Human Pluripotent Stem Cell-Based Therapies for Parkinson’s Disease: Challenges and Potential Solutions

Abstract

Over 20 years of research on human pluripotent stem cell (hPSC)-based therapies for Parkinson’s disease (PD) has recently culminated in clinical trials. The first clinical report on autologous transplantation of patient-derived hPSCs showed significant motor symptom improvement, validating the therapeutic promise of this approach. However, critical challenges remain, notably the limited engraftment and survival of donor cells. Cellular stress incurred during in vitro differentiation of hPSCs into midbrain dopaminergic progenitors and neurons contributes to the reduced survival of grafted mDA neurons. Additionally, the host brain environment at the injection sites becomes hostile to transplanted cells due to needle trauma, immune rejection, and alpha-synuclein pathology present in the brains of PD patients. This review discusses potential strategies to address both intrinsic donor cell stress and hostile host brain environment, aiming to enhance the long-term efficacy and engraftment of hPSC-based cell therapies for PD.

Graphical Abstract

INTRODUCTION

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, primarily known for its progressive motor symptoms. PD is characterized by the gradual degeneration of dopamine (DA)-producing neurons in the substantia nigra (A9 region) of the midbrain. A key pathological hallmark of PD is the abnormal aggregation of α-synuclein (α-syn), which normally plays essential roles in neurons, such as supporting synaptic transmission and regulating gene expression.¹ However, misfolded α-syn aggregates contribute to cellular dysfunctions, including mitochondrial impairment, endoplasmic reticulum stress, disruption of autophagy-lysosomal pathways, and synaptic and nuclear dysfunctions,²,³ all of which exacerbate neuronal degeneration in a self-perpetuating manner. Aggregated α-syn proteins are released into the extracellular space, where they activate inflammatory responses in glial cells through pattern recognition receptors, promoting neuroinflammation.⁴,⁵ Furthermore, aggregated α-syn can also propagate in a prion-like manner via synapses or receptor-mediated uptake by neighboring neurons, further advancing disease progression (reviewed in Brundin, et al.).⁶

Currently, PD treatment focuses on symptomatic relief through dopaminergic drugs and deep brain stimulation. Many efforts to develop disease-modifying therapies are underway, targeting toxic α-syn aggregates, neuroinflammation, and pathways that promote DA neuron degeneration. These approaches leverage antibodies, small molecules, exosomes, and gene therapies to combat the disease process. An ideal therapeutic strategy is to replace the damaged DA neurons with healthy ones through cell replacement therapy. This involves transplanting DA neuron-producing donor cells into the midbrain. PD is an optimal target for this approach due to its selective neuronal loss and localized pathology. Previous clinical trials have tested fetal midbrain tissue transplantation from aborted fetuses, with some patients showing symptom improvement for over 10 years.⁷ However, side effects such as dyskinesia emerged in some cases, and double-blind trials failed to demonstrate consistent symptom improvement with fetal tissue transplants.⁸,⁹ The inability to control the number and functionality of grafted DA neurons contributed to these inconsistent outcomes and side effects.

Stem cells capable of generating midbrain DA (mDA) neurons and self-renewing could offer an ideal alternative to fetal tissues. These cells could enable controlled DA neuron engraftment, alleviate ethical concerns, and provide a more accessible donor source for cell replacement therapy in PD.

HISTORY OF RESEARCH ON STEM CELL-BASED CELL REPLACEMENT THERAPY FOR PD

A key focus in developing stem cell, fetal ventral mesencephalic stem cell-based therapies for PD is ensuring the mDA neurogenic potential of stem cells. By definition, stem cells possess self-renewal capacity and multipotent developmental potential. However, these properties vary depending on the developmental stage of the stem cells. mDA neuron development occurs during the early stages of the developing ventral midbrain (VM), and stem cells derived from this early developmental stage efficiently generate mDA neurons.¹,² Moreover, when VM-derived stem cells are expanded to increase cell numbers, they tend to lose their inherent mDA neurogenic potential.³ These limitations of brain tissue-derived stem cells present challenges for their application in cell-based therapies for PD.

For decades, embryonic stem cells (ESCs) derived from mouse embryonic blastocysts have been widely used in generating transgenic mouse models. Unlike tissue-derived stem cells, which have limited self-renewal and developmental potential, ESCs can proliferate indefinitely while retaining pluripotency, making them ideal candidates for cell-based therapies. The potential of ESCs in PD therapy was first demonstrated when mouse ESCs (mESCs) were successfully guided to differentiate into mDA neurons by Lee, et al.⁴ This breakthrough was followed by studies showing that mESC-derived mDA neurons could restore motor function in a rat PD model,⁵ sparking further research into pluripotent stem cells (PSCs) as a source for PD therapies.

Following the validation of mESC-derived cells, the next step was transitioning to human PSCs (hPSCs), such as human ESCs (hESCs) and human induced PSCs (hiPSCs), to develop potential donor cells for PD patients (Table 1). Early efforts to differentiate hPSCs into mDA neurons were met with limited success. Traditional approaches, such as embryoid body formation⁴ and co-culture methods with stromal cells,⁶ proved inefficient for cell therapy applications, as the resulting DA neurons often lacked critical midbrain-specific markers.⁷ Midbrain-specific markers, including Nurr1, Foxa2, Lmx1a, and Engrailed-1 (En-1), are essential for PD cell therapy, as these factors are crucial for mDA neuron survival, resistance to toxic insults, and synaptic formation, all necessary for effective therapeutic outcomes.⁸,⁹,¹⁰,¹¹,¹²,¹³,¹⁴,¹⁵,¹⁶,¹⁷,¹⁸,¹⁹,²⁰

Table 1. History of Milestone Research for Stem Cell-Based PD Cell Therapy.

Year	Milestone	Researchers/institutions	Significance
199885	Derivation of hESCs	James Thomson/University of Wisconsin-Madison	First establishment of hES cell lines
200686	Grafting of hES-DA neurons	Steven Goldman/Cornell University	First report of successful grafting of hESC-derived DA precursors
200787	Development of iPSCs	Shinya Yamanaka/Kyoto University	Pioneered a method for creating patient-specific stem cells, reducing ethical concerns associated with ESCs
200988	Development of PD-iPSCs	Rudolf Jaenisch/MIT	PD-iPSC generation free of viral factors
201089	Successful differentiation of PD-iPSCs into dopaminergic neurons	Ole Isacson/Harvard University	Survival and functional effects of PD-iPSC-derived DA neurons in an animal model of PD
20117	Non-feeder-based efficient generation of hESC-derived dopaminergic neurons	Lorez Studer/MSCC	Efficient non-feeder-based generation of hESC-derived dopaminergic neurons that reverse motor dysfunction in a rodent model of 6OHDA-induced lesions
201452	hESC-derived DA progenitors are safe and efficacious in animal models	Malin Parmar/Lund University	Proof of concept demonstrating that hESC-derived dopaminergic progenitors are safe and effective in animal models of PD (similar potency to fetal neurons)
201590	Global initiative for bringing hPSC-derived DA neurons to the clinic	G-FORCE PD consortium	Establishment of a global initiative to advance human pluripotent stem cell-derived dopamine therapies into the clinic (GFORCE PD)
202091	First clinical trial using iPSC-derived DA progenitors for PD	Shiny Yamanaka/Kyoto University, Riken Center	First CiRA patient grafted with allogenic iPSC-derived DA progenitors; this milestone transitioned stem cell therapy from bench to bedside, marking a significant step toward potential treatments
202025	First hiPSC-derived autologous PD therapy	Kwang-Soo Kim/Harvard University	Evidence of safety in a patient grafted with autologous iPSC-derived dopaminergic neurons
202192	First clinical trial of hESC-derived DA neurons in patients	Lorez Studer/MSCC	Initiation of BlueRock Phase 1 trial of ESC-derived DA neuroblasts in PD patients in New York (NCT04802733 )

PD, Parkinson’s disease; hESCs, human embryonic stem cells; DA, dopamine; iPSC, induced pluripotent stem cell; hPSC, human pluripotent stem cell; hiPSCs, human induced pluripotent stem cell; CiRA, Center for iPS Cell Research and Application.

Recognizing the unique development of mDA neurons from the floor plate region,²¹,²² Studer’s group at the Sloan-Kettering Institute adapted this developmental pathway to generate mDA neurons from hESCs, resulting in cells expressing a full array of midbrain-specific markers.⁷ These hESC-derived mDA neurons demonstrated therapeutic efficacy in a rodent PD model, and Studer’s protocol, with or without modifications, remains widely used today. Further studies have since reported successful engraftment of mDA neurons and motor recovery following the transplantation of hPSC-derived mDA progenitors in humanized mouse²³ and non-human primate PD models,²⁴ bringing this approach closer to clinical application.

CURRENT PROGRESS IN CLINICAL TRANSITION OF hPSC-BASED PD THERAPY

As research on hPSC-based therapies for PD advances, clinical trials for PD patients are being launched and prepared globally (Table 2). The first clinical report on hPSC-based cell therapy for PD was published by Harvard University, detailing an autologous transplantation in a patient using patient-derived hPSCs.²⁵ Clinical assessments indicated that PD symptoms stabilized or improved at 18 to 24 months post-transplantation, demonstrating both the safety and initial efficacy of stem cell-based therapy. However, the sustained improvement of PD symptoms remains questionable. This outcome underscores the need to refine cell-based therapies to ensure more effective and durable symptomatic relief without adverse effects.

Table 2. Current Status of Clinical Trials for hPSC-Based PD Therapy.

Clinical trial ID	Trial name	Institution/company	Therapy details	Phase	Trial start	Status	Notes
NCT02452723	A study to evaluate the safety of neural stem cells (NSC) in patients with PD	Cyto Therapeutics Pty Limited	Human parthenogenetic neural stem cells (hpNSC)	Phase I	2016	Unknown	A single arm, open-label phase 1 study evaluating the safety and tolerability of hpNSC
IND17145	Transplantation of autologous mDA precursors	Harvard University	Transplantation of patient-derived iPSC DA neurons	N/A	2017	Completed	Transplantation of autologous DA neurons derived from PD patient iPSCs59
UMIN000033564	Kyoto University hPSC-based therapy	CiRA Kyoto University	Transplantation of iPSC-derived DA neurons	Phase I/II	2018	Ongoing	First-in-human trial in Japan evaluating safety and cell integration82
UMIN000033565	Kyoto trial to evaluate the safety and efficacy of tacrolimus in the iPSC-based therapy for PD	CiRA Kyoto University/Sumitomo Pharma Co., Ltd.	Transplantation of iPSC-derived DA neurons	Phase III	2018	Completed	Evaluation of the safety and efficacy of tacrolimus (immunosuppressant) in PD patients following transplantation of hiPSC-derived DA progenitors into the corpus striatum
NCT03815071	Clinical study of the safety and efficacy of autologous NSC in the treatment of PD	Allife Medical Science and Technology Co., Ltd.	iPS-NCS	Phase I	2019	Unknown	Use of autologous NSCs for the treatment of PD in China
NCT04802733	Human ESC-derived mDA neuron cell therapy (MSK-DA01) for advanced Parkinson’s disease	BlueRock Therapeutics	hESC (H9ES)-derived DA progenitor cells for PD	Phase I/II	2021	Completed	Evaluation of safety and preliminary efficacy in PD patients83
NCT05635409	STEM-PD trial: a multicentre, single arm, first in human, dose-escalation trial, investigating the safety and tolerability of intraputamenal transplantation of hES-derived DA cells for PD	Region Skane/University of Cambridge, Lund University	hESC (RC17)-derived DA progenitor cells for PD	Phase I	2022	Ongoing	Evaluation of safety and preliminary efficacy in PD patients in Europe
NCT05887466	A single center, open, single dosing, dose-escalation, phase 1/2a study to evaluate the safety and exploratory efficacy of embryonic stem cell-derived A9 dopamine progenitor cell (A9-DPC) therapy	S.Biomedics	hESC-derived DA progenitor cells for PD	Phase I/IIa	2023	Ongoing	Evaluate the safety and exploratory efficacy of hESC (SNUhESC) --derived A9 DA progenitor cell (A9-DPC) therapy

hPSC, human pluripotent stem cell; PD, Parkinson’s disease; mDA, midbrain dopamine; iPSC, induced pluripotent stem cell; hESC, human embryonic stem cell.

DONOR CELL SURVIVAL AND ENGRAFTMENT AS KEY CHALLENGES

For hPSC-derived mDA progenitors to produce therapeutic effects, the donor cells must survive, differentiate into mature mDA neurons, and form synaptic connections with host brain neurons. Among these processes, low donor cell survival poses the most significant challenge,²⁶ whereas mDA neuron differentiation/maturation and synaptic integration with host neurons are relatively efficient from the surviving donor cells.²⁷ Consistently, PET imaging of the patient’s brain post-transplantation in the clinical trial²⁵ revealed that mDA neuron engraftment levels were significantly lower than anticipated.

Factors limiting donor cell survival and engraftment

Hostile brain environment

Needle trauma

A recent study from the Harvard Group has demonstrated that most transplanted DA neurons (>90%) died during the early post-transplantation period, peaking in cell loss 1 week after implantation.²⁶ Mechanical injury from needle injection during cell implantation triggered the infiltration of host brain resident microglia and peripheral T8 lymphocytes into the graft site, leading to local inflammation around the graft. This process involves pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), which are released by the infiltrated host immune cells and induce acute DA neuron death. Another study has confirmed that TNF-α-mediated death of transplanted DA neurons shortly after transplantation and that p53 is a critical intrinsic pathway downstream of TNF-α in degenerating mDA neurons (Fig. 1).²⁸

Fig. 1. Factors limiting donor cell survival and engraftment. In PD cell transplantation, cells are injected into a pathological brain environment that already exhibits severe α-synucleinopathy, reactive oxygen species (ROS), and inflammation. The use of a transplantation needle further induces trauma, triggering the release of innate immune response factors such as pro-inflammatory cytokines and chemokines. These factors activate surrounding glial cells and facilitate the infiltration of peripheral immune cells into the brain, potentially exacerbating the inflammatory response and hindering tissue repair. Collectively, these mechanisms exert detrimental effects on the survival and integration of transplanted cells.

Host α-syn pathology

As described, misfolded α-syn aggregates in the brains of PD patients are directly toxic to DA neurons and also affect glial cells, triggering neuroinflammation. Additionally, α-syn aggregates exhibit prion-like properties, enabling neuron-to-neuron transmission. Supporting this, studies have shown the spread of α-syn pathology from host to graft in post-mortem analyses of PD patients who had undergone fetal midbrain transplantation.²⁹,³⁰ Therefore, it is crucial to consider the impact of host brain pathology on the engraftment and functionality of donor cells (Fig. 1). Nevertheless, preclinical assessments of PD cell therapy have primarily been conducted using the 6-hydroxydopamine rodent model and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine primate model—both of which feature brain regions that remain free of α-syn pathology. As a result, a critical but often overlooked challenge is that donor cells in clinical trials are inevitably placed into an inhospitable environment within the patient’s brain, marked by toxicα-syn accumulation, its propagation, and inflammation driven by the disease pathology.

Host immune rejection to donor cells

The persistence of transplanted DA neurons is significantly influenced by the host’s immune response at various levels. A critical factor for the survival of these transplanted cells is their ability to avoid graft rejection, a process governed by the host’s adaptive immune system. Studies conducted on nonhuman primates have shown that DA neurons derived from their own induced PSCs (iPSCs) provoke a significantly lower immune reaction in the brain compared to those derived from donor (allogeneic) iPSCs.³¹ The immune response of the host remains a major challenge for allogeneic cell transplants (Fig. 1). Even though the central nervous system (CNS) is often regarded as an immune-privileged area, stem cell-derived allografts introduced into the CNS can still be vulnerable to immune-mediated rejection.²⁶,³²,³³ In humanized mouse models, immune rejection has been observed in DA neuron grafts that are mismatched for human leukocyte antigen (HLA) types.²⁶,³⁴,³⁵ Additionally, the brain’s status as an immune-privileged environment appears to rely on the integrity of the blood-brain barrier (BBB). This barrier can be compromised both in the brains of individuals with PD and as a result of surgical interventions.³⁶

The surgical procedure can disrupt the BBB, undermining the immune-privileged status of the brain and potentially allowing immune cells to infiltrate. This breach necessitates the administration of immunosuppressive treatments to prevent graft rejection and support cell survival and integration.³⁷,³⁸ A recent study²⁶ has indicated that the act of transplanting cells using a needle significantly activates the host’s innate immune system, a response referred to as needle trauma. This type of neuroinflammation occurs when the initial injury disrupts the BBB, leading to the release of various immune mediators, including cytokines such as TNF-α, interleukine-1β, and IFN-γ, as well as chemokines like chemokine (C-X-C motif) ligand and C-C motif chemokine ligand.³⁹,⁴⁰ This immediate secretion of proinflammatory cytokines triggers subsequent immune responses, including the activation of glia and the robust infiltration of Iba-1⁺ microglia and major histocompatibility complex II+ cells (Fig. 1). Consequently, immunosuppressive therapies are typically recommended following transplantation. While adverse effects of stem cell transplantation for PD have not been widely reported, long-term use of immunosuppressants—particularly tacrolimus (http://dx.doi.org/10.2139/ssrn.5122485), thiopurines,⁴¹ and azathioprine⁴²—has been associated with an increased risk of certain cancers in solid organ transplant recipients.⁴³,⁴⁴

Cellular stresses in donor cells accumulated during in vitro preparation

In hPSC-based cell therapy for PD, the preparation of donor cells involves multiple in vitro processes, including the establishment, expansion, and differentiation of hESCs/iPSCs. Throughout these stages, particularly during the differentiation of hPSCs into mDA neurons, various cellular stresses can accumulate. Current protocols for differentiating hPSCs into mDA neurons begin with early VM patterning, followed by the differentiation of VM-patterned precursor cells into DA neurons. This VM patterning step, however, requires dense cell-to-cell contact⁴⁵ and the use of a combination of patterning chemicals,⁷ some of which can be toxic to cells.

These conditions lead to the accumulation of cellular stress in patterned cells, which can severely impact donor cell survival after transplantation. Although current differentiation protocols emphasize the expression of midbrain-specific factors that help resist toxic insults, achieving consistent and authentic mDA neuron differentiation remains challenging. Furthermore, these midbrain-specific factors are sensitive to oxidative and inflammatory stresses,¹¹,¹³,⁴⁶ making them prone to loss in the hostile host brain environment post-transplantation. Developing protocols that produce mDA neurons with stable expression of midbrain-specific factors, even in challenging environments, remains an important goal for enhancing donor cell resilience.

POTENTIAL SOLUTIONS

Donor cell preparation

Improving mDA neuron differentiation protocols

In current hPSC-neural differentiation protocols, neural induction and brain patterning are performed in a two-dimensional (2D) culture environment, where hPSCs are maintained at extremely high cell densities. This density is necessary for neuroectodermal induction,⁴⁵ but it also requires the use of a combination of patterning chemicals that can be variably toxic to cells.⁴⁷,⁴⁸ These harsh conditions inevitably lead to the accumulation of cellular stress in the resulting neural cells.

In our recent study,⁴⁹ we hypothesized that these stresses could be significantly reduced by performing VM patterning in a three-dimensional (3D) brain organoid culture environment, which more closely replicates the cell-cell interactions seen in the developing in vivo VM. We applied this approach, isolating and culturing neural stem cells (NSCs) from early VM-patterned organoids at day 18 in vitro. As expected, NSCs derived from the organoids (organoid-derived NSCs, or Og-NSCs) exhibited lower levels of cellular senescence, aging, and mitochondrial stress compared to those produced in 2D cultures. Consequently, Og-NSCs demonstrated robust proliferation and retained their ability to differentiate into mDA neurons, facilitating the efficient mass production of donor cells for PD cell therapy.

Moreover, mDA neurons derived from Og-NSCs consistently expressed key midbrain-specific markers, such as Foxa2, Nurr1, and En-1, and exhibited enhanced therapeutic efficacy in PD model rats. Similarly, a reproducible and efficient mDA neuron differentiation protocol has been achieved using another 3D patterning method known as the “spotting” technique.⁵⁰,⁵¹ Beyond minimizing cellular stress, further strategies are needed to enhance donor cell resistance to the inflammatory and oxidative conditions of the host brain. Recent research has identified p53 as a critical intracellular signaling molecule activated in such hostile environments.²⁸ Additional studies are needed to discover other target molecules and pathways, screen for effective therapeutic candidates, and develop methods to treat donor cells, enhancing their resilience.

Optimizing hPSC establishment

hPSCs, which include both hESCs and hiPSCs, serve as a distinctive source for the potentially limitless in vitro production of functional human cell types. In this context, hPSCs are highly promising for revolutionizing cell-based therapies for PD.

One study compared the effectiveness and potency of dopamine grafts derived from ESCs with those from fetal grafts in preclinical models.⁵² The results suggest that ESC-derived dopaminergic progenitors may perform as well as successful fetal ventral mesencephalic transplants in patients with the appropriate PD phenotype. A crucial factor in utilizing ESCs is selecting the right cell line, as some lines show a greater tendency to differentiate into specific lineages.⁵³,⁵⁴ For example, the RC17, H9, and SNUhES1-ESC lines can consistently and reliably differentiate into the A9 dopaminergic lineage and are currently undergoing evaluation in clinical trials (Table 2).

The emergence of hiPSC technology has marked a significant advancement in stem cell biology and regenerative medicine. Pioneered by Yamanaka’s group and others, hiPSCs have been successfully generated from human somatic cells using similar sets of reprogramming factors.⁵⁵,⁵⁶,⁵⁷ One major advantage of using autologous iPSCs is their potential to be recognized as “self” by the host’s immune system, potentially eliminating the need for concurrent immunosuppression.⁵⁸ However, the assumption that autologous tissues are non-immunogenic remains debated, as some studies indicate that autologous iPSC-derived products might still trigger immune responses.⁵⁹ Another concern with autologous cells is that they originate from individuals who have already developed the disease; for instance, fetal ventral mesencephalic tissue can develop PD-related pathology over time.²⁹,³⁰ Therefore, it is theoretically possible that autologous iPSC-derived dopamine cells could develop disease-related pathology, potentially compromising the transplant. Moreover, the autologous approach faces economic and logistical challenges related to producing individualized cell lines for each patient.⁶⁰,⁶¹

High-quality hPSC lines, including both ESCs and iPSCs, are developed through rigorous genomic integrity assessments and non-integrative reprogramming techniques to ensure their safety and suitability for clinical applications.⁵⁴ Safety concerns are further addressed by eliminating any remaining pluripotent cells using advanced sorting techniques and by developing immune-compatible solutions such as autologous iPSCs or universal donor lines. Comprehensive quality control measures, including the analysis of pluripotency markers and functional differentiation assays, are implemented to meet regulatory standards. Collectively, these advancements ensure that hPSC-derived therapies are safe, scalable, and effective, paving the way for groundbreaking treatments for PD and other neurodegenerative diseases.

Mitigating the hostile host brain environment

Co-transplantation of regulatory T (Treg) cells

Needle trauma from transplantation triggers immediate neuroinflammation in the host brain, which gradually subsides but initially causes significant cell death among grafted mDA neurons.²⁶ This makes the early inflammatory response a critical target for improving donor cell survival. In traumatic brain injury, research has shown that after the initial influx of inflammatory cells, Treg cells infiltrate the damaged area as part of the repair process, helping to restore homeostasis.⁶²,⁶³ Building on these insights, researchers at Harvard prepared Treg cells from peripheral blood and co-transplanted them with hPSC-derived mDA progenitor cells.²⁶ This co-transplantation approach significantly reduced the neuroinflammatory response to needle trauma by suppressing acute inflammation and immune cell infiltration. As a result, the survival of grafted mDA neurons improved, leading to enhanced therapeutic outcomes in a rodent model of PD. These findings suggest that co-transplanting Treg cells may be a promising strategy for future PD cell therapies.

Co-transplantation of astrocytes

Astrocytes, the most abundant glial cells in the brain, perform essential neurotrophic and homeostatic functions. They protect neurons from both extracellular and intracellular toxicity through mechanisms such as scavenging reactive oxygen species,⁶⁴ clearing excess excitotoxic glutamate,⁶⁵ and removing harmful lipids.⁶⁶ Additionally, astrocytes release neurotrophic factors and anti-inflammatory cytokines, further supporting neuronal health.⁶⁷

Our recent study has highlighted the therapeutic potential of astrocytes, particularly those derived from the VM, in mitigating α-syn pathology.⁶⁸,⁶⁹ These VM astrocytes act through multiple mechanisms: 1) inhibiting neuronal α-syn aggregation and transmission via paracrine signaling, 2) releasing factors that disassemble extracellular α-syn aggregates, 3) actively scavenging extracellular α-syn fibrils, and 4) stimulating neuronal autophagic clearance of α-syn through paracrine effects. Given that the transmission of α-syn pathology is a major challenge for sustaining long-term therapeutic benefits, leveraging these protective functions makes astrocyte transplantation a promising approach for neurodegenerative diseases.

Despite concerns that astrocytes might become pro-inflammatory and lose their neurotrophic properties after transplantation,⁷⁰,⁷¹,⁷²,⁷³ cumulative research suggests that cultured astrocytes generally do not undergo harmful reactivation following CNS injury. Instead, they support neurite outgrowth and minimize glial scar formation, as reviewed by.⁷⁴

Building on this evidence, we co-grafted mDA progenitor cells with astrocytes cultured from rodent brains in a PD rat model.¹⁴ This co-transplantation approach effectively reduced the hostile inflammatory environment, leading to improved mDA neuron engraftment and enhanced therapeutic outcomes. Furthermore, transplanting cultured rodent astrocytes into PD model mice alleviated α-syn pathology and protected mDA neurons from neurodegeneration.⁶⁹ These findings suggest that co-grafting astrocytes could significantly enhance mDA neuron engraftment by mitigating both needle trauma-induced acute inflammation and host brain α-syn pathology.

Strategies for targeting host immune cells

Within the first week following donor cell transplantation or even a simple needle injection, CD4/CD8 T lymphocytes, along with brain-resident immune cells such as microglia and astrocytes, are actively recruited to the grafted brain region. These immune cells release pro-inflammatory cytokines, including TNF-α and IFN-γ, which contribute significantly to the early death of grafted mDA neurons. This highlights the need for therapeutic strategies aimed at inhibiting the actions of these pro-inflammatory cytokines to improve donor cell survival.²⁸

Under normal physiological conditions, microglia and astrocytes perform neurotrophic and neurosupportive roles. However, needle trauma can polarize them into a pro-inflammatory state. Given that this glial polarization is reversible, strategies that promote a shift from a pro-inflammatory to a neurotrophic phenotype are promising. Additionally, modulating inflammatory T cell activity by recruiting host Treg cells to the graft site could further reduce immune-mediated damage and enhance graft survival.⁷⁵

Notably, even autologous cells can provoke immune reactions if the differentiated cells express antigens that are recognized as foreign.⁷⁶,⁷⁷ Evidence has also shown that allogeneic mDA cells are prone to immune rejection in the brain without immunosuppressive support.²⁵,⁷⁸ To provide broad coverage across a genetically similar population, approximately 150 HLA-matched iPSC lines would be necessary.⁷⁹ Given the current limitations, research using allogeneic cells must rely on established immunosuppressive strategies to reduce the risk of immune-mediated graft failure. The use of immunosuppressants continues to be the prevailing method for managing immune compatibility in clinical applications involving allogeneic hPSC therapies. In ongoing clinical trials targeting PD, various immunosuppressive drugs have been used: cyclosporine, azathioprine, and prednisolone in the Transneuro study (NCT01898390)⁸⁰; tacrolimus in the CiRA study (UMIN000033565)⁸¹,⁸²; a combination of tacrolimus, mycophenolate, basiliximab, and prednisolone in the NYSTEM trial (NCT04802733)⁸³; and a regimen including prednisolone, tacrolimus, basiliximab, and azathioprine in the European STEM-PD trial (NCT05635409). Recognizing that long-term immunosuppression is associated with various side effects,⁸⁴ it is important to refine immunosuppressive protocols by adjusting the type, dosage, and treatment duration to enhance consistency among patients while ensuring the safety and therapeutic effectiveness of PD cell therapies.