Simultaneous intrastriatal transplantation of autologous TREG cells and mDA cells in Parkinson's disease cell therapy

Abstract

In this specification, in particular, controllability T(T REG The invention describes compositions and methods for administering populations of cells and / or populations of midbrain dopamine (mDA) cells to a target brain (e.g., to treat Parkinson's disease and / or to reduce immune responses resulting from needle trauma during cell transplantation).

Description

[Technical Field]

[0001] Claim of priority This application claims the benefits of U.S. Provisional Application No. 63 / 500,941, filed on 9 May 2023. The entirety of the foregoing is incorporated herein by reference. [Background technology]

[0002] The major motor symptoms of Parkinson's disease (PD), including tremor, rigidity, bradykinesia, and postural instability, are caused by the selective and progressive degeneration of midbrain dopamine (mDA) neurons (mDANs) in the substantia nigra, and cell replacement therapy has become a promising treatment strategy, a concept supported by the results of previous studies on ventral fetal midbrain (VM) cell transplantation. 1~6 The clinical use of human fetal VM cells is limited by ethical and logistical considerations. mDA cells, derived from human pluripotent stem cells such as human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs), are emerging as a potential alternative cell source. 1~6 Regardless of the source of transplantable cells, a fundamental requirement of cell replacement therapy is the survival of grafted cells throughout the transplantation process. Therefore, many researchers have studied the viability of transplanted cells as a critical requirement for successful PD cell therapy, primarily using human and rodent embryonic VM cells. 7~11These initial studies revealed that the majority (80 - 99%) of transplanted mDANs undergo apoptotic cell death within 1 - 2 weeks after transplantation. In particular, these studies also showed that the number of surviving mDA neurons remains the same at later time points (e.g., 4 days, 2 weeks, and 6 weeks after transplantation), further confirming that most transplanted mDA neurons undergo early death and that new mDA neurons are not generated from transplanted VM cells. Many potential mechanisms have been proposed to explain the poor survival rate and early death of grafted mDA neurons, with most focusing on the interaction between the grafted cells and the host brain, such as inadequate supply of oxygen, glucose, or growth factors from the host brain in the immediate vicinity of the fresh graft. Therefore, numerous efforts have been made to improve graft survival using various protective agents, including calcium channel antagonists, lazaroids, caspase inhibitors, and trophic factors. 40~44 .

[0003] The use of autologous cells for transplantation can reduce the host immune response. Autologous mDA cell transplantation has been reported recently in non - human primate PD models. 12~14 . In another study, neural grafting of major histocompatibility complex (MHC) - compatible primate iPSCs reduced the host immune response and increased the survival rate of grafted mDA neurons. 15 . Furthermore, autologous hiPSC - derived mDA cells were successfully transplanted into the striatum of patients with idiopathic PD without using immunosuppressive agents, and it was shown that mice with a humanized immune system constructed from the peripheral blood mononuclear cells (PBMC) of such patients do not reject such grafts. 16 . These data indicated that graft rejection can be avoided by using autologous mDA cells, but 18 cell survival and functional recovery in the first patients, as evaluated by F - DOPA PET scan analysis and clinical motor assessment, were minimal. 16The survival rate of mDA neurons may be limited even with autologous transplantation. To compensate for this poor survival rate, some researchers are advocating transplanting a massive number of cells, such as in a recent clinical trial in Australia, where researchers are suggesting transplanting as many as 70 million cells (neuroscience.org.au / research-news / parkinsons-stem-cell-trial). Furthermore, some researchers are suggesting using cells at an earlier stage of differentiation (e.g., day 16-17) for transplantation. 17、18 While these approaches are interesting, the risks of undesirable side effects, including graft proliferation, collapse of host brain structures, and / or tumorigenesis, must be carefully investigated. [Overview of the Initiative]

[0004] This application relates to controllability T(T REG This application is at least partially based on the surprising discovery that co-administering a population of ) cells together with a population of midbrain dopamine cells (mDA cells) to the brain of a subject (e.g., a subject with Parkinson's disease) results in better outcomes for the subject (e.g., reduced immune response in the subject). Useful mDA cells may include midbrain dopamine neurons (mDANs), midbrain dopamine progenitor cells (mDAPs), and / or combinations thereof. This application also relates to T in cell therapy. REG This is at least partially based on the remarkable discovery that administering a population of cells together with a population of desired cells (e.g., neurons) reduces needle trauma (e.g., reduces the host's immune response), reduces graft proliferation, and increases the cell viability of the desired cell population.

[0005] The role of the immediate host brain tissue response via the innate immune response to surgical injury in transplants, and this is particularly important for the desired tyrosine hydroxylase (TH) within the graft. +The specific impact on the survival rate of mDA neurons had not been investigated. This specification shows that the majority (approximately 90%) of hiPSC-derived mDA neurons present in the initial population of grafted cells died within 1-2 weeks after transplantation, while residual TH cells in the graft died. - The cells were shown to survive almost entirely, whether transplanted into immunodeficient host animals, including humanized mice, or into immunocompetent host animals. The inventors believe that a key factor in this premature death of mDA neurons is the surgical trauma itself (referred to herein as “needle trauma”), which is responsible for the TH within the graft. - Compared to cells, TH + We demonstrated that this needle trauma induces an acute host neuroinflammatory response with preferential adverse effects on mDA neurons. Furthermore, we found that this needle trauma damages the host brain and causes significant death of host neuronal cells (Figure 7g).

[0006] Regulatory T cells (T REG ) is essential for maintaining normal immune tolerance and homeostasis, and is so named due to its regulation of other immune cells in order to prevent inflammatory and immune rejection responses. 19、20 Therefore, autologous T during surgical transplantation of grafts REG Simultaneous transplantation may help effectively and safely suppress the host inflammatory response to needle trauma. The inventors have developed an autologous T10. REG Simultaneous intrastriatal transplantation significantly protects both host neurons and grafted mDA neurons from needle trauma-induced death, further improving behavioral recovery in the 6-OHDA injury PD rodent model and TH within the graft. - It was found to reduce cell proliferation. REG Because it is known to suppress inflammatory responses, T REG It is used in clinical practice to control inflammation. However, T REG These are administered systemically to patients via intravenous injection, and their effects on the survival rate of mDA neurons after intrastriatal injection have not been elucidated. Therefore, the inventors have developed an ex vivo-grown autologous CD4 + CD25+ T REG Adoption is carried out by intravenous injection of approximately 1,000,000 units. 27、28 Using the conventional T REG The effectiveness of the therapy was verified (Foxp3 + , 68.8±4.07%) (Figure 9b), and this was compared to direct intrastriatal transplantation. Adoptive transplantation was MHCII + It reduced cell infiltration by approximately 50%, but that was T REG The efficiency was significantly lower than that of 20,000 direct intrastriatal transplants (Figure 1n). In summary, only 2% of the cells were used, but T REG Direct intrastriatal injection is far more effective than conventional adoption transfers.

[0007] Therefore, in this specification, in particular, T REG A method for treating Parkinson's disease (PD) in patients is described, which involves the simultaneous transplantation of cells and mDA neurons into the patient's brain. REG Cells and mDA neurons can be transplanted simultaneously or directly, one after the other. REG Cells and mDA neurons REG It may be a cell or an autologous mDA neuron. Alternatively, T REG Cells and mDA neurons may be homogeneous or heterogeneous. REG Cells and mDA neurons can be transplanted into the striatum. REG Cells and mDA cells can be transplanted in quantities of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0008] In various embodiments of methods for treating PD, T REG Cells and mDA cells were transplanted simultaneously, T REG Cells and mDA cells are directly transplanted one after another, T REG The cells are autologous T REG cells, allogeneic T REG Cells, or heterologous T cells REG mDA cells are cells, and mDA cells are autologous mDA cells, allogeneic mDA cells, or heterologous mDA cells, TREG The cells and mDA cells are transplanted into the striatum.

[0009] Furthermore, in this specification, T REG Also described are methods for reducing and / or suppressing a needle-induced host immune response in a patient receiving cell transplantation, including co-transplanting cells with the cells to be transplanted. In some embodiments, the cells to be transplanted may be mDA cells (e.g., mDA neurons, mDA progenitor cells, and mixtures thereof). In some embodiments, T REG Cells and mDA cells can be transplanted simultaneously or directly, one after the other. In some embodiments, T REG Cells and mDA cells are autologous T cells. REG Cells (i.e., patient-derived) and autologous mDA cells may be used. In some embodiments, patient-derived T cells are used. REG The cells can be used with mDA cells, which may be homogeneous or heterogeneous. In some embodiments, T REG Cells and mDA cells can be transplanted into the striatum. In some embodiments, T REG Cells and mDA cells can be transplanted in quantities of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0010] In various embodiments, T REG Cells and mDA cells were transplanted simultaneously, T REG Cells and mDA cells are directly transplanted one after another, T REG The cells are autologous T REG cells, allogeneic T REG Cells, or heterologous T cells REG mDA cells are cells, and mDA cells are autologous mDA cells, allogeneic mDA cells, or heterologous mDA cells, T REG The cells and mDA cells are transplanted into the striatum.

[0011] Furthermore, T REGThe unexpected finding was that simultaneous transplantation significantly rescued host brain cells caused by needle trauma compared to the control group (Figures 7e, 7f, 7g; Figures 1h-1k). REG Simultaneous transplantation has been shown to protect the patient's (host) neurons from death.

[0012] Furthermore, in this specification, in particular, T REG A method for improving the growth of mDA cells from a graft at the time of transplantation to a patient is described, which includes co-transplanting cells together with transplanted mDA cells, and co-transplantation results in a higher proportion of TH+ cells in the graft compared to a graft of mDA cells alone. In some embodiments, T REG Cells and mDA neurons can be transplanted simultaneously or directly, one after the other. In some embodiments, T REG Cells and mDA neurons REG These may be cells and autologous mDA neurons. In some embodiments, T REG The cells and mDA cells may be homogeneous or heterogeneous. In some embodiments, T REG Cells and mDA neurons can be implanted in the striatum. In some embodiments, T REG Cells and mDA neurons can be transplanted in quantities of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0013] In various embodiments, T REG Cells and mDA cells were transplanted simultaneously, T REG Cells and mDA cells are directly transplanted one after another, T REG The cells are autologous T REG cells, allogeneic T REG Cells, or heterologous T cells REG mDA cells are cells, and mDA cells are autologous mDA cells, allogeneic mDA cells, or heterologous mDA cells, T REG The cells and mDA cells are transplanted into the striatum.

[0014] Furthermore, TH -Abnormal cell growth can disrupt the patient's brain structure and cause adverse effects and / or side effects. In this regard, T REG Simultaneous transplantation of cells and mDA cells is possible for the overall size of the graft and Ki67 + It offers advantages by reducing the number of proliferating cells and enhances the safety of cell therapy.

[0015] Furthermore, the inventors found that needle trauma preferentially kills not only mDA neurons but also other types of neurons, such as GABAergic neurons, compared to non-neuronal proliferating cells. Therefore, T REG Simultaneous transplantation may be beneficial not only to the survival rate of mDA neurons but also to the survival rate of other types of neurons, and can be applied in a way that is beneficial to both the efficacy and safety of cell replacement therapy for Parkinson's disease and other neurodegenerative diseases.

[0016] Finally, and importantly, the inventors have found that co-implantation of specific antibodies against pro-inflammatory cytokines (such as FDA-approved infliximab (anti-TNF-α), canakinumab (anti-IL-1β), and emaparmab (anti-IFN-γ)), or co-implantation of antibodies against injury-associated molecular pattern (DAMP) molecules (such as anti-HMGB1 Ab and anti-IL-1α Ab), also exhibits protective effects against needle trauma-induced death of host brain cells and grafted mDA neurons. Furthermore, the inventors have found that administration of anti-inflammatory drugs (such as cyclosporine A, dexamethasone, and FK506) also exhibits protective effects. Therefore, these methods / reagents, either alone or in combination, also exhibit protective effects. REG It can be used instead.

[0017] In some embodiments, this specification provides a method for treating a subject having Parkinson's disease (PD), wherein the controllable T(T) REGThe Specified Description describes a method comprising administering a population of cells and a population of midbrain dopamine (mDA) cells to the brain of a subject. In some embodiments, the Specified Description describes a method for treating a subject having Parkinson's disease (PD), comprising administering to the subject a population of midbrain dopamine (mDA) cells and antibodies (Ab) (optionally, antibodies that conjugate pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally, anti-TNF-α Ab (optionally infliximab), anti-IL-1β Ab (optionally canakinumab), anti-IFN-γ Ab (optionally emaparmab), anti-HMGB1 Ab, anti-IL-1α Ab).

[0018] In some embodiments, this specification provides a method for reducing an immune response in a subject during cell transplantation (e.g., an immune response induced by needle trauma), wherein the subject is given an effective amount of T REG Methods are described that involve administering a population of cells together with a second population of cells ("transplant cells," optionally mDA cells). In some embodiments, the Specified Description describes methods for reducing an immune response in a subject during cell transplantation (e.g., an immune response induced by needle trauma), which involves administering to the subject an effective amount of antibody (Ab) (optionally, an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (optionally infliximab), optionally anti-IL-1β Ab (optionally canakinumab), optionally anti-IFN-γ Ab (optionally emaparmab), optionally anti-HMGB1 Ab, or anti-IL-1α Ab) together with a population of cells (optionally mDA cells).

[0019] In some embodiments, the foregoing describes a method for increasing the cell viability of transplanted cells (optionally mDAN) in cell transplantation and / or reducing the abnormal proliferation of cells (optionally TH- cells, Ki67+ cells, and / or cells other than mDAN) during cell transplantation, wherein an effective amount of T REGThe description includes a method for co-transplanting a population of cells together with a second population of cells ("transplant cells," optionally mDA cells). In some embodiments, the Specified Method increases the cell viability of transplant cells (optionally mDA cells) in cell transplantation and / or cells (optionally TH cells, Ki67 cells) during cell transplantation. + A method for reducing the abnormal proliferation of cells (and / or cells other than mDANs) is described, comprising administering an effective amount of antibody (Ab) (optionally, an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally, anti-TNF-α Ab (optionally, infliximab), anti-IL-1β Ab (optionally, canakinumab), anti-IFN-γ Ab (optionally, emaparmab), anti-HMGB1 Ab, or anti-IL-1α Ab) to a subject together with a second population of cells (optionally, mDA cells).

[0020] In some embodiments of the methods described herein, the transplanted cells are mDA cells. In some embodiments of the methods described herein, the second population of cells is a population of mDA cells. In some embodiments of the methods described herein, the population of mDA cells comprises midbrain dopamine neurons (mDANs) and midbrain dopamine progenitor cells (mDAPs). In some embodiments of the methods described herein, the population of mDA cells comprises at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% mDAPs and at least about 5, 10, 15, 20, or 25% mDANs.

[0021] In some embodiments of the methods described herein, the mDA cell:Treg cell ratio is at least about 1:1, 1:2, 1:3, 1:4, or 1:5. REG Cells and mDA cells are administered to the subject simultaneously and optionally mixed together in a single composition. In some embodiments of any of the methods described herein, T REGCells are first administered to a subject, followed by administration of mDA cells to the subject. In some embodiments of any of the methods described herein, mDA cells are first administered to the subject, followed by T REG cells being administered to the subject. In some embodiments of any of the methods described herein, T REG cells and mDA cells are transplanted as a single composition in a single dose. In some embodiments of any of the methods described herein, T REG cells and mDA cells are transplanted as a single composition in repeated doses.

[0022] In some embodiments of any of the methods described herein, T REG cells and mDA cells are autologous T REG cells and autologous mDA cells. In some embodiments of any of the methods described herein, T REG cells and mDA cells are allogeneic T REG cells and allogeneic mDA cells. In some embodiments of any of the methods described herein, T REG cells are autologous T REG cells, syngeneic T REG cells, or allogeneic T REG cells. In some embodiments of any of the methods described herein, mDA cells are autologous mDA cells, syngeneic mDA cells, or allogeneic mDA cells. In some embodiments of any of the methods described herein, the transplanted cells (optionally mDA cells) are autologous transplanted cells, syngeneic transplanted cells (optionally mDA cells), or allogeneic transplanted cells (optionally mDA cells).

[0023] In some embodiments of any of the methods described herein, autologous mDA is obtained by in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into mDA cells, and the hiPSCs are derived from cells from the subject. In some embodiments of any of the methods described herein, T REG cells and mDA cells are syngeneic T REGAutologous mDA cells are obtained by in vitro differentiation of human pluripotent stem cells (hPSCs), and optionally human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into mDA cells.

[0024] In some embodiments of any of the methods described herein, T REG Cells and mDA cells are administered into the striatum, optionally by intrastriatal injection.

[0025] In some embodiments of any of the methods described herein, T REG The cells are transplanted into the striatum. In some embodiments of the methods described herein, mDA cells are transplanted into the striatum. In some embodiments of the methods described herein, the transplanted cells (optionally mDA cells) are transplanted into the striatum.

[0026] In some embodiments of the methods described herein, simultaneous transplantation results in a graft with a higher TH content in the graft compared to a graft without simultaneous transplantation. + An increase in cells is observed. In some embodiments of any of the methods described herein, simultaneous transplantation produces a graft with TH in the graft compared to a graft without simultaneous transplantation. + Cell:TH - Increased cell ratio and / or TH in grafts + Cell: Ki67 + An increase in the cell ratio is observed. In some embodiments of any of the methods described herein, simultaneous transplantation produces a graft with a higher Ki67 content in the graft compared to a graft without simultaneous transplantation. + Cell reduction and / or TH in grafts - A decrease in the number of cells is observed.

[0027] In some embodiments of any of the methods described herein, T REG The cells and transplanted cells (optionally mDA cells) are transplanted simultaneously. In some embodiments of any of the methods described herein, T REGThe cells and transplanted cells (optionally mDA cells) are transplanted separately. In some embodiments of any of the methods described herein, T REG Cells are first administered to the subject, followed by the administration of transplanted cells (optionally selected mDA cells) to the subject. In some embodiments of any of the methods described herein, transplanted cells (optionally selected mDA cells) are first administered to the subject, followed by T REG Cells are administered to the target. In some embodiments of any of the methods described herein, the ratio of transplanted cells to Treg cells is at least about 1:1, 1:2, 1:3, 1:4, or 1:5. In some embodiments of any of the methods described herein, T REG Cells and transplanted cells are administered to the subject simultaneously and optionally mixed together in a single composition. In some embodiments of any of the methods described herein, T REG Cells and transplanted cells are administered to the subject as a single dose in a single composition. In some embodiments of any of the methods described herein, T REG Cells and transplanted cells are administered to the subject in repeated doses in a single composition.

[0028] In some embodiments of the methods described herein, an effective amount of T REG Cells and an effective amount of mDA cells are administered to the subject. In some embodiments of any of the methods described herein, T is administered to the subject. REG The amount of cells and mDA cells is sufficient to improve one or more symptoms of PD in the subject, thereby treating PD. In some embodiments of any of the methods described herein, the subject is administered T REG The amount of cells is sufficient to reduce or eliminate the amount (and / or percentage) of at least one pro-inflammatory cytokine (e.g., TNF-α, IL-1β, and / or IFN-γ) and / or at least one damage-associated molecular pattern (DAMP, e.g., HMGB1) at and / or near the injection site.

[0029] As used herein, cell transplantation includes any therapy in which a population of cells is administered to a subject (e.g., via injection, infusion, graft / transplantation), such as cell therapy.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the use of the present invention. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0031] Other features and advantages of the present invention will become apparent from the detailed description and drawings below, as well as from the claims. [Brief explanation of the drawing]

[0032] [Figure 1-1]Figures 1a–1n. Effects of needle trauma-induced neuroinflammation on transplanted cells. 1a–1g, NSG or C4-hu mice were sacrificed 14 days after intrastriatal transplantation of C4-mDAP. The total number and percentage of TH+mDA neurons were determined by immunofluorescence staining with anti-TH / Hoechst 33342 pre-transplant (1a, 1c, 1d) or immunohistochemical staining with anti-TH / hNUCLEI antibody post-transplant (1b, 1c, 1d) (one-way ANOVA, Tukey's post-hoc test; n=4 per group). The level of inflammatory cell infiltration was confirmed by immunofluorescence staining with anti-hIba1 (1e), anti-hCD45 (1f), and anti-hCD4 (1g) antibodies. 1h–1k, inhibition of needle trauma-induced host inflammatory response using TREG. Fischer 344 rats were sacrificed 7 days after simultaneous intrastriatal transplantation in TP medium with or without autologous TREGs (20,000 cells / rat) derived from each rat. The level of inflammatory cell infiltration into the needle marks was determined by immunohistochemical staining with anti-Iba1(1h) and anti-MHCII(i) antibodies. 1j, The level of needle-induced neuroinflammation was determined by immunofluorescence staining with anti-IFN-γ antibody. 1k, The level of neuroinflammation-induced cell death due to needle trauma was analyzed by the TUNEL assay (Student's t-test; n=5 per group). 1l-1n, Advantages of simultaneous transplantation with autologous TREGs. The level of inflammatory cell infiltration into the needle marks was determined by immunohistochemical staining with anti-MHCII antibody. 1l, The inhibitory effect on needle-induced neuroinflammation was compared 7 days after simultaneous transplantation with syngeneic and autologous TREGs (20,000 cells / rat). 1m, inhibition of needle trauma-induced neuroinflammation was measured 7 days after co-implantation of 2,000, 20,000, or 100,000 autologous TREGs per rat. 1n, adoptive transfer of ex vivo-grown autologous TREGs (million cells / rat) obtained from each rat or intrastriatal transplantation of ex vivo-grown autologous TREGs (20,000 cells / rat) was compared 7 days later (one-way ANOVA, Tukey's post-hoc test; n=5 per group). Each error bar represents mean ± sem. ns, not significant; **, P<0.01; ***, P<0.001. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 1-4] (As stated above.) [Figure 1-5] (As stated above.) [Figure 1-6] (As stated above.) [Figure 1-7] (As stated above.) [Figure 2-1] Figures 2a-2f. Pro-inflammatory cytokine-induced immunogenicity in transplanted C4-mDAP. a-c, C4-mDAP was incubated in vitro with 100 ng / ml IFN-γ for 2 days. The level of IFN-γ-induced immunogenicity was determined by flow cytometry analysis using anti-HLA-ABC / HLA-DR antibody (2a), anti-CD80 / CD86 / CD40 / PD-L1 / PD-L2 / CD47 antibody (2b), and by Western blot analysis using anti-Zg16 / Hormad1 antibody (2c). 2d-2f, MLR-like co-culture assay using C4-mDAP and C4 / K2-PBMC. 2d, Level of T cell activation measured by flow cytometry analysis using anti-CD3 / CD69 antibody. 2e, Level of immunogenicity-induced cell death determined by flow cytometry analysis using anti-hNCAM / Annexin-V staining. 2f. Levels of IFN-γ secretion measured by ELISA. ‡; Activated with Dynabeads® Human T-Activator (one-way ANOVA, Tukey's post-hoc test; n=3). Each error bar represents mean ± sem. **, P<0.01; ***, P<0.001. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 3-1]Figures 3a–3l. Pro-inflammatory cytokine-induced cell death of C4-mDAP. 3a–3i, C4-mDAP was incubated in vitro with 100 ng / ml IFN-γ for 7 days. The level of IFN-γ-induced cell death was determined by Western blotting with anti-cleavage caspase-3 antibody (3a), flow cytometry analysis with annexin-V / 7-AAD staining (3b), and TUNEL assay (3c) (Student's t-test; n=3, biologically independent experiments). TH levels were assayed by immunofluorescence staining (3d), measuring the number of total cells (3e) or TH+ cells (3f), mean TH+ fluorescence intensity (3g), and TH+ neurite length (3h) (Student's t-test; n≧4). 3i, apoptotic cell death (annexin-V+) was analyzed by gating TH+ and TH- cells using flow cytometry (one-way ANOVA, Tukey's post-hoc test; n=6). 3j-3l, C4-mDAP with or without TREG was co-cultured for 7 days under in vitro inflammatory conditions (with or without 100 ng / ml IFN-γ). 3j, immunofluorescence staining of TH and Ki67. Percentage of TH+ (3k) and Ki67+ (3l) cells in total cells (one-way ANOVA, Tukey's post-hoc test; n=5). Scale bar: 100 μm. Each error bar represents mean ± sem. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Figure 3-4] (As stated above.) [Figure 3-5] (As stated above.) [Figure 4-1]Figures 4a–4h. Effect of co-transplantation of TREG with C4-mDAP in a xenogeneic PD model. 4a–4h, Fischer 344 rats were sacrificed 2 or 20 weeks after co-transplantation of autologous TREG (20,000 cells / rat) and C4-mDAP with or without CsA (control) in the striatum. 4a, 4b, Amphetamine-induced turnover scores were determined every 4 weeks over 20 weeks post-transplant (two-way, one-way ANOVA, Tukey's post-hoc test; n=5 per group). 4c, At 2 weeks, the level of inflammatory cell infiltration was determined by immunohistochemical staining with anti-MHCII antibody. 4d, The level of neuroinflammation-induced cell death was analyzed by TUNEL assay at 2 weeks. 4e, 4g, The total number of C4-mDAP was confirmed by hNUCLEI staining at 2 weeks (4e) and 20 weeks (4g). The total number of 4f, 4h, and TH+ cells was determined by immunofluorescence staining after 2 weeks (4f) and 20 weeks (4h), respectively (one-way ANOVA, Tukey's post-hoc test; n=5 per group). Each error bar represents the mean ± sem. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 4-2] (As stated above.) [Figure 4-3] (As stated above.) [Figure 4-4] (As stated above.) [Figure 5-1]Figures 5a-5m. In vivo effects of co-transplantation of TREG with C4-mDAP. 5a-5m, NSG mice were sacrificed 2 or 20 weeks after intrastriatal co-transplantation of C4-mDAP with or without autologous C4-TREG (20,000 cells / mouse). 5a, Amphetamine-induced rotation test was performed every 4 weeks for 24 weeks post-transplant (two-way ANOVA, Bonferroni post-hoc test). 5b-5d, Behavioral assessments using amphetamine-induced rotation test (5b), apomorphine-induced rotation test (5c), and cylinder test (5d) at 20 weeks post-transplant (one-way ANOVA, Tukey post-hoc test; n=5 per group). The total number of grafted C4-mDAP (hNUCLEI+) cells (e) and graft volume (hNCAM+) (5f) were analyzed at 2 and 20 weeks, respectively (Student's t-test; n=5 per group). The percentages of Ki67+ and SOX1+PAX6+Ki67+ cells (5g) and the correlation between Ki67+ cell percentage and graft volume (5h) (Student's t-test; n=5 per group) were examined for 5g and 5h. The total number of TH+ cells (5i) was examined at 2 and 20 weeks, respectively (Student's t-test; n=5 per group). Images of A9-like or A10-like cells were shown for 5j. 5k, Quantification of TH+ neurons co-expressing GIRK2+ALDH1A1+(A9) or calbindin+(A10) (Student's t-test; n=5 per group). 5l, Percentage of NeuN+ / hNUCLEI+ cells (Student's t-test; n=5 per group). 5m, Images of DA synaptic connections in host striatum identified by TH+hSYP-DARPP32+ staining. Each error bar represents mean ± sem. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 5-4] (As stated above.) [Figure 5-5] (As stated above.) [Figure 5-6] (As stated above.) [Figure 5-7] (As stated above.) [Figure 5-8] (As stated above.) [Figure 6-1] Figures 6a-6i: Effects of needle trauma-induced neuroinflammation on transplanted postmittal neurons. 6a-6e: NSG mice were sacrificed 14 days after intrastriatal transplantation of B1 / H9-mDAP. The total number and percentage of TH+mDA neurons were determined by immunofluorescence staining with anti-TH / Hoechst 33342 before transplantation (6a, 6c, 6e) or immunohistochemical staining with anti-TH / hNUCLEI antibody after transplantation (6b, 6d, 6e) (n=4 per group). 6f-6i: NSG mice were sacrificed 14 days after intrastriatal transplantation of GABAergic progenitor cells derived from C4-iPSC / B1-iPSC / H9-ESC. The total number and percentage of C4-GABAergic neurons (6f), B1-GABAergic neurons (6g), or H9-GABAergic neurons (6h) were determined by immunofluorescence staining using anti-VGAT / Hoechst 33342 antibody pre-transplant and anti-VGAT / Nkx2.1 / hNUCLEI antibody post-transplant (6i) (n=4 per group). Each error bar represents the mean ± sem. [Figure 6-2] (As stated above.) [Figure 6-3] (As stated above.) [Figure 7-1]Figures 7a-7g: Early time-dependent analysis of inflammatory response to needle trauma. 7a-7d, TP medium was injected into the striatum of Fischer 344 rats sacrificed 1, 2, 3, 5, and 7 days after surgery. The level of needle trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-TNF-α (7a) and IL-1β (7b) antibodies, with staining quantified in 7a' and 7b' (bar graphs in the left panel, respectively). 7c, astrocytes were stained with anti-GFAP antibody, with staining quantified in 7c' (bar graph in the left panel). Post-injection imaging and quantitative analysis of Iba-1+ cells (yellow box: resident microglia, red box: infiltrating Iba-1- cells) (7d, 7d') (n=5 per group). 7e-7g, Inhibition of needle trauma-induced host inflammatory response and cell death using TREG. Fischer 344 rats were sacrificed 2 or 7 days after co-intrastriatal transplantation of TP medium with or without autologous TREGs (20,000 cells / rat) derived from each rat. The level of needle trauma-induced neuroinflammation was determined by immunofluorescence staining with anti-TNF-α (7e, 7e') antibodies and anti-IL-1β (7f, 7f') antibodies (Student's t-test; n=3 per group). The levels of 7g, 7g', and needle trauma-induced neuronal cell death were determined by immunohistochemical staining with anti-NeuN antibodies (Student's t-test; n=5 per group). Each error bar represents the mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 7-2] (As stated above.) [Figure 7-3] (As stated above.) [Figure 7-4] (As stated above.) [Figure 7-5] (As stated above.) [Figure 7-6] (As stated above.) [Figure 7-7] (As stated above.) [Figure 7-8] (As stated above.) [Figure 8-1]Figures 8a-8d: Long-term time-dependent analysis of cellular responses to needle trauma. 8a-8d, TP medium was injected into the striatum of Fischer 344 rats, and the rats were sacrificed 4, 7, 14 days, and 1, 3, and 6 months after surgery. The level of inflammatory cell infiltration into the needle wound was determined by immunohistochemical staining using anti-Iba1 (8a) and anti-MHCII (8b) antibodies. 8c, the level of needle trauma-induced neuroinflammation was determined by immunofluorescence staining using anti-IFN-γ antibody. 8d, the level of cell death due to needle trauma was analyzed by the TUNEL assay (n=5 per group). Mean values ​​are connected by the red line. [Figure 8-2] (As stated above.) [Figure 8-3] (As stated above.) [Figure 8-4] (As stated above.) [Figure 9-1] Figures 9a-9g: TREG function after ex vivo proliferation. 9a, 9b, levels of selected CD4+CD25+Foxp3+ T cells (nTREG) (9a) and ex vivo-proliferated rat CD4+CD25+Foxp3+ T cells (proliferating TREG) (9b) were analyzed by flow cytometry staining. 9c, TREG suppression assays of Tconv cell proliferation based on CFSE dilution in the presence of rat nTREG or proliferating TREG in 1:1 and 1:2 ratios (TREG:Tconv) were analyzed by flow cytometry (Student's t-test; n=3). 9d-9f, interaction between rat TREG (rTREG) and C4-mDAP. C4-mDAP and rTREG were co-incubated for 72 hours in 5:1 and 1:1 ratios. The levels of TH(9d), Foxa2(9e), or Lmx1a(9f) mRNA expression in C4-mDAP were determined by quantitative real-time PCR and normalized to actin (one-way ANOVA, Tukey's post-hoc test; n=3 biologically independent experiments). The levels of (9g) isolated human CD4+CD127lowCD25+Foxp3+TREG were analyzed by flow cytometry. Each error bar represents mean ± sem.ns, not significant. [Figure 9-2] (As stated above.) [Figure 9-3](As stated above.) [Figure 10-1] Figures 10a-10b: Localization and function of autologous TREGs after co-transplantation. 10a, 10b, After co-transplantation of autologous TREGs (20,000 cells / rat) in TP medium and autologous TREGs in Fischer 344 rats, (10a) TREG localization was determined by immunofluorescence staining with anti-Foxp3 antibody 1, 2, 3, 5, and 7 days after surgery (one-way ANOVA, Tukey's post-hoc test; n=5 per group). 10b, The level of inflammatory cell infiltration into the needle site was determined by immunohistochemical staining with anti-MHCII antibody (two-way ANOVA, Bonferroni's post-hoc test; n=5 per group). Each error bar represents mean ± sem. **, P<0.01; ***, P<0.001. [Figure 10-2] (As stated above.) [Figure 11-1] Figures 11a-11e: Inhibition of needle trauma-induced neuroinflammation by CsA treatment. 11a-11e: Fischer 344 rats were sacrificed 7 days after co-intrastriatal transplantation of TP medium with TREG (20,000 cells / rat) or CsA treatment (ip). 11a: Schematic diagram of the experimental method. The level of inflammatory cell infiltration into the needle wound was determined by immunohistochemical staining using anti-Iba1 (11b) and anti-MHCII (11c) antibodies. 11d: The level of needle trauma-induced neuroinflammation was determined by immunofluorescence staining using anti-IFN-γ antibody. 11e: The level of neuroinflammation-induced cell death due to needle trauma was analyzed by TUNEL assay (one-way ANOVA, Tukey's post-hoc test; n=5 per group). Each error bar represents the mean ± sem. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 11-2] (As stated above.) [Figure 11-3] (As stated above.) [Figure 11-4] (As stated above.) [Figure 11-5] (As stated above.) [Figure 12-1]Figures 12a-12p: In vitro effects of pro-inflammatory cytokine treatment on C4-mDAP. 12g of C4-mDAP was incubated in vitro for 2 days with or without 20 ng / ml TNF-α, 10 ng / ml IL-1β, or 100 ng / ml IFN-γ. The level of TNF-α / IL-1β-induced immunogenicity was determined by flow cytometry analysis using anti-HLA-ABC / HLA-DR / CD80 / CD86 / CD40 / PD-L1 / PD-L2 / CD47 antibodies (12a) and by Western blot analysis using anti-Zg16 / Hormad1 antibodies (12b). TH levels were assessed by immunofluorescence staining (12c), the number of total cells (12d) or TH+ cells (12e), the mean TH+ fluorescence intensity (12f), and TH+ neurite length (12g) (Student's t-test; n≧4). 12h, 12i, and C4-mDAP were incubated in vitro with 20 ng / ml TNF-α or 10 ng / ml IL-1β for 7 days. The level of TNF-α / IL-1β-induced cell death was determined by flow cytometry analysis of annexin-V / 7-AAD staining (12h) and TUNEL assay (12i) (one-way ANOVA, Tukey's post-hoc test; biologically independent experiments of n=3). 12j–12m C4-mDAP with or without TREG was co-cultured in vitro under inflammatory conditions (with or without 20 ng / ml TNF-α or 10 ng / ml IL-1β) for 7 days. Immunofluorescence staining for 12j, TH, and FoxA2 was performed. The percentage of TH+(12k) and FoxA2+(12m) cells in total cells, as well as TH+ neurite length (l), were measured (one-way ANOVA, Tukey's post-hoc test; n≧3). 12n, 12n', and NSG mice were sacrificed 14 days after simultaneous intrastriatal transplantation of C4-mDAP with or without anti-IFN-γ mAb. The percentage of TH+mDA neurons was determined post-transplant by immunohistochemical staining with anti-TH / hNUCLEI antibody (Student's t-test; n=5 per group). C4-mDAP cells with or without 12o, 12p, TREG, and anti-TGF-β1 or mouse IgG1 isotype control antibodies were co-cultured for 7 days.The level of proliferation was determined by immunofluorescence staining with anti-Ki67 antibody (12o), and staining was quantified at 12p (one-way ANOVA, Tukey's post-hoc test; n=5). Each error bar represents the mean ± sem. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 12-4] (As stated above.) [Figure 12-5] (As stated above.) [Figure 12-6] (As stated above.) [Figure 12-7] (As stated above.) [Figure 12-8] (As stated above.) [Figure 13-1] Figures 13a–13e: TREG co-transplantation suppresses inflammatory cell infiltration and C4-mDAP immunogenicity in a xenogeneic PD model. 13a–13e: Fischer 344 rats were sacrificed 2 weeks after intrastriatal co-transplantation of TREG and C4-mDAP with or without CsA. Levels of CD11b+ / CD11c+ (13a), NKp46+ (13b), CD19+ (13c), and CD4+ / CD8+ (13d, 13d' (lower panel)) cells were assessed by immunofluorescence staining. Most of the yellow dots seen in the +TREG, +CsA, and +TREG+CsA groups in 13d' and 13d are nonspecific signals and not actual cells (yellow arrows). (13e) HLA class I / II expression was examined by immunofluorescence staining (n=5 per group). [Figure 13-2] (As stated above.) [Figure 13-3] (As stated above.) [Figure 13-4] (As stated above.) [Figure 13-5] (As stated above.) [Figure 13-6] (As stated above.) [Figure 14-1]Figures 14a-14k: Co-transplantation of TREG suppresses C4-mDAP proliferation in a xenogeneic PD model. 14a-14k, Fischer 344 rats were sacrificed 20 weeks after co-transplantation of TREG and C4-mDAP with or without CsA into the striatum. 14a, 14b, Stereoanalytic estimation of graft volume by hNCAM+ staining. Number of Ki67+ cells in hNUCLEI+ cells (14c, 14d) (Student's t-test, n=5 per group). e, Correlation between Ki67+ cell count and graft volume. 14f, Number of TH+ and FoxA2+ cells was analyzed by immunofluorescence staining (Student's t-test, n=20). Images of 14g-14k neurons (14g, NeuN+), astrocytes (14h, hGFAP+), VLMCs (i, hCOL1A1+), oligodendrocytes (14j, OLIG2+), and microglia (14k, hIba-1+). Scale bar: 100 μm. Each error bar represents the mean ± sem. *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 14-2] (As stated above.) [Figure 14-3] (As stated above.) [Figure 14-4] (As stated above.) [Figure 14-5] (As stated above.) [Figure 14-6] (As stated above.) [Figure 15-1]Figures 15a-15h: Effects of co-transplantation of TREG with C4-mDAP in an autologous C4-humanized PD model. 15a-15h: C4-humanized mice were sacrificed 8 weeks after intrastriatal co-transplantation of C4-mDAP with or without autologous C4-TREG (20,000 cells / mouse). 15a: Schematic diagram of the experimental design. 15b: Survival rate of C4-humanized mice. Total number of transplanted C4-mDAP (c), graft volume (15d), and TH+ cells (15g) were assayed. Number of Ki67+ cells (15e) (Student's t-test; n=5) and correlation between Ki67+ cell count and hNUCLEI+ cell count (15f). At 15 h, hCD4+ T cell levels were confirmed by immunofluorescence staining (Student's t-test; all data passed the Shapiro-Wilk normality test; n=2 (-TREG) and n=3 (+TREG) per group). Each error bar represents the mean ± sem. *, P<0.05. [Figure 15-2] (As stated above.) [Figure 15-3] (As stated above.) [Figure 15-4] (As stated above.) [Figure 16-1] Figures 16a-16d: In vivo effects of co-transplantation of TREG with C4-mDAP. 16a-16d: NSG mice were sacrificed 20 weeks after co-transplantation of C4-mDAP with or without autologous C4-TREG (20,000 cells / mouse) into the striatum. 16a, 16b: Images (16a) and quantitative assessment (16b) of hSYP+ in the DL STR (Student's t-test; n=5 per group). 16c, 16d: Images (16c) and quantitative assessment (16d) of graft-derived TH+ fiber density in (i) the cingulate cortex (CTX), (ii) the perinasal CTX, (iii) the DL STR, and (iv) the ventrolateral (VL) STR. T: Transplant (Student's t-test; n=3 per group). Each error bar represents mean ± sem. *, P<0.05;**, P<0.01;***, P<0.00. [Figure 16-2] (As stated above.) [Figure 16-3] (As stated above.) [Figure 17-1]Figures 17a–17e: Function and related mechanisms of TREG. 17a–17c, Expression levels of TGF-β receptor (17a), SIRPα (17b), and galectin-1 (17c) in C4-mDAP were analyzed by flow cytometry. 17d, 17e, NSG mice were sacrificed 8 weeks after simultaneous intrastriatal transplantation of C4-mDAP with mitomycin C-treated C4-T naive (20,000 cells / mouse) or C4-TREG (20,000 cells / mouse). Total number of transplanted C4-mDAP (hNUCLEI+) (17d) and graft volume (hNCAM+) (17e) were analyzed at 8 weeks, respectively (Student's t-test; n=4 per group). Each error bar represents mean ± sem. **, P<0.01. [Figure 17-2] (As stated above.) [Figure 17-3] (As stated above.) [Figure 18] Dynamic profile of molecular and cellular changes after needle trauma. During needle injection, the brain suffers physical damage, leading to the rupture of resident cells such as neurons, astrocytes, microglia, and oligodendrocytes. This rupture is caused by the impact of the needle, causing these cells to burst. Subsequently, the ruptured cells rapidly release injury-associated molecular patterns (DAMPs), which affect neighboring cells and induce the production and secretion of cytokines and chemokines due to activation. Among the cells that sense this response, neutrophils are rapidly recruited to the injury site and play a crucial role in immediately removing the debris. Simultaneously, astrocytes and microglia become progressively activated and migrate to the injury area over time. By approximately day 3, peripheral monocytes infiltrate, and depending on the severity of the brain injury, T cells and B cells also infiltrate and participate in the repair process. This series of inflammatory processes is important for the removal of cellular debris resulting from needle trauma and promotes essential steps for the repair and homeostasis of the injury area. However, at the same time, this event appears to cause substantial damage and death to engrafted mDANs. [Figure 19]Three distinct stages of hPSC-based CRT when mDAN may die. Schematic diagram of the three stages where mDAN may die. Stage 1 involves in vitro differentiation of hPSCs into mDA cells, primarily containing mDAP and mDAN, using optimized procedures. Stage 2 encompasses the retrieval and cryopreservation of in vitro differentiated mDA cells, emphasizing the critical steps of cryopreservation, storage, and thawing. Stage 3 involves final in vivo transplantation and consists of surgical transplantation, early (less than 2 weeks) and late (more than 2 weeks) stages of graft establishment. Potential challenges and considerations in each stage, including cell viability, immune response, and environmental factors, are discussed for a comprehensive understanding of the optimized process in hPSC-based CRT for PD. [Figure 20] Strategies targeting adaptive immunity. Generally, allogeneic transplantation using mDA cells derived from hESCs still requires immunosuppression, whereas autologous transplantation using mDA cells derived from hiPSCs is characterized by immune tolerance and eliminates the need for immunosuppressants. Alternative strategies to the use of autologous cells include the application of HLA-matched hiPSCs to reduce the risk of graft rejection through ongoing efforts to establish an HLA-matched iPSC bank encompassing a diverse range of donors. Another approach focuses on the development of “universal donor stem cells,” incorporating genetic modifications such as CRISPR / Cas9-mediated knockout of HLA class I and II components, along with lentiviral overexpression of immune receptor CD47 or HLA-E / G transgenes. [Figure 21]Strategies targeting innate immunity. In CRT, the standard procedure involves injecting cells into the brain using a needle that induces needle trauma and subsequent secretion of various innate immune response factors, including DAMP, pro-inflammatory / anti-inflammatory cytokines, and chemokines. These factors activate surrounding glial cells, causing infiltration of peripheral immune cells into the brain and positively contributing to damage repair. However, they also exert detrimental effects on grafted cells, leading to severe cell death in mDANs. Potential strategies to enhance grafted cell viability include obtaining autologous TREG cells from the patient, increasing their quantity and functionality, and confirming improved therapeutic effects through simultaneous transplantation into the brain. Furthermore, exploring the efficacy of inhibitors, neutralizing antibodies, and encapsulation methods to enhance grafted cell viability within the inflammatory microenvironment, combined with the application of TREG technology, represents a promising direction for future advances in CRT. [Figure 22] Antibodies targeting inflammatory cytokines reduce MHC-II+ cell infiltration. The level of inflammatory cell infiltration at needle marks was determined by immunohistochemical staining using anti-MHCII antibodies. [Modes for carrying out the invention]

[0033] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, and its prevalence continues to rise with the aging of the global population. Specific degeneration of midbrain dopamine neurons (mDAN; mDA cells) in the substantia nigra is central to the pathophysiology of PD. Therefore, cell replacement therapy (CRT) has emerged as a promising treatment approach, initially supported by various open-label clinical studies using fetal ventral midbrain (fVM) cells. Despite the favorable initial results, fVM cell therapy has inherent and logistical limitations that have hindered its transition to a standard treatment for PD. Recent efforts in the field of cell therapy have shifted their focus to the use of human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, in order to overcome existing challenges. However, regardless of the transplantable cell source (e.g., xenogeneic, allogeneic, or autologous), the poor and volatile survival rate of transplanted dopamine cells remains a major barrier. Evidence is emerging that highlights the crucial role of the post-transplant host immune response in the impact of transplanted mDAN survival, clearly indicating that this is an important area for further research. We investigated the functional effects of the host immune response on the survival and efficacy of grafted dopamine cells, explored potential strategic approaches to modulate the host immune response, and aimed to achieve optimal results in the future clinical application of CRT for PD.

[0034] Lessons from fetal ventral midbrain (fMV) transplantation research The clinical outcomes of fVM-based CRT varied considerably, and it was sometimes associated with graft-induced dyskinesia (GID). Beginning in the late 1980s, stem cell scientists attempted CRT using fVM tissue excised from aborted embryos (usually 6-9 weeks old) because aborted embryos showed the most promising results among various dopamine-producing cells. 6~13 In these early open-label trials using fVM tissue, some patients showed enhanced motor function, reduced rigidity and tremor, shorter "off" periods, reduced medication use, or, in a small number of cases, complete discontinuation. 18Some patients showed significant long-term improvements, including enhanced dopamine uptake, as revealed by F-DOPA PET scan analysis (Table 1). 14~35 Subsequent postmortem studies of deceased transplant patients further supported these prospective clinical outcomes, revealing successful engraftment of transplanted mDANs with robust growth and innervation into the host striatum, and demonstrating that engrafted mDANs functionally integrate into the neural circuits in the patients' brains. In summary, these successful clinical data provided a “proof of concept” for CRT in PD. Despite these prospective results, subsequent double-blind, sham-controlled studies showed that the clinical benefit was not statistically significant and that it is not recommended as a treatment for PD. 36~38 Furthermore, approximately 30% of transplant patients (18 out of 56) developed the serious side effect of Gender Identity Disorder (GID). 36~38 This significantly diminished the initial enthusiasm for the fetal cell-based approach. These inconsistent and disappointing results, along with ethical, medical, and practical limitations, made it impossible to implement this approach as a standard treatment for PD. 6~11 .

[0035] Poor and variable survival rates in transplant mDANs may underlie the inconsistent clinical outcomes observed in fVM-based CRTs. Why have the clinical outcomes of fVM transplantation been inefficient and variable? Over the past several decades, more than 400 PD patients have undergone fVM transplantation, and this has been thoroughly analyzed. 6~13 Furthermore, stem cell scientists conducted extensive preclinical studies using rat and mouse embryonic VM tissue with three objectives: (1) to understand the inefficient and variable clinical outcomes of fVM transplantation, (2) to elucidate the biological factors affecting transplant cell viability, and (3) to enhance the viability of transplanted mDANs for successful future CRT. 39~41These clinical and preclinical studies revealed key insights and a variety of potential factors contributing to inconsistent clinical outcomes. These factors encompass a range of variables, including varying patient ages and clinical conditions; diverse fetal cell preparation methods; heterogeneous and variable fetal conditions (typically requiring 6–8 fetuses per patient); variable immunosuppressive regimens; and target sites for transplantation (putamen, caudate nucleus, and / or substantia nigra) (Table 1). 6~14、18~31、35、37、38、42 While all of these donor-specific and / or host-specific factors are likely to play a significant role in clinical outcomes, we hypothesize that the poor and variable survival rates of grafted mDANs in the graft directly contribute to inefficient clinical outcomes. Indeed, the survival rate problem of grafted mDANs has been a focus of CRT research since the concept of fVM transplantation, due to the limited supply of aborted fetuses, as outlined in the insightful overview by Brundin and colleagues. 43 In 31 independent studies using rat embryo VM transplantation, the survival rate of grafted mDAN ranged from 0.7 to 23.3%, with a mean of 6.86%. Similarly, in studies of human VM graft transplantation in thymus-deficient rats... 44、45 and in postmortem studies of human fVM transplantation 9、12、13、22、23、27、37、38、42 A consistent survival rate range of 5–10% was reported. In conclusion, the key lesson from previous clinical and preclinical studies is that grafted mDANs are highly inefficient and variable, which is likely the underlying cause of the poor and inconsistent clinical outcomes observed.

[0036] [Table 1-1]

[0037] [Table 1-2]

[0038] When and why do grafted mDANs die during fVM transplantation? As mentioned above, the survival rate of transplanted mDANs has been extensively studied in preclinical studies using rodent embryonic VM cells to model human fVM transplantation. 43、46~52 A striking feature of these studies is that the majority of mDANs die immediately after transplantation, usually within a week, and the number of surviving mDANs in the graft does not increase or change at later stages. This observation is quite surprising considering that embryonic VM cells contain a considerable number of early progenitor cells in addition to already differentiated mDANs. These data suggest two possibilities: (1) progenitor cells in fVM have very limited ability to proliferate and differentiate, and / or (2) the host environment does not support their proliferation and differentiation into mDANs. Based on these preclinical studies, Brundin and colleagues proposed four distinct stages in which mDANs may die. 43 In Stage 1 (embryonic detachment), mDANs may die due to hypoxic and hypoglycemic damage that occurs during embryo removal from maternal blood supply. In Stage 2 (cell preparation), mDANs may die due to axonal transection and other traumatic damage caused by mechanical dissociation. In Stage 3 (intrastriatal injection), mDANs may die during the transplantation procedure and the period immediately following graft rejection, and in Stage 4 (graft maturation), mDANs may die during maturation and innervation in the host brain.

[0039] Extensive research has been conducted to understand the reasons for the poor survival rate of grafted mDANs and their premature death after transplantation, revealing a variety of possibilities. When mDANs excised from embryos are transplanted into the striatum under PD conditions, they become deficient in nutrients and growth factors essential for their survival. Consistent with this concept, cell pretreatment with growth factors such as basic fibroblast growth factor (bFGF) and glial cell line-derived neurotrophic factor (GDNF) significantly enhanced the survival rate of grafted mDANs. In particular, bFGF pretreatment resulted in approximately a twofold increase in survival rate. 53、54On the other hand, continuous delivery via co-transplantation of bFGF-overexpressing fibroblasts resulted in a tenfold increase, promoting more robust and rapid behavioral recovery. 55 Furthermore, since neuronal injury is often associated with excitotoxicity, oxidative stress, and calcium imbalance, researchers have explored the pathways and molecules associated with these phenomena. Among these, calcium channel blockers (such as flunarizine) and lipid peroxidation inhibitors (such as lasaroids) have shown noteworthy effects on mDAN survival rates. 56~58 Furthermore, inhibition of cell death pathways, such as caspase inhibitors (e.g., Ac-YVAD-cmk), significantly enhanced mDAN survival. 51 .

[0040] When do mDANs derived from hPSCs die during the transplantation process? The primary advantage of hPSCs lies in their ability to produce an unlimited supply of transplantable cell sources such as mDANs. Perhaps due to this advantage, cell viability in hPSC-based CRTs has not received sufficient attention and remains under-examined, unlike fVM-based CRTs. Given the significant differences in cellular, developmental, and proliferative properties between fVMs and hPSCs, both similar and different factors must be considered for successful CRTs in PD. To address these issues, we investigated the cell viability problem of hPSC-based CRTs using several rodent models, including wild-type (with immunosuppression) and thymus-deficient rats, as well as immunodeficient NOD SCID gamma (NSG) mice and humanized NSG mice. 84 Interestingly, the majority (approximately 90%) of mDANs (derived from both hESCs and hiPSCs) die within the first 1-2 weeks after transplantation, which is remarkably similar to fVM transplantation. 43、46~52Furthermore, this study revealed that the transplantation procedure itself induces an acute host inflammatory response. Surprisingly, even when only culture medium was injected without any cells, a host immune response was induced, and this was shown to be induced by the host's innate immune response. The immune response pattern was remarkably similar to that of traumatic brain injury (TBI) (Figure 18). 87、88 Therefore, the inventors referred to this phenomenon as "needle trauma." 84 This needle trauma appears to physically damage the host brain, causing acute cell death of host neuronal cells around the injection site, which in turn triggers the immediate secretion of pro-inflammatory cytokines (e.g., TNF and IL-1β) near the needle site, activation of astrocytes / microglia, and Iba-1β. + and major histocompatibility complex (MHC) II + This is thought to induce a subsequent immune response, including robust infiltration of inflammatory cells, which peaked on day 7, then declined at 1 month and disappeared at 6 months. Importantly, this needle trauma preferentially caused the death of most mDANs, rather than midbrain dopamine progenitor cells (mDAPs), within the graft. Furthermore, unlike fVM-based CRT, the total number of mDANs (and grafted cells) increased significantly during the later stages, suggesting that some mDAPs proliferated in the host brain after transplantation and differentiated into new mDANs. 84 In light of these new findings, and in conjunction with insights from previous fVM transplantation research, 43、46~52 The hPSC-derived mDAN appeared to die during one of the three stages of the hPSC-based CRT procedure (Figure 19).

[0041] In Stage 1, hESCs / hiPSCs are cultured and then differentiated in vitro over a specific period using an optimized procedure (Figure 19). Various laboratories use diverse optimized protocols. 65、89~96On the other hand, these methods typically utilize dual inhibition of SMAD targeting BMP and TGFβ signaling, as well as dual activation of WNT and SHH signaling, based on previous developmental studies. This in vitro differentiation process results in the generation of mDA cells, primarily mDAP and mDAN, ranging from 60–95% and 5–25%, respectively, depending on the individual protocol. 65、89~96 These protocols differ in detailed methods and culture medium components. For example, some protocols utilize 2D monolayer culture, while others use a combination of 2D and 3D cultures, such as embryoid bodies and suspension neurosphere cultures. mDA cells differentiated in vitro may lose viability during the differentiation process. For example, we have observed that a substantial portion of cells die and / or undergo apoptosis in uniformly distributed monolayer cultures, and that by dividing the monolayer into smaller isolated portions, as known as the “spotting method” described previously (e.g., International Publication No. 2020 / 237104), the percentage of these unhealthy or apoptotic cells during the in vitro differentiation process is significantly reduced. 65、97 Step 1 is similar to the initial steps of fVM-based CRT, where the donor embryo is excised and prepared as either a cell suspension or a tissue block for transplantation. 43 Previous studies have demonstrated that cell viability is substantially influenced by various conditions and types of graft media used to prepare fVM-derived cells. 98~100Therefore, in vitro differentiation conditions and culture media should be rigorously tested and optimized to maximize viability. This optimization is crucial because non-viable / apoptotic components of these final cell products can persist until transplantation and negatively impact clinical outcomes. For example, in conjunction with needle trauma-induced neuroinflammation, these dead or apoptotic components of transplanted cell products can trigger further host immune responses in stage 3. While FACS procedures can be designed to eliminate dead / dying cells, this may impose further adverse effects on viable cells.

[0042] In step 2, mDA cells differentiated in vitro are harvested and cryopreserved in liquid nitrogen until transplantation (Figure 19). Recent studies have shown that hPSC-derived mDA cells can be cryopreserved without losing their viability, cellular phenotype, and in vivo function. 65、93、101~103 Therefore, most groups plan to incorporate this cryopreservation step in hPSC-based CRT. Stage 2 involves three key steps: (i) harvesting and cryopreserving mDA cells differentiated in vitro using optimal cryopreservation medium in cryovials, (ii) storing multiple cryovials in liquid nitrogen, and (iii) thawing the cryovials and filling surgical syringes with mDA cells before transplantation. Theoretically, mDA cells may undergo cell death at any of these steps. For example, while hPSC-derived mDA cells have been reported to maintain their viability and function, the period during which they can be stored in liquid nitrogen without compromising their viability and function remains an unclear area. Given that needle trauma has been observed to preferentially affect mDANs over mDAPs, the effects of cryopreservation and subsequent handling are likely to vary significantly depending on the cell type. 84 Therefore, re-examining this problem and systematically analyzing the viability of various cell types at each step may be crucial for the final optimization of the process.

[0043] Stage 3 constitutes the final in vivo transplantation step and includes (i) surgical transplantation, (ii) the initial stage of graft establishment (less than 2 weeks), and (iii) the later stage (more than 2 weeks) (Figure 19). The host immune response is at work in these steps and can lead to mDA cell death. During the initial step of graft injection, mDA cells may retain viability in the syringe for a limited period (e.g., 10-20 minutes). However, prolonged delays in the injection process that exceed clinical procedure standards can lead to the death of some mDA cells (particularly mDANs) even before they are injected into the host brain. Therefore, it is desirable to minimize delays in the surgical procedure. In particular, the surgical procedure itself appears to induce an acute innate immune response, resulting in a defiant neuroinflammatory environment. Thus, injected mDANs are subjected to early preferential cell death. Furthermore, an adaptive immune response may occur between the grafted cells and the host immune system, which can lead to graft rejection in the absence of immunosuppression. In the later stages of engraftment, Stage 3, surviving mDA cells undergo maturation, differentiation, and integration into the host brain, establishing new functional circuits. However, some of the newly emerging and / or maturing mDA cells are likely to die even at this later stage due to a variety of factors, including persistent host immune responses, insufficient essential factors (e.g., blood supply, growth factors, and oxygen), as well as unfavorable PD environments characterized by neuroinflammation, oxidative stress, and α-synucleinopathy.

[0044] When human inducible pluripotent stem cell (hiPSC)-derived mDA cells are transplanted into the striatum of immunodeficient NOD SCID gamma or humanized mice, only a small percentage (less than 10%) of transplanted tyrosine hydroxylase (TH) is observed. + mDA neurons survived for two weeks after transplantation. In contrast, residual TH neurons survived. - Most of the graft cells survived. Surprisingly, autoregulatory T cells (T REG Transplantation of mDA cells significantly altered the response to needle trauma caused by transplantation, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, T REGFurthermore, simultaneous intrastriatal transplantation of hiPSC-derived mDA cells significantly protected grafted mDA neurons from needle trauma-related death and substantially improved treatment outcomes in a 6-OHDA-injured PD rodent model. REG Simultaneous transplantation with TH - It suppresses the undesirable proliferation of graft cells, and TH + This resulted in smaller grafts with a higher proportion and higher absolute number of neurons. In summary, these data highlight the importance of the initial inflammatory response to surgical injury in the difference in the viability of graft cellular components, and autologous T14 grafts with iPSC-derived mDA grafts. REG The simultaneous transplantation of these neurons is suggested to effectively reduce needle trauma-induced death of mDA neurons.

[0045] Treatment method Therefore, this method provides a clinically applicable personalized cell therapy for PD that increases the viability of grafted cells. In some embodiments, this specification describes methods for treating PD, methods for increasing cell viability in cell transplantation, methods for increasing the growth of transplanted cells, and / or methods for reducing abnormal proliferation during cell transplantation.

[0046] Since needle trauma is likely to be a significant factor in the success of cell therapy for all cell types, methods for increasing graft viability in cell therapy and / or reducing the immune response in subjects receiving cell therapy are also described herein.

[0047] In some embodiments, the method described herein involves administering an effective amount of T to subjects requiring administration (e.g., subjects receiving cell therapy and / or subjects with (or at risk of developing) PD). REG This involves administering a population of cells together with a second population of cells (e.g., mDA cells).

[0048] In some embodiments, the methods described herein involve administering an effective amount of antibody (Ab) (e.g., an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canakinumab), anti-IFN-γ Ab (e.g., emaparmab), anti-HMGB1 Ab, and anti-IL-1α Ab) together with a second population of cells (e.g., mDA cells) to a subject in need of administration (e.g., a subject receiving cell therapy and / or a subject with (or at risk of developing) PD).

[0049] In some embodiments, the method described herein is T REG Administration of a population of cells, Administration of a population of mDA cells, Administration of Abs that bind pro-inflammatory cytokines (e.g., anti-TNF-α Ab, anti-IL-1β Ab, anti-IFN-γ Ab), Administration of Abs that bind to damage-associated molecular patterns (DAMPs) (e.g., anti-HMGB1 Ab, anti-IL-1α Ab), T REG Administration of a composition comprising a population of cells and a second population of cells (e.g., mDA cells), and / or Administration of a composition comprising an Ab that binds pro-inflammatory cytokines (e.g., anti-TNF-α Ab, anti-IL-1β Ab, anti-IFN-γ Ab) or an Ab that binds DAMP molecules (e.g., anti-HMGB1 Ab, anti-IL-1α Ab) and a population of cells (e.g., mDA cells) to a target requiring administration. Includes.

[0050] Such subjects (for example, subjects receiving cell therapy, subjects identified as benefiting from cell therapy, subjects with PD, or subjects at risk of developing PD) can be identified by skilled healthcare professionals using methods known in the art.

[0051] T is useful in this method REGThe cell populations are described herein and are known in the art. Useful T REG Cells may express one or more of FoxP3, CD25, CD47, CD45, CD4, CD8, and CD127. See also U.S. Patent Application Publication No. 2023 / 0323299, No. 2023 / 0047159, No. 2020 / 0330515, and References 163-167. Accordingly, the methods herein may include obtaining a sample containing T cells (e.g., blood or PBMC cells) from a subject, isolating Treg cells, growing the Treg cells, and administering the cells. In some embodiments, primary somatic cells are obtained from a subject to be treated (e.g., a subject having (or at risk of developing) PD), while in some embodiments, cells are obtained from a different subject, in some embodiments, from a subject of the same species as the subject to be treated, preferably from an immunologically compatible subject.

[0052] Methods described herein may include, for example, the use of induced pluripotent stem cells (hiPSCs) that are similar to neurogenic floor plate cells, which are known in the art or can be generated using methods described herein. In some embodiments, a method for generating hiPSCs may include obtaining a population of primary somatic cells from a subject, e.g., a subject undergoing cell therapy, a subject with Parkinson's disease (PD), a subject at risk of developing PD, and / or a subject requiring treatment for PD. Preferably, the subject is a mammal, e.g., human.

[0053] The method may include obtaining primary somatic cells, generating a population of cells containing mDA cells, and administering the cells. In some embodiments, primary somatic cells are obtained from subjects to be treated (e.g., subjects with (or at risk of developing) PD), while in some embodiments, the cells are obtained from different subjects, in some embodiments, from subjects of the same species as the subjects to be treated, preferably from immunologically compatible subjects. Preferably, mDA cells are cells expressing one, two, or more mDA markers (e.g., FOXA2, OTX2, LMX1A, and EN1, e.g., FOXA2 and LMX1A) (optionally, TH cells co-expressing FOXA2, LMX1A, and NURR1). + The method described herein is sufficient to produce a population that includes cells and optionally expresses one, two, or more mDAN markers (e.g., TH, DAT, and PITX3), but does not include cells expressing SOX1, PAX6, and KI67.

[0054] Populations of mDA cells useful in this method are described herein and are known in the art. These mDA cell populations useful in the method described herein are derived from hESCs / hiPSCs and can subsequently be differentiated in vitro over a specific period using an optimized procedure (Figure 19). 65、89~96In some embodiments, the population of mDA cells includes mDAP and mDAN. In some embodiments, the population of mDA cells includes at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% mDAP and at least about 5, 10, 15, 20, or 25% mDAN. See also U.S. Patent Publication Nos. 2022 / 0243174, 2018 / 0371422, 2012 / 0128655, 2013 / 0052268, 2016 / 0002604, 2014 / 0199274, and 2009 / 0226401, as well as U.S. Patent Nos. 11,898,169, 11,001,809, 9,657,273, and 9,750,768, and references 61-65 and 89-96.

[0055] In some embodiments, the methods described herein reduce the immune response in subjects induced by needle trauma during cell transplantation and / or treat PD. In some embodiments, the methods described herein treat TH in grafts. + To increase the number and / or percentage of cells (e.g., T REG (Compared to grafts in which neither the cell population nor the pro-inflammatory cytokine-targeting Ab is simultaneously transplanted.) In some embodiments, the methods described herein involve TH in the graft. + Cell:TH - Increase the cell ratio and / or TH in the graft + Cell: Ki67 + Increase the ratio of cells (for example, T REG (Compared to grafts in which neither the cell population nor the pro-inflammatory cytokine-targeting Ab is simultaneously transplanted.) In some embodiments, the method described herein involves Ki67 in the graft. + Reduce the number and / or percentage of cells, and / or TH in the graft. - Reduce the number and / or percentage of cells (e.g., T REG(Compared to grafts in which neither the cell population nor the pro-inflammatory cytokine-targeting agent (Ab) was simultaneously transplanted.)

[0056] In some embodiments, the cells, antibodies, and compositions described herein can be administered to subjects described herein. In some embodiments, any administration step of any of the methods described herein includes systemic, parenteral, intravenous, intracerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular administration. In some embodiments, the administration step includes intravenous, intracerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular injection. In some embodiments, the administration step includes direct injection into the substantia nigra pars compacta of the brain. In some embodiments, the administration step includes direct introduction of cell therapy and / or antibodies into the brain or cerebrospinal fluid (CSF) of the subject.

[0057] In some embodiments, cells, antibodies, and compositions can be administered using methods known in the art. In some embodiments, cells are administered by being implanted bilaterally or unilaterally into one or more of the affected areas of the brain, for example, using magnetic resonance imaging-guided stereotactic brain surgery, for example, in the caudate nucleus, putamen, and substantia nigra. See, for example, Garitaonandia et al., Stem Cells Dev. 2018 Jul 15;27(14):951-957, Kikuchi et al., Nature 548:592-596 (31 August 2017), MOrizane et al., Nature Communications 8:385 (2017), and Sonntag et al., Prog Neurobiol. 2018 Sep;168:1-20.

[0058] In some embodiments, the cells, antibodies, and compositions described herein may be administered in single or repeated doses.

[0059] In the Party embodiment, T REGCells and transplanted cells (e.g., mDA cells) are administered to the subject simultaneously. In some embodiments, T REG Cells and transplanted cells (e.g., mDA cells) are administered separately. In some embodiments, T REG Cells are first administered to the target, followed by the administration of transplanted cells (e.g., mDA cells). In some embodiments, transplanted cells (e.g., mDA cells) are first administered to the target, followed by T REG The cells are administered to the target.

[0060] In some embodiments, antibodies (Ab) (e.g., antibodies that conjugate pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canakinumab), anti-IFN-γ Ab (e.g., emaparmab), anti-HMGB1 Ab, anti-IL-1α Ab) and transplanted cells (e.g., mDA cells) are administered to the subject simultaneously. In some embodiments, antibodies (Ab) (e.g., antibodies that conjugate pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canakinumab), anti-IFN-γ Ab (e.g., emaparmab), anti-HMGB1 Ab, anti-IL-1α Ab) and transplanted cells (e.g., mDA cells) are administered separately. In some embodiments, an antibody (Ab) (e.g., an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canakinumab), anti-IFN-γ Ab (e.g., emaparmab), anti-HMGB1 Ab, anti-IL-1α Ab) is administered to the subject first, followed by the administration of transplanted cells (e.g., mDA cells). In some embodiments, transplanted cells (e.g., mDA cells) are administered to the subject first, followed by the administration of an antibody (Ab) (e.g., an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules, optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canakinumab), anti-IFN-γ Ab (e.g., emaparmab), anti-HMGB1 Ab, anti-IL-1α Ab). In some embodiments, the antibody and cells are administered intrastriatically to the patient. In some embodiments, cells are administered to the subject using one administration method (optionally, intrastriatal), and antibodies are administered to the subject using a different administration method (optionally, intravenously).

[0061] In the Party embodiment, T REG The cells are autologous T REG cells, allogeneic T REG Cells, or heterologous T cells REGIt is a cell. In some embodiments, the transplanted cell (e.g., mDA cell) is an autologous mDA cell, an allogeneic transplanted cell (e.g., mDA cell), or a xenogeneic transplanted cell (e.g., mDA cell). In some embodiments, T REG The cell is transplanted into the striatum. In some embodiments, the transplanted cell (e.g., mDA cell) is transplanted into the striatum.

[0062] Compositions comprising an antibody (Ab) (e.g., an antibody that binds a pro-inflammatory cytokine or a damage-associated molecular pattern (DAMP) molecule, optionally an anti-TNF-α Ab (e.g., infliximab), an anti-IL-1β Ab (e.g., canakinumab), an anti-IFN-γ Ab (e.g., emapalumab), an anti-HMGB1 Ab, an anti-IL-1α antibody) and methods of administering such compositions are well known in the art.

[0063] In some embodiments, the administration step includes systemic, parenteral, intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intrashell, intrahippocampal, intrastriatal, or intraventricular administration. In some embodiments, the administration step includes intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intrashell, intrahippocampal, intrastriatal, or intraventricular injection. In some embodiments, the administration step includes direct injection into the substantia nigra pars compacta of the brain. In some embodiments, the administration step includes introducing the cell therapy and / or the antibody directly into the subject's brain or cerebrospinal fluid (CSF).

[0064] See also U.S. Patent Application Publication Nos. 2022 / 0243174, 2018 / 0371422, 2012 / 0128655, 2013 / 0052268, 2016 / 0002604, 2014 / 0199274, and 2009 / 0226401, as well as U.S. Pat. Nos. 11,898,169, 11,001,809, 9,657,273, and 9,750,768.

[0065] Needle trauma is known in the art, but needle trauma is of particular concern in cell therapies that clog needles or require larger diameter needles to administer without damaging the cell therapy. Needle characteristics include inner diameter and outer diameter, length, rigidity, and bevel design. Deep subcortical target structures such as the caudate nucleus or putamen require long, thin needles / cannulas (usually 19 cm or longer) of sufficient rigidity to penetrate the target site without damaging the overlying structures. Shorter needles (8 - 10 cm) require direct brain exposure, which is more invasive (Amer, M.H., et al. (2017) Translational considerations in injectable cell - based therapeutics for neurological applications: concepts, progress and challenges. npj Regen Med 2, 23, Kondziolka, D., et. al. (2011) Injection parameters affect cell viability and implant volumes in automated cell delivery for the brain. Cell Transplant. 20, 1901 - 1906).

[0066] The needles used to administer cell grafts may be at least 30G, 28G, 26G, 24G, 22G, 20G, 18G or thicker, and / or have an outer diameter (O.D.) of at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 mm (step - wise outer diameters between any of these parameters, e.g., 1.5 - 0.5 mm).

[0067] Needle parameters are known in the art and can be determined by skilled medical practitioners using methods known in the art. See also the following: · Song et al., JCI 2020, Park et al., Nature 2023:26G, outer diameter (OD), 0.464mm, • Doi et al., Nature Communication 2020: 22G, OD, 0.718 mm and 24G, OD, 0.566 mm, · Kriks et al., Nature 2011:28G, 0.362mm, · Schweitzer et al., NEJM 2020:22G, OD; 0.72mm, • Freed et al., NEJM 2001: Tissue implants were positioned using stainless steel guide cannulas with stepped outer diameters ranging from 1.5 to 0.6 mm. A round stylet was included in the cannula's perforation while it was passing through to the posterior end of the putamen. · Spencer et al., NEJM 1992:1 mm, and • Freed et al., NEJM 1992: A 1.5 mm cannula extended from its support in the stereotactic device to the surface of the brain, where an internal stylet with an outer diameter of 0.46 or 0.64 mm penetrated the putamen.

[0068] During needle injection, the brain suffers physical damage, causing rupture of resident cells such as neurons, astrocytes, microglia, and oligodendrocytes. This rupture, caused by the impact of the needle, triggers the rapid release of damage-associated molecular patterns (DAMPs) in these cells, which affect neighboring cells and induce the production and secretion of cytokines and chemokines resulting from activation.

[0069] The data presented herein are for the first time to show that this needle trauma is directly involved in the death of host brain cells and a significant increase in inflammation near the injection site, and is a major obstacle to success in cell therapy (e.g., the success of grafts in cell transplantation).

[0070] Host immune response in hPSC-based CRT The survival of engrafted mDA cells is greatly influenced by various levels of the host immune response. A fundamental requirement for the survival of transplanted cells is to avoid graft rejection, a process regulated by the host's adaptive immune system. Traditionally, certain specialized organs, such as the brain and eyes, have been considered to have immune privilege. 104 This may explain why early fVM transplantation studies did not use immunosuppressants (Table 1). Conversely, from a counter-perspective, it has been suggested that even autologous cells can trigger an immune response when differentiated cell products express immunogenic antigens. 83、105 To investigate this controversial issue of immunogenicity in autologous versus allogeneic brain grafts, the inventors performed transplantation experiments in the striatum of NSG mice, patient-humanized NSG mice, and allogeneic-humanized NSG mice, containing patient-derived and allogeneic mDA cells. The inventors' findings revealed that autologous mDA cells were rejected in allogeneic-humanized mice, while autologous mDA cells were accepted in autologous-humanized mice. 61 Allogeneic mDA cells were shown to be highly likely to be rejected without immunosuppression when transplanted into the brain. It is noteworthy that the brain's immune privilege may require an intact blood-brain barrier (BBB), which is evident in the PD brain. 106 Furthermore, these cells can be damaged by surgical procedures. In addition, recent research by the inventors suggests that the host immune system interacts not only with grafted cells but also with surgical instruments (i.e., needles), clearly demonstrating the crucial role of the host innate immune response in the process, as described below. 84 .

[0071] Adaptive immune response and immunosuppression Adaptive immune responses play a crucial role in the rejection of allogeneic or xenografts. 107This adaptive immunity is characterized by a precise and highly sensitive response of T and B cells to MHC, also known as HLA, which is expressed by all nucleated cells. Even slight differences in the expression of HLA molecules (or more) by grafted cells can trigger aggressive rejection by the body. Therefore, even intracerebral transplantation of allogeneic mDA cells requires considerable immunosuppression. In fact, all current and future clinical trials involving allogeneic hPSC-derived mDA cells are planned to use substantial levels of immunosuppression. 90、93、95、108 It is crucial to recognize that long-term immunosuppression is associated with a variety of side effects, including increased susceptibility to infections and malignancies, along with additional costs and inconveniences. Previous studies have demonstrated a correlation between infection risk and the dose and duration of immunosuppressants. 109、110 When used in combination with other immunosuppressants, the risk and severity of infection increased. 111 Furthermore, immunosuppression by cyclosporine is known to increase the risk of malignant tumors such as lymphoma or skin cancer. 112、113 Other possible side effects include loss of appetite, vomiting, nausea, increased hair growth, and tremors, but these symptoms usually subside as the body adapts to the immunosuppressant. Given these various side effects, researchers are actively exploring ways to optimize the route of administration, dosage, and duration of immunosuppression. For example, a promising strategy involves leveraging topical immunosuppression to avoid systemic side effects associated with standard immunosuppression. 114 .

[0072] Innate immune response Innate immunity constitutes the body's initial line of defense and includes physical, chemical, and cellular mechanisms to rapidly counter or eliminate foreign antigens introduced from external sources. 107、115 Unlike acquired immunity, which is acquired through exposure to invaders, innate immunity is present from birth and relies on antigen-nonspecific defense mechanisms. The primary functions of innate immunity are to directly eliminate pathogens and, most importantly, to rapidly mobilize immune cells to the site of infection and inflammation.116、117 This is achieved by sensing pathogens and producing cytokines (e.g., TNFα, IL-1β, IFNγ) and chemokines (e.g., CXCL, CCL). Furthermore, innate immunity responds to physical damage caused by external mechanical forces. A typical example is neuroinflammation induced by TBI, where initial traumatic injury triggers disruption of both macrobarriers (e.g., skin) and microbarriers (e.g., cell membranes), leading to the secretion of various immune molecules. 118 .

[0073] Recent research by the inventors has shown that transplantation procedures using needles significantly induce the host's innate immune response, which has been referred to as "needle trauma" due to its similarity to TBI (Figure 18). 84 Primary TBI occurs immediately after impact and involves damage to the brain's physical structures, including meningeal and neuronal contusions, axonal shearing, and vascular damage. Secondary TBI develops gradually and involves various cellular processes such as blood-brain barrier (BBB) ​​dysfunction, excitotoxicity, mitochondrial dysfunction, oxidative stress, inflammation, and cell loss. 115 Needle trauma similarly induces these downstream pathways, and further elucidation may provide promising molecular targets to mitigate its impact on the viability of grafted mDA cells, thereby potentially enhancing the outcomes of hPSC-based CRT (see below). It should be noted that PD patients typically exhibit both high levels of encephalitis and systemic inflammation compared to healthy individuals. 119~121 This high level of inflammation in PD patients may have a more pronounced adverse effect on CRT outcomes. Furthermore, it is essential to recognize that the systemic immune system varies considerably between individuals, and these immunological differences can lead to differences in treatment outcomes. However, it is important to recognize that the primary factor affecting the survival rate of transplanted mDANs is inflammation induced by brain damage caused by the transplantation procedure itself. Therefore, further research efforts are needed to fully understand the distinct roles of intrinsic inflammatory status and surgery-induced inflammation in determining the survival rate of grafted mDANs.

[0074] Promising strategies for enhancing cell viability for successful hPSC-based CRT While hPSC-based CRT offers the advantage of generating an unlimited number of transplantable cells, it also faces the challenge of poor survival rates for hPSC-derived mDANs, as has been evident from previous preclinical studies (Table 2). 96、122~131 Therefore, the development of novel strategies to enhance the survival rate of mDANs in CRT is inevitable. One promising approach is to modify the host immune response to create a more favorable environment for transplanted cells.

[0075] Strategies targeting adaptive immunity Avoiding graft rejection is a prerequisite for the survival rate of grafted cells. Therefore, all allografts require a considerable level of immunosuppression, but the duration of immunosuppression required for each patient remains unclear. Non-human primates 132~134 and humans 61 Recent studies in this field have demonstrated that autologous transplantation does not require immunosuppression. However, this autologous approach involves substantial time and cost. Alternative strategies that reduce the need for immunosuppression include the use of HLA-matched iPSCs. Evidence supporting this approach comes from recent primate studies showing that transplantation of mDA cells derived from HLA-matched primate iPSCs reduces the host immune response and increases mDA survival. 12 Therefore, numerous groups are striving to establish HLA-matched hiPSCs as a bank from common HLA homozygous donors, aiming to minimize graft rejection after transplantation and reduce time and effort compared to the autologous approach (Figure 20). 135、136 Professor Yamanaka's group estimated that hiPSC lineages derived from approximately 140 specific HLA homozygous donors are sufficient to cover up to 90% of the Japanese population. 137 Due to genetic diversity, large-scale hPSC banks are currently being established in the United States to encompass diverse ethnic groups such as European Americans, African Americans, Hispanics, and Asians.138 Despite these efforts, even with HLA-matched cells, immune responses can still occur through indirect pathways triggered by HY minor histocompatibility antigens or through innate immunity mediated by natural killer (NK) cells. 139、140 .

[0076] Efforts to develop "universal donor stem cells" that can avoid immune rejection are attracting significant attention. 141、142 This approach primarily aims to eliminate HLA molecules that are critical to adaptive immunity and graft rejection in donor cells. Early attempts in 2013 aimed to eliminate HLA molecules that specifically target HLA class I, primarily expressing β2-microtubulin (B2M), which is structurally non-polymorphic heavy chain across most cell types. 143、144 However, cells lacking HLA class I, generated by genome engineering approaches, showed limitations because they were susceptible to lysis by NK cells via the "missing self" response. 145 In particular, when the CD94 / NGK2A complex on NK cells associates with any HLA class I molecule, including HLA-E, which is recognized for its minimal pleomorphism and expression only in B2M knockout cells, the lysis mechanism is avoided. 145、146 In recent years, through the process of technological development and optimization, Schrepfer and his colleagues 147They devised a method to generate low immunogenic donor iPSCs through the following three steps: (1) CRISPR / Cas9-mediated knockout of the B2M gene (a component of HLA class I), (2) lentiviral overexpression of the CIITA gene (a master regulator of HLA class II), and (3) lentiviral overexpression of the immune receptor CD47 transgene. Schrepfer and colleagues demonstrated that various cell types (e.g., endothelial cells, smooth muscle cells, and cardiomyocytes) derived from these manipulated donor iPSCs effectively avoided immune rejection in completely HLA-incompatible host animals and survived for extended periods without immunosuppression. Furthermore, Akitsu Hotta and colleagues created a pseudo-homozygous iPSC system by using CRISPR-Cas9 to bi-allelically disrupt HLA-A and HLA-B while retaining a single HLA-C allele. 148 Twelve HLA-C-retaining iPSC lines with HLA class II knockout are estimated to be immunologically compatible with over 90% of the world's population. While these strategies are promising for iPSC-based regenerative medicine applications, it remains unclear whether these manipulated cells will retain full functionality and low immunogenicity after transplantation into patients. 149 Extensive genetic manipulation (e.g., ectopic CD47 expression) can lead to unintended adverse effects such as oncogenic transformation and increased susceptibility to infection. 150、151 Furthermore, if cells are infected with a virus, rapid removal may not be feasible, highlighting the need for the development of genetically engineered kill switches. However, this strategy could potentially induce unintended adverse reactions, and further research is needed to refine and implement safety strategies. 152、153 Furthermore, even if these genetic modifications successfully avoid immune rejection through adaptive immunity, the transplanted graft may still be susceptible to the innate immune response caused by needle trauma.

[0077] Strategies targeting innate immunity The host innate immune response induced by TBI or needle trauma initiates inflammation within minutes of injury. This response is characterized by the secretion and upregulation of injury-associated molecular patterns (DAMPs), cytokines, and chemokines, immune cell infiltration (e.g., neutrophils and myeloid cells), followed by activation of glial cells (astrocytes and microglia), and leukocyte recruitment. 107、115、154 Numerous studies have demonstrated that these molecules, including DAMP, cytokines, and chemokines, are acutely secreted within 6 hours after TBI. 155 Therefore, various research attempts regarding therapeutic development in TBI have sought to target innate immunity. These studies have revealed that blocking these molecules using specific inhibitors has a significant effect in treating TBI in animal models. 156~159 It is interesting to determine whether these inhibitors and / or neutralizing antibodies can produce similar effects in needle trauma and improve the viability of engrafted mDA cells. This approach justifies future investigation (Figure 21). Furthermore, considering that needle trauma acutely induces the secretion of various cytokines, chemokines, and DAMP (within minutes to hours post-injury), an interesting approach involves delaying cell grafting after needle insertion (without cells). Indeed, previous studies have shown that delaying the injection of dopaminergic cell suspension to more than one hour after cannula insertion significantly increases neuronal viability. 160、161 .

[0078] Regulatory T cells (T REG ) plays a crucial role in maintaining immune tolerance and homeostasis. 162、163 . Regulatory T cells (T REG It is involved in numerous autoimmune and inflammatory diseases and is clinically used to improve survival rates in various organ transplants through adoptive transfer after ex vivo proliferation. 164 At TBI, REG It infiltrates the site of injury after initial inflammatory cell infiltration and promotes the repair process.165、166 Therefore, private T REG It was hypothesized that this could mitigate the innate immune response induced by needle trauma and enhance the survival rate of grafted mDANs. REG In verifying the effects 164、167 Approximately 1,000,000 ex vivo-grown autologous T cells REG It is administered intravenously, MHCII + It caused a modest but significant reduction in the innate immune response, as indicated by a decrease in cell infiltration. However, due to the rapid onset of needle trauma-induced neuroinflammation in the brain, the adoptive transfer method was T REG It can be inefficient because it takes time for them to infiltrate and become functional. On the other hand, only 2% (20,000) of T REG Direct intrastriatal transplantation of cells resulted in more rapid and significant behavioral improvements in a rodent model of PD, with potent suppression of needle trauma-induced inflammation and a significant increase in the survival rate of grafted mDANs (Table 2). 84 Nevertheless, the mDAN rescue was incomplete, and REG This suggests that further optimization of simultaneous intrastriatal transplantation is needed. One possible approach is to use a hydrogel system before transplantation. REG and includes encapsulating mDA cells (Figure 21) 168~170 Furthermore, considering that needle trauma can occur in CRT for other CNS and non-CNS diseases, REG It is of great interest to investigate whether simultaneous transplantation can similarly reduce needle trauma-induced inflammation and generally enhance the viability of desired therapeutic cell products. 171 .

[0079] Strategies targeting long-term differentiation and maturation in the later stages of graft establishment. Even if grafted mDA cells manage to evade the initial immune attack after transplantation and survive the early stages, they face a suboptimal environment, unlike early brain development where all essential nutrients, oxygen, growth factors, associated transcription factors, and developmental signals are supplied or induced in precise temporal and spatial patterns. Instead, the new environment for grafted mDA cells is characterized by elevated neuroinflammation, limited / absent supply of essential factors, and / or pathological conditions of aged Parkinson's disease such as alpha-synucleinopathy. 172、173 In fact, recent single-cell RNA sequencing analyses have revealed that grafted cells often exhibit inappropriate differentiation into mDANs, instead favoring differentiation into alternative cell types such as astrocytes and vascular leucomeninge cells. 174 These inappropriate and heterogeneous graft cell populations may be non-functional and potentially induce undesirable immunogenicity. This challenge is exacerbated by the lengthy process by which these mDA cells mature into mDANs through neuronal growth, re-innervate the host brain, and ultimately establish a new functional network. This process can take a considerable amount of time, ranging from 4–6 months in rodent brains to 1–3 years in human brains (Table 1). Therefore, developing and implementing therapeutic strategies aimed at enhancing the viability, differentiation, and / or maturation of grafted mDA cells is critical. In support of this idea, pre-treatment of fVM cells with growth factors such as bFGF and GDNF increased the viability of grafted mDANs by approximately twofold. 53、54 On the other hand, long-term supply of bFGF resulted in a tenfold increase in the number of surviving mDANs. 55 Furthermore, recent studies have demonstrated that pretreatment with GDNF or viral delivery enhances the survival and differentiation of hPSCs (Table 2). 123、175~177 Based on these promising findings, promising strategies involve promoting a sustained supply of relevant growth factors, which could improve not only survival rates but also the differentiation of new mDANs from the graft.

[0080] [Table 2-1]

[0081]

Table 2-2

[0082]

Table 2-3

[0083] Since the pathological feature of PD is the selective loss of mDAN in the substantia nigra, CRT has been the focus of extensive research over the past 40 years. In particular, fVM-based CRT has provided not only proof of concept but also valuable lessons. Among these, the most important lesson is that the survival rate of transplanted mDAN is very limited, which may underlie the variable and often inefficient clinical outcomes. Despite extensive research efforts, the molecular and cellular mechanisms underlying the acute and extensive death of transplanted mDAN are still only partially understood.

[0084] Recent advances in stem cell technology offer promising prospects for hPSC-based CRT, with various hPSC sources (e.g., autologous hiPSCs, HLA-matched hiPSCs, and allogeneic hESCs) being explored for the scalable production of transplantable mDA cells in ongoing or upcoming clinical trials. However, several critical issues must be addressed for the successful implementation of hPSC-based CRT. First, improving transplant mDA survival is a top priority, drawing lessons from fVM-based CRT. Second, it is essential to address the multiple stages and steps of CRT where mDA cells are at risk of death. Third, understanding and manipulating both the host's adaptive and innate immune responses is critical, with recent evidence highlighting the role of surgical procedures in inducing the host's innate immune response. Future research efforts should focus on developing effective strategies for manipulating host immune responses to enhance transplant mDA survival and improve clinical outcomes.

[0085] While grafted mDANs may survive after transplantation, it is important to note that they face an unfavorable PD host environment, including neuroinflammation and a limited supply of essential factors. These challenges make long-term survival and maturation of mDANs extremely difficult, and further research is needed in this area. Furthermore, PD patients often exhibit degeneration of other neurons besides mDANs, such as noradrenergic and / or serotonergic neurons. 178~181 This contributes to various non-motor deficits. Therefore, even the most successful hPSC-based CRT may not provide a "cure" for PD. Instead, it is expected to be an integral component of a comprehensive treatment strategy that complements other approaches such as novel drug treatments (e.g., anti-inflammatory and neuroprotective agents) and gene therapy.

[0086] Compositions for cell therapy As stated above, the method described herein is as follows: T REG A group of cells, A population of mDA cells, Antibodies that bind pro-inflammatory cytokines (e.g., anti-TNF-α Ab, anti-IL-1β Ab, anti-IFN-γ Ab), or antibodies that bind DAMP molecules (e.g., anti-HMGB1 Ab, anti-IL-1α Ab), T REG A composition comprising a population of cells and a second population of cells (e.g., mDA cells) (the cells may be mixed together in a single composition or administered separately), and / or A composition comprising an antibody (e.g., an antibody that conjugates pro-inflammatory cytokines or damage-associated molecular patterns (DAMP) molecules, optionally anti-TNF-α Ab, anti-IL-1β Ab, anti-IFN-γ Ab, anti-HMGB1 Ab, anti-IL-1α Ab) and a population of cells (e.g., mDA cells) (the antibody and cells may be mixed together in a single composition or administered separately). This includes using a composition containing one or more of the above on an object that requires its use.

[0087] T is useful in this method REG The cell populations are described herein and are known in the art. Useful T REG Cells can express one or more of FoxP3, CD25, CD47, CD45, CD4, CD8, and CD127. See also U.S. Patent Application Publication Nos. 2023 / 0323299, 2023 / 0047159, 2020 / 0330515, and references 163–167.

[0088] Populations of mDA cells useful in this method are described herein and are known in the art. These mDA cell populations useful in the method described herein are derived from hESCs / hiPSCs and can subsequently be differentiated in vitro over a specific period using an optimized procedure (Figure 19). 65、89~96In some embodiments, the population of mDA cells includes mDAP and mDAN. In some embodiments, the population of mDA cells includes at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% mDAP and at least about 5, 10, 15, 20, or 25% mDAN. A useful population of mDA cells is cells expressing one, two, or more mDAP markers (e.g., FOXA2, OTX2, LMX1A, and EN1, e.g., FOXA2 and LMX1A) (optionally, TH cells co-expressing FOXA2, LMX1A, and NURR1). + The sample may include cells expressing one, two, or more mDAN markers (e.g., TH, DAT, and PITX3), and optionally may not include cells expressing SOX1, PAX6, and / or KI67. See also U.S. Patent Publication Nos. 2022 / 0243174, 2018 / 0371422, 2012 / 0128655, 2013 / 0052268, 2016 / 0002604, 2014 / 0199274, and 2009 / 0226401, as well as U.S. Patent Nos. 11,898,169, 11,001,809, 9,657,273, and 9,750,768, and references 65 and 89-96.

[0089] In some embodiments, the methods described herein involve the administration of a composition comprising a mixture of cell types (e.g., a composition comprising both a population of Treg cells and a population of mDA cells). The methods described herein have demonstrated that significantly smaller numbers of Treg cells need to be administered together with mDA cells, in contrast to when Treg cells are administered systemically (e.g., IV). Thus, the ratio of Treg cells to mDA cells may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0090] The methods and compositions described herein may be used for, or may contain, other cell types, including other neuronal types. Numerous neuronal differentiation protocols are known in this field, for example, Salimi et al., Mol Biol Rep. 2014 Mar;41(3):1713-21, Gunhnlar et al., Molecular Psychiatry 23:1336-1344(2018), Trilck et al., Methods Mol Biol. 2016;1353:233-59, Zhang et al., Stem Cell Res Ther. 2018 Mar 15;9(1):67, D'Aiuto et al., Organogenesis. 2014;10(4):365-77, Marton and Ioannidis, Stem Cells Translational Medicine 2019;8:366-374, and Bell et al. Bio-protocol 9(5):e3188(2019). See DOI:10.21769 / BioProtoc.3188, Bianchi et al., Stem Cell Research 32:126-134(2018).

[0091] In some embodiments, a composition comprising any of the cells and antibodies described herein may include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes salines, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for pharmaceutically acceptable administration. In some embodiments, the composition is formulated to be suitable for its intended route of administration.

[0092] Methods for formulating appropriate pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and the book series Drugs and the Pharmaceutical Sciences: A Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and osmotic regulators such as sodium chloride or glucose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0093] Suitable compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippani, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow for easy injection. The composition must be stable under manufacturing and storage conditions and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, it is preferable to include isotonic agents in the composition, such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride. Long-term absorption of the injectable composition can be achieved by including absorption retarders, such as aluminum monostearate and gelatin, in the composition. Useful solutions and compositions may include any of the solutions, buffers, and stabilizers disclosed herein, for example, the composition and / or method may involve using a hydrogel system known in the art to hydrate cells (e.g., T) before administration. REG This may include encapsulating cells and / or mDA cells (Figure 21). 168~170 .

[0094] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredient and any further desired components from their pre-sterile filtered solution.

[0095] In some embodiments, compositions such as sustained-release formulations containing implants and microencapsulated delivery systems are prepared using carriers that protect cells and / or antibodies from rapid elimination from the body. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. Nanoparticles (1-1,000 nm) and microparticles (1-1,000 μm), such as nanospheres and microspheres, as well as nanocapsules and microcapsules, can also be used. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811, Bourges et al., Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Invest Opth Vis Sci 44:3562-9 (2003), Bourges et al., Intraocular implants for extended drug delivery: therapeutic applications. Adv Drug Deliv Rev 58:1182-1202 (2006), Ghate et al., Ocular drug delivery. Expert Opin Drug Deliv 3:275-87 (2006), and Short, Safety Evaluation of Ocular Drug Delivery Formulations: Techniques and Practical Considerations. Toxicol Pathol 36(1):49-62 (2008).

[0096] Any of the compositions described herein may be contained in a container, kit, pack, or dispenser, along with instructions for administration.

[0097] Dosage An "effective dose" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic dose is an amount that achieves a desired therapeutic effect. This amount may be the same as, or different from, a prophylactic effective dose, which is the amount required to prevent the onset of a disease or disease symptom. An effective dose may be administered in one or more doses, applications, or dosages. The therapeutic effective dose (i.e., effective dosage) of a therapeutic compound depends on the selected therapeutic compound. The composition may be administered once or multiple times per day, including once every other day, or once or multiple times per week. It will be understood by those skilled in the art that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health status and / or age, and other pre-existing diseases, may influence the dose and timing required to effectively treat the subject. Furthermore, the treatment of a subject with a therapeutically effective dose of a therapeutic compound described herein may include a single treatment or a series of treatments.

[0098] References (for detailed explanations only)

[0099] [Table 3-1]

[0100] [Table 3-2]

[0101] [Table 3-3]

[0102] [Table 3-4]

[0103] Table 3-5

[0104] Table 3-6

[0105] Table 3-7

[0106] Table 3-8

[0107] Table 3-9

[0108] Table 3-10

[0109] Table 3-11

[0110] Table 3-12

[0111] Table 3-13

[0112] Table 3-14

[0113] [Table 3-15] [Examples]

[0114] The present invention is further described in the following embodiments, but these do not limit the scope of the invention as described in the claims.

[0115] Methods used in the examples and experiments animal Fischer 344 (8-10 weeks old, male) rats, Sprague Dolly (8-10 weeks old, male) rats, and NOD SCID gamma (NOD.Cg-Prkdc SCID Il2rg tm1Wjl (SzJ, 8-10 weeks old, male) mice were purchased from Charles River or The Jackson Laboratory and housed in individually ventilated micro-isolation cages under specific pathogen-free (SPF) conditions at the Mailman Research Center Animal Care Facility of McLean Hospital. All animals were maintained on a 12-hour:12-hour light / dark cycle and had free access to food and water. The animal studies were conducted in accordance with National Institutes of Health guidelines and approved by McLean Hospital's Institutional Animal Care and Use Committee (2015N000001 and 2015N000002).

[0116] C4-mDAP differentiation (C4-hiPSC) from hiPSCs in PD patients The inventors have previously described the efficient generation of mDA precursor cells (mDAPs) by differentiating human induced pluripotent stem cells (hiPSCs) using their novel "spotting"-based method. 21、22In short, C4-hiPSCs were seeded into 12 Matrigel-coated spots (spots on a grid of six intersections of lines) and retained in those plates from D1 to D14 of differentiation. To pattern differentiated cells to the bottom plate and mDAP, 10 μM SB431542 (D1-8), 200 nM LDN193189 (D1-12), 100 ng / ml SHH (D2-10), 100 ng / ml FGF8 (D2-10), 2 μM purmorphamine (D2-10), and 1 μM CHIR99021 (D4-12) were added to the culture medium. On day 9, cells were treated with 40 μM quercetin for 16 hours to eliminate residual hiPSCs. On D15 of differentiation, the cells were dissociated into single cells and re-seed at a rate of approximately 2.5 million cells per dish in induction medium supplemented with 20 ng / ml BDNF, 20 ng / ml GDNF, 500 μM dbcAMP, 200 μM ascorbic acid, 10 ng / ml TGF-β3, and 10 μM DAPT on poly-L-ornithine / fibronectin / laminin-coated dishes (D12-15). mDAP cells (D15 cells) were supplied every two days and maintained without subculturing until day 28. More detailed information on C4-hiPSC reprogramming and C4-mDAP differentiation has been previously described. 16、21、22 B1-mDAP or H9-mDAP were generated from independent hiPSC lines (B1) and H9-hESC lines, respectively, derived from separate sporadic PD patients, using the same in vitro differentiation procedure. All cells were validated for mycoplasma contamination every four weeks using the Venor GeM Mycoplasma Detection Kit (MP0025, Sigma-Aldrich). All experiments were performed using cells that had been shown to be mycoplasma-negative.

[0117] In vitro differentiation of hiPSCs and hESCs into GABAergic neurons and progenitor cells The present inventors, Liu et al. 54As described, hiPSCs and hESCs were differentiated into GABAergic progenitor cells. Briefly, C4-hiPSCs, B1-hiPSCs, or H9-hESCs were trypsin-treated and grown as suspension spheres in low-adhesion flasks from differentiation D0 to D14. To pattern the differentiated cells into GABAergic progenitor cells, 2 mM L-glutamine (D0-14), 10 μM β-mercaptoethanol (D0-14), 100 nM LDN193189 (D0-14), 10 μM SB431542 (D0-7), 5 μM IWP2 (D0-7), and 0.1 μM SAG (D0-21) were added to KSR medium. From day 14, cells were grown in N2AA medium containing 100 ng / ml FGF8 (D14-21), 10 ng / ml GDNF, and 10 ng / ml BDNF. Three weeks after differentiation, cIN spheres were trypsin-treated and seeded on plates coated with poly-L-ornithine / fibronectin in B27GB medium, then harvested and transplanted on appropriate days.

[0118] Surgical procedure Animals were anesthetized with isoflurane using the SomnoSuite anesthesia system (Kent Scientific Corporation, Torrington, CT, USA), and stereotactic brain surgery was performed using a stereotactic brain fixation frame (David KOPF Instruments, Tujunga, CA, USA) equipped with a Micro4 microsyringe pump controller (World Precision Instruments, Sarasota, FL, USA). Massive unilateral lesions of the substantia nigra striatal pathway (loss of more than 95% of striatal DA) were produced by stereotactic brain injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (rats) or substantia nigra pars compacta (mice). Rats were injected with 10 mg / kg of desipramine 15 minutes prior to anesthesia to protect noradrenergic projections. Two microliters of 6-OHDA (7.5 mg / ml in 0.2% ascorbic acid and 0.9% physiological saline) were injected using a 2.5 μl Hamilton syringe (Hamilton Company, Reno, NV). Coordinates were calculated referring to Bregma: anterior-posterior (AP), -4.0; lateral-medial (ML), -1.3; and dorsal-ventral (DV), -7.0. REG For intrastriatal implantation of C4-mDAP (or TP medium only, control) with or without the presence of cyclosporine A, one 2 μl deposit was placed at the following coordinates: AP, +0.8; ML, -3.0; and DV, -5.5. 100,000 C4-mDAP cells were implanted at a rate of 0.4 μl / min through a 10 μl Hamilton syringe fitted with a Blunt 26G, 0.75-inch needle. After injection, the needle was held in the brain for 5 minutes, then slowly withdrawn over 5 minutes, the incised skin was sealed using Autoclip® Surgical Suture (Fine science tools, Foster City, CA), and the animals were monitored on a warming pad until recovery. Rats in the CsA injection group were given daily injections of cyclosporine A (10 mg / kg, intraperitoneal), starting the day before C4-mDAP implantation.

[0119] Mice were injected with 25 mg / kg desipramine 15 minutes prior to anesthesia to protect noradrenergic projections. One microliter of 6-OHDA (3.0 mg / ml in 0.2% ascorbic acid and 0.9% saline) was injected using a 2.5 μl Hamilton syringe (Hamilton Company, Reno, NV). Coordinates were calculated referring to Bregma: anterior-posterior (AP), -2.9; medial-lateral (ML), -1.1; and dorsal-ventral (DV), -4.5. REG For intrastriatal implantation of C4-mDAP with or without the following coordinates, one 2 μl deposit was placed at AP, +0.5; ML, -1.8; and DV, -3.5. 100,000 C4-mDAPs were implanted at a rate of 0.4 μl / min through a 10 μl Hamilton syringe fitted with a Blunt 26G, 0.75-inch needle. After injection, the needle was held in the brain for 5 minutes, then slowly withdrawn over 5 minutes. The incised skin was sealed using Autoclip® Surgical Suture (Fine science tools, Foster City, CA), and the animals were monitored on a warming pad until recovery.

[0120] In each animal experiment, all groups used the same batch of C4-mDAP (Figures 4a-4h: Batch 1, Figures 5a-5m: Batch 2, Figures 15a-15h: Batch 3). In all animal experiments, the same experimental set was transplanted on the same day. For example, in the Fischer 344 animal experiment (Figures 4a-4h), the four groups analyzed at week 2 were transplanted on the same day (D0), while the same four groups analyzed at week 20 were transplanted the following day (D+1).

[0121] Humanized mouse The PD mouse model was induced by injecting 6-OHDA into the substantia nigra of NSG mice (as described above). PBMCs were isolated from patient (C4) blood and injected intraperitoneally into PD mice to generate C4-hu mice reconstituted with the immune system of patient C4. More detailed information is described previously. 16 .

[0122] Rotation test Rotational behavior was induced in animals that had been successfully injured using 6-OHDA by intraperitoneal administration of D-amphetamine, an indirect presynaptic DA agonist (4 mg / kg / rat, 5 mg / kg / mouse). Rotational behavior was induced by subcutaneous administration of apomorphine, a direct postsynaptic DA agonist (1 mg / kg / mouse). Rotational bias was recorded for 30 or 90 minutes using an automated system (SD Instruments, San Diego, CA). The number of whole-body rotations toward the lesion side was counted as a positive value, and only animals showing a net ipsilateral rotation of 6 or more rotations per minute were considered to have successfully injured their tails.

[0123] Cylinder test To measure forelimb motor asymmetry, mice were placed in a glass cylinder (20 cm in diameter) and recorded touching the wall with their feet at least 30 times for 10 minutes. The 10-minute recordings of the mice's behavior were analyzed by two researchers, blinded to the experimental group, by counting the number of wall touches with the left foot, right foot, and both feet. The results were calculated as the average number of touches using the right foot and as a percentage of the average number of total touches.

[0124] T REG Isolation Rat T REG It was isolated from the blood of Fischer 344 rats. Human T REG PBMCs were isolated from patient blood (C4) or PBMCs (K2) purchased from STEMCELL®. Blood samples were collected from the jugular vein into anticoagulation collection tubes, and PBMCs were isolated using SepMate® tubes by density gradient centrifugation. Rat PBMCs were stained with APC conjugate anti-CD4 (550057, BD) and PE conjugate anti-CD25 (554866, BD) antibodies according to the manufacturer's instructions and isolated using a FACS sorting machine. Human PBMCs were stained with EasySep® human CD4 according to the manufacturer's instructions. + CD127 low CD25 +Regulatory T cells were stained using a regulatory T cell isolation kit (18063, STEMCELL Technologies) and isolated using an EasySep® magnet. REG and T ナイーブ The cells were maintained in complete medium (RPMI1640 containing 10% heat-inactivated FBS and 100 μg / ml penicillin / streptomycin). REG :Fischer 344 (Donor, A) / Fischer 344 (Recipient, A'); Homemade T REG :Fischer 344 (donor / recipient, same rat).

[0125] ex vivo T REG Proliferation Isolated rat CD4 + CD25 + T cells were cultured in a 96-well round-bottom plate (2 × 10⁻¹⁰ 4 Cells were activated using plate-bound anti-rat CD3 / CD28 (5 μg / ml each) and rIL-2 (50 U / ml) (1 × 10⁶ cells / well). Cells were increased by changing or adding culture medium every 2-3 days. 6 Cells were maintained at a concentration of [number] cells / ml. The cells were reactivated on day 10 using initial activation conditions and maintained with IL-2. Proliferating cells were harvested at various time points.

[0126] T REG Suppression assay Standard rat CD4 + T cells (T conv Rat T100 was labeled with CellTrace® CFSE (Life Technologies, Invitrogen®) and stimulated with plate-bound anti-CD3 antibody and soluble CD28 antibody. REG In a 1:1 or 1:2 ratio, CFSE-labeled T conv The cells were added to a 96-well round-bottom plate on which they had been seeded. The cells were kept in complete culture medium for 48 hours. conv Cellular CFSE dilutions were analyzed by flow cytometry.

[0127] Flow cytometry To promote intracellular staining of Foxp3 (320012, Biolegend), CD4 + CD25 + T cells were fixed and permeabilized using Foxp3 fixation / permeabilization buffer according to the manufacturer's instructions. C4-mDAP was dissociated using Accutase, filtered through a 70 μm cell strainer to prepare single-cell suspensions, fixed with 4% formaldehyde, and stained with PE conjugate anti-HLA-ABC (560168, BD) and FITC conjugate anti-HLA-DR (555811, BD) or FITC conjugate anti-CD80 (560926, BD), PE conjugate anti-CD86 (560957, BD), PE conjugate anti-CD40 (568581, BD), PE conjugate anti-PD-L1 (568080, BD), PE conjugate anti-PDL2 (558066, BD), and PE conjugate anti-CD47 (568090, BD) antibodies. A fluorescent dye-compatible isotype control was used and subtracted during analysis.

[0128] TUN EL assay Cell death was determined using the Abcam TUNEL assay kit (ab66110, Abcam) according to the manufacturer's instructions. Frozen brain sections were fixed in 4% formaldehyde for 15 minutes and washed twice with PBS. Sections were incubated in 20 μg / ml proteinase K at room temperature for 5–15 minutes, washed twice, and then incubated for 1 hour in a dark, humidified incubator at 37°C with a DNA labeling solution containing TdT reaction enzyme and Br-dUTP. Labeled sections were washed with rinse buffer, incubated with an antibody solution containing anti-BrdU-Red antibody, and counterstained with DAPI. Major sites surrounding areas damaged by needle trauma were selected, and TUNEL assays were performed using 5 rats per group and 2–3 sections per rat. - Cells were counted. C4-mDAP cultured in vitro was treated for 7 days with or without 100 ng / ml IFN-γ, 20 ng / ml TNF-α, or 10 ng / ml IL-1β, and cell death was confirmed by the same method. TUNEL+ The number of cells was calculated in 3 to 5 randomly selected regions within the field, and the same experiment was repeated independently three times. Cell counting was performed by two researchers who were blinded to the assignment of experimental groups.

[0129] Apoptosis assay The proportion of apoptotic cells was determined using the 7-aminoactinomycin D (7-AAD) and FITC conjugate annexin V detection kit (640922, Biolegend) according to the manufacturer's instructions. C4-mDAP was dissociated using Accutase, filtered through a 70 μm cell strainer to prepare a single-cell suspension, washed twice with PBS, and incubated in the dark for 15–30 minutes with annexin-V-FITC and 7-AAD, annexin-V-FITC and APC-hNCAM, or annexin-V-FITC and PE-TH in binding buffer. The reaction was stopped by adding 4 volumes of binding buffer, washed twice with PBS, and analyzed by flow cytometry. See Figure 7a for the gating strategies used in the cell death assay.

[0130] Mixed lymphocyte reaction (MLR)-like co-culture assay PBMCs (C4 or K2) and C4-mDAP were co-cultured in 96-well plates for 3–5 days in ratios of 1:1, 1:2, or 1:10 (C4-mDAP:PBMC). Dynabeads® human T-activator (‡) was used as a positive control. Subsequently, PBMCs were harvested, stained with APC-hCD3 and PE-hCD69 antibodies, and analyzed by flow cytometry to determine the growth profile. C4-mDAP was harvested, stained with APC-hNCAM antibody and Annexin-V-PE, and analyzed by flow cytometry to determine the cell death profile. REG A C4-mDAP that has or does not have T REGC4-mDAP cells were co-cultured for 7 days under in vitro inflammatory conditions with a suppressor inspector (130-092-909, Miltenyi Biotech). C4-mDAP cells with or without Treg cells, and anti-TGFβ1 or mouse IgG1 isotype control antibodies were also co-cultured for 7 days.

[0131] ELISA After co-culturing PBMCs and C4-mDAP for 3–5 days, aliquots of the culture supernatant were obtained, and the amount of secreted IFN-γ was measured by ELISA according to the manufacturer's instructions (DIF50, R&D Systems).

[0132] Quantitative real-time PCR Total RNA was isolated from C4-mDAP using the GeneJET RNA purification kit (Thermo Fisher Scientific) according to the manufacturer's instructions. cDNA synthesis was performed using the iScript® cDNA synthesis kit (Bio-Rad). The following primers were used: TH Forward: 5'-CGGGCTTCTCGGACCAGGTGTA-3' (Sequence ID 1), Reverse: 5'-CTCCTCGGCGGTGTACTCCACA-3' (Sequence ID 2), Foxa2 forward: 5'-GGTGCTTTGGCTGACTTTTT-3' (Sequence ID 3), Reverse: 5'-GTTGCTCACGGAGGAGTAGC-3' (Sequence ID 4), Lmx1a forward: 5'-AGGGTGACGAGTTTGTCCTGA-3' (Sequence ID 5), Reverse: 5'-CATCGCTTTTGCCTGAGTCTG-3' (Sequence ID 6), and Actin forward: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO: 7) Reverse: 5'-CTCCTTAATGTCACGCACGAT-3' (Sequence ID 8) I used it.

[0133] For qRT-PCR, the inventors used SsoAdvanced® Universal SYBR Green Supermix, and the reaction was performed using the CFX Connect® Real-Time System (Bio-Rad, Hercules, CA). Relative expression was calculated using the comparative Ct method with C4 fibroblasts as a reference control (value = 1).

[0134] Western blot Cells were dissolved in RIPA lysis buffer (Sigma) (with a protease inhibitor), and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). Equivolute 6× loading buffer was added to each sample, which was then boiled for 10 minutes and loaded onto a 4–12% Bis-Tris Plus gel. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane. The membranes were probed with anti-cleavage caspase-3 (9661S, CST), anti-caspase-3 (14220T, CST), anti-Zg16 (NBP1-58007, Novus Biologicals), anti-Hormad1 (13917, Proteintech), or anti-β-actin (ab8227, Abcam) antibodies diluted 1:1000 in blocking solution (PBS containing 0.1% BSA), and subsequently incubated with horseradish peroxidase conjugate secondary antibody (Amersham). The bound antibodies were visualized using ECL (Amersham). See Figures 6a-6i for gel source data.

[0135] immunocytochemistry C4-mDAP cells cultured in 6, 12, 24, and 48-well plates were treated with 100 ng / ml IFN-γ, 20 ng / ml TNF-α, or 10 ng / ml IL-1β for 2 and 7 days, washed with PBS, and fixed with 4% formaldehyde in PBS for 10 minutes. The cells were incubated in a blocking solution (0.3% Triton X-100 and 1% horse serum in PBS) at room temperature for 1 hour, and then incubated overnight at 4°C in the same solution with anti-TH antibody. Next, the cells were incubated with Alexa Flour 488 or 568 conjugate secondary antibody and Hoechst 33342 or DAPI for nuclear staining at room temperature for 1 hour. Cell images were acquired by fluorescence microscopy (KEYENCE, Osaka, Japan), and data for specific cell populations were determined from the microscopic images using ImageJ software.

[0136] immunohistochemistry After the experiment was completed, the animals were induced into deep anesthesia by intraperitoneal injection of ketamine / xylazine, followed by intracardiac perfusion at a flow rate of 10 ml / min with ice-cold PBS for 10 minutes, and then with 4% formaldehyde for 20 minutes. The brains were removed and post-fixed overnight in 4% formaldehyde at 4°C, followed by continuous incubation in 20% and 30% sucrose. The sucrose-injected brains were embedded in OCT compound and frozen at -83°C, and coronal sections (30 μm) covering the entire striatum were sequentially collected (Leica CM1950, Buffalo Grove, IL). Free-floating coronal sections (including needle marks or entire grafts) were incubated for 1 hour in blocking solution (0.3% Triton X-100 and 1% horse serum in PBS), and then incubated overnight at 4°C with anti-MHCII (554926, BD), anti-Iba1 (ab178846, Abcam), anti-TH (P40101, Pel-Freez Biologicals), anti-hNCAM (sc-106, Santa Cruz), and anti-hNUCLEI (ab191181, Abcam) antibodies. The stained brains were washed and stained with peroxidase conjugate secondary antibodies for 1 hour. Finally, the sections were visualized using the DAB peroxidase substrate kit according to the manufacturer's instructions. Images in which the needle marks or grafts were clearly visible were selected.

[0137] Immunofluorescence Free-floating coronary brain sections were processed for antigen retrieval as needed and pre-incubated for 1 hour at room temperature in a blocking solution (0.3% Triton X-100, 5% normal donkey serum, and 3% BSA in PBS). Brain sections were treated with anti-IFN-γ (ab216642, ab9657, Abcam) (506702, BioLegend), anti-hIba1 (ab178680, Abcam), anti-hCD4 (ab133616, Abca). m), anti-hCD45 (ab40763, Abcam), anti-Foxp3 (ab22510, Abcam), anti-TH (AB1542, Sigma-Aldrich), anti-CD11b (MA180560, Thermo Fisher Scientific), anti-CD11c (PA5-90208, Thermo Fisher Scientific), anti-NKp46 (MABF1970, EMD Millipore), anti-CD19 (14-0194-82, Thermo Fisher Scientific), anti-rCD4 (ab237722, Abcam), anti-rCD8 (550298, BD), anti-HLA-ABC (ab70328, Abcam), anti-HLA-DR (ab92511, Abcam), anti-FoxA2 (89000721, Fisher Scientific), anti-Ki67 (ab16667, Abcam), anti-CD47 (ab260419, Abcam), anti-NeuN (ab279297, Abcam), anti-hGFAP (DPABY-745, Creative Diagnostics), anti-hCOL1A1 (AF6220, R&D Systems), anti-OLIG2 (AB9610, Sigma-Aldrich), anti-VGAT (PA5-63808, Thermo Fisher Scientific) and anti-Nkx2.1 (MA5-33074, Thermo Fisher The cells were incubated overnight at 4°C in the same solution with the (Scientific) antibody, washed three times with PBS, and then incubated with Alexa Fluor 488, Alexa 568, or Alexa 647 conjugate secondary antibody with DAPI or Hoechst 33342 at room temperature for 1 hour. Sections stained with the secondary antibody alone were processed and photographed under the same conditions and used as negative controls.

[0138] statistical analysis Microsoft Excel software (Microsoft Corp.) and GraphPad Prism v9 software were used for all statistical analyses, and the specific tests used are described in the figure captions. Unless otherwise noted, all experiments were performed using biological triple replicates, and the exact number of samples (n) used can be found in the figure captions. Two researchers, blinded to the experimental group assignments, counted the number of stained cells or MFI in the whole brain section of each mouse / rat, or in the most visible section of the needle marks of each mouse / rat. When comparing two groups where all data passed the Shapiro-Wilk normality test, or within a single group, Student's t-test (unpaired two-tailed test) was used, while multi-group comparisons were performed using two-way ANOVA followed by Bonferroni post-hoc test, or one-way ANOVA followed by Tukey's test. Values ​​p<0.05 were considered statistically significant, and asterisks in the figures indicate p-value significance: * p<0.05, ** p<0.01, *** p<0.001.

[0139] [Example 1] TH after cell transplantation + Rapid and preferential loss of mDA neurons To model the early survival rate of human mDA neurons in an in-house setting, the inventors have described the following: 16 Next, midbrain dopamine progenitor cells (mDAPs) derived from (C4) iPSCs of PD patients were transplanted into the striatum of mice humanized with the patient's own immune cells. The C4-iPSCs were transplanted using the inventors' established spotting-based procedure. 21、22 Regarding the differentiation protocol for iPSC-derived mDA, which was performed in vitro for 28 days. 21~24 A typical example is approximately 15% TH +C4-mDAP containing mDA neurons was generated (Figure 1a, Figure 1d). The inventors transplanted a total of 100,000 C4-mDAPs into the striatum of immunologically humanized mice (C4-hu, reconstituted with patient PBMCs) and immunodeficient NOD SCID gamma (NSG) mice. The animals were sacrificed two weeks after transplantation, and graft survival was examined by immunohistochemistry using antibodies against human nuclei (hNUCLEI) and TH, respectively, to determine viable cells and TH. + The total number of mDA neurons was detected (Figure 1b). The inventors found 85,901±7,460 and 74,455±3,842 hNUCLEI neurons in C4-hu and NSG mice, respectively. + Cells were detected (Figure 1c). In striking contrast, only 627±279 and 942±171 TH cells were found in C4-hu and NSG mice, respectively. + Cells survived, accounting for less than 1% of transplanted cells (or less than 10% of transplanted mDA neurons). These data demonstrate that mDA neurons within the graft die predominantly early (i.e., within two weeks post-transplantation) in relatively immunodeficient mice, with or without human immune system rearrangement, while the remainder of the transplanted cells are largely preserved. To address whether the vulnerability of mDA neurons is specific to this patient (C4), we transplanted further in vitro differentiated cells from independent hiPSC lines (B1; derived from separate sporadic PD patients) and H9 hESC lines and found that this phenomenon is a common feature (Figures 6a-6e). In particular, C4-hu mice showed a greater number of human cells in the graft compared to NSG mice (hNUCLEI + (Figure 1c), this may be due to immune cell infiltration from the humanized immune system as a secondary response to initial neuroinflammation (as observed in traumatic brain injury (TBI)). Supporting this, immunohistochemical analysis showed that human Iba-1 + (hIba-1 + ; macrophages / microglia), hCD45 + (White blood cells) and hCD4 +It was revealed that a considerable number of immune cells derived from the humanized host immune system, such as T lymphocytes, accumulate near the graft in C4-hu mice, but not in NSG mice (Figures 1e-1g). To address whether early cell death is specific to mDA neurons, the inventors introduced vesicular GABA transporters (VGATs), which are another neuronal lineage derived from two hiPSC (C4 and B1) and one hESC (H9) system. + By examining GABAergic neurons, the inventors found that these neurons also exhibited early cell death after transplantation (Figures 6f-6i). Therefore, while more neuronal lineages need to be examined to generalize this conclusion, the inventors' data suggest that early cell death after transplantation may be common for postmittal neurons.

[0140] [Example 2] Needle trauma and autologous T in immunocompetent rats REG The effects of simultaneous transplantation While the above data revealed important insights into early post-transplant events, these immunodeficient mice differ considerably from actual clinical situations and may limit the validity of immune response analysis. Therefore, we used Fischer 344 rats to investigate brain changes induced by surgical procedures. When we injected only culture medium (without any cells) into the striatum of Fischer 344 rats, we found acute pro-inflammatory cytokine release and immediate activation of astrocytes / microglia at the injection site (Figures 7a-7d), indicating that the host brain's response to penetrating brain injury (hereinafter referred to as "needle trauma") is similar to the well-known effects of other forms of TBI using closed head trauma models. 25、26 The inventors then demonstrated the ability to reproduce the host response at various time points (4 days to 6 months after injection) and showed that Iba-1 + and MHCII + We found that inflammatory cells showed robust infiltration near the needle marks, reaching a peak number on day 7, then decreasing at 1 month, and becoming undetectable at 6 months (Figures 8a and 8b). In particular, interferon-gamma (IFN-γ) +Cells showed a peak at 4 days after injection, the earliest point in time analyzed, indicating that needle trauma induced immediate neuroinflammation in the host brain, which also gradually decreased over time (Figure 8c). Needle trauma-induced death of host brain cells near the injection site, as examined by TUNEL staining, peaked at 7 days after injection (Figure 8d). Because needle trauma induces substantial neuroinflammation-induced death of host brain cells, we also found that transplanted cells, particularly mDA neurons, also induced significant neuroinflammation-induced death, as demonstrated by our data (Figures 1b-1d; Figure 8d) and previous studies using VM cell transplantation. 7~10 This led us to hypothesize that the transplanted cells may be susceptible to neuroinflammation-induced cell death immediately after transplantation.

[0141] In TBI, after initial inflammatory cell infiltration, T REG It is well established that it ultimately infiltrates the damaged area as part of the repair process and restores homeostasis. 25、26 Therefore, the inventors of this invention have developed their own T REG We hypothesized that simultaneous transplantation of autologous CD4 could be an effective approach to reduce acute neuroinflammation caused by needle trauma. To address this, we introduced autologous CD4 + CD25 + T REG (Foxp3 + (85.5 ± 3.12%) were isolated from individual Fischer 344 rats by FACS sorting of peripheral blood obtained via the jugular vein (Figure 9a), 20,000 cells were injected into the striatum of the same rats, and each brain was analyzed on day 2 or day 7. REG Transplantation significantly suppressed the acute induction of pro-inflammatory cytokines (TNF-α, IL-1β) on day 2 and the infiltration of inflammatory cells on day 7, and was associated with robust protection of host neurons from death compared to the control group (Figures 1h-1k, 7e-7g), demonstrating their significant protective effects. Next, we investigated autologous T14. REG Paired isogeneic T REG We compared the protective effects of rat autoT REG and related T REG MHCII has similar efficiency. + We observed that it inhibited the infiltration of inflammatory cells (Figure 1l). Autologous TREG MHCII + Inflammatory cell infiltration was suppressed in a dose-response manner, and the maximum effect was reached when 20,000 cells were transplanted (Figure 1m). REG It was initially detected near the needle mark and gradually disappeared within a week, but MHCII + It effectively inhibited the infiltration of inflammatory cells (Figures 10a-10b).

[0142] The inventors also obtained approximately 1,000,000 ex vivo-grown autologous CD4 cells. + CD25 + T REG Conventional T by intravenous injection for adoptive transfer REG therapy 27、28 The effect was verified (Foxp3 + , 68.8±4.07%) (Figure 9b). Adoptions are MHCII + It reduced cell infiltration by approximately 50%, but that was due to 20,000 T cells. REG The efficiency was lower than direct intrastriatal transplantation (Figure 1n). In particular, natural T REG (nT REG ) and proliferation T REG No functional difference was observed between the two (Figure 9c). Finally, the inventors investigated whether administration of cyclosporine A (CsA), a typical immunosuppressant, could suppress the inflammatory effects of needle trauma. When administered daily until the animals were slaughtered, CsA treatment suppressed the host inflammatory response (e.g., hIb-1). + MHCII + , IFN-γ + , and TUNEL + It significantly inhibited cells, but a single T REG It was not as effective as transplantation (Figures 11a-11e).

[0143] [Example 3]: Analysis of immunogenicity in vitro To investigate the effects of neuroinflammation in more detail, the inventors of the present invention developed C4-mDAP 16、21The cells were treated with pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ). First, the inventors examined the expression of molecules related to immunogenicity. 29~31 We found that HLA class I and / or II molecules were robustly induced after 2 days of treatment with IFN-γ (Figure 2a) or TNF-α, but not with IL-1β (Figure 12a). In contrast, the expression of T cell costimulatory molecules (CD80, CD86, and CD40) and immune checkpoint molecules (PD-L1, PD-L2, and CD47) was not altered by treatment with these cytokines (Figure 2b, Figure 12a). Furthermore, immunogenicity factors Zg16 and Hormad1 32、33 The expression of these molecules was neither detected nor induced (Figure 2c, Figure 12b), suggesting that pro-inflammatory cytokines, with the exception of HLA molecules, do not significantly alter the expression of immunogenicity-related molecules in C4-mDAP. The inventors then analyzed the immune response in vitro using a mixed lymphocyte reaction (MLR)-like co-culture assay with autologous or allogeneic PBMCs. When C4-mDAP and allogeneic PBMCs (K2-PBMCs) were co-cultured, T cell activity was significantly induced and further enhanced by IFN-γ treatment (Figure 2d). Furthermore, significant apoptotic cell death in C4-mDAP occurred when co-cultured with K2-PBMCs (Figure 2e). In striking contrast, T cell activity was not induced when co-cultured with autologous PBMCs (C4-PBMCs), either in the absence or presence of IFN-γ, and no cell death was observed even in the presence of IFN-γ. Furthermore, enzyme-linked immunosorbent assay (ELISA) confirmed that incubation of C4-mDAP with allogeneic but non-autologous PBMCs induced IFN-γ secretion (Figure 2f). In summary, our data demonstrate that allogeneic but non-autologous PBMCs, when co-incubated with C4-mDAP, induce T cell activation, which contradicts our previous findings on rejection / survival patterns in allogeneic and autologous transplantation studies using humanized mice. 16This is consistent with the findings. Therefore, the inventors conclude that the premature death of mDA neurons was not caused by the immunogenicity of the transplanted autologous mDAP.

[0144] [Example 4]: Effects of pro-inflammatory cytokines on mDAP in vitro The inventors then investigated inflammation-induced cell death in vitro by treating C4-mDAP with pro-inflammatory cytokines for 2 or 7 days. After 2 days of treatment with IFN-γ, C4-mDAP exhibited morphological changes accompanied by reduced TH expression and shortened neurite length as measured by mean fluorescence intensity (MFI), but cell death was unaffected (Figures 12c-12g). However, 7 days after IFN-γ treatment, cleaved caspase-3 was detected, which, as examined by annexin-V / 7AAD staining, was associated with approximately 12% further apoptotic cell death (Figures 3a, 3b). The TUNEL assay also revealed approximately 15% cell death in C4-mDAP (Figure 3c). Treatment with TNF-α showed a similar effect, while IL-1β showed a weaker effect on C4-mDAP (Figures 12h-12i). Furthermore, cells and TH + Total number of cells, TH expression level, and TH + All neurite lengths were significantly reduced (Figures 3d-3h). In contrast, FoxA2 expression remained unchanged (Figures 12j-12m). In particular, consistent with our in vivo data (Figures 1b, 1c), treatment with pro-inflammatory cytokines for 7 days induced a loss of approximately 15% of the total cell number, but TH + Cell loss is much greater (over 80%), TH + mDA neurons are TH-mediated against pro-inflammatory cytokine-induced cell death. - This suggested that it was more fragile than cells. To support this, the inventors treated C4-mDAP with IFN-γ and used flow cytometry to TH + and TH - When they were analyzed by gating the cells, most TH +While the cells entered apoptosis (programmed cell death), TH - The cells were unaffected (Figure 3i). The inventors also used autologous T13. REG We investigated whether co-incubation with TH could rescue apoptotic cell death induced by IFN-γ. As shown in Figures 3j and 3k, + The cells are autologous T REG In the presence of TH, cells were almost completely rescued from cell death. In summary, our data shows that pro-inflammatory cytokines acutely induced by needle trauma in vivo are resuscitated in vitro. + This could lead to preferential loss of mDA neurons, which is T REG It has been shown that it can be rescued by co-incubation. However, simultaneous intrastriatal transplantation using neutralizing IFN-γ antibody is not possible. + The cells could not be rescued (Figure 12n), indicating that individual pro-inflammatory cytokines could not replicate the complexity of neuroinflammation in an intact brain.

[0145] Interestingly, the inventors also developed their own T REG Co-incubation with Ki67 + It significantly reduced the percentage of cells, but TH + We found that it did not alter the percentage of cells (Figure 3l). This is in line with previous studies demonstrating the role of TGF-β1 signaling in regulating neuronal proliferation and the size of specific brain regions. 34、35 Based on this, the inventors of the present invention, T REG We hypothesized that TGF-β1 secreted by may be the basis for our observations. To support this possibility, we found that treatment with an anti-TGF-β1 antibody resulted in Ki67 + We found that the percentage of cells was significantly restored (Figure 12o, Figure 12p).

[0146] The inventors also investigated the inhibition of needle trauma-induced host inflammatory responses using neutralizing monoclonal antibodies. Post-needle trauma in NSG dKO mice, the degree of inflammation was assessed by the degree of MHC-II-positive cell infiltration as described herein. Specifically, Fischer 344 rats were sacrificed 7 days after co-infusion into the striatum of TP medium containing or without monoclonal antibodies (anti-TNF-α, anti-IL-1β, anti-IFN-γ, anti-HMGB1, anti-IL1α) (2 mg / rat) derived from each rat. The level of inflammatory cell infiltration into the needle wound was determined by immunohistochemical staining using anti-MHC-II antibodies (one-way ANOVA, Tukey's post-hoc test; n=4 per group). Each error bar represents the mean ± sem. *** P<0.001 (Figure 22). All of the antibodies tested (anti-TNF-α, anti-IL-1β, anti-IFN-γ, anti-HMGB1, and anti-IL-1α) significantly suppressed inflammation (Figure 22). Our data demonstrate that the suppression of inflammation correlates with the protection of transplanted dopamine neurons, and these data strongly suggest that treatment with these antibodies leads to better survival rates of mDANs after transplantation.

[0147] [Example 5]: Autologous T in heterogeneous PD models REG Therapeutic effects of simultaneous transplantation Based on the above data, the inventors have determined that the host's autologous T1 REG We investigated whether simultaneous intrastriatal transplantation of C4-mDAPs protects transplanted mDA neurons from needle trauma-induced cell death during cell therapy (both allogeneic and autologous). Since C4-mDAPs are heterogeneous to rats, we first investigated whether their gene expression is heterogeneous in rat T4-mDAPs in a 1:1 or 5:1 mixed ratio. REG The researchers verified and confirmed that the drug remained unchanged after 72 hours of co-incubation with C4-mDAP and autologous rat T REGThe C4-mDAP was simultaneously implanted into the striatum of 6-OHDA-injured Fischer 344 rats, with or without CsA treatment, and amphetamine-induced spinning behavior was monitored monthly after implantation (Figure 4a). As expected, implantation of C4-mDAP without CsA did not improve spinning behavior (Figure 4b). Interestingly, C4-mDAP without CsA and autologous T14 were not affected. REG Simultaneous transplantation of CsA also did not improve rotational behavior. When rats were administered CsA daily, C4-mDAP transplantation significantly reduced rotational behavior at 20 weeks post-transplantation, and this effect was observed in autologous T REG It was further enhanced by simultaneous transplantation with [another organism].

[0148] T for grafts REG To verify the initial effects, the inventors performed histological analysis two weeks after transplantation. When only C4-mDAP was transplanted, substantial infiltration and cell death of inflammatory cells were detected by MHCII and TUNEL staining, respectively, and these were observed with CsA treatment and / or T REG Simultaneous transplantation significantly increased rescue (Figure 4c, Figure 4d). A similar pattern was observed in myeloid cells (CD11b). + / CD11c + ), NK cells (NKp46 + ), B cells (CD19 + ), and T cells (CD4 + / CD8 + This was observed in other inflammatory cells, including (Figures 13a-13d). Furthermore, the expression of immunogenicity-related molecules (HLA I and II) was observed in CsA treatment and / or T REG Simultaneous transplantation significantly reduced the incidence (Figure 13e). hNUCLEI + Immunohistochemical analysis of cells revealed CsA and / or T REG Simultaneous transplantation resulted in the survival of the majority of grafted cells, while C4-mDAP alone (-CsA, -T) did not. REG Grafts composed of ) were shown to be rejected (Figure 4e). Therefore, TH + The cells were almost completely eliminated when only C4-mDAP was transplanted (Figure 4f). Interestingly, CsA was injected daily, and TREG Although it was injected only once, each treatment prevented graft rejection at this 2-week mark, and TH + Cell preservation, T REG Higher efficiency was observed when using [this method] (Figures 4e and 4f).

[0149] Next, the inventors analyzed the grafts (C4-mDAP in Fischer 344 rats) 20 weeks after transplantation (after behavioral studies, as shown in Figure 4a). As expected from the 2-week graft analysis, all grafts contained only C4-mDAP (-CsA, -T). REG ) was rejected when transplanted (Figure 4g). In particular, T REG Even when transplanted simultaneously, all grafts were rejected, and the difference from the 2-week result was T REG This suggested that CsA alone could only provide short-term graft protection. However, in this heterogeneous model, the graft survived with daily CsA administration and approximately 2,500 TH cells. + Cells were detected (Figure 4h). Surprisingly, the inventors found that T REG In grafts using simultaneous transplantation, graft size and hNUCLEI + Both the total number of cells, REG The grafts were significantly smaller and contained less Ki67 than those that did not undergo simultaneous transplantation. + We found that it was associated with cells (Figure 4g, Figures 14a-14d). Ki67 + There was a strong correlation between cell number and graft volume (Figure 14e). However, the inventors of TH + When cells are examined specifically, TH + The total number of cells is T REG If T is transplanted simultaneously, REG Compared to grafts without simultaneous transplantation, the number of FoxA2 grafts increased by almost double (Figure 4h), whereas FoxA2 grafts... - Cell numbers were similar or slightly decreased (Figure 14f). Recent studies 36、37 This generally agrees with T REGBoth grafts using simultaneous transplantation and grafts not using simultaneous transplantation contained neurons, glial cells (astrocytes, oligodendrocyte lineage cells, and microglia), and vascular leulomyelitis cells (VLMCs) (Figures 14g to 14k). Our analysis revealed that Ki67 + Except for a significant decrease in cell number, T REG No significant changes in cell type composition were detected after simultaneous transplantation.

[0150] [Example 6]: Autonomous T in the Autonomous PD Model REG Therapeutic effects of simultaneous transplantation The inventors then administered mDAP and T to patient-immunized humanized mice from the same patient. REG By simultaneously porting T in the custom configuration REG The therapeutic effect was investigated (C4-T REG (Figure 9g). For this purpose, we used C4-hu mice to produce a PD model by stereotactic injection of 6-OHDA into the substantia nigra, and C4-mDAP and C4-T REG The grafts were simultaneously transplanted into the striatum (Figure 15a). Unfortunately, these mice did not survive for more than two months after transplantation (Figure 15b). Previous studies have shown that humanized mice typically die from graft-versus-host disease (GVHD) 30–90 days after injection of human PBMCs into NSG mice. 38、39 Therefore, this early death of C4-hu mice is likely due to the development of GVHD. Although the inventors were unable to conduct behavioral tests and long-term graft analysis, the inventors observed that C4-T2 mice were 2 months post-transplant. REG The effect of simultaneous transplantation was investigated. Stereochemical analysis of the graft revealed that hNUCLEI + The total number of cells and graft volume were greater in the T group compared to the group using only C4-mDAP. REG The simultaneous transplant group showed a lower level (Figure 15c, Figure 15d), which was due to Ki67 + It correlated with the number of cells (Figure 15e, Figure 15f). In contrast, TH + The number of cells is T REG The amount was higher in the simultaneous transplant group (Figure 15g). REGAt this point, it was no longer detectable (Figure 15h). Therefore, the inventors of the present invention have determined that self-T REG Although the long-term benefits of simultaneous transplantation could not be verified, these humanized mouse data were consistent with the data observed in Fischer 344 rats, and autologous T REG This supports the concept that simultaneous transplantation of mDA neurons protects them from the acute host inflammatory response to needle trauma, thereby improving their survival rate.

[0151] The inventors then used unmodified NSG mice to develop C4-mDAP and C4-T REG The therapeutic effect of simultaneous transplantation was investigated. First, the inventors generated unilateral 6-OHDA-injured NSG mice that consistently exhibited more than 6 amphetamine-induced ipsilateral rotations per minute throughout the entire study period (24 weeks). Because the NSG mice were relatively immunodeficient, the grafts were not rejected even without immunosuppression. When the inventors transplanted 100,000 C4-mDAP cells into the striatum of these mice, rotational behavior was significantly reduced at 20 weeks post-transplantation (less than 6 rotations per minute) (Figure 5a). REG When C4-mDAP was transplanted simultaneously, the reduction in rotation began earlier (16 weeks post-transplant) and showed greater improvement in both amphetamine-induced and apomorphine-induced rotation tests compared to the group receiving C4-mDAP alone (Figures 5b and 5c). Furthermore, all transplanted mice showed significant improvement in forelimb function in the cylinder test at 20 weeks post-transplant (Figure 5d).

[0152] The inventors compared the histology and immunohistochemistry of grafts between a group receiving C4-mDAP alone and a group receiving simultaneous transplantation. In the second week, hNUCLEI + The total number of cells is C4-T REG The initial number of cells was approximately 100,000, with or without simultaneous transplantation, and these increased to approximately 673,000 and 401,000 cells, respectively, at 20 weeks post-transplantation (Figure 5e). Consistent with these data, the graft volume at 20 weeks post-transplantation was 8.53 ± 0.73 mm² in the C4-mDAP-only group and the simultaneous transplantation group, respectively.3 and 5.11±0.57mm 3 (Figure 5f), and that is Ki67 + Percentage of cells (0.79 ± 0.06%; +T) REG It correlated with :0.27±0.04%) (Figure 5g, Figure 5h). TH + The total number of cells was higher at 2 weeks post-transplant (1,825±287;+T) despite the smaller overall graft volume. REG :4, 427±189) and 20 weeks (5, 421±534;+T REG In both cases (9 and 523±835), the values ​​were significantly higher in the grafts of the simultaneous transplant group (Figure 5i). + Among neurons, GIRK2 + ALDH1A1 - A9 and Calvinzine + The percentage of A10-like DA subtypes was not altered by simultaneous transplantation (Figure 5j, Figure 5k). The ratio of neurons to total cells (NeuN) + / hNUCLEI + Cells were significantly higher in grafts from mice with simultaneous transplantation (27.39 ± 1.99%, +T, respectively). REG :49.93±1.96%) (Figure 5l). Furthermore, the inventors found that the simultaneously transplanted grafts produced a higher density of graft-derived human synaptophysin (hSYP) in the dorsolateral striatum (DL STR). + It was found to contain synapses (5.54±0.65%;+T REG : 13.64±1.30%, Figure 16a, Figure 16b). T REG Higher levels of TH at the graft-host boundary in simultaneously transplanted brains. + hSYP + Neuron terminals are their preferred target, host dendritic spines (DARPP32 + It was identified on the neuron and was consistent with the synaptic connection with the host striatal neuron (Figure 5m). The inventors identified TH + When the integration of transplanted C4-mDAP cells into the host brain was evaluated using innervation density, TH in the striatum +Fiber density in the co-grafted group was DL STR (13.50 ± 0.75%; +T REG :24.38±1.24) and VL STR(15.96±0.94;+T REG Both were higher (55.50 ± 1.07%) (Figure 16c, Figure 16d).

[0153] [Example 7]: Summary of experimental data In the 1980s and 1990s, numerous researchers studied the viability of transplanted cells as a crucial requirement for successful PD cell therapy, primarily using human and rodent embryonic VM cells. 7~11 These early studies revealed that the majority (80–99%) of transplanted mDA neurons undergo apoptotic cell death within 1–2 weeks post-transplantation. Notably, these studies also showed that the number of viable mDA neurons remained largely the same at later points in time (e.g., 4 days, 2 weeks, and 6 weeks post-transplantation), further supporting the view that most transplanted mDA neurons suffer premature death and that new mDA neurons are not generated from transplanted VM cells. Numerous potential mechanisms have been proposed to explain the poor survival rate and premature death of grafted mDA neurons, largely focusing on interactions between grafted cells and the host brain, such as inadequate supply of oxygen, glucose, or growth factors from the host brain located immediately adjacent to the fresh graft. Therefore, numerous efforts have been made to improve graft survival using a variety of protective factors, including calcium channel antagonists, lasaroids, caspase inhibitors, and trophic factors. 40~44 In contrast to these extensive early studies using fetal tissue, the viability of mDA cells in grafts derived from hiPSCs / hESCs has not been systematically studied. We have previously demonstrated that the use of autologous iPSC sources addresses the problem of cell-mediated rejection without the use of immunosuppressants. 16 On the other hand, it has been demonstrated that the immediate activation of the innate immune system as part of the inflammatory response to surgical injury cannot be overcome by this method.

[0154] In this study, the inventors found that penetrating trauma in surgical transplants is similar to that described in other models of TBI. 25、26、45、46 It induces a host inflammatory response, including a robust innate immune response similar to that of TH, and this immediate inflammatory response leads to the desired therapeutic use of the graft. + This indicates that it is specifically harmful to cellular components. In line with early survival rate studies using embryonic VM cells, the inventors have shown that transplanted TH + Less than 10% of neurons survive in the early stages of transplantation, while TH - We found that the cells were largely viable, which was validated in independent experiments using two hiPSC systems (C4 and B1) and one hESC system (H9). In vitro studies using pro-inflammatory cytokines (e.g., IFN-γ, TNF-α, and IL-1β) showed that this cytokine attack was TH + It preferentially induces apoptosis (programmed cell death) in neurons, and this is autologous T1 REG Co-incubation with TH has been shown to significantly rescue cells. However, in contrast to VM cell transplantation, TH + The number of cells and other cells increases substantially at later stages. The inventors have shown that mDAP derived from hiPSC / hESCs has a higher potential for differentiation and proliferation than embryonic VM cells, and that new TH cells are produced in the graft after transplantation. + Neurons - It was hypothesized that these cells were generated from mDAP cells. Supporting this concept, our previous research has shown that neural progenitor cells derived from mouse ES cells exhibit much higher proliferation and differentiation potential than those derived from mouse embryonic VM cells. 47、48 .

[0155] Furthermore, our data indicates that the T19 is autologous to the host. REG Simultaneous intrastriatal transplantation of cells significantly improves the inflammatory response to needle trauma, and TH + It was revealed that this promotes the survival of mDA neurons and similarly reduces the death of host brain cells. Although the activation of inflammatory and immune cells caused by needle trauma is nonspecific, TH in the graft -Compared to cells, TH + Because it is preferentially harmful to neurons, it is harmful regardless of whether the grafted cells are autologous, allogeneic, or heterogeneous. Our data show that autologous T cells are harmful to the host. REG This has been shown to effectively mitigate the needle trauma-induced process in both autologous and xenografts, leading to better recovery of motor impairment. Therefore, the inventors have demonstrated in recent rodent models of TBI that this method has demonstrated. 49 Thus, the patient's T REG We hypothesize that simultaneous transplantation of autologous T cells could improve clinical outcomes in both autologous and allogeneic cell transplantation scenarios. However, autologous T cells... REG Simultaneous transplantation enabled only short-term survival of xenografts in Fischer 344 rats, but not long-term survival. Therefore, systemic immunosuppression is likely still necessary for long-term graft survival in allogeneic cell transplantation.

[0156] While further research is certainly needed, the inventors of the present invention believe that T REG We hypothesize that it exerts neuroprotective effects through multiple direct and / or indirect pathways as follows: Firstly, T REG Immunosuppressive cytokines (e.g., TGF-β1) secreted by directly affect mDAP. This is supported by previous studies showing that mature mDA neurons express TGF-β receptors. 50 Furthermore, TGF-β1 protects mDA neurons from pro-inflammatory cytokine (e.g., IFN-γ)-driven neurotoxicity. 51 This was shown. The inventors confirmed that C4-mDAP also expresses the TGF-β receptor (Figure 17a). Secondly, T REGAs shown in our data, this can indirectly regulate neuroinflammation by suppressing the expression of pro-inflammatory cytokines by surrounding resident brain cells (e.g., astrocytes and microglia) in response to needle trauma (Figures 1j, 7e, and 7f). Thirdly, these effects may help prevent the infiltration of inflammatory cells from the periphery (Figures 1h to 1n and 10b). Furthermore, recent studies have shown that intercellular interactions (e.g., CD47-SIRPα and CD45-galectin 1) can directly protect mDA neurons from MPTP neurotoxicity. 52、53 The inventors found that C4-mDAP expresses both SIRPα and galectin-1 (Figures 17b and 17c), and that T expresses CD47 and CD45. REG However, this suggests that mDA cells can be directly protected through these intercellular contact mechanisms.

[0157] Unexpected findings in this study are T REG Simultaneous transplantation is TH - It substantially reduces cell-related graft proliferation and improves growth from grafts, along with a higher ratio of TH + This resulted in a significantly lower graft volume in terms of cell content. REG This raises the question of how simultaneous transplantation affects the cell fate determination of transplant progenitor cells in the graft. REG To determine whether the effect may be due to a dilution effect, the inventors compared C4-T, which are very similar in size but functionally different. REG and C4-naive T cells (C4-T ナイーブ The simultaneous transplantation of the same number of C4-T REG Or C4-T ナイーブ When transplanted simultaneously with C4-mDAP, the cell count and size of the graft are C4-T REG The size was smaller only when it was transplanted simultaneously (Figure 17d, Figure 17e), strongly suggesting that this was not due to a dilution effect. Since C4-mDAP expresses the TGF-β receptor (Figure 17a), T REGGraft cell proliferation can be controlled through the secretion of TGF-β, which is known to regulate neuronal cell proliferation. 34、35 As supporting evidence, treatment with anti-TGF-β1 antibody showed that Ki67 + The percentage of cells was significantly restored (Figure 12o, Figure 12p). These T REG Since the effects are not related to the source of mDA cells (autologous vs. allogeneic) themselves, they are also relevant to allogeneic grafting scenarios. In contrast to other strategies for immunosuppression involving systemic administration, T REG Simultaneous transplantation is a site-specific strategy and is unlikely to induce harmful systemic side effects. Furthermore, in the acute phase (results at 2 weeks), T REG A single dose of T was beneficial, as was daily CsA. REG Our demonstration that the cells themselves did not persist in long-term grafts suggests that the benefit is related to the acute-phase regulation of the innate immune response, and that subsequent administration of these cells is not required to sustain the effect. Therefore, the strategy reduces the duration and extent of the risk of adverse effects.

[0158] In summary, our research suggests that early survival of specific mDA neurons in grafts is strongly influenced by the host innate immune response, while long-term graft survival is more susceptible to adaptive immune responses, thus revealing a two-stage involvement of the immune system in graft survival. mDAP and T REG Simultaneous transplantation modulates the host immune response induced by needle trauma, and TH - By significantly protecting mDANs while suppressing cell proliferation, it is beneficial to both the efficacy and safety of cell replacement therapy for PD and other neurodegenerative disorders.

[0159] References (all sections except for detailed explanations)

[0160] [Table 4-1]

[0161] [Table 4-2]

[0162] [Table 4-3]

[0163] [Table 4-4]

[0164] [Table 4-5]

[0165] Other Embodiments While the present invention has been disclosed in specific embodiments, it will be understood by those skilled in the art that certain substitutions, modifications, and / or omissions can be made to embodiments without departing from the spirit of the invention. Accordingly, the foregoing descriptions are intended to be merely illustrative and should not limit the scope of the invention. All references, scientific papers, patent publications, and any other documents cited herein are incorporated by reference with respect to the substance of their disclosures.

[0166] Although the present invention has been described in detail, it should be understood that the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are contained within the following claims.

Claims

1. A method for treating a subject with Parkinson's disease (PD), wherein the controlled T(T) ＲＥＧ The method comprising administering a population of ) cells and a population of midbrain dopamine (mDA) cells to the target brain.

2. The method according to claim 1, wherein the population of mDA cells includes midbrain dopamine neurons (mDANs) and midbrain dopamine progenitor cells (mDAPs).

3. Said T ＲＥＧ The method according to claim 1 or 2, wherein the cells and the mDA cells are administered to the subject simultaneously and optionally mixed together in a single composition.

4. Said T ＲＥＧ The cells are first administered to the subject, followed by the mDA cells being administered to the subject, or the mDA cells are first administered to the subject, followed by the T ＲＥＧ The method according to claim 1 or 2, wherein cells are administered to the subject.

5. Said T ＲＥＧ Cells and the mDA cells, autologous T ＲＥＧ The method according to any one of claims 1 to 4, wherein the cells are autologous mDA cells.

6. The method according to claim 5, wherein the autologous mDA is obtained by in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into mDA cells, and the hiPSCs are derived from cells from the subject.

7. Said T ＲＥＧ Cells and the mDA cells are allogeneic T ＲＥＧ The method according to any one of claims 1 to 5, wherein the cells and allogeneic mDA cells are, optionally, obtained by in vitro differentiation of human pluripotent stem cells (hPSCs), optionally human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) into mDA cells.

8. Said T ＲＥＧ cells and said mDA cells are heterologous T ＲＥＧ cells and heterologous mDA cells, the method according to any one of claims 1 to 5.

9. Said T ＲＥＧ The method according to any one of claims 1 to 8, wherein cells and mDA cells are administered into the striatum by intrastriatal injection, optionally.

10. A method for treating a subject having Parkinson's disease (PD), comprising administering to the subject a population of midbrain dopamine (mDA) cells and an antibody (Ab) (optionally, an antibody that conjugates pro-inflammatory cytokines or injury-associated molecular pattern (DAMP) molecules, optionally, anti-TNF-α Ab (optionally infliximab), anti-IL-1β Ab (optionally canakinumab), anti-IFN-γ Ab (optionally emaparmab), anti-HMGB1 Ab, or anti-IL-1α Ab).

11. A method for reducing the immune response in a subject during cell transplantation (e.g., an immune response induced by needle trauma, an immune response at the site of administration), wherein the subject is given an effective amount of T ＲＥＧ The method comprising administering a population of cells together with a second population of cells.

12. The method according to claim 11, wherein the second population of cells is a population of mDA cells.

13. Said T ＲＥＧ The method according to claim 12, wherein the cells and the mDA cells are transplanted simultaneously.

14. Said T ＲＥＧ The cells and the mDA cells were transplanted separately. Said T ＲＥＧ The cells are first administered to the subject, followed by the mDA cells being administered to the subject, or The mDA cells are first administered to the subject, followed by the T ＲＥＧ The method according to claim 12, wherein cells are administered to the subject.

15. Said T ＲＥＧ Cells, autologous T ＲＥＧ cells, allogeneic T ＲＥＧ Cells, or heterologous T cells ＲＥＧ The method according to any one of claims 11 to 14, wherein the cell is a cell.

16. The method according to any one of claims 12 to 15, wherein the mDA cells are autologous mDA cells, allogeneic mDA cells, or heterogeneic mDA cells.

17. Said T ＲＥＧ The method according to any one of claims 11 to 16, wherein cells are transplanted into the striatum.

18. The method according to any one of claims 12 to 17, wherein the mDA cells are transplanted into the striatum.

19. A method for reducing an immune response in a subject induced by needle trauma during cell transplantation, comprising administering to the subject an effective amount of antibody (Ab) (optionally, an antibody conjugating a pro-inflammatory cytokine or injury-associated molecular pattern (DAMP) molecule; optionally, anti-TNF-α Ab (optionally infliximab), anti-IL-1β Ab (optionally canakinumab), anti-IFN-γ Ab (optionally emaparmab), anti-HMGB1 Ab, or anti-IL-1α Ab) together with a population of cells (optionally mDA cells).

20. A method for increasing cell viability in cell transplantation, increasing the growth of transplanted cells, or reducing abnormal proliferation during cell transplantation, wherein an effective amount of T ＲＥＧ The method comprising simultaneously transplanting a population of cells together with a second population of cells ("transplant cells," optionally mDA cells).

21. The method according to claim 20, wherein the transplanted cells are mDA cells.

22. Simultaneous transplantation produces a graft, and compared to grafts that do not undergo simultaneous transplantation, the graft has TH ＋ An increase in cells was observed. Simultaneous transplantation produces a graft, and compared to grafts that do not undergo simultaneous transplantation, the graft has TH － Cell: TH － Increased cell ratio and / or TH in grafts ＋ Cell: Ki67 － An increase in the cell ratio is observed, and / or Simultaneous transplantation produces a graft, and compared to grafts that do not undergo simultaneous transplantation, the graft has Ki67 ＋ Cell reduction and / or TH in grafts － A decrease in cells is observed. The method according to claim 20 or 21.

23. Said T ＲＥＧ The method according to any one of claims 20 to 22, wherein the cells and the transplanted cells (optionally mDA cells) are transplanted simultaneously.

24. Said T ＲＥＧ The cells and the transplanted cells (optionally selected mDA cells) are transplanted separately. Said T ＲＥＧ The cells are first administered to the subject, followed by the transplanted cells (optionally selected mDA cells) being administered to the subject, or The transplanted cells (optionally selected mDA cells) are first administered to the subject, followed by the T ＲＥＧ The method according to any one of claims 20 to 22, wherein cells are administered to the subject.

25. Said T ＲＥＧ Cells, autologous T ＲＥＧ cells, allogeneic T ＲＥＧ Cells, or heterologous T cells ＲＥＧ The method according to any one of claims 20 to 24, wherein the cell is a cell.

26. The method according to any one of claims 20 to 25, wherein the transplanted cells (optionally mDA cells) are autologous transplanted cells, allogeneic transplanted cells (optionally mDA cells), or xenotransplanted cells (optionally mDA cells).

27. Said T ＲＥＧ The method according to any one of claims 20 to 26, wherein cells are transplanted into the striatum.

28. The method according to any one of claims 20 to 27, wherein the transplanted cells (optionally selected mDA cells) are transplanted into the striatum.

29. A method for increasing cell viability in cell transplantation, increasing the growth of transplanted cells, or reducing abnormal proliferation during cell transplantation, comprising administering to the subject an effective amount of an antibody (Ab) (optionally, an antibody conjugating a pro-inflammatory cytokine or a damage-associated molecular pattern (DAMP) molecule, optionally, anti-TNF-α Ab (optionally infliximab), anti-IL-1β Ab (optionally canakinumab), anti-IFN-γ Ab (optionally emaparmab), anti-HMGB1 Ab, or anti-IL-1α Ab) and a population of cells (optionally mDA cells).

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