neuroinflammation and protect neurons in a mouse model of Parkinson’s disease. J. Neuroimmune Pharmacol. 15, 224–237 (2020).

    Article  PubMed  Google Scholar 

  51. Liu, D. et al. Impact of schizophrenia GWAS loci converge onto distinct pathways in cortical interneurons vs glutamatergic neurons during development. Mol. Psychiatry 27, 4218–4233 (2022).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all members of the Molecular Neurobiology Laboratory for discussion. This work was supported by NIH grants (NS127391 and OD024622), the Parkinson’s Cell Therapy Research Fund at McLean Hospital and Massachusetts General Hospital, the Masson Family Endowed Scholar in Neurosurgery and the George A. Lopez, MD Endowed Chair in Neurosurgery. T.M.H. has received support from the NIH NINDS grant K23NS099380.

Author information

Author notes

  1. These authors contributed equally: Tae-Yoon Park, Jeha Jeon

Authors and Affiliations

  1. Molecular Neurobiology Laboratory, Department of Psychiatry and McLean Hospital, Harvard Medical School, Belmont, MA, USA

    Tae-Yoon Park, Jeha Jeon, Nayeon Lee, Jisun Kim, Bin Song, Dongxin Liu, Young Cha, Minseon Kim, Pierre Leblanc & Kwang-Soo Kim

  2. Program in Neuroscience, Harvard Medical School, Belmont, MA, USA

    Tae-Yoon Park, Jeha Jeon, Nayeon Lee, Jisun Kim, Bin Song, Dongxin Liu, Young Cha, Minseon Kim, Pierre Leblanc & Kwang-Soo Kim

  3. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

    Jung-Ho Kim & Sang-Kyou Lee

  4. Good T Cells, Inc., Seoul, Republic of Korea

    Sang-Kyou Lee

  5. Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Todd M. Herrington

  6. Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Bob S. Carter, Jeffrey S. Schweitzer & Kwang-Soo Kim

  7. Harvard Stem Cell Institute, Harvard Medical School, Belmont, MA, USA

    Kwang-Soo Kim

Authors
  1. Tae-Yoon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeha Jeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nayeon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jisun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bin Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jung-Ho Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sang-Kyou Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dongxin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Young Cha
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Minseon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Pierre Leblanc
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Todd M. Herrington
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Bob S. Carter
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Jeffrey S. Schweitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Kwang-Soo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.-S.K. and T.-Y.P. conceived the study. T.-Y.P., J.J. and N.L. designed experiments. T.-Y.P. performed in vitro and in vivo experiments, including generating humanized and PD model mice, and most of the data preparation. J.J. performed animal surgeries and behavioural analyses. N.L., Y.C. and M.K. performed the differentiation of C4-, B1- and H9-mDAPs and D.L. performed the differentiation of C4-, B1- and H9-GABAergic cells, including immunocytochemistry staining. Y.C. performed the reprogramming of C4- and B1-iPS cells. T.-Y.P., J.-H.K. and S.-K.L. performed the isolation of T cells and Treg cells and functional assays. T.-Y.P., J.J., P.L. and J.S.S. performed the humanized mice study. T.-Y.P., N.L. and J.K. performed MLR-like co-culture assays, TUNEL assays, ELISA and qPCR. T.-Y.P., N.L., B.S. and M.K. performed the cytokine-induced cell death experiment. T.-Y.P., J.-H.K., S.-K.L. and P.L. performed the FACS analysis of mDAPs and Treg cells. M.K. performed the western blots. T.-Y.P., N.L., J.K., Y.C., M.K. and D.L. performed the immunocytochemistry, immunohistochemistry and immunofluorescence staining. T.-Y.P., N.L., J.K., M.K. and D.L. performed stereological cell counting. P.L., T.M.H., B.S.C. and J.S.S. contributed to data analyses, interpretation and editing the manuscript. All authors reviewed and discussed the manuscript. K.-S.K. and T.-Y.P. wrote the paper and K.-S.K. supervised all aspects of the work and approved the final manuscript.

Corresponding author

Correspondence to Kwang-Soo Kim.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Agnete Kirkeby, Asuka Morizane, Qizhi Tang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Influence of needle-trauma-induced neuroinflammation on transplanted post-mitotic neurons.

a–e, NSG mice were killed 14 days after intra-striatal transplantation of B1/H9-mDAPs. The total number and percentage of TH+ mDANs were determined by immunofluorescence staining with anti-TH/Hoechst 33342 before transplantation (a,c,e) or immunohistochemistry staining with anti-TH/hNUCLEI antibodies after transplantation (b,d,e). (n = 4 per group). f–i, NSG mice were killed 14 days after intra-striatal transplantation of C4-iPS cell/B1-iPS cell/H9-ES cell-derived GABAergic progenitors. The total number and percentage of C4- (f), B1- (g) or H9- (h) GABAergic neurons were determined by immunofluorescence staining with anti-VGAT/Hoechst 33342 before transplantation and anti-VGAT/NKX2.1/hNUCLEI antibodies after transplantation (i). (n = 4 per group). Each error bar represents mean ± s.e.m.

Source data

Extended Data Fig. 2 Early time-dependent analysis of inflammatory responses to the needle trauma.

a–d, TP medium was injected into the striatum of Fischer 344 rats that were killed on days 1, 2, 3, 5 and 7 after surgery. The level of needle-trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-TNF (a) and IL-1β (b) antibodies, and staining is quantified in a′ and b′. c, Astrocytes were stained with anti-GFAP antibody, and staining is quantified in c′. Representative images and quantitative analysis of IBA1+ cells (yellow box: brain-resident microglia, red box: infiltrated IBA1+ cells) (d, d′) after injection. (n = 5 per group). e–g, Inhibition of needle trauma-induced host inflammatory response and cell death using Treg cells. Fischer 344 rats were killed 2 or 7 days after intra-striatal co-transplantation of TP medium with or without autologous Treg cells from each rat (20,000 cells per rat). The level of needle-trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-TNF (e,e′) and -IL-1β (f,f′) antibodies. (Two-tailed unpaired t-test; n = 3 per group). g,g′, The level of needle-trauma-induced neuronal cell death was determined by immunohistochemical staining with anti-NeuN antibody. (Two-tailed unpaired t-test; n = 5 per group). Each error bar represents mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 3 Long-term time-dependent analysis of cellular responses to the needle trauma.

a–d, TP medium was injected into the striatum of Fischer 344 rats and rats were killed on days 4, 7 and 14 and 1, 3 and 6 months after surgery. The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-IBA1 (a) and -MHCII (b) antibodies. c, The level of needle-trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-IFNγ antibody. d, The level of cell death by needle trauma was analysed by TUNEL assay. (n = 5 per group). The mean value was connected by a red line. Each error bar represents mean ± s.e.m.

Source data

Extended Data Fig. 4 Treg cell function after ex vivo expansion.

a,b, The levels of sorted CD4+CD25+FOXP3+ T cells (nTreg) (a) and ex-vivo-expanded rat CD4+CD25+FOXP3+ T cells (expanded Treg) (b) were analysed by flow cytometry staining. c, Treg suppression assay of Tconv cell proliferation based on CFSE dilution in the presence of rat nTreg cells or expanded Treg cells at 1:1 and 1:2 ratio (Treg:Tconv) and analysed by flow cytometry. (Two-tailed unpaired t-test; n = 3 biologically independent samples). d–f, Interaction between rat Treg cells (rTreg) and C4-mDAPs. C4-mDAPs and rTreg cells were co-incubated for 72 h at a ratio of 5:1 and 1:1. The levels of TH (d), FOXA2 (e) or LMX1A (f) mRNA expression in C4-mDAPs were determined by qPCR and normalized to actin. (One-way ANOVA, Tukey’s post-hoc test; n = 3 biologically independent experiments). g, The levels of isolated human CD4+CD127lowCD25+FOXP3+ Treg cells were analysed by flow cytometry. Each error bar represents mean ± s.e.m. ns, not significant.

Source data

Extended Data Fig. 5 Localization and function of autologous Treg cells after co-transplantation.

a, b, After intra-striatal co-transplantation of Fischer 344 rats with TP medium and autologous Treg cells (20,000 cells per rat), (a) the localization of Treg cells was determined by immunofluorescence staining with anti-FOXP3 antibody at 1, 2, 3, 5 and 7 days after surgery. (One-way ANOVA, Tukey’s post-hoc test; n = 5 per group). b, The level of inflammatory cell infiltration into needle track was determined by immunohistochemical staining with anti-MHCII antibody. (Two-way ANOVA, Bonferroni’s multiple comparisons post-hoc test; n = 5 per group). Each error bar represents mean ± s.e.m. **P < 0.01, ***,P < 0.001.

Source data

Extended Data Fig. 6 Inhibition of needle-trauma-induced neuroinflammation by treatment with CsA.

a–e, Fischer 344 rats were killed 7 days after intra-striatal co-transplantation of TP medium with Treg cells 20,000 cells per rat) or CsA treatment (intraperitoneally). a, Schematic overview of experimental method. The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-IBA1 (b) and -MHCII (c) antibodies. d, The level of needle-trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-IFNγ antibody. e, The level of neuroinflammation-induced cell death by needle trauma was analysed by TUNEL assay. (One-way ANOVA, Tukey’s post-hoc test; n = 5 per group). Each error bar represents mean ± s.e.m. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 7 In vitro effects of treatment with pro-inflammatory cytokines on C4-mDAPs.

a–g, C4-mDAPs were incubated in vitro with or without 20 ng/ml TNF, 10 ng/ml IL-1β or 100 ng/ml IFNγ for 2 days. The level of TNF/IL-1β-induced immunogenicity was determined by flow cytometry analysis with anti-HLA-ABC/HLA-DR/CD80/CD86/CD40/PD-L1/PD-L2/CD47 antibodies (a) and by western blot analysis with anti-ZG16/HORMAD1 antibodies (b). TH levels were assessed by immunofluorescence staining (c), the number of total cells (n = 4) (d) or TH+ cells (n = 4) (e), TH+ mean fluorescence intensity (n = 5) (f), and TH+ neurite length (n = 7) (g). (Two-tailed unpaired t-test; biologically independent experiments). h,i, C4-mDAPs were incubated in vitro with 20 ng/ml TNF or 10 ng/ml IL-1β for 7 days. The level of TNF-/IL-1β-induced cell death was determined by flow cytometry analysis of Annexin-V/7-AAD staining (h) and TUNEL assay (i). (One-way ANOVA, Tukey’s post-hoc test; n = 3 biologically independent experiments). j–m, C4-mDAPs with or without Treg cells were co-cultured for 7 days under in vitro inflammatory conditions (with or without 20 ng/ml TNF or 10 ng/ml IL-1β). j, Immunofluorescence staining of TH and FOXA2. Percentages of TH+ (n = 3) (k) and FOXA2+ (n = 3) (m) cells among total cells, and TH+ neurite length (n = 4) (l) were measured. (One-way ANOVA, Tukey’s post-hoc test; biologically independent samples). n,n’, NSG mice were killed 14 days after intra-striatal co-transplantation of C4-mDAPs with or without anti-IFNγ monoclonal antibody. The percentage of TH+ mDANs was determined by immunohistochemistry staining with anti-TH/hNUCLEI antibodies after transplantation. (Two-tailed unpaired t-test; n = 5 per group). o,p, C4-mDAPs with or without Treg cells and anti-TGFβ1 or mouse IgG1 isotype control antibody were co-cultured for seven days. The level of proliferation was determined by immunofluorescence staining with anti-Ki67 antibody (o) and staining is quantified in p. (One-way ANOVA, Tukey’s post-hoc test; n = 5 biologically independent samples). In box plots in d–g, the centre line represents the median, box edges represent the interquartile range, and error bars represent maximum and minimum values. Each error bar represents mean ± s.e.m. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 8 Treg co-transplantation suppresses inflammatory cell infiltration and C4-mDAP immunogenicity in a xenogeneic model of PD.

a–e, Fischer 344 rats were killed two weeks after intra-striatal co-transplantation of C4-mDAPs with or without Treg cells and CsA. The levels of CD11b+/CD11c+ (a), NKp46+ (b), CD19+ (c), and CD4+/CD8+ (d,d′) cells were assessed through immunofluorescence staining. d′, Most of the yellow dots seen in the +Treg cells, +CsA, and +Treg cells + CsA groups in d are nonspecific signals, not real cells (yellow arrows). e, Expression of HLA class I/II was examined through immunofluorescence staining. (n = 5 per group).

Extended Data Fig. 9 Treg co-transplantation suppresses C4-mDAP proliferation in a xenogeneic model of PD.

a–k, Fischer 344 rats were killed 20 weeks after intra-striatal co-transplantation of C4-mDAPs with or without Treg cells and CsA. a,b, Stereological estimation of graft volume through hNCAM+ staining. Number of Ki67+ cells (c,d) among hNUCLEI+ cells. (Two-tailed unpaired t-test; n = 5 per group). e, Correlation between Ki67+ cell number and graft volume. (Two-tailed correlation). f,f′, The number of TH+ and FOXA2+ cells was analysed by immunofluorescence staining. (Two-tailed unpaired t-test; Mann–Whitney post-hoc test; n = 20 per group). g–k, Representative images of neuronal cells (g, NeuN+), astrocytes (h, hGFAP+), vascular leptomeningeal cells (i, hCOL1A1+), oligodendrocyte lineage cells (j, OLIG2+) and microglia (k, hIBA1+). (5 rats per group were identified). In box plots in b,d,f′, the centre line represents the median, box edges represent the interquartile range, and error bars represent maximum and minimum values. Scale bars: 100 μm. Each error bar represents mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 10 Effects of Treg co-transplantation with C4-mDAPs in an autologous C4-humanized model of PD.

a–h, C4-humanized mice were killed eight weeks after intra-striatal co-transplantation of C4-mDAPs with or without autologous C4-Treg cells (20,000 cells per mouse). a, Schematic overview of experimental design. b, Survival of C4-humanized mice. Total number of transplanted C4-mDAPs (c), graft volume (d) and TH+ cells (g) were assayed. Number of Ki67+ (e) and correlation between Ki67+ and hNUCLEI+ cell number (f). (Two-tailed correlation). h, The levels of hCD4+ T cells in all three mice in +Treg cells group were confirmed through immunofluorescence staining. (Two-tailed unpaired t-test; Mann–Whitney post-hoc test; n = 2 (−Treg cells) or 3 (+Treg cells) per group).

Source data

Extended Data Fig. 11 In vivo effects of Treg co-transplantation with C4-mDAPs.

a–d, NSG mice were killed 20 weeks after intra-striatal co-transplantation of C4-mDAPs with or without autologous C4-Treg cells (20,000 cells per mouse). a,b, Representative images (a) and quantitative assessment (b) of hSYP+ within the DL STR. (Two-tailed unpaired t-test; n = 5 per group). c,d, Representative images (c) and quantitative assessment (d) of graft-derived TH+ fibre density in the (i) cingulate cortex (CTX), (ii) perirhinal CTX, (iii) DL STR, and (iv) ventrolateral (VL) STR. T, transplant. (Two-tailed unpaired t-test; n = 3 per group). In box plots in b, the centre line represents the median, box edges represent the interquartile range, and error bars represent maximum and minimum values. Each error bar represents mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 12 Functions of Treg cells and related mechanisms.

a–c, The expression level of TGFβ receptor (a), SIRPα (b) and galectin-1 (c) in C4-mDAPs was analysed by flow cytometry. d,e, NSG mice were killed eight weeks after intra-striatal co-transplantation of C4-mDAPs with mitomycin-C-treated C4-Tnaive (20,000 cells per mouse) or C4-Treg cells (20,000 cells per mouse). The total number of transplanted C4-mDAPs (hNUCLEI+) (d) and the graft volume (hNCAM+) (e) were analysed after eight weeks. (Two-tailed unpaired t-test; n = 4 per group). Each error bar represents mean ± s.e.m. **P < 0.01.

Source data

Supplementary information

Supplementary Figures

This file contains the uncropped blots (Supplementary Fig. 1) and the gating strategies used for cell death analysis and cell sorting (Supplementary Fig. 2).

Reporting Summary

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Park, TY., Jeon, J., Lee, N. et al. Co-transplantation of autologous Treg cells in a cell therapy for Parkinson’s disease. Nature 619, 606–615 (2023). https://doi.org/10.1038/s41586-023-06300-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-023-06300-4

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing