In vivo striatum co-transplantation of autologous TREG and MDA cells in Parkinson's disease cell therapy

Abstract

Described herein, inter alia, are compositions and methods of use for administering a population of regulatory T (TREG) cells and / or a population of midbrain dopamine (mDA) cells to the brain of a subject (e.g., treating Parkinson's disease and / or reducing immune responses due to acupuncture wounds during cell transplantation).

Description

[0001] CLAIM OF PRIORITY

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 500,941, filed May 9, 2023. The entire contents of the foregoing application are incorporated herein by reference. BACKGROUND

[0003] The cardinal motor symptoms of Parkinson’s disease (PD), including tremor, rigidity, bradykinesia, and postural instability, are caused by selective and progressive degeneration of midbrain dopamine (mDA) neurons (mDANs) in the substantia nigra, making cell replacement therapy a promising therapeutic strategy, a concept supported by results from previous fetal ventral midbrain (VM) cell transplantation studies 1-6 . Clinical use of human fetal VM cells is limited by ethical and procedural levels, and mDA cells derived from human pluripotent stem cells such as human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) are emerging as potential alternative cell sources 1-6 . Regardless of the source of transplantable cells, a fundamental requirement for cell replacement therapy is the survival of grafted cells during the transplantation process. Thus, many investigators have studied the survival of transplanted cells as a critical requirement for successful PD cell therapy, primarily using human and rodent fetal VM cells 7-11 . These early studies revealed that the vast majority (80-99%) of transplanted mDANs undergo apoptotic cell death within 1-2 weeks post-transplantation. Notably, these studies also revealed that the number of surviving mDA neurons remained constant at later time points (e.g., at 4 days, 2 weeks, and 6 weeks post-transplantation), further supporting the notion that most transplanted mDA neurons die early and that no new mDA neurons are generated from transplanted VM cells. Numerous potential mechanisms have been proposed to explain the poor survival and early death of transplanted mDA neurons, which have primarily focused on the interactions between the transplanted cells and the host brain, such as a lack of proper oxygen, glucose, or growth factor supply from the host brain in the immediate vicinity of the new graft. Thus, many efforts have been made to improve graft survival using different protective factors, including calcium channel antagonists, lazaroids, caspase inhibitors, and trophic factors 40-44 .

[0004] Transplantation using autologous cells can reduce the host immune response. Autologous mDA cell transplantation in a non-human primate PD model has recently been reported 12-14In another study, neurografting of major histocompatibility complex (MHC)-matched primate iPSCs reduced the host immune response and increased the survival of transplanted mDA neurons. 15 Furthermore, autologous hiPSC-derived mDA cells were successfully transplanted into the striatum of sporadic PD patients without the use of immunosuppressants, and mice with a humanized immune system constructed from the patient's peripheral blood mononuclear cells (PBMCs) did not reject such grafts. 16 Although these data show that using autologous mDA cells can avoid graft rejection, as can be achieved through... 18 The first patient assessed by F-DOPA PET scan analysis and clinical motor evaluation showed only moderate (modest) cell survival and functional recovery. 16 This indicates that even in autologous transplantation, the survival of mDA neurons can be limited. To compensate for this poor survival, some researchers have proposed transplanting large numbers of cells, such as a recent clinical trial in Australia that proposed transplanting up to 70 million cells (neuroscience.org.au / research-news / parkinsons-stem-cell-trial). Furthermore, some researchers have suggested using cells from earlier differentiation stages (e.g., days 16-17) for transplantation. 17,18 While these approaches are of interest, the risks of unintended side effects, including graft overgrowth, damage to host brain structures, and / or tumor formation, need to be carefully examined. Summary of the Invention

[0005] This application is based at least in part on the following surprising discovery: the modulating T(T) REG Co-administration of a cell population with a midbrain dopamine cell (mDA cell) population to the brain of a subject (e.g., a subject with Parkinson's disease) resulted in better outcomes for the subject (e.g., a reduced immune response in the subject). The useful mDA cells may comprise midbrain dopamine neurons (mDANs), midbrain dopamine progenitor cells (mDAPs), and / or combinations thereof. This application is also based at least in part on the surprising finding that administration of T cells with a desired cell (e.g., neuronal) population in cell therapy… REG Cell populations reduce needle trauma (e.g., reduce the host's immune response), reduce graft overgrowth, and increase cell survival of the desired cell population.

[0006] The role of the immediate response of host brain tissue to the surgical trauma via innate immune responses, and how this affects, in particular, the survival of tyrosine hydroxylase (TH) -containing cells within the graft + The survival of mDA neurons has not been specifically studied. Here we show that the vast majority (~90%) of hiPSC-derived mDA neurons present in the initial population of transplanted cells die within 1-2 weeks post-transplantation, while the remaining TH - cells in the graft mostly survive, whether they are transplanted into immunodeficient or immunocompetent host animals, including humanized mice. We show that an important factor for this early death of mDA neurons is the surgical trauma itself (herein referred to as "needle trauma"), which triggers an acute host neuroinflammatory response with preferential detrimental effects on TH - cells in the graft compared to TH + cells. Moreover, we find that this needle trauma impairs the host brain and significantly kills host neuronal cells (Figure 7g).

[0007] Regulatory T cells (T REG ) are indispensable for maintaining normal immune tolerance and homeostasis, and are so named because of their ability to regulate other immune cells to control inflammatory and immune rejection responses 19,20 . Thus, co-transplantation of autologous T REG cells during surgical implantation of the graft can effectively and safely help suppress the host inflammatory response to needle trauma. We find that intrastriatal co-transplantation of autologous T REG cells significantly protects both host neurons and transplanted mDA neurons from this needle trauma-induced death, further improving behavioral recovery in a 6-OHDA-lesioned PD rodent model and reducing TH - cell proliferation within the graft. Because T REG cells are known to suppress inflammatory responses, they are used to control inflammation in clinical settings. However, T REG cells are administered systemically to patients by intravenous injection, and their effects on the survival of mDA neurons after intrastriatal injection are unknown. Thus, we test the effects of a conventional T 27,28 cell therapy using adoptively transferred REG autologous CD4 + CD25 + T REG cells (Foxp3 +, 68.8 ± 4.07%) to be implemented (Figure 9b) and compared to direct intrastriatal transplantation. While adoptive transfer reduced the infiltration of MHCII + cells by approximately 50%, it was significantly less efficient than direct intrastriatal transplantation of 20,000 T REG cells (Figure In). In short, while using only 2% of the cells, T REG direct intrastriatal injection of T

[0008] Accordingly, described herein, inter alia, are methods of treating Parkinson's disease (PD) in a patient by co-transplanting T REG cells and mDA neurons into the brain of the patient. The T REG cells and mDA neurons can be transplanted simultaneously or one after the other. The T REG cells and mDA neurons can be autologous T REG cells and autologous mDA neurons. Alternatively, the T REG cells and mDA neurons can be allogeneic, or xenogeneic. The T REG cells and mDA neurons can be transplanted intrastriatally. The T REG cells and mDA cells can be transplanted in amounts of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0009] In various embodiments of the method of treating PD: the T REG cells and mDA cells are transplanted simultaneously; the T REG cells and mDA cells are transplanted one after the other; the T REG cells are autologous T REG cells, allogeneic T REG cells, or xenogeneic T REG cells; the mDA cells are autologous mDA cells, allogeneic mDA cells, or xenogeneic mDA cells; the T REG cells and mDA cells are transplanted intrastriatally.

[0010] Also described herein are methods of reducing and / or suppressing host immune responses triggered by needle trauma in a patient undergoing cell transplantation, comprising co-transplanting T REG cells with the cells to be transplanted. In some embodiments, the cells to be transplanted can be mDA cells (e.g., mDA neurons, mDA progenitor cells, and mixtures thereof). In some embodiments, the T REG cells and mDA cells can be transplanted simultaneously or one after the other. In some embodiments, the T REG cells and mDA cells can be autologous T REGCells (i.e., from a patient) and autologous mDA cells. In some embodiments, the patient-derived T REG Cells can be used with mDA cells, which can be allogeneic or xenogeneic. In some embodiments, the T REG Cells and mDA cells can be transplanted intrastriatally. In some embodiments, the T REG Cells and mDA cells can be transplanted in amounts of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0011] In various embodiments: the T REG Cells and mDA cells are transplanted simultaneously; the T REG Cells and mDA cells are transplanted one after the other; the T REG Cells are autologous T REG Cells, allogeneic T REG Cells, or xenogeneic T REG Cells; the mDA cells are autologous mDA cells, allogeneic mDA cells, or xenogeneic mDA cells; the T REG Cells and mDA cells are transplanted intrastriatally.

[0012] Furthermore, it was unexpectedly found that co-transplantation of T REG significantly rescued the death of host brain cells caused by needle trauma (Figures 7e, 7f, 7g; Figures lh- lk) compared to control groups, indicating that co-transplantation of T REG will protect patient (host) neurons from death.

[0013] Also described herein, inter alia, is a method of improving outgrowth from a graft of mDA cells transplanted into a patient, comprising co-transplanting T REG Cells with the transplanted mDA cells, wherein the co-transplantation results in a high proportion of TH+ cell content in the graft compared to a graft of mDA cells alone. In some embodiments, the T REG Cells and mDA neurons can be transplanted simultaneously or one after the other. In some embodiments, the T REG Cells and mDA neurons are autologous T REG Cells and autologous mDA neurons. In some embodiments, the T REG Cells and mDA cells are allogeneic or xenogeneic. In some embodiments, the T REG Cells and mDA neurons can be transplanted intrastriatally. In some embodiments, the T REG Cells and mDA neurons can be transplanted in amounts of 5,000 to 100,000 cells and 2 million to 20 million mDA cells, respectively.

[0014] In various embodiments: the T REG The cells and mDA cells are transplanted simultaneously;T REG The cells and mDA cells are transplanted one after another;T REG The cells are autologousT REG The cells are allogeneicT REG The cells are xenogeneicT REG The cells; the mDA cells are autologous, allogeneic, or xenogeneic;T REG The cells and mDA cells are transplanted intrastriatally.

[0015] Furthermore, the abnormal ectopic growth of TH-cells can destroy the brain structure of the patient and can cause adverse reactions and / or side effects. In this regard, the T REG The co-transplantation of the cells and mDA cells has advantages by reducing the overall size of the graft and the number of Ki67+ proliferating cells and enhancing the safety of the cell therapy.

[0016] Furthermore, we found that, compared to non-neuronal proliferating cells, the needle trauma not only preferentially kills mDA neurons, but also kills other types of neurons, such as GABAergic neurons. Therefore, the T REG The co-transplantation of the T

[0017] Finally, but equally importantly, we found that the co-transplantation of specific antibodies against pro-inflammatory cytokines (FDA-approved: Infliximab (anti-TNF-a), Canakinumab (anti-IL-1b), Epratuzumab (anti-IFN-g)) or co-transplantation with antibodies against damage-associated molecular pattern (DAMP) molecules (e.g., anti-HMGB1 Ab, anti-IL-1a Ab) also showed protective effects against the needle trauma-induced death of host brain cells and transplanted mDA neurons. Furthermore, we found that the administration of anti-inflammatory drugs (e.g., cyclosporine A, dexamethasone, and FK506) showed protective effects. Therefore, these methods / agents can also be used instead of the T REG .

[0018] In some embodiments, described herein are methods of treating a subject having Parkinson’s disease (PD), the method comprising transplanting into the subject T REG) cell population and midbrain dopamine (mDA) cell population are administered to the brain of a subject. In some embodiments, described herein are methods of treating a subject having Parkinson’s disease (PD), comprising administering to the subject a midbrain dopamine (mDA) cell population and an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or damage-associated molecular pattern (DAMP) molecule; optionally an anti-TNF-a Ab (optionally infliximab), an anti-IL-1b Ab (optionally canakinumab), an anti-IFN-g Ab (optionally ipilimumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab).

[0019] In some embodiments, described herein are methods of reducing the immune response in a subject during a cell transplantation procedure (e.g., an immune response triggered by a needle trauma), comprising administering to the subject an effective amount of a T REG cell population with a second cell (“transplanted cell”; optionally mDA cell) population. In some embodiments, described herein are methods of reducing the immune response in a subject during a cell transplantation procedure (e.g., an immune response triggered by a needle trauma), comprising administering to the subject an effective amount of an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or damage-associated molecular pattern (DAMP) molecule; optionally an anti-TNF-a Ab (optionally infliximab), an anti-IL-1b Ab (optionally canakinumab), an anti-IFN-g Ab (optionally ipilimumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab) with a cell (optionally mDA cell) population.

[0020] In some embodiments, described herein are methods of increasing cell survival of transplanted cells (optionally mDAN) in a cell transplantation and / or reducing overgrowth of cells (optionally TH-cells, Ki67+ cells, and / or cells other than mDAN) in a cell transplantation procedure, comprising administering to the subject an effective amount of a T REG cell population with a second cell (“transplanted cell”; optionally mDA cell) population. In some embodiments, described herein are methods of increasing cell survival of transplanted cells (optionally, mDAN) in a cell transplantation and / or reducing overgrowth of cells (optionally, TH-cells, Ki67 +A method of treating a subject having a disease or disorder associated with excessive growth of cells, and / or cells other than mDANs, the method comprising administering to the subject an effective amount of an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or damage-associated molecular pattern (DAMP) molecule; optionally, an anti-TNF-a Ab (optionally, infliximab), an anti-IL-1b Ab (optionally, canakinumab), an anti-IFN-g Ab (optionally, ipilimumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab) and a population of second cells (optionally, mDA cells).

[0021] In some embodiments of any of the methods described herein, the transplanted cells are mDA cells. In some embodiments of any of the methods described herein, the population of second cells is a population of mDA cells. In some embodiments of any 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 any 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.

[0022] In some embodiments of any of the methods described herein, the ratio of mDA cells: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, the T REG The cells and mDA cells are administered to the subject simultaneously, optionally mixed together in a single composition. In some embodiments of any of the methods described herein, the T REG The cells are administered to the subject first, followed by administration of the mDA cells to the subject. In some embodiments of any of the methods described herein, the mDA cells are administered to the subject first, followed by administration of the T REG The cells are administered to the subject first, followed by administration of the mDA cells to the subject. In some embodiments of any of the methods described herein, the mDA cells are administered to the subject first, followed by administration of the T REG The cells and mDA cells are transplanted as a single composition in a single dose. In some embodiments of any of the methods described herein, the T REG The cells and mDA cells are transplanted as a single composition in repeated doses.

[0023] In some embodiments of any of the methods described herein, the T REG The cells and mDA cells are autologous T REG The cells and mDA cells are autologous T REG The cells and mDA cells are xenogeneic T REGCells and allogeneic mDA cells. In some embodiments of any of the methods described herein, the T REG Cells are autologous T REG Cells, allogeneic T REG Cells, or allogeneic T REG Cells. In some embodiments of any of the methods described herein, the mDA cells are autologous mDA cells, allogeneic 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, allogeneic transplanted cells (optionally mDA cells), or allogeneic transplanted cells (optionally mDA cells).

[0024] In some embodiments of any of the methods described herein, the autologous mDA is obtained by in vitro differentiation of human induced pluripotent stem cells (hiPSCs) into mDA cells, wherein the hiPSCs are derived from cells from the subject. In some embodiments of any of the methods described herein, the T REG Cells and mDA cells are allogeneic T REG Cells and allogeneic mDA cells, optionally wherein the autologous mDA is 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.

[0025] In some embodiments of any of the methods described herein, the T REG Cells and mDA cells are intrastriatally administered, optionally by intrastriatal injection.

[0026] In some embodiments of any of the methods described herein, the T REG Cells are transplanted intrastriatally. In some embodiments of any of the methods described herein, the mDA cells are transplanted intrastriatally. In some embodiments of any of the methods described herein, the transplanted cells (optionally mDA cells) are transplanted intrastriatally.

[0027] In some embodiments of any of the methods described herein, the co-transplantation results in a graft, and wherein the TH + Cells are increased in the graft compared to a graft without co-transplantation. In some embodiments of any of the methods described herein, the co-transplantation results in a graft, and wherein the TH + Cells:TH - Cells are increased and / or the TH + Cells:Ki67 +Cell proportion is increased. In some embodiments of any of the methods described herein, the co-transplant produces a graft, and wherein the proportion of Ki67 + cells and / or TH - cells is reduced in the graft compared to a graft without co-transplant.

[0028] In some embodiments of any of the methods described herein, the T REG cells and the transplanted cells (optionally mDA cells) are transplanted simultaneously. In some embodiments of any of the methods described herein, the T REG cells and the transplanted cells (optionally mDA cells) are transplanted separately. In some embodiments of any of the methods described herein, the T REG cells are administered to the subject first, followed by the transplanted cells (optionally mDA cells) being administered to the subject. In some embodiments of any of the methods described herein, the transplanted cells (optionally mDA cells) are administered to the subject first, followed by the T REG cells being administered to the subject. In some embodiments of any of the methods described herein, the ratio of transplanted cells: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, the T REG cells and the transplanted cells are administered to the subject simultaneously, optionally mixed together in a single composition. In some embodiments of any of the methods described herein, the T REG cells and the transplanted cells are administered to the subject in a single composition in a single dose. In some embodiments of any of the methods described herein, the T REG cells and the transplanted cells are administered to the subject in a single composition in repeated doses.

[0029] In some embodiments of any 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, the amount of T REG cells and mDA cells administered to the subject is sufficient to ameliorate one or more symptoms of PD in the subject, thereby treating PD. In some embodiments of any of the methods described herein, the amount of T REG cells administered to the subject is sufficient to reduce or eliminate the amount (and / or percentage) of at least one pro-inflammatory cytokine (e.g., TNF-a, IL-1b, and / or IFN-g) and / or, and / or at least one damage-associated molecular pattern (DAMP; e.g., HMGB1) at and / or near the injection site.

[0030] As used herein, cell transplantation includes any therapy in which a population of cells is administered (e.g., via injection, infusion, graft / transplantation) to a subject, such as a cell therapy.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use of the present application; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0032] Other features and advantages of the present application will be apparent from the following detailed specification, the drawings, and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0033] FIGS. 1a-1n. Effect of needle trauma-induced neuroinflammation on grafted 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 immunofluorescent staining with anti-TH / Hoechst 33342 before transplantation (1a, 1c, 1d) or immunohistochemical staining with anti-TH / hNuclei antibody after transplantation (1b, 1c, 1d). 1h-1k, The level of inflammatory cell infiltration was confirmed by immunofluorescent staining with anti-hIba1 (1e), anti-hCD45 (1f), and anti-hCD4 (1g) antibodies. 1h-1k, The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1h) and anti-MHCII (i) antibodies. 1j, The level of needle trauma-induced neuroinflammation was determined by immunofluorescent staining with anti-IFN-γ antibody. 1k, The level of cell death induced by needle trauma-induced neuroinflammation was analyzed by TUNEL assay. (Student’s t-test; n=4 per group). 1l-1n, Autologous T cells were intrastriatally co-transplanted with C4-mDAP (20k cells / rat) in Fischer 344 rats. The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1l) and anti-MHCII (1m) antibodies. The level of needle trauma-induced neuroinflammation was determined by immunofluorescent staining with anti-IFN-γ antibody (1n). (Student’s t-test; n=5 per group). + mDA neurons. (One-way ANOVA, Tukey’s post-hoc test; n=4 per group). The level of inflammatory cell infiltration was confirmed by immunofluorescent staining with anti-hIba1 (1e), anti-hCD45 (1f), and anti-hCD4 (1g) antibodies. 1h-1k, The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1h) and anti-MHCII (i) antibodies. 1j, The level of needle trauma-induced neuroinflammation was determined by immunofluorescent staining with anti-IFN-γ antibody. 1k, The level of cell death induced by needle trauma-induced neuroinflammation was analyzed by TUNEL assay. (Student’s t-test; n=5 per group). 1l-1n, Autologous T cells were intrastriatally co-transplanted with C4-mDAP (20k cells / rat) in Fischer 344 rats. The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1l) and anti-MHCII (1m) antibodies. The level of needle trauma-induced neuroinflammation was determined by immunofluorescent staining with anti-IFN-γ antibody (1n). (Student’s t-test; n=5 per group). REG Inhibition of needle trauma-induced host inflammatory response. Fischer 344 rats were intrastriatally co-transplanted with C4-mDAP (20k cells / rat) and TP medium with or without autologous T cells (20k cells / rat). The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1l) and anti-MHCII (1m) antibodies. (Student’s t-test; n=5 per group). REG Inhibition of needle trauma-induced host inflammatory response. Fischer 344 rats were intrastriatally co-transplanted with C4-mDAP (20k cells / rat) and TP medium with or without autologous T cells (20k cells / rat). The level of inflammatory cell infiltration into the needle track was determined by immunohistochemical staining with anti-Iba1 (1l) and anti-MHCII (1m) antibodies. (Student’s t-test; n=5 per group). REGAdvantages of co-transplantation. The level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-MHCII antibody. 1l, in syngeneic and autologous T REG Seven days after co-transplantation of (20k cells / rat) cells, the inhibitory effect on neuroinflammation induced by acupuncture trauma was compared. 1m, with 2k, 20k, or 100k autologous T cells per rat. REG The inhibition of acupuncture-induced neuroinflammation was measured 7 days post-transplantation. The levels of amplified autologous T cells obtained from each rat in vitro were compared after 7 days. REG Adoptive transfer or in vitro expansion of autologous T cells (1,000 k cells / rat) REG striatal transplantation (20k cells / rat). (One-way ANOVA, Tukey post-hoc test; n=5 per group). Each error bar represents the mean ± sem. ns, not significant; P < 0.01; P < 0.001.

[0034] Figures 2a-2f. Immunogenicity induced by pro-inflammatory cytokines in transplanted C4-mDAP. ac, C4-mDAP was incubated with 100 ng / ml IFN-γ in vitro for 2 days. The level of IFN-γ-induced immunogenicity was determined by flow cytometry analysis with anti-HLA-ABC / HLA-DR (2a), anti-CD80 / CD86 / CD40 / PD-L1 / PD-L2 / CD47 antibody (2b), and by Western blot analysis with anti-Zg16 / Hormad1 antibody (2c). 2d-2f, MLR-like co-culture assays using C4-mDAP and C4 / K2-PBMCs. 2d, Levels of T cell activation were measured by flow cytometry analysis with anti-CD3 / CD69 antibody. 2e, Levels of immunogenicity-induced cell death were determined by flow cytometry analysis with anti-hNCAM / annexin-V staining. 2f, the level of IFN-γ secretion as measured by ELISA. ; using Dynabeads TM Human T-activator activation. (One-way ANOVA, Tukey post-hoc test; n=3). Each error bar represents the mean ± sem. P < 0.01; P < 0.001.

[0035] Figures 3a-3l. Pro-inflammatory cytokine-induced cell death in C4-mDAP. 3a-3i, C4-mDAP were incubated with 100 ng / ml IFN-γ in vitro for 7 days. The level of IFN-γ-induced cell death was determined by western blotting with anti-cleaved caspase-3 antibody (3a), flow cytometry analysis of annexin-V / 7-AAD staining (3b), and TUNEL assay (3c). (Student’s t-test; n = 3 biological independent experiments). TH levels were analyzed by immunofluorescence staining (3d), and measuring total cell number (3e) or TH + cell number (3f), TH + mean fluorescence intensity (3g), and TH + neurite length (3h). (Student’s t-test; n > 4). 3i, Apoptotic cell death (annexin-V + ) was analyzed using flow cytometry by gating TH - and TH + cells. (One-way ANOVA, Tukey’s post-hoc test; n = 6). 3j-3l, C4-mDAP were co-cultured with or without T REG cells in vitro under inflammatory conditions (with or without 100 ng / ml IFN-γ) for 7 days. 3j, Immunofluorescence staining of TH and Ki67. The percentage of TH + (3k) and Ki67 + (3l) cells in total cells. (One-way ANOVA, Tukey’s post-hoc test; n = 5). Scale bars: 100 pm. Each error bar represents mean ± s.e.m. ns, not significant; P < 0.05, P < 0.01; P < 0.001.

[0036] Figures 4a-4h. Effect of T REG cells co-transplanted with C4-mDAP in a xenogeneic PD model. 4a-4h, T REGFischer 344 rats were sacrificed 2 or 20 weeks after co-transplantation of C4-mDAP (20k cells / rat) and CsA (control) into the striatum. 4a, 4b: Amphetamine-induced rotation scores were determined every 4 weeks post-transplantation for 20 weeks. (Two-way ANOVA, Tukey 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: At 2 weeks, the level of neuroinflammatory-induced cell death was analyzed by TUNEL assay. 4e, 4g: At 2 weeks (4e) and 20 weeks (4g), the total number of C4-mDAP was confirmed by hNUCLEI staining. 4f, 4h: TH was determined by immunofluorescence staining at 2 weeks (4f) and 20 weeks (4h), respectively. + Total number of cells. (One-way ANOVA, Tukey 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.

[0037] Figure 5a-5m. T REG In vivo effects of co-transplantation with C4-mDAP. 5a-5m, with or without autologous C4-T. REG NSG mice were sacrificed 2 or 20 weeks after co-transplantation of C4-mDAP (20k cells / mouse) into the striatum. 5a. Amphetamine-induced rotation test was performed every 4 weeks post-transplantation for 24 weeks. (Two-way ANOVA, Bonferroni post-hoc test). 5b-5d. Behavioral assessments were conducted at 20 weeks post-transplantation using the amphetamine-induced rotation test (5b), the apomorphine-induced rotation test (5c), and the cylinder test (5d). (One-way ANOVA, Tukey post-hoc test; n=5 per group). 5e and 5f. The total number of transplanted C4-mDAP cells (hNUCLEI) was analyzed at 2 and 20 weeks, respectively. + (e) and graft volume (hNCAM) + (5f). (Student t-test; n=5 per group). 5g, 5h, Ki67 + and SOX1 + PAX6 + Ki67 + Percentage of cells (5g) and Ki67 + Correlation between cell percentage and graft volume (5h). (Student t-test; n=5 per group). TH was examined at 2 and 20 weeks, respectively.+ Total cell count. (Student t-test; n=5 per group). 5j, images of A9- or A10-like cells. 5k, cells co-expressing GIRK2. + ALDH1A1 + (A9) or calcium-binding protein + (A10) of TH + Quantitative analysis of neurons. (Student t-test; n=5 per group). 5l, NeuN + / hNUCLEI + Percentage of cells. (Student t-test; n=5 per group). 5m, through TH + hSYP + DARPP32 + Images of DA synaptic connections in the host striatum identified by staining. Each error bar represents the mean ± sem. ns, not significant; P < 0.05 P < 0.01; P < 0.001.

[0038] Figures 6a-6i: Effects of needle-induced neuroinflammation on transplanted postmitotic neurons. 6a-6e: NSG mice were sacrificed 14 days after intrastriatal transplantation with B1 / H9-mDAP. TH was determined by immunofluorescence staining with anti-TH / Hoechst 33342 before transplantation (6a, 6c, 6e) or by immunohistochemical staining with anti-TH / hNUCLEI antibody after transplantation (6b, 6d, 6e). + Total number and percentage of mDA neurons. (n=4 per group). 6f-6i, NSG mice were sacrificed 14 days after striatal 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 (6i) with anti-VGAT / Hoechst 33342 prior to transplantation and anti-VGAT / Nkx2.1 / hNUCLEI antibody post-transplantation. (n=4 per group). Each error bar represents mean ± sem.

[0039] Figures 7a-7g: Early time-dependent analysis of the inflammatory response to acupuncture trauma. 7a-7d: TP medium was injected into the striatum of Fischer 344 rats euthanized on days 1, 2, 3, 5, and 7 post-surgery. The level of neuroinflammation induced by acupuncture trauma was determined by immunofluorescence staining with anti-TNF-α (7a) and IL-1β (7b) antibodies, and quantitative staining was performed in 7a' and 7b' (bars in the left figure, respectively). 7c: Astrocytes were stained with anti-GFAP antibody, and quantitative staining was performed in 7c' (bars in the left figure). Iba-1 injection was performed after the procedure. + Cells (yellow box: resident microglia of the brain, red box: infiltrating Iba-1 cells) + Images and quantitative analysis of cells (7d, 7d'). (n=5 per group). 7e-7g, using T REG Inhibits host inflammatory response and cell death induced by acupuncture trauma. With or without autologous T cells from each rat. REG Fischer 344 rats were sacrificed 2 or 7 days after striatal co-transplantation with TP medium (20 kJ / rat). The level of needle-induced neuroinflammation was determined by immunofluorescence staining with anti-TNF-α (7e, 7e') and anti-IL-1β (7f, 7f') antibodies (Student t-test; n=3 per group). The level of needle-induced neuronal death was determined by immunohistochemical staining with anti-NeuN antibody (7g, 7g') (Student t-test; n=5 per group). Each error bar represents mean ± sem. P < 0.05; P < 0.01; P < 0.001.

[0040] Figures 8a-8d: Long-term time-dependent analysis of cellular responses to needle puncture trauma. In Figures 8a-8d, TP medium was injected into the striatum of Fischer 344 rats, and the rats were sacrificed on days 4, 7, and 14 and at 1, 3, and 6 months post-surgery. The level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-Iba1 (8a) and anti-MHCII (8b) antibodies. In Figure 8c, the level of neuroinflammation induced by needle puncture trauma was determined by immunofluorescence staining with anti-IFN-γ antibody. In Figure 8d, the level of cell death induced by needle puncture trauma was analyzed by TUNEL assay. (n=5 per group). Mean values ​​are connected by red lines.

[0041] Figures 9a-9g: T after in vitro amplification REGFunction. 9a, 9b, analysis of sorted CD4 by flow cytometry staining. + CD25 + FoxP3 + T cells (nT cells) REG (9a) and in vitro amplified rat CD4 + CD25 + FoxP3 + T cells (expanded T cells) REG (9b) level. 9c, in 1:1 and 1:2 ratios (T REG :T conv In rat nT REG or amplified T REG T based on CFSE dilution method exists conv T cells proliferate REG Inhibition was measured and analyzed by flow cytometry. (Student t-test; n=3). 9d-9f, rat T REG (rT REG The interaction between C4-mDAP and rT. REG Co-incubated at 5:1 and 1:1 ratios for 72 hours. The expression levels of TH (9d), Foxa2 (9e), or Lmx1a (9f) mRNA in C4-mDAP were determined by quantitative real-time PCR and normalized to actin. (One-way ANOVA, Tukey post-hoc test; n=3 biologically independent experiments). (9g) Isolated human CD4 was analyzed by flow cytometry. + CD127 low CD25 + FoxP3 + T REG The level. Each error bar represents the mean ± sem. ns, not significant.

[0042] Figures 10a-10b: Autologous T cells after co-transplantation REG Location and function. 10a, 10b, using TP medium and autologous T REG Following co-transplantation of Fischer 344 rats into the striatum at 20k cells / rat, (10a) T cells were determined by immunofluorescence staining with anti-Foxp3 antibody at days 1, 2, 3, 5, and 7 post-surgery. REG Location of inflammatory cells. (One-way ANOVA, Tukey post-hoc test; n=5 per group). 10b, the level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-MHCII antibody. (Two-way ANOVA, Bonferroni post-hoc test; n=5 per group). Each error bar represents the mean ± sem. P < 0.01; P < 0.001.

[0043] Figures 11a-11e: Inhibition of acupuncture-induced neuroinflammation by CsA treatment. 11a-11e, in TP medium with T REG Fischer 344 rats were sacrificed 7 days after striatal co-transplantation with (20k cells / rat) or CsA-treated (ip). 11a, Schematic overview of experimental methods. The level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-Iba1 (11b) and anti-MHCII (11c) antibodies. 11d, The level of needle-induced neuroinflammation was determined by immunofluorescence staining with anti-IFN-γ antibody. 11e, The level of cell death induced by needle-induced neuroinflammation was analyzed by TUNEL assay. (One-way ANOVA, Tukey post-hoc test; n=5 per group). Each error bar represents mean ± sem. ns, not significant; P < 0.05 P < 0.01; P < 0.001.

[0044] Figures 12a-12p: In vitro effects of pro-inflammatory cytokine treatment on C4-mDAP. 12a-12g: 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 with anti-HLA-ABC / HLA-DR / CD80 / CD86 / CD40 / PD-L1 / PD-L2 / CD47 antibody (12a) and by Western blot analysis with anti-Zg16 / Hormad1 antibody (12b). Immunogenicity was assessed by immunofluorescence staining (12c), total cell count (12d), or TH... + Cell count (12e), TH + Average fluorescence intensity (12f) and TH +Th1 levels were assessed using neurite length (12g). (Student t-test; n≥4). C4-mDAP was incubated with 20 ng / ml TNF-α or 10 ng / ml IL-1β in vitro for 7 days at 12h and 12i. The level of TNF-α / IL-1β-induced cell death was determined by flow cytometry analysis with annexin-V / 7-AAD staining (12h) and TUNEL assay (12i). (One-way ANOVA, Tukey post-hoc test; n=3 biologically independent experiments). 12j-12m, with or without T1... REG C4-mDAP was co-cultured for 7 days under in vitro inflammatory conditions (with or without 20 ng / ml TNF-α or 10 ng / ml IL-1β). Immunofluorescence staining for TH and FoxA2 was performed. Total TH levels in cells were measured. + (12k) and FoxA2 + The percentage of (12m) cells and TH + Neuronal spur length (l). (One-way ANOVA, Tukey post-hoc test; n≥3). 12n, 12n', NSG mice were sacrificed 14 days after striatal co-transplantation with or without anti-IFN-γ mAb C4-mDAP. TH was determined post-transplantation by immunohistochemical staining with anti-TH / hNUCLEI antibody. + Percentage of mDA neurons. (Student t-test; n=5 per group). 12o, 12p, with or without T. REG C4-mDAP cultured with anti-TGF-β1 or mouse IgG1 isotype control antibodies for 7 days was performed. Proliferation levels were determined by immunofluorescence staining with anti-Ki67 antibody (12o), and quantitative staining was performed at 12p. (One-way ANOVA, Tukey post-hoc test; n=5). Each error bar represents mean ± sem. ns, not significant; P < 0.05 P < 0.01; P < 0.001.

[0045] Figures 13a-13e: T REG Co-transplantation suppressed inflammatory cell infiltration and C4-mDAP immunogenicity in xenograft PD models. 13a-13e, in the presence or absence of T REG Two weeks after co-transplantation of C4-mDAP with CsA into the striatum, Fischer 344 rats were sacrificed. CD11b was assessed by immunofluorescence staining. + / CD11c + (13a), NKp46+ (13b), CD19 + (13c), and CD4 + / CD8 + (13d, 13d' (see below)) Cell levels. 13d', in 13d+T cells. REG +CsA and +T REG Most of the yellow dots seen in the +CsA group are nonspecific signals rather than real cells (yellow arrows). (13e) HLA class I / II expression was examined by immunofluorescence staining. (n=5 per group).

[0046] Figures 14a-14k: T REG Co-transplantation inhibits C4-mDAP proliferation in xenograft PD models. 14a-14k, in the presence or absence of T REG Fischer 344 rats were sacrificed 20 weeks after co-transplantation of C4-mDAP with CsA into the striatum. 14a and 14b were then analyzed via hNCAM. + Stereoscopic estimation of the volume of stained grafts. hNUCLEI + Ki67 in cells + Cell count (14c, 14d). (Student t-test; n=5 per group). e, Ki67 + Correlation between cell number and graft volume. 14f, analysis of TH by immunofluorescence staining. + and FoxA2 + Cell count. (Student t-test; n=20). 14g-14k, neurons (14g, NeuN) + ), astrocytes (14h, hGFAP) + VLMC(i, hCOL1A1) + Oligodendrocytes (14j, OLIG2) + ), and microglia (14kJ, hIba-1) + Image of ( ). Scale bar: 100 μm. Each error bar represents the mean ± sem. P < 0.05; P < 0.01; P < 0.001.

[0047] Figures 15a-15h: T REG The role of co-transplantation with C4-mDAP in autologous C4 humanized PD models. 15a-15h, with or without autologous C4-T REGEight weeks after co-transplantation of C4-mDAP (20 kC cells / mouse) into the striatum, C4-humanized mice were sacrificed. 15a, Schematic overview of experimental design. 15b, Survival of C4-humanized mice. The total number of transplanted C4-mDAP (c), graft volume (15d), and TH were measured. + Cells (15g). Ki67 + The number (15e) (Student t-test; n=5) and Ki67 + With hNUCLEI + Correlation between cell numbers (15f). hCD4 was confirmed by immunofluorescence staining at 15h. + T cell levels. (Student t-test; all data passed Shapiro-Wilk normality test; n=2 for each group (-T) REG ) and n=3 (+T REG Each error bar represents the average value ± sem. P < 0.05.

[0048] Figures 16a-16d: T REG In vivo effects of co-transplantation with C4-mDAP. 16a-16d, with or without autologous C4-T REG NSG mice were sacrificed 20 weeks after co-transplantation of C4-mDAP (20k cells / mouse) into the striatum. hSYP was then incorporated into 16a and 16b DL STRs. + Images (16a) and quantitative assessment (16b). (Student t-test; n=5 per group). 16c, 16d, graft-derived TH in (i) cingulate cortex (CTX), (ii) perinasal CTX, (iii) DL STR, and (iv) ventrolateral (VL) STR. + Images (16c) and quantitative assessment (16d) of fiber density. T, transplant. (Student t-test; n=3 per group). Each error bar represents mean ± sem. P < 0.05; P < 0.01; P < 0.00.

[0049] Figures 17a-17e: T REG Functions and related mechanisms. 17a-17c: Flow cytometry was used to analyze the expression levels of TGF-β receptor (17a), SIRPα (17b), and galactolectin-1 (17c) in C4-mDAP. 17d, 17e: The expression levels of C4-T cells in C4-mDAP treated with mitomycin C and C4-T cells were analyzed.幼稚 (20k cells / mouse) or C4-T REG NSG mice were sacrificed 8 weeks after co-transplantation of 20k cells / mouse into the striatum. The total number of transplanted C4-mDAP cells (hNUCLEI) was analyzed after 8 weeks. + (17d) and graft volume (hNCAM) + (17e). (Student t-test; n=4 per group). Each error bar represents the mean ± sem. P < 0.01.

[0050] Figure 18: Dynamic distribution of molecular and cellular changes following acupuncture trauma. Following acupuncture injection, the brain undergoes physical injury, leading to the rupture of resident cells such as neurons, astrocytes, microglia, and oligodendrocytes. This rupture is caused by the impact of the needle, resulting in the bursting of these cells. Subsequently, the ruptured cells rapidly release damage-associated molecular patterns (DAMPs), which affect neighboring cells and trigger 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, playing a crucial role in the rapid removal of debris. Simultaneously, astrocytes and microglia become increasingly activated over time and migrate towards the injury area. By approximately day three, peripheral monocytes infiltrate, and depending on the severity of brain injury, T and B cells may also infiltrate, participating in repair functions. This cascading inflammatory process is essential for removing cellular debris caused by acupuncture trauma, a key step in promoting repair and homeostasis in the injured area. However, at the same time, the event appears to have caused substantial damage and death to the engrafted mDAN.

[0051] Figure 19 This section outlines three distinct phases of hPSC-based CRT when mDNA death is a potential concern. A schematic diagram illustrates these three phases. Phase 1 involves the in vitro differentiation of hPSCs into mDA cells, primarily containing mDAP and mDNA, using an optimized procedure. Phase 2 includes the harvesting and cryopreservation of the in vitro differentiated mDA cells, highlighting the critical steps of cryopreservation, storage, and thawing. In Phase 3, the final in vivo transplantation occurs, comprising surgical transplantation, early (<2 weeks), and late (>2 weeks) graft establishment. Potential challenges and considerations for each phase, including cell viability, immune response, and environmental factors, are discussed to provide a comprehensive understanding of the optimized process for hPSC-based CRT in PD.

[0052] Figure 20Strategies targeting adaptive immunity. Typically, allogeneic transplantation using mDA cells derived from hESCs still requires immunosuppression, while autologous transplantation using mDA cells from hiPSCs is characterized by immune tolerance, eliminating the need for immunosuppressants. Alternative strategies to utilizing autologous cells involve applying HLA-matched hiPSCs to reduce the risk of graft rejection, with ongoing efforts to build a library of HLA-matched iPSCs from a wide variety of donors. Another approach focuses on developing “universal donor stem cells” that incorporate genetic modifications such as CRISPR / Cas9-mediated knockout of HLA class I and II components, and lentiviral overexpression of the immune receptor CD47 or HLA-E / G transgenes.

[0053] Figure 21 Strategies targeting the innate immune system. In CRT, the standard procedure involves injecting cells into the brain using a needle, which induces needle-induced 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 and lead to the infiltration of peripheral immune cells into the brain, positively contributing to damage repair. However, they also have a detrimental effect on transplanted cells, leading to severe cell death in mDAN. Potential strategies to enhance the survival of transplanted cells involve obtaining autologous T cells from the patient. REG Cells were increased in number and function, and the therapeutic effect was demonstrated through co-transplantation into the brain. Additionally, T... REG The application of these technologies to explore the efficacy of inhibitors, neutralizing antibodies, and encapsulation methods to enhance graft cell survival in the inflammatory microenvironment provides a promising direction for the future advancement of CRT.

[0054] Figure 22 Antibodies targeting inflammatory cytokines reduce MHC-II. + Cell infiltration. The level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-MHCII antibody. Detailed Implementation

[0055] Parkinson's disease (PD) represents the second most common neurodegenerative disorder after Alzheimer's disease, and its prevalence continues to rise with the aging of the global population. At the heart of the pathophysiology of PD is the specific degeneration of midbrain dopamine neurons (mDANs; mDA cells) in the substantia nigra. Therefore, cell replacement therapy (CRT) has emerged as a promising treatment approach, initially supported by various open-label clinical studies employing fetal ventral midbrain (fVM) cells. Despite initially favorable results, fVM cell therapy has inherent and operational limitations that hinder its transition to a standard treatment for PD. Recent efforts in the field of cell therapy have shifted their focus to utilizing human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, to overcome existing challenges. However, poor and variable survival of implanted dopamine cells remains a major obstacle, regardless of the source of transplantable cells (e.g., xenogeneic, allogeneic, or autologous). Emerging evidence highlights the crucial role of the post-transplant host immune response in influencing the survival of implanted mDANs, underscoring an important area for further research. The inventors investigated the functional outcomes of host immune response on the survival and efficacy of transplanted dopamine cells, and explored potential strategic approaches to modulate host immune response for optimal results in future clinical applications of CRT for PD.

[0056] Lessons learned from fetal ventral midbrain (fMV) transplantation studies

[0057] Clinical outcomes of fVM-based CRT vary widely and are occasionally associated with graft-induced dyskinesia (GID).

[0058] Beginning in the late 1980s, stem cell scientists began using fVM tissue dissected from aborted embryos (usually 6 to 9 weeks old) for CRT, as they showed the most promising results in a variety of dopamine-producing cells. 6-13 In these early open-label trials using fVM tissue, some patients showed significant and long-term improvements, including enhanced motor function, reduced rigidity and tremor; reduced "off" periods; reduction in medication use, or in rare cases, complete cessation; and enhanced dopamine uptake, such as... 18 As demonstrated by F-DOPA PET scan analysis (Table 1). 14-35Subsequent post-mortem studies of deceased transplant patients further supported these positive clinical results, revealing successful engraftment of the implanted mDAN, its robust extraterrestrial growth, and its neural innervation of the host striatum, demonstrating that the engrafted mDAN functionally integrates into the neural circuits of the patient's brain. In summary, these successful clinical data provided a "proof of concept" for CRT in PD. Despite these positive results, subsequent double-blind, sham-controlled studies showed that the clinical benefit was not statistically significant and it is not recommended as a treatment for PD. 36-38 In addition, approximately 30% of the transplant patients (18 out of 56 patients) experienced serious side effects: GID. 36-38 This significantly dampened the initial enthusiasm for fetal cell-based methods. These inconsistent and disappointing results, along with ethical, medical, and practical limitations, make this approach unfeasible as a standard treatment for PD. 6-11

[0059] Poor and variable survival rates of implanted mDANs may underlie the inconsistent clinical outcomes observed in fVM-based CRT.

[0060] Why are the clinical outcomes of fVM transplantation inefficient and variable? Over the past few decades, more than 400 PD patients have undergone fVM transplantation and their cases have been thoroughly analyzed. 6-13 In addition, stem cell scientists have conducted extensive preclinical studies using rat and mouse embryonic VM tissues with three objectives: (1) to understand the inefficient and variable clinical outcomes of fVM transplantation, (2) to elucidate the biological factors affecting the survival of transplanted cells, and (3) to enhance the survival of implanted mDANs in order to successfully conduct CRT in the future. 39-41 These clinical and preclinical studies have revealed important insights and a variety of potential factors leading to inconsistent clinical outcomes. These factors include a range of variables, including different patient ages and clinical statuses; different fetal cell preparation methods; heterologous and variable fetal status (typically requiring 6 to 8 fetuses per patient); variable immunosuppressive regimens; and transplantation target sites (putamen, caudate nucleus, and / or substantia nigra) (Table 1). 6-14,18-31,35,37,38,42 While all these donor and / or host-specific factors may play a significant role in clinical outcomes, we hypothesize that poor and variable survival of the transplanted mDAN in the graft directly leads to inefficient clinical outcomes. Indeed, due to the limited supply of aborted fetuses, the survival of this transplanted mDAN has been a focal point of CRT research since the concept of fVM transplantation was introduced. As outlined in the insightful review by Brundin et al., 43In 31 independent studies using rat embryonic VM transplantation, the survival rate of transplanted mDAN ranged from 0.7% to 23.3%, with an average of 6.86%. Similarly, studies on human VM transplantation in athymic rats... 44,45 In autopsy studies of human fVM transplantation, a consistent survival range between 5% and 10% was reported. 9,12,13,22,23,27,37,38,42 In summary, a key lesson learned from previous clinical and preclinical studies is the high inefficiency and variable survival of graft mDANs, which may be the root cause of the observed poor and inconsistent clinical outcomes.

[0061] Table 1: List of clinical trials using human fetal tissue

[0062]

[0063]

[0064] As mentioned above, the survival of transplanted mDANs has been extensively studied in preclinical studies using rodent embryonic VM cells to mimic human fVM transplantation. 43,46-52 A striking feature of these studies is that most mDANs die shortly after transplantation (usually within a week), and the number of surviving mDANs in the graft does not increase or change later. This observation is quite surprising, given that embryonic VM cells contain a large number of early progenitor cells in addition to differentiated mDANs. These data suggest two possibilities: (1) the progenitor cells in fVMs have a very limited capacity for proliferation and differentiation 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 at which mDANs may die during this period. 43 In stage 1 (embryo removal), mDAN may die due to hypoxia and hypoglycemia injury that occurs during the removal of the embryo from the maternal blood supply. In stage 2 (cell preparation), mDAN may die due to axotomy caused by mechanical dissociation and other traumatic injuries. In stage 3 (intrastriatal injection), mDAN may die during implantation and in the immediate period after graft injection, and in stage 4 (graft maturation), mDAN may die during maturation and innervation in the host brain.

[0065] Extensive research has been conducted to understand the poor survival of transplanted mDANs and the reasons for their early death after transplantation, revealing several possibilities. When mDANs dissected from embryos are transplanted into the striatum under PD conditions, they become deprived of nutrients and growth factors essential for their maintenance. Consistent with this concept, pretreatment of cells with growth factors such as basic fibroblast growth factor (bFGF) and glial cell line-derived neurotrophic factor (GDNF) significantly enhanced the survival of transplanted mDANs. Notably, while bFGF pretreatment resulted in an approximately 2-fold increase in survival, 53,54 However, continuous delivery via co-transplantation of fibroblasts overexpressing bFGF resulted in a 10-fold increase, promoting more robust and rapid behavioral recovery. 55 Furthermore, since neuronal damage is often associated with excitotoxicity, oxidative stress, and calcium imbalance, researchers have explored pathways and molecules related to these phenomena. Among them, calcium channel blockers (such as flunarizine) and lipid peroxidation inhibitors (such as lazaro-like drugs) have shown significant effects on mDAN survival. 56-58 In addition, inhibition of cell death pathways, such as caspase inhibitors (e.g., Ac-YVAD-cmk), significantly enhances mDAN survival. 51

[0066] When do hPSC-derived mDANs die during the transplantation process?

[0067] The main advantage of hPSCs lies in their ability to generate an unlimited supply of transplantable cell sources, such as mDNA. Perhaps precisely because of this advantage, unlike fVM-based CRT, cell survival in hPSC-based CRT has not received sufficient attention and remains poorly studied. Given the significant differences in cellular, developmental, and proliferative characteristics between fVMs and hPSCs, both similarities and differences must be considered to ensure successful CRT in PD. To address these issues, we investigated cell survival in hPSC-based CRT using several rodent models, including wild-type (immunosuppressed) and athymic rats, as well as immunocompromised NOD SCIDγ (NSG) mice and humanized NSG mice. 84 Interestingly, most (approximately 90%) mDANs (derived from both hESC and hiPSC) die within the first 1-2 weeks after transplantation, which is very similar to fVM transplantation. 43,46-52 Furthermore, this study revealed that the transplantation process itself triggers an acute host inflammatory response. Notably, even when only culture medium was injected without any cells, a host immune response was triggered, indicating that it was induced by the host's innate immune response. This is because the pattern of immune response is very similar to the immune response pattern of traumatic brain injury (TBI). Figure 18 ), 87,88We call this phenomenon "acupuncture trauma". 84 This acupuncture trauma appears to physically damage the host brain, leading to acute cellular death of host neurons around the injection path. This is thought to trigger subsequent immune responses such as the immediate secretion of pro-inflammatory cytokines (e.g., TNF and IL-1β), activation of astrocytes / microglia, and Iba-1 near the needle tract. + Major Histocompatibility Complex (MHC) II + Robust infiltration of inflammatory cells was observed, peaking on day 7, then declining at 1 month and disappearing at 6 months. Importantly, this needle-induced trauma preferentially led to the death of most mDANs in the graft rather than midbrain dopamine progenitor cells (mDAPs). Furthermore, unlike fVM-based CRT, the total number of mDANs (and transplanted cells) increased significantly at a later stage, indicating that some mDAPs proliferated and differentiated into new mDANs in the post-transplant host brain. 84 In light of these new findings, combined with insights from previous fVM porting research... 43,46-52 It appears that the mDAN from the hPSC source dies during one of the three stages of the hPSC-based CRT procedure. Figure 19 ).

[0068] In Phase 1, hESC / hiPSCs are cultured and then differentiated in vitro at specific times using optimized procedures. Figure 19 Although different laboratories employ different optimization schemes, 65,89-96 However, based on previous developmental studies, these methods typically employ dual inhibition of SMAD targeting BMP and TGFb signaling, and dual activation of WNT and SHH signaling. This in vitro differentiation process results in the generation of mDA cells, which primarily contain mDAP and mDAN in the ranges of 60-95% and 5-25%, respectively, depending on the individual protocol. 65,89-96 These protocols differ in their detailed methods and culture medium compositions. For example, some protocols utilize 2D monolayer methods, while others employ a combination of 2D and 3D cultures, such as embryoid and floating neurosphere cultures. There is a possibility that mDA cells differentiated in vitro may lose viability during differentiation. For instance, we observed that a significant proportion of cells died and / or became apoptotic on uniformly distributed monolayer cultures, and that dividing the monolayer into smaller, segregated portions, termed the previously described “spotting method” (e.g., WO 2020 / 237104), significantly reduced the percentage of these unhealthy or apoptotic cells during in vitro differentiation. 65,97 Phase 1 is similar to the initial steps of fVM-based CRT, in which donor embryos are dissected and prepared into cell suspensions or tissue blocks for transplantation. 43Previous studies have shown that cell viability is substantially affected by the different conditions and types of culture media used to prepare fVM-derived cells for transplantation. 98-100 Therefore, in vitro differentiation conditions and culture media should be rigorously tested and optimized to maximize viability. This optimization is crucial because the non-viable / apoptotic components of these final cell products persist until transplantation and can negatively impact clinical outcomes. For example, combined with needle prick-induced neuroinflammation, these dead or apoptotic components of the transplanted cell products may trigger additional host immune responses in stage 3. Although FACS procedures can be designed to exclude dead / dying cells, they may have additional harmful effects on living cells.

[0069] In stage 2, in vitro differentiated mDA cells were 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, cell phenotype, and in vivo function. 65,93,101-103 Therefore, most research groups plan to incorporate this cryopreservation step into hPSC-based CRT. Phase 2 comprises three key steps: (i) harvesting and cryopreserving in vitro differentiated mDA cells using the optimal cryopreservation medium in cryovials, (ii) storing multiple cryovials in liquid nitrogen, and (iii) thawing the cryovials and loading the mDA cells into surgical syringes prior to transplantation. Theoretically, mDA cells can 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 duration for which they can be stored in liquid nitrogen without impairing their viability and function remains an area of ​​uncertainty. Given that needle prick trauma has been observed to affect mDA cells more preferentially than mDAP, it seems reasonable that the effects of cryopreservation and subsequent treatment vary significantly depending on cell type. Therefore, re-examining this issue and systematically analyzing the viability of different cell types at each step may be crucial for the ultimate optimization of the process.

[0070] Phase 3 constitutes the final in vivo transplantation step and includes (i) surgical transplantation, (ii) early (<2 weeks) graft establishment, and (iii) late (>2 weeks) graft establishment. Figure 19The host immune response plays a role in these steps and can lead to the death of mDA cells. During the initial steps of graft injection, mDA cells can remain viable in the syringe for a limited period of time (e.g., 10–20 minutes). However, prolonged delays during injection (exceeding the normal time in clinical procedures) can lead to the death of a subset of mDA cells (especially mDAN), even before they are injected into the host brain. Therefore, it is recommended to minimize delays during the surgical procedure. Notably, the surgical procedure itself appears to trigger an acute innate immune response, culminating in a harsh neuroinflammatory environment. The injected mDAN thus undergoes early and preferential cell death. Furthermore, adaptive immune responses can occur between the transplanted cells and the host immune system, potentially leading to graft rejection in the absence of immunosuppression. In the later stages of colonization in stage 3, surviving mDA cells undergo maturation, differentiation, and integration into the host brain to establish new functional circuits. However, it seems reasonable that a subset of new and / or mature mDAN may die even in this later stage due to various factors such as a sustained host immune response, insufficient essential factors (e.g., blood supply, growth factors, and oxygen), and an unfavorable PD environment characterized by neuroinflammation, oxidative stress, and alpha-synucleinopathy.

[0071] When mDA cells derived from human induced pluripotent stem cells (hiPSCs) are transplanted into the striatum of immunodeficient NOD SCID γ or humanized mice, only a small percentage (<10%) of tyrosine hydroxylase (TH) is implanted. + mDA neurons survived for 2 weeks after transplantation. Conversely, most of the remaining TH neurons... - Transplanted cells survived. Notably, autologous regulatory T cells (T cells) also survived. REG Transplantation of mDA cells significantly altered the response to acupuncture trauma induced by mDA cell transplantation, suppressing acute neuroinflammation and immune cell infiltration. Furthermore, T… REG Intrastral co-transplantation of hiPSC-derived mDA cells significantly protected transplanted mDA neurons from needle-induced traumatic death and significantly improved treatment outcomes in a rodent model of PD with 6-OHDA injury. REG Co-transplantation also suppressed TH - Unintended proliferation of transplanted cells resulted in TH cells with a higher proportion and a higher absolute number. + A more compact graft of neurons. In summary, these data highlight the importance of the initial inflammatory response to surgical injury in the differential survival of the cellular components of the graft and demonstrate the role of autologous T cells. REG Co-transplantation with iPSC-derived mDA grafts effectively reduced acupuncture trauma-induced mDA neuron death.

[0072] Treatment

[0073] Therefore, this method provides a clinically applicable personalized cell therapy for PD that increases the viability of transplanted cells. In some embodiments, this document describes methods for treating PD, methods for increasing cell survival in cell transplantation, methods for increasing the extracellular growth of transplanted cells, and / or methods for reducing overgrowth during cell transplantation.

[0074] Since needle trauma can be a significant factor in the success of cell therapy for all cell types, this article also describes methods to increase graft viability and / or reduce immune responses in subjects receiving cell therapy.

[0075] In some implementations, the methods described herein include administering an effective amount of T to a subject in need (e.g., a subject with cell therapy and / or a subject with PD (or at risk of developing PD)). REG Cell populations and second cell populations (e.g., mDA cells).

[0076] In some implementations, the methods described herein include administering an effective amount of an antibody (Ab) (e.g., an antibody that binds to pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules; optionally, an anti-TNF-α Ab (e.g., infliximab), an anti-IL-1β Ab (e.g., cannabinoid), an anti-IFN-γ Ab (e.g., epavali), an anti-HMGB1 Ab, and an anti-IL-1α Ab) to a second cell population (e.g., mDA cells) to a subject in need (e.g., a subject with cell therapy and / or a subject with PD (or at risk of developing PD)).

[0077] In some implementations, the methods described herein include:

[0078] Apply T REG Cell population;

[0079] Apply mDA to a cell population;

[0080] Administer anti-inflammatory cytokines (ABS) that bind to pro-inflammatory cytokines (e.g., anti-TNF-α ABS, anti-IL-1β ABS, anti-IFN-γ ABS);

[0081] Administer Abs that bind to damage-associated molecular patterns (DAMPs) (e.g., anti-HMGB1 Abs, anti-IL-1α Abs);

[0082] Application containing T REG A composition of a cell population and a second cell (e.g., mDA cells); and / or

[0083] A composition comprising an Ab (anti-inflammatory cytokine Ab, anti-IL-1β Ab, anti-IFN-γ Ab) or an Ab (anti-HMGB1 Ab, anti-IL-1α Ab) binding to DAMP molecules and a population of cells (e.g., mDA cells) is administered to a subject in need.

[0084] Such subjects (e.g., subjects receiving cell therapy, such as those identified as benefiting from cell therapy, subjects with PD, or subjects at risk of developing PD) can be identified by healthcare professionals using methods known in the art.

[0085] T is used in this method REG Cell populations are described herein and are known in the art. Useful T REG The cells may express one or more of FoxP3, CD25, CD47, CD45, CD4, CD8, and CD127. See also US 2023 / 0323299, US 2023 / 0047159, US 2020 / 0330515, and references 163-167. Therefore, the methods described herein may include obtaining a sample containing T cells (e.g., blood or PBMC cells) from a subject, isolating Treg cells, expanding Treg cells, and administering the cells. In some embodiments, primary cells are obtained from a subject to be treated (e.g., a subject with PD (or at risk of developing PD), but in some embodiments, cells are obtained from a different subject, and in some embodiments, cells are obtained from a subject of the same species as the subject to be treated (preferably an immune-matched subject).

[0086] The methods described herein may include the use of induced pluripotent stem cells (hiPSCs), such as neural-derived basal plate cells that can be generated using methods known in the art or described herein. In some embodiments, methods for generating hiPSCs may include obtaining a population of primary cells from a subject (e.g., a subject undergoing cell therapy, a subject with PD, a subject at risk of developing PD, and / or a subject requiring treatment for PD). Preferably, the subject is a mammal, such as a human.

[0087] The method may include obtaining primary somatic cells; generating a cell population containing mDA cells; and administering the cells. In some embodiments, primary somatic cells are obtained from a subject to be treated (e.g., a subject with PD (or at risk of developing PD) WO20254), but in some embodiments, cells are obtained from a different subject, and in some embodiments, cells are obtained from a subject of the same species as the subject to be treated (preferably an immune-matched subject). Preferably, the mDA cells are generated by the method described herein, which is sufficient to generate TH cells containing expression of one, two, or more mDAP markers (e.g., FOXA2, OTX2, LMX1A, and EN1, such as FOXA2 and LMX1A; optionally co-expressing FOXA2, LMX1A, and NURR1). + The cells (cells) may include, optionally, cells expressing one, two, or more mDAN markers (e.g., TH, DAT, and PITX3), but exclude populations of cells expressing SOX1, PAX6, and KI67.

[0088] The mDA cell populations used in this method are described herein and are known in the art. The mDA cell populations used in the methods described herein can be derived from hESC / hiPSC and subsequently differentiated in vitro over a specific period using an optimization procedure. Figure 19 ). 65,89-96 In some embodiments, the mDA cell population comprises mDAP and mDAN. In some embodiments, the mDA cell population comprises 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 US 2022 / 0243174; US 2018 / 0371422; US 2012 / 0128655; US 2013 / 0052268; US 2016 / 0002604; US 2014 / 0199274; and US 2009 / 0226401; and US 11,898,169; US 11,001,809; US 9,657,273; and US 9,750,768; and references 61-65 and 89-96.

[0089] In some embodiments, the methods described herein reduce the immune response in subjects triggered by needle prick trauma during cell transplantation and / or treatment of PD. In some embodiments, the methods described herein increase TH in the graft. + The number and / or percentage of cells (e.g., compared to those without co-transplanted T cells) REG(Compared to grafts containing cell populations or Abs targeting pro-inflammatory cytokines). In some embodiments, the methods described herein increase TH in the graft. + Cell: TH - The proportion of cells and / or an increase in TH in the graft + Cell: Ki67 + Cell ratio (e.g., compared to T cells without co-transplantation) REG Compared to grafts containing cell populations or Abs targeting pro-inflammatory cytokines. In some embodiments, the methods described herein reduce Ki67 in grafts. + The number and / or percentage of cells and / or reduction of TH in the graft - The number and / or percentage of cells (e.g., compared to those without co-transplanted T cells) REG (Compared to cell populations or grafts targeting pro-inflammatory cytokines).

[0090] In some embodiments, the cells, antibodies, and compositions described herein may be administered to the subjects described herein. In some embodiments, the administration steps of any of the methods described herein include systemic, parenteral, intravenous, intracerebral, intraspinal, intrathecal, intracisional, intraputaminal, intrahippocampal, intrastriatal, or intraventricular administration. In some embodiments, the administration steps include intravenous, intracerebral, intraspinal, intrathecal, intracisional, intraputaminal, intrahippocampal, intrastriatal, or intraventricular injection. In some embodiments, the administration steps include direct injection into the substantia nigra pars compacta. In some embodiments, the administration steps include direct introduction of cell therapy and / or antibodies into the brain or cerebrospinal fluid (CSF) of the subject.

[0091] In some embodiments, cells, antibodies, and compositions may be administered using methods known in the art. In some embodiments, cells are administered by, for example, direct implantation into the affected area of ​​the subject's brain or its vicinity using magnetic resonance imaging-guided stereotactic surgery, such as bilateral or unilateral implantation into one or more of 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); Sonntag et al., Prog Neurobiol.2018 Sep; 168:1-20.

[0092] In some implementations, the cells, antibodies, and compositions described herein may be administered in a single dose or in repeated doses.

[0093] In some implementations, T REG The cells and the transplanted cells (e.g., mDA cells) are administered to the subject simultaneously. In some implementations, T... REG The cells and the transplanted cells (e.g., mDA cells) are administered separately. In some embodiments, T cells are first administered... REG Cells are administered to the subject, followed by the administration of transplanted cells (e.g., mDA cells) to the subject. In some embodiments, the transplanted cells (e.g., mDA cells) are administered to the subject first, followed by the administration of T cells. REG The cells were administered to the subject.

[0094] In some embodiments, the antibody (Ab) (e.g., an antibody that binds to pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules; optionally, an anti-TNF-α Ab (e.g., infliximab), an anti-IL-1β Ab (e.g., cannabinoids), an anti-IFN-γ Ab (e.g., epavadumab), an anti-HMGB1 Ab, or an anti-IL-1α Ab) and the transplanted cells (e.g., mDA cells) are administered to the subject simultaneously. In some embodiments, the antibody (Ab) (e.g., an antibody that binds to pro-inflammatory cytokines or damage-associated molecular pattern (DAMP) molecules; optionally, an anti-TNF-α Ab (e.g., infliximab), an anti-IL-1β Ab (e.g., cannabinoids), an anti-IFN-γ Ab (e.g., epavadumab), an anti-HMGB1 Ab, or an anti-IL-1α Ab) and the transplanted cells (e.g., mDA cells) are administered separately. In some embodiments, an antibody (Ab) (e.g., an antibody that binds to pro-inflammatory cytokines or damage-associated molecular patterns (DAMP) molecules; optionally, an anti-TNF-α Ab (e.g., infliximab), an anti-IL-1β Ab (e.g., cannabinoids), an anti-IFN-γ Ab (e.g., epavadumab), an anti-HMGB1 Ab, or an anti-IL-1α Ab) is first administered to the subject, followed by administration of transplanted cells (e.g., mDA cells) to the subject. In some embodiments, the antibody and cells are administered to the patient intrastriatally. In some implementations, 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, intravenous).

[0095] In some implementations, T REG The cell is an autologous T cell REG Cells, allogeneic T cells REG Cells, or xenogeneic T cells REG Cells. In some embodiments, the transplanted cells (e.g., mDA cells) are autologous mDA cells, allogeneic transplanted cells (e.g., mDA cells), or xenogeneic transplanted cells (e.g., mDA cells). In some embodiments, T... REG Cells are transplanted into the striatum. In some embodiments, the transplanted cells (e.g., mDA cells) are transplanted into the striatum.

[0096] Compositions comprising antibodies (Abs) (e.g., antibodies that bind to pro-inflammatory cytokines or damage-associated molecular patterns (DAMP) molecules; optionally anti-TNF-α Ab (e.g., infliximab), anti-IL-1β Ab (e.g., canatumab), anti-IFN-γ Ab (e.g., epavamab), anti-HMGB1 Ab, anti-IL-1α Ab) and methods of administering said compositions are well known in the art.

[0097] In some embodiments, administration includes systemic, parenteral, intravenous, intracerebral, intraspinal, intrathecal, intracranial, intraventricular, putamen, hippocampal, striatum, or intraventricular administration. In some embodiments, administration includes intravenous, intracerebral, intraspinal, intrathecal, intracranial, putamen, hippocampal, striatum, or intraventricular injection. In some embodiments, administration includes direct injection into the substantia nigra pars compacta. In some embodiments, administration includes direct introduction of cell therapy and / or antibodies into the subject's brain or cerebrospinal fluid (CSF).

[0098] See also US 2022 / 0243174; US 2018 / 0371422; US 2012 / 0128655; US 2013 / 0052268; US 2016 / 0002604; US 2014 / 0199274; and US 2009 / 0226401; and US 11,898,169; US 11,001,809; US 9,657,273; and US 9,750,768.

[0099] Acupuncture trauma is known in the art, but it is particularly concerning for cell therapy because it requires needles with larger diameters to be applied without clogging or damaging the cell therapy. Needle characteristics include inner and outer diameters, length, stiffness, and bevel design. Deep subcortical target structures, such as the caudate nucleus or striatum, require sufficiently rigid, long, and thin needles / cannulas (typically 19 cm or longer) to penetrate the target site without damaging overlying structures. Shorter needles (8–10 cm) would require direct brain exposure, which is more invasive (Amer, MH, 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).

[0100] Needles used for administering cell grafts may be at least 30G, 28G, 26G, 24G, 22G, 20G, 18G or greater and / or have an outer dimension (OD) 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, including a gradient outer diameter between any of these parameters (e.g., 1.5 to 0.5 mm).

[0101] Needle parameters are known in the art and can be determined by a skilled healthcare professional using methods known in the art. See also,

[0102] • Song et al., JCI 2020, Park et al., Nature 2023: 26G, external dimensions (OD), 0.464mm;

[0103] • Doi et al., Nature Communications 2020: 22G, OD, 0.718mm and 24G, OD, 0.566mm;

[0104] • Kriks et al., Nature 2011: 28G, 0.362mm;

[0105] • Schweitzer et al., NEJM 2020: 22G, OD; 0.72mm;

[0106] • Freed et al., NEJM 2001: Placement of tissue implants using stainless steel guide cannulas with varying outer diameters from 1.5 to 0.6 mm. A round-tipped inner core needle is embedded in the lumen of the cannula during insertion into the posterior tip of the putamen.

[0107] • Spencer et al., NEJM 1992: 1mm; and

[0108] • Freed et al., NEJM 1992: A 1.5-mm cannula extends from its support in a stereotactic device to the brain surface, with an inner core needle of 0.46 or 0.64 mm outer diameter penetrating the putamen.

[0109] Following needle injection, the brain suffers physical damage, leading to the rupture of resident cells such as neurons, astrocytes, microglia, and oligodendrocytes. This rupture, caused by the impact of the needle, results in these cells rapidly releasing damage-associated molecular patterns (DAMPs), which affect neighboring cells and trigger the production and secretion of cytokines and chemokines due to activation.

[0110] The data presented in this article are the first to show that this needle prick trauma directly leads to a significant increase in host brain cell death and inflammation near the injection site, which poses a substantial obstacle to the success of cell therapy (e.g., graft success in cell transplantation).

[0111] Host immune response in hPSC-based CRT

[0112] The survival of colonized mDA cells is significantly influenced by various levels of host immune responses. A fundamental requirement for transplanted cell survival is avoiding graft rejection, a process regulated by the host's adaptive immune system. Traditionally, certain specialized organs, such as the brain and eye, have been considered immune-exempt. 104 This could explain why early fVM transplantation studies did not use immunosuppressants (Table 1). Instead, a contrasting view suggests that even autologous cells can elicit an immune response if the differentiated cell products express immunogenic antigens. 83,105To investigate the controversial issue of immunogenicity of autologous and allogeneic brain transplants, we conducted transplantation experiments involving the transplantation of patient-derived, allogeneic mDA cells into the striatum of NSG mice, patient-humanized NSG mice, and allogeneic humanized NSG mice. Our findings revealed that while autologous mDA cells were rejected in allogeneic humanized mice, they were accepted in autologous humanized mice. 61 This indicates that when allogeneic mDA cells are transplanted into the brain, they may be rejected without immunosuppression. It is noteworthy that immune immunity in the brain may require an intact blood-brain barrier (BBB), which is present in PD-positive brains. 106 Both the transplanted cells and surgical instruments can potentially damage them. Furthermore, our recent research indicates that the host immune system interacts not only with the transplanted cells but also with the surgical instruments (i.e., the needle), highlighting the crucial role of the host's innate immune response in this process. 84 As described below.

[0113] Adaptive immune response and immunosuppression

[0114] Adaptive immune responses play a crucial role in the rejection of allogeneic or xenografts. 107 This adaptive immunity is characterized by a precise and sensitive response of T and B cells to MHC (also known as HLA), expressed by all nucleated cells. Even slight differences in HLA molecule expression in transplanted cells can trigger active clearance in the body. Therefore, even for intracerebral transplantation of allogeneic mDA cells, significant immunosuppression is required. In fact, all current and future clinical trials involving allogeneic hPSC-derived mDA cells are planned to use strong immunosuppression. 90,93,95,108 It is important to acknowledge that long-term immunosuppression is associated with a variety of side effects, including susceptibility to infections and malignancies, and also incurs additional costs and inconvenience. Previous studies have demonstrated a correlation between infection risk and the dosage and duration of immunosuppressants. 109,110 When used in combination with other immunosuppressants, the risk and severity of infection increase. 111 In addition, immunosuppression with cyclosporine is known to increase the risk of malignant tumors such as lymphoma or skin cancer. 112,113 Other side effects may include loss of appetite, vomiting, nausea, hirsutism, and tremors, although these symptoms usually subside as the body adapts to the immunosuppressant. Given these diverse side effects, researchers are actively exploring ways to optimize the route, dosage, and duration of immunosuppression. For example, promising strategies involve using local immunosuppression to avoid the systemic side effects associated with standard immunosuppression. 114

[0115] Innate immune response

[0116] The innate immune system forms the body’s first line of defense, including physical, chemical, and cellular mechanisms, to rapidly counteract or eliminate foreign antigens introduced from external sources. 107,115 Unlike adaptive immunity, which is acquired through exposure to invaders, innate immunity is present at birth and depends on antigen-nonspecific defense mechanisms. The main function of innate immunity is to directly eliminate pathogens, and most importantly, to rapidly recruit immune cells to sites of infection and inflammation. 116,117 This is achieved by sensing pathogens and producing cytokines (e.g., TNFα, IL1β, IFNγ) and chemokines (e.g., CXCL, CCL). Additionally, the innate immune response responds to physical damage caused by external mechanical forces. A representative example is neuroinflammation induced by TBI, where initial traumatic damage triggers disruption of both macroscopic barriers (e.g., skin) and microscopic barriers (e.g., cell membranes), leading to the secretion of various immune molecules. 118

[0117] Our recent research indicates that needle-based transplantation procedures significantly trigger a host innate immune response, which we term "needle-induced trauma" due to its similarity to total invasive biopsy (TBI). Figure 18 ). 84 Primary TBI occurs immediately after the shock, damaging the brain's physical structure, including meningeal and neuronal contusions, axonal shearing, and vascular injury. Secondary TBI develops gradually, involving various cellular processes such as BBB disruption, excitotoxicity, mitochondrial dysfunction, oxidative stress, inflammation, and cell loss. 115 Since acupuncture trauma similarly induces these downstream pathways, further elucidation could provide promising molecular targets to mitigate their impact on the survival of transplanted mDA cells, potentially enhancing the outcomes of hPSC-based CRT (see below). Notably, PD patients typically exhibit elevated levels of both brain and systemic inflammation compared to healthy individuals. 119-121 This elevated inflammatory state in PD patients may have a more significant adverse impact on CRT outcomes. Furthermore, it is crucial to recognize the significant variations in the systemic immune system among individuals, including those with PD, and that these immune differences may lead to variations in treatment outcomes. However, it is important to acknowledge that the primary factor influencing the survival of transplanted mDANs is inflammation triggered by brain injury caused by the transplant procedure itself. Therefore, further research efforts are needed to fully understand the distinct roles of intrinsic inflammatory states and surgery-induced inflammation in determining the survival of transplanted mDANs.

[0118] Potential strategies for enhancing cell survival in successful hPSC-based CRTs

[0119] While hPSC-based CRT offers the advantage of generating an unlimited number of transplantable cells, it also faces the challenge of poor survival of hPSC-derived mDAN, as demonstrated by previous preclinical studies (Table 2). 96,122-131 Therefore, it is necessary to develop novel strategies to enhance mDAN survival during CRT. One promising approach is to modify the host immune response, thereby creating a more favorable environment for the transplanted cells.

[0120] Strategies targeting adaptive immunity

[0121] The survival of transplanted cells is predicated on avoiding graft rejection. Therefore, all allogeneic transplants require significant levels of immunosuppression, although the duration of immunosuppression required for each patient remains uncertain. Recent studies in non-human primates... 132-134 and people 61 Studies have demonstrated that autologous transplantation does not require immunosuppression. However, this autologous approach is time-consuming and expensive. Alternative strategies to mitigate the need for immunosuppression involve the use of HLA-matched iPSCs. Supporting evidence for this approach comes from recent primate studies showing that transplantation of mDA cells from HLA-matched primate iPSCs reduces the host immune response and increases mDA survival. 12 Therefore, many groups are working to establish HLA-matched hiPSCs as libraries from common HLA homozygous donors, aiming to minimize post-transplant graft rejection and reduce time and effort compared to autologous methods. Figure 20 ). 135,136 Professor Yamanaka's team estimates that the hiPSC line, derived from approximately 140 unique HLA homozygous donors, is sufficient to cover up to 90% of the Japanese population. 137 Due to genetic diversity, a large-scale hPSC database is currently being established in the United States to cover different ethnic groups, such as European Americans, African Americans, Hispanics, and Asians. 138 Despite these efforts, immune responses may still occur even with HLA-matched cells, due to indirect pathways caused by HY minor histocompatibility antigens or innate immunity caused by natural killer (NK) cells. 139,140

[0122] Efforts to develop "universal donor stem cells" that avoid immune rejection have attracted widespread attention. 141,142 This method primarily aims to eliminate HLA molecules in donor cells, which are crucial for adaptive immunity and graft rejection. The initial attempt in 2013 aimed to eliminate HLA molecules that specifically target HLA class I, which are primarily expressed across most cell types as β2-tubulin (B2M) (a structurally non-polymorphic heavy chain). 143,144However, cells lacking the HLAI class generated through genome engineering methods exhibit limitations because they are susceptible to lysis by NK cells via the "missing self" response. 145 It is noteworthy that the CD94 / NGK2A complex on NK cells circumvents the cleavage mechanism when it binds to any HLA class I molecule, including HLA-E, which is known for its minimal polymorphism and expression only in B2M knockout cells. 145,146 Recently, through the process of technology development and optimization, Schrepfer and his colleagues 147 A method for generating low-immunogenic donor iPSCs was designed through three steps: (1) CRISPR / Cas9-mediated knockout of the B2M gene (a component of HLA class I), (2) the CIITA gene (a master regulator of HLA class II), and (3) lentiviral overexpression of the immune receptor CD47 transgene. They demonstrated that various cell types derived from these engineered donor iPSCs (e.g., endothelial cells, smooth muscle cells, and cardiomyocytes) effectively circumvented the immune rejection of completely HLA-mismatched host animals and survived long-term without immunosuppression. Additionally, Akitsu Hotta and colleagues created pseudohomozygous iPSC lines using CRISPR-Cas9 by disrupting the HLA-A and HLA-B biallelic alleles while retaining a single HLA-C allele. 148 It is estimated that 12 HLA-C-preserved and HLA-II knockout iPSC lines are immunocompatible with >90% of the world's population. While these strategies hold promise for iPSC-based regenerative medicine applications, it remains uncertain whether these engineered cells will retain full functionality and low immunogenicity after transplantation into patients. 149 Extensive genetic manipulation (e.g., ectopic CD47 expression) can lead to unforeseen adverse effects, such as oncogenic transformation and impaired immune responses. 150,151 Furthermore, rapid removal may be impractical when cells become infected with a virus, prompting the need to develop gene integration for kill switches. However, this strategy could potentially induce unintended adverse reactions, necessitating further research to refine and implement safer strategies. 152,153 Furthermore, even if these genetic alterations successfully circumvent immune rejection through adaptive immunity, the transplanted graft may still be susceptible to the innate immune response induced by needle prick trauma.

[0123] Strategies targeting innate immunity

[0124] The host's innate immune response triggered by TBI or needle prick trauma induces inflammation within minutes of injury. This response is characterized by the secretion and upregulation of damage-associated molecular patterns (DAMPs), cytokines, chemokines, immune cell infiltration (e.g., neutrophils and myeloid cells), and subsequent activation of glial cells (astrocytes and microglia) and recruitment of leukocytes. 107,115,154 Numerous studies have demonstrated that these molecules, including DAMP, cytokines, and chemokines, are secreted dramatically within 6 hours after TBI. 155 Therefore, various research efforts have attempted to target the innate immune system for therapeutic development in TBI. These studies have revealed the significant therapeutic effects of using specific inhibitors to block those molecules in animal models. 156-159 It is worth exploring whether these inhibitors and / or neutralizing antibodies can produce similar effects in needle prick wounds and improve the survival of colonized mDA cells. This approach warrants further investigation. Figure 21 Furthermore, given that acupuncture trauma dramatically triggers the secretion of various cytokines, chemokines, and DAMP (within minutes to hours after injury), an attractive strategy could involve delaying cell grafting immediately after needle insertion (when no cells are present). Indeed, previous studies have shown that neuronal survival is significantly increased when dopaminergic cell suspensions are delayed for more than one hour after cannula insertion. 160,161

[0125] Regulatory T cells (T cells) REG It plays an important role in maintaining immune tolerance and homeostasis. 162,163 They are involved in many autoimmune and inflammatory diseases and have been used clinically to improve survival rates in various organ transplants through adoptive transfer after ex vivo expansion. 164 In TBI, T REG Infiltrate the damaged area after initial inflammatory cell infiltration to promote the repair process. 165,166 Therefore, assuming self-T REG It can reduce the innate immune response triggered by needle prick trauma and enhance the survival of transplanted mDNA. In the conventional adoptive transfer test T... REG When it is in effect, 164,167 Approximately 1,000,000 ex vivo expanded autologous T cells were injected intravenously. REG This leads to a moderate but significant reduction in the innate immune response, such as MHCII. + The reduced infiltration of cells is indicated. However, due to the rapid onset of neuroinflammation induced by acupuncture trauma in the brain, adoptive transfer may be inefficient because it requires time for T cells to become responsive. REG It infiltrates and functions. On the other hand, only 2% (20,000) of TREG Direct intrastriatal transplantation of cells resulted in robust suppression of needle-induced inflammation and significantly increased survival of transplanted mDANs (Table 2), with faster and more significant behavioral improvements in rodent models of PD. 84 However, the rescue of mDAN is incomplete, indicating that T REG Further optimization of striatal co-transplantation is necessary. One potential approach could involve encapsulating the T-cell tissue with a hydrogel system prior to transplantation. REG and mDA cells ( Figure 21 ). 168-170 Furthermore, considering that needlestick trauma can also occur during CRT for other CNS and non-CNS diseases, it is of significant research importance to test T... REG Could co-transplantation similarly reduce needle-induced inflammation and, overall, enhance the survival of desired therapeutic cell products? 171

[0126] Strategies aimed at establishing long-term differentiation and maturation in the later stages of grafting.

[0127] Even if transplanted mDA cells manage to evade the initial immune attack after transplantation and survive early, they face a suboptimal environment different from early brain development, where all essential nutrients, oxygen, growth factors, related transcription factors, and developmental signals are provided or induced in a precise temporal and spatial manner. Instead, the new environment for transplanted mDA cells is characterized by aging PD pathologies, such as elevated neuroinflammation, limited / absent supply of essential factors, and / or synucleinopathy. 172,173 In fact, recent single-cell RNA sequencing analysis has revealed that transplanted cells often exhibit insufficient differentiation toward mDNA, but instead are more likely to differentiate toward alternative cell types such as astrocytes and vascular meningeal cells. 174 These inadequate and heterologous transplanted cell populations can be nonfunctional and can potentially induce undesirable immunogenicity. This challenge is exacerbated by the lengthy process required for these mDA cells to mature into mDANs with neuronal extraterrestrial growth, re-innervate into the host brain, and ultimately establish functional new networks. This process can take a considerable amount of time, ranging from 4–6 months in the rodent brain to 1–3 years in the human brain (Table 1). Therefore, it is crucial to develop and implement therapeutic strategies aimed at enhancing the survival, differentiation, and / or maturation of transplanted mDA cells. Supporting this idea, pretreatment of fVM cells with growth factors such as bFGF and GDNF increased the survival of transplanted mDANs by approximately 2–fold. 53,54 Long-term supply of bFGF led to a 10-fold increase in the number of surviving mDANs. 55Furthermore, 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, potential strategies involve promoting a sustained supply of relevant growth factors, which could not only improve survival but also differentiate new mDANs from grafts.

[0128] Table 2: List of preclinical studies using PSC-derived mDA cells

[0129]

[0130]

[0131]

[0132] Because selective loss of mDNA in the substantia nigra is a hallmark pathological feature of PD, transplantation resuscitation (CRT) has been a focus of extensive research over the past four decades. In particular, fVM-based CRT has not only provided proof of concept but also valuable lessons. Among these, the most important is the very limited survival of implanted mDNA, which may be a potential cause of variable and often inefficient clinical outcomes. Despite extensive research, the molecular and cellular mechanisms of acute and large-scale mDNA death after transplantation remain poorly understood.

[0133] Recent advances in stem cell technology have opened the door to hPSC-based clinical trials, where various hPSC sources (e.g., autologous hiPSCs, HLA-matched hiPSCs, and allogeneic hESCs) are being explored in ongoing or upcoming clinical trials for the scalable production of transplantable mDA cells. However, several key issues must be addressed to successfully realize hPSC-based CRT. First, learning from lessons learned with fVM-based CRT, improving the survival of implanted mDA cells is paramount. Second, addressing the various stages and steps of CRT where mDA cells are at risk of dying is essential. Third, understanding and manipulating both the host's adaptive and innate immune responses is crucial, with recent evidence highlighting the role of surgical manipulation in triggering the host's innate immune response. Future research should focus on developing effective strategies to manipulate the host's immune response to enhance the survival of implanted mDA cells and improve clinical outcomes.

[0134] It is important to note that although transplanted mDNA can survive post-transplantation, they face unfavorable PD host environments, including neuroinflammation and a limited supply of essential factors. These challenges make prolonged survival and maturation very difficult, requiring further research. Additionally, PD patients often exhibit degeneration of other neurons besides mDNA, such as noradrenergic and / or serotonergic neurons.178-181 This leads 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 part of a comprehensive treatment strategy, complementing other approaches such as novel drug therapies (e.g., anti-inflammatory and neuroprotective drugs) and gene therapy.

[0135] Compositions for cell therapy

[0136] As described above, the methods described herein include using compositions comprising any one or more of the following:

[0137] T REG Cell population;

[0138] mDA cell population;

[0139] Antibodies that bind to pro-inflammatory cytokines (e.g., anti-TNF-α Ab, anti-IL-1β Ab, anti-IFN-γ Ab) or bind to DAMP molecules (e.g., anti-HMGB1 Ab, anti-IL-1α Ab);

[0140] Includes T REG A composition of a cell population and a second cell (e.g., mDA cells) population, wherein the cells may be mixed together into a single composition or applied individually; and / or

[0141] Compositions comprising antibodies (e.g., antibodies that bind to 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), wherein the antibodies and cells may be mixed together as a single composition or administered separately to a subject in need.

[0142] T is used in this method REG 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 US 2023 / 0323299, US 2023 / 0047159, US 2020 / 0330515, and references 163-167.

[0143] The mDA cell populations used in this method are described herein and are known in the art. The mDA cell populations used in the methods described herein can be derived from hESC / hiPSC and subsequently differentiated in vitro over a specific period using an optimization procedure. Figure 19 ). 65,89-96In some embodiments, the mDA cell population comprises mDAP and mDAN. In some embodiments, the mDA cell population comprises 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. Useful mDA cell populations may include those 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). + Cells expressing one, two, or more mDAN markers (e.g., TH, DAT, and PITX3); and optionally, excluding cells expressing SOX1, PAX6, and / or KI67. See also US 2022 / 0243174; US 2018 / 0371422; US 2012 / 0128655; US 2013 / 0052268; US 2016 / 0002604; US 2014 / 0199274; and US 2009 / 0226401; and US 11,898,169; US 11,001,809; US 9,657,273; and US 9,750,768; and references 65 and 89-96.

[0144] In some embodiments, the methods described herein include administering 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 demonstrate that a significantly smaller number of Treg cells need to be administered together with mDA cells compared to systemic (e.g., IV) administration of Treg cells. Therefore, the ratio of Treg cells to mDA cells can be approximately 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.

[0145] The methods and compositions described herein can be used and comprise other cell types, including other neuronal types. Many neuronal differentiation protocols are known in the art; see, 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; Bell et al. Bio-protocol 9(5): e3188 (2019). DOI: 10.21769 / BioProtoc.3188; Bianchi et al., StemCell Research 32:126-134 (2018).

[0146] In some embodiments, compositions comprising any of the cells and antibodies described herein may contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, as well as isotonic agents and absorption delay agents, etc., that are compatible with drug administration. In some embodiments, the composition is formulated to be compatible with its intended route of administration.

[0147] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate; and agents for adjusting tension such as sodium chloride or dextran. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0148] Compositions suitable for injectable applications may include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid sufficient for easy injection. It should be stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol), and liquids. For example, appropriate flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be caused by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition. Useful solutions and compositions can contain any solutions, buffers, and stabilizers disclosed herein; for example, the composition and / or method may include encapsulating cells (e.g., T...) using a hydrogel system known in the art prior to administration. REG Cells and / or mDA cells) Figure 21 ). 168-170

[0149] Sterile injectable solutions can be prepared by incorporating the desired amount of an active compound with one or more of the ingredients listed above, as needed, into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile solvent containing an alkaline dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0150] In some embodiments, the composition is prepared with a carrier that will protect cells and / or antibodies from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, 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 (including liposomes that target selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. Nanoparticles (1 to 1,000 nm) and microparticles (1 to 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, for example, as described below: 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).

[0151] Any of the compositions described herein may be included in a container, kit, package, or dispenser along with instructions for use.

[0152] dose

[0153] "Effective amount" is an amount sufficient to achieve a beneficial or desired outcome. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a preventive effective amount, which is the amount necessary to prevent the onset of disease or disease symptoms. Effective amounts can be administered, applied, or dosed once or multiple times. The therapeutically effective amount (i.e., effective dose) of a therapeutic compound depends on the selected therapeutic compound. Compositions can be administered from once or multiple times daily to once or multiple times weekly; including every other day. Those skilled in the art will understand that certain factors can influence the dose and duration required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, prior treatment, the subject's overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic compound described herein may include a single treatment or a series of treatments.

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[0336] Example

[0337] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0338] Methods used in the examples and experiments

[0339] animal

[0340] Fischer 344 (8-10 weeks old, male) rats, Sprague Dawley (8-10 weeks old, male) rats, and NODSCID γ (NOD.Cg-Prkdc) rats were used. SCID Il2rg tm1Wjl / SzJ (8-10 weeks old, male) mice were purchased from Charles River or The Jackson Laboratory and housed under specific pathogen-free (SPF) conditions in individually ventilated miniature cages at the Mailman Research Center Animal Care Facility at McLean Hospital. All animals were kept on a 12-hour:12-hour light / dark cycle with free access to food and water. Animal studies were conducted in accordance with National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of McLean Hospital (2015N000001 and 2015N000002).

[0341] C4-mDAP differentiation from hiPSCs (C4-hiPSCs) of PD patients

[0342] We previously described the efficient generation of mDA progenitor cells (mDAPs) from the differentiation of human induced pluripotent stem cells (hiPSCs) using our novel "spotting"-based method. 21,22 In short, C4-hiPSCs were plated on 12 spots coated with a matrix gel (spots on a grid of 6 intersecting lines) and held in those plates from differentiation D1 to D14. To induce differentiation of cells into a substrate 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, to eliminate remaining hiPSCs, the cells were treated with 40 μM quercetin for 16 hours. At differentiation day 15, cells were dissociated into single cells and re-coated at approximately 2.5 million cells / plate onto poly-L-ornithine / fibronectin / lamin-coated culture dishes (days 12-15) 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. mDAP cells (day 15 cells) were cultured with the medium changed every 2 days and maintained without passage until day 28. More detailed descriptions of C4-hiPSC reprogramming and C4-mDAP differentiation have been previously provided. 16,21,22 B1-mDAP or H9-mDAP were generated from an independent hiPSC line derived from another sporadic PD patient (B1), and the H9-hESC line, respectively, using the same in vitro differentiation procedure. Mycoplasma contamination of all cells was tested every four weeks using the Venor GeM Mycoplasma Detection Kit (MP0025, Sigma-Aldrich). All experiments were performed using cells that showed a mycoplasma-negative result.

[0343] hiPSCs and hESCs differentiated in vitro into GABAergic neurons and progenitor cells.

[0344] We differentiated hiPSCs and hESCs into GABAergic progenitor cells, as Liu et al. 54As described above. In short, C4-hiPSCs, B1-hiPSCs, or H9-hESCs were trypsinized and grown from differentiation D0 to D14 in low-adhesion culture flasks as suspension spheres. To pattern the differentiated cells as 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. Starting 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 spheroids were trypsinized, plated on poly-L-ornithine / fibronectin-coated plates in B27GB medium, and harvested and transplanted on appropriate dates.

[0345] Surgical procedure

[0346] Animals were anesthetized with isoflurane using the SomnoSuite anesthesia system (Kent Scientific Corporation, Torrington, CT, USA), and stereotactic surgery was performed using a stereotactic instrument (David KOPF Instruments, Tujunga, CA, USA) equipped with a Micro4 microinfusion pump controller (World Precision Instruments, Sarasota, FL, USA). Severe unilateral lesions (>95% striatal DA loss) of the nigrostriatal pathway were induced by stereotactic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain tract (rat) or the pars compacta of the substantia nigra (mouse). Fifteen minutes prior to anesthesia, rats were injected with 10 mg / kg desipramine to protect noradrenergic projections. Two microliters of 6-OHDA (7.5 mg / ml in 0.2% ascorbic acid and 0.9% saline) were injected using a 2.5 μl Hamilton syringe (Hamilton Company, Reno, NV). Calculate coordinates based on the anterior fontanelle: anteroposterior (AP), -4.0; medial-lateral (ML), -1.3; and dorsoventral (DV), -7.0. For positions with or without a T... REGIn striatal implantation of C4-mDAP (or TP medium only; control) was performed by placing a 2 μl deposit at the following coordinates: AP, +0.8; ML, -3.0; and DV, -5.5. C4-mDAP (100,000) was implanted at a rate of 0.4 μl / min using a 10 μl Hamilton syringe equipped with a 26 G, 0.75-inch blunt needle. Following injection, the needle was left in the brain for 5 minutes, then slowly withdrawn over a 5-minute period using an Autoclip. ® Surgical sutures (Fine Science Tools, Foster City, CA) were used to suture the incised skin, and the animals were placed on warm pads for monitoring until recovery. Rats in the CsA injection group received daily injections of cyclosporine A (10 mg / kg, ip) starting one day before transplantation with C4-mDAP.

[0347] Fifteen minutes prior to anesthesia, mice were injected with 25 mg / kg desipramine to protect noradrenergic nerve 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 with reference to the anterior fontanelle: anteroposterior (AP), -2.9; medial-lateral (ML), -1.1; and dorsoventral (DV), -4.5. For mice with or without T… REG For striatal implantation of C4-mDAP, a 2 μl deposit was placed at the following coordinates: AP, +0.5; ML, -1.8; and DV, -3.5. C4-mDAP (100,000) was implanted at a rate of 0.4 μl / min using a 10 μl Hamilton syringe equipped with a 26G, 0.75-inch blunt needle. After injection, the needle was left in the brain for 5 minutes, and then slowly withdrawn over a 5-minute period using an Autoclip. ® Surgical sutures (Fine Science Tools, Foster City, CA) were used to suture the cut skin, and the animal was placed on a warm pad for monitoring until recovery.

[0348] All groups in each animal experiment used the same batch of C4-mDAP (Figs. 4a-4h; batch 1, Figs. 5a-5m; batch 2, Figs. 15a-15h; batch 3). In all animal experiments, the same experimental groups were transplanted on the same day. For example, in the Fischer 344 animal experiments (Figs. 4a-4h), the four groups analyzed at 2 weeks were transplanted on the same day (D0), and the same four groups analyzed at 20 weeks were transplanted on the second day (D+1).

[0349] Humanized mice

[0350] A PD mouse model was induced by injecting 6-OHDA into the substantia nigra of NSG mice (described above). PBMCs were isolated from the blood of patients (C4) and injected intraperitoneally into PD mice to generate C4-hu mice reconstituted with the immune system of patient C4. More detailed information has been previously described. 16 .

[0351] Rotation test

[0352] Intraperitoneal administration of D-amphetamine (an indirect presynaptic dopamine agonist) (4 mg / kg / rat, 5 mg / kg / mouse) induced rotational behavior in animals successfully induced by 6-OHDA. Subcutaneous administration of apomorphine (a direct-acting postsynaptic dopamine agonist) (1 mg / kg / mouse) induced rotational behavior. Rotational deviations were 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 positive, and only animals exhibiting more than 6 net ipsilateral rotations per minute were considered successfully induced.

[0353] cylinder test

[0354] To measure asymmetry in forelimb use, mice were placed in a glass cylinder (20 cm in diameter) for 10 minutes, and the number of times their paws touched the cylinder wall was recorded, with a minimum of 30 touches. Two researchers unaware of the experimental group analyzed the 10-minute video recordings of mouse behavior by counting the number of times the left paw, right paw, and both paws touched the cylinder wall simultaneously. The results were calculated as the percentage of the average number of touches using the right paw and the average number of total touches.

[0355] T REG Separation

[0356] Rat T cells were isolated from the blood of Fischer 344 rats. REG From the patient's blood (C4) or purchased from STEMCELL TM (K2) isolated human T from PBMC REG Blood samples were collected from the jugular vein into an anticoagulant collection tube and processed using SepMate. TM Rat PBMCs were separated by density gradient centrifugation. Following the manufacturer's instructions, rat PBMCs were stained with APC-conjugated anti-CD4 antibody (550057, BD) and PE-conjugated anti-CD25 antibody (554866, BD) and separated using a FACS sorting system. EasySep was used according to the manufacturer's instructions. TM Human CD4 + CD127 low CD25 +Human PBMCs were stained using a regulatory T cell isolation kit (18063, STEMCELL Technologies) and EasySep was used. TM Magnet separation. The separated T... REG and T 幼稚 Maintain in complete culture medium (RPMI 1640 containing 10% heat-inactivated FBS and 100 μg / ml penicillin / streptomycin). Homologous T REG Fischer 344 (donor, A) / Fischer 344 (recipient, A'); autologous T REG Fischer 344 (donor / recipient, same rat).

[0357] isolated T REG Amplification

[0358] isolated rat CD4 + CD25 + T cells were detected in a 96-well round-bottom plate (2x10⁻¹²). 4 Cells were cultured in wells and activated with plate-bound anti-rat CD3 / CD28 (5 μg / ml each) and rIL-2 (50 U / ml). Cells were maintained at 1 x 10⁻⁶ cells / well by changing or adding culture medium every 2–3 days. 6 Cells / ml concentration. Cells were reactivated on day 10 using the initial activation conditions and maintained with IL-2. Expanded cells were harvested at different time points.

[0359] T REG Inhibition assay

[0360] Rats' conventional CD4 + T cells (T cells) conv Using CellTrace TM CFSE (Life Technologies,Invitrogen TM The antibodies were labeled and stimulated with plate-bound anti-CD3 and soluble CD28 antibodies. Rat T1 cells were then... REG Add to CFSE-labeled T cells at a ratio of 1:1 or 1:2 conv Cells were stored in 96-well round-bottom plates in complete culture medium for 48 hours. T cells were analyzed by flow cytometry. conv CFSE dilution of cells.

[0361] Flow cytometry

[0362] To facilitate intracellular staining of Foxp3 (320012, Biolegend), CD4 was fixed and permeabilized using Foxp3 fixation / permeabilization buffer according to the manufacturer's instructions. +CD25 + T cells. C4-mDAP was dissociated with Accutase and filtered through a 70 μm cell filter to prepare single-cell suspensions, fixed with 4% formaldehyde, and stained with PE-conjugated anti-HLA-ABC (560168, BD) and FITC-conjugated anti-HLA-DR (555811, BD) or FITC-conjugated anti-CD80 (560926, BD), PE-conjugated anti-CD86 (560957, BD), PE-conjugated anti-CD40 (568581, BD), PE-conjugated anti-PD-L1 (568080, BD), PE-conjugated anti-PDL2 (558066, BD), and PE-conjugated anti-CD47 (568090, BD) antibodies. Fluorescent dye-matched isotype controls were used and subtracted during analysis.

[0363] TUNEL Measurement

[0364] 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 at room temperature in 20 μg / ml proteinase K for 5–15 minutes, washed twice, and then incubated for 1 hour in a dark, humidified 37°C incubator with a DNA-labeled solution containing TdT reactive enzyme and Br-dUTP. Labeled sections were washed with rinsing buffer, incubated with an antibody solution containing anti-BrdU-Red antibody, and counterstained with DAPI. A main field of view was selected around the area of ​​needle prick injury, and TUNEL assays were performed using 5 rats per group and 2–3 sections per rat. + Cells were counted. Cultured C4-mDAP cells were 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 using the same method. TUNEL was calculated in 3–5 randomly selected regions of the field of view. + Cell counts were performed, and the same experiment was independently repeated three times. Cell counts were conducted by two researchers who were unaware of the assignment of the experimental groups.

[0365] Apoptosis assay

[0366] The proportion of apoptotic cells was determined using the 7-aminoactinomycin D (7-AAD) and FITC-conjugated annexin-V assay kit (640922, Biolegend) according to the manufacturer's instructions. C4-mDAP was dissociated with Accutase and filtered through a 70 μm cell filter to prepare a single-cell suspension, washed twice with PBS, and incubated in the dark for 15–30 min in binding buffers containing annexin-V-FITC and 7-AAD, annexin-V-FITC and APC-hNCAM, or annexin-V-FITC and PE-TH. 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 strategy used in the cell death assay.

[0367] Mixed lymphocyte reaction (MLR)-like co-culture assay

[0368] PBMC (C4 or K2) and C4-mDAP were co-cultured in 96-well plates at a ratio of 1:1, 1:2, or 1:10 (C4-mDAP:PBMC) for 3–5 days. Dynabeads TM Human T-activator ( ) were used as positive controls. PBMCs were then harvested, stained with APC-hCD3 and PE-hCD69 antibodies, and analyzed by flow cytometry to determine proliferation profiles. C4-mDAP was harvested, stained with APC-hNCAM antibody and annexin-V-PE, and analyzed by flow cytometry to determine cell death profiles. Cells with or without C4-T were also analyzed. REG C4-mDAP and T REG The inhibitor test (130-092-909, Miltenyi Biotech) was co-cultured for 7 days under in vitro inflammatory conditions. C4-mDAP containing Treg cells (+Treg inhibitor test) or without Treg cells, along with anti-TGFβ1 or mouse IgG1 isotype control antibodies, were co-cultured for 7 days.

[0369] ELISA

[0370] After co-culturing PBMC and C4-mDAP for 3–5 days, aliquots of the culture supernatant were obtained to measure the secretion of IFN-γ by ELISA according to the manufacturer’s instructions (DIF50, R&D Systems).

[0371] Quantitative Real-Time PCR

[0372] Total RNA was isolated from C4-mDAP using the GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. iScript was used. TMcDNA synthesis was performed using a cDNA synthesis kit (Bio-Rad). The following primers were used:

[0373] Forward TH: 5'-CGGGCTTCTCGGACCAGGTGTA-3' (SEQ ID NO:1);

[0374] Reverse: 5'-CTCCTCGGCGGTGTACTCCACA-3'(SEQ ID NO:2);

[0375] Foxa2 forward: 5'-GGTGCTTTGGCTGACTTTTT-3' (SEQ ID NO:3);

[0376] Reverse: 5'-GTTGCTCACGGAGGAGTAGC-3' (SEQ ID NO:4);

[0377] Lmx1a forward: 5'-AGGGTGACGAGTTTGTCCTGA-3' (SEQ ID NO:5);

[0378] Reverse: 5'-CATCGCTTTTGCCTGAGTCTG-3' (SEQ ID NO:6); and

[0379] Actin forward: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO:7);

[0380] Reverse: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID NO:8).

[0381] For qRT-PCR, we used SsoAdvanced TM Universal SYBR Green Supermix, and in CFXConnect TM The reaction was performed on a 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).

[0382] Protein blot

[0383] Cells were lysed in RIPA lysis buffer (Sigma) (containing protease inhibitors), and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). An equal volume of 6X loading buffer was added to each sample, which was then boiled for 10 minutes and loaded onto a 4–12% Bis-Tris Plus gel. Proteins were electrophoresed and then transferred to a polyvinylidene fluoride membrane. The membrane was probed with antibodies against lysed 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) diluted 1:1000 in blocking solution (PBS containing 0.1% BSA), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Amersham). The bound antibodies were visualized using ECL (Amersham). See Figures 6a-6i for gel-source data.

[0384] Immunocytochemistry

[0385] 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 min. Cells were incubated at room temperature in blocking solution (0.3% Triton X-100 and 1% horse serum in PBS) for 1 hour, followed by overnight incubation at 4°C with anti-TH antibody in the same solution. Cells were then incubated at room temperature with Alexa Flour 488 or 568 conjugated secondary antibody and Hoechst 33342 or DAPI for nuclear staining for 1 hour. After obtaining cell images by fluorescence microscopy (KEYENCE, Osaka, Japan), data on specific cell populations were determined from the microscopic images using ImageJ software.

[0386] Immunohistochemistry

[0387] After the experiment, animals were induced into deep anesthesia by intraperitoneal injection of ketamine / toluidine. They were then perfused intracardiacly with ice-cold PBS at a flow rate of 10 ml / min for 10 minutes, followed by intracardiac perfusion 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-infused brains were embedded in an OCT compound, frozen at -83°C, and coronal sections (30 μm) covering the entire striatum were continuously collected (Leica CM1950, Buffalo Grove, IL). Free-floating coronal sections (including needle tracts or entire grafts) were incubated for 1 hour in blocking solution (0.3% Triton X-100 and 1% horse serum in PBS), followed by overnight incubation at 4°C with antibodies against MHCII (554926, BD), Iba1 (ab178846, Abcam), TH (P40101, Pel-Freez Biologicals), hNCAM (sc-106, Santa Cruz), and hNUCLEI (ab191181, Abcam). The stained brain tissue was washed and stained with peroxidase-conjugated secondary antibody for 1 hour. Finally, the sections were visualized using the DAB peroxidase substrate kit according to the manufacturer's instructions. Images in which needle tracts or grafts were clearly visible were selected.

[0388] Immunofluorescence

[0389] Free-floating coronary brain slices were processed as needed for antigen retrieval and pre-incubated at room temperature for 1 hour in blocking solution (0.3% Triton X-100, 5% normal donkey serum, and 3% BSA in PBS). Brain slices were treated with the same solutions containing anti-IFN-γ (ab216642, ab9657, Abcam) (506702, BioLegend), anti-hIba1 (ab178680, Abcam), anti-hCD4 (ab133616, Abcam), 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), and anti-HLA-ABC. Anti-HLA-DR (ab70328, 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 Scientific) antibodies were incubated overnight at 4°C, washed three times with PBS, and then incubated with Alexa Fluor 488-conjugated secondary antibodies, Alexa 568-conjugated secondary antibodies, or Alexa... The 647-conjugated secondary antibody was incubated with DAPI or Hoechst 33342 at room temperature for 1 hour. Sections stained with the secondary antibody separately were processed and photographed under the same conditions and used as negative controls.

[0390] Statistical analysis

[0391] Microsoft Excel (Microsoft Corp.) and GraphPad Prism v9 software were used for all statistical analyses, and the specific tests used are described in the legend. Unless otherwise stated, all experiments were performed in biological triplicate, and the exact number (n) of samples used can be found in the legend. Two researchers unaware of the experimental group assignments counted the number of stained cells or MFIs in all brain slices or the most visible slice of the needle tract in each mouse / rat. Student's t-test (unpaired two-tailed) was used when comparing two groups or within a group, all data having passed the Shapiro-Wilk normality test; for multiple group comparisons, two-way ANOVA followed by Bonferroni post-hoc test, or one-way ANOVA followed by Tukey's test, was used. Values ​​p < 0.05 were considered statistically significant, and an asterisk in the figure indicates significance of the p-value. p < 0.05; p < 0.01; p < 0.001.

[0392] Example 1: TH after cell transplantation + Rapid and preferential loss of mDA neurons

[0393] To mimic the early survival of human mDA neurons in their autologous environment, we transplanted (C4) iPSC-derived midbrain dopamine progenitor cells (mDAP) from PD patients into the striatum of mice humanized with the patients' autologous immune cells, as described above. 16 C4-iPSC uses the speckle-based program we built. 21,22 After 28 days of in vitro differentiation, it produced a product containing approximately 15% TH. + C4-mDAP of mDA neurons (Fig. 1a, 1d), which is relevant to the mDA differentiation protocol derived from iPSCs. 21-24 This is typical. We transplanted a total of 100,000 C4-mDAP cells into the striatum of immune humanized mice (C4-hu; reconstructed from patient PBMCs) and immunodeficient NODSCID γ (NSG) mice. Animals were sacrificed two weeks post-transplantation, and viable cells and TH were detected using antibodies against the human nucleus (hNUCLEI) and TH, respectively. + The total number of mDA neurons was determined by immunohistochemistry to examine graft survival (Figure 1b). We detected 85,901 ± 7,460 and 74,455 ± 3,842 hNUCLEI neurons in C4-hu and NSG mice, respectively. +Cells (Fig. 1c). In stark contrast, only 627 ± 279 and 942 ± 171 TH cells survived in C4-hu and NSG mice, respectively. + Cells, representing <1% of transplanted cells (or <10% of transplanted mDA neurons). These data indicate that even in relatively immunodeficient mice with or without human immune system remodeling, mDA neurons within the graft die disproportionately and early (i.e., within 2 weeks post-transplantation), while the remaining transplanted cells largely survive. To investigate whether the vulnerability of mDA neurons is specific to this patient (C4), we transplanted additional in vitro differentiated cells from independent hiPSC lines (B1; derived from a single sporadic PD patient) and H9 hESC lines and found that this phenomenon was a universal feature (Figs. 6a-6e). Notably, C4-hu mice showed a higher number of human cells (hNUCLEI) in the graft compared to NSG mice (Fig. 1c). + This could be due to the infiltration of immune cells from the humanized immune system as a secondary response to the initial neuroinflammation (as observed in traumatic brain injury (TBI)). Supporting this, immunohistochemical analysis revealed a large number of immune cells from the humanized host immune system, such as human Iba-1. + (hIba-1 + Macrophages / microglia), hCD45 + (white blood cells) and hCD4 + (T lymphocytes) accumulated near the graft in C4-hu mice, but not in NSG mice (Fig. 1e-1g). To investigate whether initial cell death is specific to mDA neurons, we tested another neuronal lineage derived from two hiPSC (C4 and B1) and one hESC (H9) lineage of vesicular GABA transporter (VGAT). + We found that these neurons exhibited similar early cell death after transplantation (Figs. 6f-6i). Therefore, while more neuronal lineages need to be tested to generalize this conclusion to a wider range of scenarios, our data suggest that initial cell death after transplantation may be common for post-mitotic neurons.

[0394] Example 2: Autologous T cells in immunocompetent rats REG The role of acupuncture trauma and co-transplantation

[0395] While the data above reveal important insights into early post-transplant events, these immunodeficient mice may differ significantly from actual clinical settings, thus limiting the validity of immune response analyses. Therefore, we used Fischer 344 rats to investigate changes in the brain induced by surgical manipulation. When we injected only culture medium (without any cells) into the striatum of Fischer 344 rats, we observed acute release of pro-inflammatory cytokines and immediate activation of astrocytes / microglia at the injection site (Figs. 7a–7d), indicating a host brain response to penetrating brain injury (referred to here as “needle puncture trauma”) using a closed head injury model. 25,26 This replicates the well-known effects of other forms of TBI. We then analyzed host responses at different time points (4 days to 6 months post-injection) and found that Iba-1 + and MHCII + Inflammatory cells showed robust infiltration near the needle tract, peaking in number on day 7, then declining at 1 month and becoming undetectable at 6 months (Figs. 8a, 8b). Notably, interferon-γ (IFN-γ) was also observed. + Cellular activity peaked on day 4 post-injection (the earliest time point analyzed), indicating that needle trauma triggered immediate neuroinflammation in the host brain, which gradually decreased over time (Fig. 8c). Needle trauma-induced death of host brain cells near the injection site, as examined by TUNEL staining, peaked on day 7 post-injection (Fig. 8d). Given that needle trauma triggers extensive neuroinflammation-induced death of host brain cells, we hypothesize that implanted cells, particularly mDA neurons, may also undergo neuroinflammation-induced death shortly after transplantation, consistent with our data (Figs. 1b-1d; Fig. 8d) and previous studies using VM cell transplantation. 7-10 .

[0396] In TBI, it is generally accepted that after the initial inflammatory cell infiltration, T... REG Ultimately, it penetrates the damaged area as part of the repair process and returns to a steady state. 25,26 Therefore, we assume that the self-T REG Co-transplantation could be an effective method to reduce acute neuroinflammation caused by acupuncture trauma. To investigate this, we isolated autologous CD4 from each individual Fischer 344 rat using peripheral blood obtained via the jugular vein via FACS sorting (Fig. 9a). + CD25 + T REG (Foxp3 + (85.5 ± 3.12%), 20,000 cells were injected into the striatum of the same rat, and each brain was analyzed on day 2 or 7. Compared with the control group (Fig. 1h-1k, Fig. 7e-7g), TREG 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, which was associated with robust protection against host neuronal death, demonstrating their significant protective effect. We then compared autologous T cells with... REG With the same T REG The protective effect was observed in rat autologous and syngeneic T cells. REG Suppressing MHCII with similar efficiency + Infiltration of inflammatory cells (Fig. 1l). Autologous T cells REG Inhibition of MHCII in a dose-response manner + Inflammatory cell infiltration was observed, reaching its maximum effect when 20,000 cells were transplanted (Fig. 1m). Initially, injected T cells were detected near the needle tract. REG It gradually disappeared within a week, but effectively inhibited MHCII. + Infiltration of inflammatory cells (Figs. 10a-10b).

[0397] We also administered approximately 1,000,000 ex vivo amplified autologous CD4 cells via intravenous injection. + CD25 + T REG (Foxp3 + (68.8 ± 4.07%) to test the use of adoption transfer 27,28 Regular T REG The role of the therapy (Figure 9b). Although adoptive transfer will MHCII + Cell infiltration was reduced by approximately 50%, but its efficiency was lower than 20,000 T. REG Direct striatal transplantation (Fig. 1n). It is noteworthy that natural T... REG (nT REG ) and amplified T REG There were no functional differences between them (Figure 9c). Finally, we tested whether administration of cyclosporine A (CsA) (a typical immunosuppressant) could suppress the inflammatory effects of needle prick trauma. When administered daily until animal sacrifice, CsA treatment significantly suppressed the host inflammatory response (e.g., hIba-1). + MHCII + IFN-γ + and TUNEL + (cells), but not as good as a single T cell. REG The transplant was effective (Figures 11a-11e).

[0398] Example 3: Analysis of in vitro immunogenicity

[0399] To investigate the role of neuroinflammation in more detail, we treated C4-mDAP with pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ). 16,21 First, we examined the expression of molecules associated with immunogenicity. 29-31 Furthermore, treatment with IFN-γ (Fig. 2a) or TNF-α instead of IL-1β (Fig. 12a) for 2 days robustly induced HLA class I and / or II molecules. Conversely, the expression of T cell co-stimulatory molecules (CD80, CD86, and CD40) and immune checkpoint molecules (PD-L1, PD-L2, and CD47) was not altered by treatment with these cytokines (Fig. 2b, Fig. 12a). Additionally, the immunogenic factors Zg16 and Hormad1 were not detected or induced. 32,33 The expression of these molecules (Fig. 2c, Fig. 12b) indicates that pro-inflammatory cytokines do not significantly alter the expression of immunogenicity-related molecules in C4-mDAP other than HLA molecules. We then analyzed the in vitro immune response using a mixed lymphocyte reaction (MLR)-like co-culture assay with autologous or allogeneic PBMCs. When C4-mDAP was co-cultured with allogeneic PBMCs (K2-PBMCs), T cell activity was significantly induced and further enhanced by IFN-γ treatment (Fig. 2d). Furthermore, significant apoptotic cell death of C4-mDAP occurred when co-cultured with K2-PBMCs (Fig. 2e). In stark contrast, no T cell activity was induced when co-cultured with autologous PBMCs (C4-PBMCs) 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 not autologous, PBMCs triggered IFN-γ secretion (Fig. 2f). In summary, our data indicate that allogeneic, rather than autologous, PBMCs trigger T cell activation when co-incubated with C4-mDAP, consistent with our previous findings using humanized mice. 16 The findings regarding rejection / survival patterns are consistent with those found in allogeneic and autologous transplantation studies. Therefore, we conclude that early death of mDA neurons is not caused by the immunogenicity of the transplanted autologous mDAP.

[0400] Example 4: Effects of pro-inflammatory cytokines on mDAP in vitro

[0401] We then examined inflammation-induced cell death in vitro by treating C4-mDAP with pro-inflammatory cytokines for 2 or 7 days. After 2 days of IFN-γ treatment, C4-mDAP exhibited morphological changes with reduced TH expression, as examined by mean fluorescence intensity (MFI) and shortened neurite length, but cell death was unaffected (Fig. 12c-12g). However, after 7 days of IFN-γ treatment, cleaved caspase-3 was detected, accompanied by approximately 12% additional apoptotic cell death, as examined by annexin-V / 7AAD staining (Fig. 3a, 3b). TUNEL assays also revealed approximately 15% cell death in C4-mDAP (Fig. 3c). Treatment with TNF-α showed a similar effect, while IL-1β showed a weaker effect on C4-mDAP (Fig. 12h-12i). Furthermore, cells and TH... + Total number of cells, TH expression level, and TH + Neuronal neurite length was significantly reduced (Figs. 3d-3h). Conversely, FoxA2 expression remained unchanged (Figs. 12j to 12m). Notably, consistent with our in vivo data (Figs. 1b, 1c), treatment with pro-inflammatory cytokines for 7 days induced approximately 15% loss of total cell number, but significantly greater loss of TH. + Cell loss (>80%) indicates TH + mDA neurons compared to TH - Cells are more susceptible to cell death induced by pro-inflammatory cytokines. Supporting this is the effect of IFN-γ treatment on C4-mDAP and flow cytometry via gated TH cells. + and TH - When analyzing these cells, most TH + Cells enter apoptosis and die, while TH... - Cells were unaffected (Fig. 3i). We also tested with autologous T cells. REG Can co-incubation with TH rescue IFN-γ-triggered apoptotic cell death? As shown in Figures 3j and 3k, TH + Cells in autologous T REG In their presence, they were almost completely rescued from cell death. In summary, our data indicate that pro-inflammatory cytokines acutely induced by acupuncture trauma in vivo can induce TH in vitro. + The preferential loss of mDA neurons can be detected by T... REG Co-incubation was attempted to rescue TH. However, striatal co-transplantation with neutralizing IFN-γ antibodies failed to rescue TH. + Cells (Fig. 12n) show that a single pro-inflammatory cytokine cannot reproduce the complexity of neuroinflammation in the complete brain.

[0402] Interestingly, we also found that it was related to self-T REGCo-incubation significantly reduced Ki67 + The percentage of cells, although it did not change TH + Percentage of cells (Figure 3l). Based on previous studies showing the role of TGF-β1 signaling in controlling neuronal proliferation and the size of specific brain areas. 34,35 We speculate that it is caused by T REG The secretion of TGF-β1 may be the reason for our observation. Supporting this possibility is that we found that treatment with anti-TGF-β1 antibody significantly restored Ki67. + Percentage of cells (Fig. 12o, 12p).

[0403] We also investigated the inhibition of host inflammatory responses induced by needle prick trauma using neutralizing monoclonal antibodies. Following needle prick trauma in NSG dKO mice, the degree of inflammation was examined by the extent of MHC-II-positive cell infiltration as described herein. Specifically, Fischer 344 rats were sacrificed 7 days after striatal co-transplantation in TP medium with or without monoclonal antibodies (anti-TNF-α, anti-IL-1β, anti-IFN-γ, anti-HMGB1, anti-IL1α) (2 mg / rat) from each rat. The level of inflammatory cell infiltration into the needle tract was determined by immunohistochemical staining with anti-MHC-II antibody. (One-way ANOVA, Tukey post-hoc test; n=4 per group). Each error bar represents mean ± sem. P<0.001 Figure 22 All tested antibodies (anti-TNF-α, anti-IL-1β, anti-IFN-γ, anti-HMGB1, and anti-IL-1α) significantly inhibited inflammation. Figure 22 Since our data indicate that suppression of inflammation is associated with protection of implanted dopamine neurons, these data strongly suggest that treatment with these antibodies will lead to better survival of transplanted mDANs.

[0404] Example 5: Autologous T REG The therapeutic effects of co-transplantation in xenogeneic PD models

[0405] Based on the above data, we tested the host-autologous T... REG Whether striatal co-transplantation will protect transplanted mDA neurons from needle-induced cell death during cell therapy (both allogeneic and autologous). Since C4-mDAP is xenogeneic in rats, we first tested and confirmed this by mixing it with rat T cells at a 1:1 or 5:1 ratio. REG Co-incubation for 72 hours did not alter their gene expression (Figs. 9d-9f). Next, we will use C4-mDAP and autologous rat T... REGC4-mDAP was co-transplanted into the striatum of each 6-OHA-damaged Fischer 344 rat with or without CsA treatment, and amphetamine-induced rotational behavior was monitored monthly post-transplantation (Fig. 4a). As expected, transplantation of C4-mDAP without CsA failed to improve rotational behavior (Fig. 4b). Interestingly, C4-mDAP without CsA and autologous T... REG Co-transplantation of C4-mDAP did not improve rotational behavior. When rats were administered CsA daily, C4-mDAP transplantation significantly reduced rotation 20 weeks post-transplantation, and this effect was mediated by autologous T... REG The co-transplantation further enhances it.

[0406] To test T REG For the early effects of the graft, we performed histological analysis 2 weeks post-grafting. When C4-mDAP was grafted alone, MHCII and TUNEL staining detected extensive infiltration of inflammatory cells and cell death, respectively, which was confirmed by CsA treatment and / or T… REG Co-transplantation resulted in significant salvage (Figs. 4c, 4d). This was particularly effective for myeloid cells (CD11b). + / CD11c + NK cells (NKp46) + ), B cells (CD19) + ), and T cells (CD4) + / CD8 + Similar patterns were observed in other inflammatory cells (Figs. 13a-13d). Furthermore, treatment with CsA and / or T... REG Co-transplantation significantly reduced the expression of immunogenicity-related molecules (HLA I and II) (Figure 13e). hNUCLEI + Immunohistochemical analysis of cells showed CsA and / or T REG Co-transplantation resulted in the survival of most transplanted cells, while only C4-mDAP(-CsA, -T) cells survived. REG The graft composed of C4-mDAP was rejected (Figure 4e). Therefore, when only C4-mDAP was transplanted, TH was almost completely eliminated. + Cells (Fig. 4f). Interestingly, despite daily injections of CsA and T... REG Only one injection is needed, but each treatment is administered at this 2-week timeframe to prevent graft rejection and reserve TH. + Cells, of which T REG The observed efficiency was higher than that of the CsA treatment (Figures 4e and 4f).

[0407] Next, we analyzed the grafts (C4-mDAP into Fischer 344 rats) 20 weeks post-transplantation (following the behavioral studies, shown in Figure 4a). As expected from the 2-week graft analysis, when only C4-mDAP (-CsA, -T) was transplanted... REG When co-transplanted T, all grafts were rejected (Fig. 4g). It is noteworthy that even when co-transplanted T... REG At that time, all grafts were rejected, and the difference from the results at 2 weeks indicated that individual T REG It can only provide short-term graft protection. However, in this xenograft model, the graft survived with daily CsA application, and approximately 2,500 TH were detected. + Cells (Fig. 4h). Surprisingly, we found that in cells with T... REG Among the co-transplanted grafts, graft size and hNUCLEI + The total number of cells was significantly smaller, accompanied by a decrease compared to the absence of T cells. REG Ki67 with fewer co-transplanted grafts + Cells (Fig. 4g, Fig. 14a-14d). Ki67 + A strong correlation exists between cell number and graft volume (Fig. 14e). However, when we specifically examined TH... + When cells are present, compared to those without T cells REG Compared to co-transplanted grafts, when co-transplanted T REG At that time, TH + The total number of cells increased almost twofold (Fig. 4h), while FoxA2 + Cell numbers were similar or slightly reduced (Fig. 14f). This is consistent with recent studies. 36,37 The overall consistency is that there is a T and there is no T. REG The co-transplanted grafts all contained neurons, glial cells (astrocytes, oligodendrocyte lineage cells, and microglia), and vascular leptomeningeal cells (VLMCs). Figure 14g -14k). Besides Ki67 + Aside from a significant decrease in cell number, our analysis did not detect any abnormalities in T cells. REG Significant changes in cell type composition were detected during co-transplantation.

[0408] Example 6: Autologous T in Autologous PD Model REG The therapeutic effects of co-transplantation

[0409] Next, we will use the same patients' mDAP and T... REG (C4-T REG Figure 9g) was used to examine T in co-transplanted humanized mice from patients. REGThe therapeutic effect in the autologous environment. To this end, we used C4-hu mice by stereotactically injecting 6-OHDA into the substantia nigra and incorporating C4-mDAP and C4-T... REG Co-transplantation into the striatum was used to generate a PD model (Fig. 15a). Unfortunately, none of these mice survived more than two months after transplantation (Fig. 15b). This early death in C4-hu mice is likely due to graft-versus-host disease (GVHD), as previous studies have shown that humanized mice typically die from GVHD 30–90 days after human PBMCs are injected into NSG mice. 38,39 Although we were unable to perform behavioral tests and long-term graft analysis, we investigated C4-T 2 months post-transplantation. REG The role of co-transplantation. Stereoscopic analysis of the grafts showed that, compared with the C4-mDAP-only group, hNUCLEI... + The total number of cells and the graft volume at T REG Smaller in the co-transplantation group (Figs. 15c, 15d), which is consistent with Ki67 + Cell number correlation (Figs. 15e, 15f). Conversely, TH... + Cell count in T REG Higher levels were observed in the co-transplantation group (Figure 15g). T was no longer detected at this point. REG (Figure 15h). Therefore, although we cannot test autologous T... REG The long-term benefits of co-transplantation are evident, but these data from humanized mice are consistent with those observed using Fischer 344 rats and support the view that co-transplantation of autologous T cells... REG It protects mDA neurons from the acute host inflammatory response to acupuncture trauma and allows for improved survival.

[0410] We then used unmodified NSG mice to study C4-mDAP and C4-T. REG The therapeutic effect of co-transplantation. First, we generated NSG mice with unilateral 6-OHDA lesions that consistently exhibited >6 amphetamine-induced ipsilateral rotations per minute throughout the test period (24 weeks). Since NSG mice are relatively immunodeficient, no graft rejection occurred despite the absence of immunosuppression. When we transplanted 100,000 C4-mDAPs into the striatum of these mice, rotational behavior was significantly reduced (<6 rotations per minute) starting at 20 weeks post-transplantation (Fig. 5a). When co-transplanted C4-T… REG At that time, the reduction in rotation began earlier (16 weeks post-transplantation) and showed greater improvement in both amphetamine-induced and apomorphine-induced rotation tests compared to the C4-mDAP-only group (Figs. 5b, 5c). Furthermore, all transplanted mice showed significantly improved forelimb function in the cylinder test at 20 weeks post-transplantation (Fig. 5d).

[0411] We compared the histological and immunohistochemical characteristics of the grafts between the C4-mDAP-only group and the co-grafting group. At 2 weeks, hNUCLEI + The total number of cells in the absence or presence of C4-T REG In the co-transplantation case, the number of cells was approximately 100,000, which increased to approximately 673,000 and 401,000 cells, respectively, at 20 weeks post-transplantation (Fig. 5e). Consistent with these data, the graft volumes at 20 weeks post-transplantation were 8.53 ± 0.73 and 5.11 ± 0.57 mm in the C4-mDAP-only group and the co-transplantation group, respectively. 3 (Figure 5f), which is related to Ki67 + Percentage of cells (0.79±0.06%; +T) REG : 0.27±0.04%) related (Figure 5g, 5h). TH + The total number of cells was 1,825 ± 287 at 2 weeks post-transplantation. REG : 4,427±189) and 20 weeks (5,421±534; +T REG The values ​​of 9,523 ± 835 were significantly higher in the co-transplantation group, despite a smaller total graft volume (Fig. 5i). In TH... + In neurons, GIRK2 + ALDH1A1 + A9-like and calcium-binding proteins + The percentage of A10-like DA subtypes was not altered by co-transplantation (Fig. 5j, 5k). The ratio of neurons to total cells (NeuN) + / hNUCLEI + Cells were significantly higher in co-transplanted mouse grafts (27.39 ± 1.99%, respectively); +T REG 49.93±1.96% (Fig. 5l). Furthermore, we found that co-transplanted grafts contained a higher density of graft-derived human synaptophysin (hSYP) in the dorsolateral striatum (DLSTR). + Synapse (5.54±0.65%; +T) REG : 13.64±1.30%; Figures 16a, 16b). At T REG At the graft-host boundary of the co-transplanted brain, in the host dendritic spine (DARPP32) + Higher levels of TH were identified on neurons. + hSYP + At the neuronal terminals, host dendritic spines are their preferred targets, consistent with the synaptic connections of host striatal neurons (Fig. 5m). When we use TH... +When assessing the integration of transplanted C4-mDAP cells into the host brain, the striatum contains TH... + Fiber density in the co-transplantation group was DL STR (13.50±0.75%; +T). REG :24.38±1.24) and VL STR(15.96±0.94;+T REG The values ​​were all higher in the range of 55.50±1.07% (Figures 16c and 16d).

[0412] Example 7: Summary of Experimental Data

[0413] In the 1980s and 1990s, many researchers investigated the survival of transplanted cells as a key requirement for successful PD cell therapy, primarily using human and rodent embryonic VM cells. 7-11 These early studies revealed that the vast majority (80-99%) of transplanted mDA neurons undergo apoptotic cell death within 1-2 weeks post-transplantation. Notably, these studies also revealed that the number of surviving mDA neurons subsequently remained unchanged at later time points (e.g., 4 days, 2 weeks, and 6 weeks post-transplantation), further supporting the view that most transplanted mDA neurons die early and no new mDA neurons are generated from the transplanted VM cells. Many potential mechanisms have been proposed to explain the poor survival and early death of transplanted mDA neurons, primarily focusing on interactions between transplanted cells and the host brain, such as a lack of adequate oxygen, glucose, or growth factor supply 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 factors, including calcium channel antagonists, lazaro-like drugs, caspase inhibitors, and trophic factors. 40-44 In contrast to the extensive early studies of these fetal-derived tissues, the survival of mDA cells in grafts derived from hiPSCs / hESCs has not been systematically investigated. While we have previously demonstrated that using autologous iPSC sources resolves cell-mediated rejection without immunosuppression. 16 However, the immediate activation of the innate immune system, as part of the inflammatory response to surgical injury, cannot be overcome by this measure.

[0414] Here, we demonstrate that surgical implantation and penetrating trauma induce a host inflammatory response, including a robust innate immune response similar to that described in other models of TBI. 25,26,45,46 Furthermore, this immediate inflammatory response is essential for the therapeutic TH treatment of the graft. + The cellular components are particularly harmful. Consistent with early survival studies using embryonic VM cells, we found that less than 10% of transplanted TH cells... + Neurons survive in the early stages of transplantation, while TH- Most cells survived, as confirmed in independent experiments using two hiPSC lines (C4 and B1) and one hESC line (H9). In vitro studies using pro-inflammatory cytokines (e.g., IFN-γ, TNF-α, and IL-1β) demonstrated that this cytokine challenge preferentially triggers TH. + Neuronal apoptosis is a form of cell death that occurs through interaction with autologous T cells. REG Co-incubation resulted in significant rescue. However, unlike VM cell transplantation, TH... + The number of both hiPSCs and other cells increased significantly at later time points. We hypothesize that mDAPs derived from hiPSCs / hESCs have greater differentiation and proliferation potential than embryonic VM cells, and that TH cells from the graft increase significantly after transplantation. - New TH cells are generated in mDAP cells + Neurons. Supporting this concept is our previous research, which has shown that neural precursors derived from mouse ES cells exhibit significantly greater expansion and differentiation potential than those derived from mouse embryonic VM cells. 47,48 .

[0415] Our data also reveal that, in relation to the host's own T... REG Intrastral co-transplantation significantly improved the inflammatory response to acupuncture trauma and promoted TH. + The survival of mDA neurons was improved, and the death of host brain cells was also reduced. Since the inflammation and immune cell activation induced by acupuncture trauma are nonspecific, but related to TH in the graft... - Cellular comparison with TH + Neurons are preferentially detrimental, therefore regardless of whether the graft cells are autologous, allogeneic, or xenogeneic, they are all detrimental. Our data indicate that, compared to the host's own T cells... REG This effectively mitigates the needle-induced trauma process in both autologous and xenograft grafts, leading to better improvement in motor deficits. Therefore, we hypothesize that the patient's T... REG Co-transplantation can improve clinical outcomes in autologous and allogeneic cell transplantation protocols, as recently demonstrated in a rodent model of TBI. 49 However, due to autologous T REG Co-transplantation only allows for short-term, not long-term, survival of xenografts in Fischer 344 rats; long-term graft survival in allogeneic cell transplantation may still require systemic immunosuppression.

[0416] Although further research is needed, we speculate that T REG It exerts its neuroprotective effect through several direct and / or indirect pathways, including T. REGSecreted immunosuppressive cytokines (e.g., TGF-β1) directly affect mDAP. Supporting this is the fact that previous studies have shown that mature mDA neurons express TGF-β receptors. 50 Furthermore, TGF-β1 protects mDA neurons from neurotoxicity driven by pro-inflammatory cytokines (e.g., IFN-γ). 51 We confirmed that C4-mDAP also expresses the TGF-β receptor (Figure 17a). Secondly, T... REG Neuroinflammation can be indirectly modulated by inhibiting the expression of pro-inflammatory cytokines in response to acupuncture trauma in resident brain cells (e.g., astrocytes and microglia), as shown in our data (Fig. 1j, Fig. 7e, 7f). Third, these effects can help prevent the infiltration of inflammatory cells from the periphery (Fig. 1h-1n, Fig. 10b). Furthermore, recent studies have shown that cell-cell interactions (e.g., CD47-SIRPα and CD45-galactosyllectrin 1) can directly protect mDA neurons from MPTP neurotoxicity. 52,53 We found that C4-mDAP expresses both SIRPα and galactolectin-1 (Figures 17b and 17c), indicating that T cells expressing CD47 and CD45... REG These cell-to-cell contact mechanisms can directly protect mDA cells.

[0417] An unexpected finding in this study was that T REG Co-transplantation significantly reduced the risk of complications associated with TH. - Cell-associated graft expansion resulted in significantly smaller graft volume and a higher proportion of TH. + Cell content, and improved extracellular growth from the graft. This led to T REG How co-transplantation affects cell fate determination of transplanted progenitor cells in the graft. To determine T... REG To determine whether the effect on graft proliferation might be due to dilution, we compared C4-T... REG and C4 naive T cells (C4-T) 幼稚 The co-portage of C4-T cells, which are very similar in size but different in function. REG Or C4-T 幼稚 When co-transplanted with C4-mDAP, only when C4-T REG During co-transplantation, the cell number and size of the grafts were smaller (Figs. 17d, 17e), strongly suggesting that this was not due to dilution. Because C4-mDAP expresses the TGF-β receptor (Fig. 17a), T... REG Transplanted cell proliferation can be controlled by secreting TGF-β, which is known to control nerve cell proliferation. 34,35 Supporting this is the fact that treatment with anti-TGF-β1 antibody significantly restored Ki67.+ Percentage of cells (Fig. 12o, 12p). Due to these T cells... REG Their effects are independent of the origin of the mDA cells themselves (autologous or allogeneic), therefore they are also applicable to allogeneic transplantation protocols. Compared to other immunosuppressive strategies involving systemic administration, T... REG Co-transplantation is a site-specific strategy that is unlikely to cause harmful systemic side effects. Furthermore, we demonstrate that in the acute phase (2-week results), a single administration of T... REG It has benefits such as daily CsA, and T REG The cells themselves are not present in long-term grafts, suggesting that the benefits are related to the acute-phase regulation of the innate immune response, and that subsequent administration of these cells is not required to maintain the effects. Therefore, this strategy reduces the time and extent of risk of adverse effects.

[0418] In summary, our study demonstrates that the survival of initial specific mDA neurons in grafts is strongly influenced by the host's innate immune response, while long-term graft survival is affected by the adaptive immune response, revealing a two-stage involvement of the immune system in graft survival. mDAP and T REG Co-transplantation modulated the host immune response triggered by acupuncture trauma and significantly protected mDAN while inhibiting TH. - Cell proliferation will benefit the efficacy and safety of cell replacement therapy for PD and other neurodegenerative diseases.

[0419] References (all parts except for the detailed embodiments)

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[0474] Other embodiments

[0475] While the invention has been disclosed in certain embodiments, those skilled in the art will understand that certain substitutions, modifications, and / or omissions may be made to the embodiments without departing from the spirit of the invention. Therefore, the foregoing description is intended to be exemplary only and should not limit the scope of the invention. The substance of all disclosures in all references, scientific articles, patent publications, and any other documents cited herein is incorporated herein by reference.

[0476] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method for treating a subject with Parkinson's disease (PD), the method comprising administering a regulatory T (T) agent... REG The cell population and the midbrain dopamine (mDA) cell population were administered to the brain of the subject.

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

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

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

5. The method of any one of the preceding claims, wherein the T REG cells and the mDA cells are autologous T REG cells and autologous mDA cells.

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

7. The method of any one of claims 1 to 5, wherein the T REG cells and the mDA cells are allogeneic T REG cells and allogeneic mDA cells, optionally wherein the autologous mDA are 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. The method of any one of claims 1 to 5, wherein the T REG cells and the mDA cells are xenogeneic T REG cells and xenogeneic mDA cells.

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

10. A method of treating a subject having Parkinson’s disease (PD), the method comprising administering to the subject a population of midbrain dopamine (mDA) cells and an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or damage-associated molecular pattern (DAMP) molecule; optionally an anti-TNF-a Ab (optionally infliximab), an anti-IL-1b Ab (optionally canakinumab), an anti-IFN-g Ab (optionally ipafrumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab).

11. A method of reducing an immune response (e.g., an immune response triggered by a needle stick wound, an immune response at a site of administration) in a subject during a cell transplant procedure, the method comprising administering to the subject an effective amount of T REG a population of cells with a second population of cells.

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

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

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

15. The method of any one of claims 11 to 14, wherein the T REG cells are autologous T REG cells, allogeneic T REG cells, or xenogeneic T REG cells.

16. The method of any one of claims 12-15, wherein the mDA cells are autologous mDA cells, allogeneic mDA cells, or xenogeneic mDA cells.

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

18. The method of any one of claims 12-17, wherein the mDA cells are transplanted intrastriatally.

19. A method of reducing the immune response of a subject triggered by needle trauma during a cell transplantation procedure, comprising administering to the subject an effective amount of an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or damage-associated molecular pattern (DAMP) molecule; optionally an anti-TNF-a Ab (optionally infliximab), an anti-IL-1b Ab (optionally canakinumab), an anti-IFN-g Ab (optionally ipafrumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab) with a population of cells (optionally mDA cells).

20. A method of increasing cell survival in a cell transplantation or increasing the outgrowth of transplanted cells or reducing overgrowth during a cell transplantation, the method comprising co-transplanting an effective amount of T REG a population of cells with a population of second cells ("transplanted cells"; optionally mDA cells).

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

22. The method of claim 20 or claim 21, wherein the co-transplantation results in a graft, and wherein TH + cells in the graft are increased compared to a graft without co-transplantation. wherein the co-transplantation results in a graft, and wherein TH + Cells: TH - Increased proportion of cells; and / or + Cells: Ki67 + Increased proportion of cells; and / or wherein co-transplantation results in a graft, and wherein there is a reduction in Ki67 + TH in the graft compared to a graft without co-transplantation - TH in the graft.

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

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

25. The method of any one of claims 20-24, wherein the T REG cells are autologous T REG cells, allogeneic T REG cells, or xenogeneic T REG cells.

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

27. The method of any one of claims 20-26, wherein the T REG Cells are transplanted into the striatum.

28. The method of any one of claims 20-27, wherein the transplanted cells (optionally mDA cells) are transplanted intrastriatally.

29. A method of increasing cell survival in cell transplantation or increasing the outgrowth of transplanted cells or reducing overgrowth during cell transplantation, the method comprising administering to the subject an effective amount of an antibody (Ab) (optionally, an antibody that binds a proinflammatory cytokine or a damage-associated molecular pattern (DAMP) molecule; optionally, an anti-TNF-a Ab (optionally infliximab), an anti-IL-1b Ab (optionally canakinumab), an anti-IFN-g Ab (optionally ipilimumab), an anti-HMGB1 Ab, or an anti-IL-1a Ab) to a population of cells (optionally mDA cells).

Images