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.
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All data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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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.
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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.
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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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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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
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DOI: https://doi.org/10.1038/s41586-023-06300-4
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