Challenges involved in cell therapy for Parkinson’s disease using human pluripotent stem cells

Neurons derived from human pluripotent stem cells (hPSCs) provide a valuable tool for studying human neural development and neurodegenerative diseases. The investigation of hPSC-based cell therapy, involving the differentiation of hPSCs into target cells and their transplantation into affected regions, is of particular interest. One neurodegenerative disease that is being extensively studied for hPSC-based cell therapy is Parkinson’s disease (PD), the second most common among humans. Various research groups are focused on differentiating hPSCs into ventral midbrain dopaminergic (vmDA) progenitors, which have the potential to further differentiate into neurons closely resembling DA neurons found in the substantia nigra pars compacta (SNpc) after transplantation, providing a promising treatment option for PD. In vivo experiments, where hPSC-derived vmDA progenitor cells were transplanted into the striatum or SNpc of animal PD models, the transplanted cells demonstrated stable engraftment and resulted in behavioral recovery in the transplanted animals. Several differentiation protocols have been developed for this specific cell therapy. However, the lack of a reliable live-cell lineage identification method presents a significant obstacle in confirming the precise lineage of the differentiated cells intended for transplantation, as well as identifying potential contamination by non-vmDA progenitors. This deficiency increases the risk of adverse effects such as dyskinesias and tumorigenicity, highlighting the importance of addressing this issue before proceeding with transplantation. Ensuring the differentiation of hPSCs into the target cell lineage is a crucial step to guarantee precise therapeutic effects in cell therapy. To underscore the significance of lineage identification, this review focuses on the differentiation protocols of hPSC-derived vmDA progenitors developed by various research groups for PD treatment. Moreover, in vivo experimental results following transplantation were carefully analyzed. The encouraging outcomes from these experiments demonstrate the potential efficacy and safety of hPSC-derived vmDA progenitors for PD cell therapy. Additionally, the results of clinical trials involving the use of hPSC-derived vmDA progenitors for PD treatment were briefly reviewed, shedding light on the progress and challenges faced in translating this promising therapy into clinical practice.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease in humans.It is caused by the specific loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain.PD is characterized by motor symptoms, such as tremor, muscular stiffness, and bradykinesia, but it is also associated with cognitive impairment, sleep disturbances, depression, and a weakened sense of smell (Poewe et al., 2017).Various methods have been used to treat PD.Drug therapy, deep brain stimulation (DBS), gene therapy, and cell therapy are currently available treatments.Levodopa, dopamine agonists, and monoamine oxidase-B (MAO-B) inhibitors are representative drugs used in an attempt to increase low dopamine levels in PD patients (Connolly and Lang, 2014).These drug therapies are known to improve the motor symptoms of patients, but long-term treatment with levodopa or dopamine agonists can worsen the patient's symptoms due to drug tolerance and neurotoxicity.Additionally, nausea, daytime somnolence, and edema are possible adverse effects of these treatments (Connolly and Lang, 2014).DBS is a surgical therapy that alleviates symptoms by inserting electrodes into movement-controlling regions of the brain, such as the subthalamic nucleus (STN) or globus pallidus internus (GPi), for electrical stimulation (Bronstein et al., 2011).Patients who have acquired medication resistance due to longterm pharmacological therapy may benefit from DBS.The advantage of DBS is that the patient may reduce their medicine dosage and switch the electrodes on and off as required.As the DBS-implanted brain areas are also involved with emotions, adverse effects, such as emotional disorders and manic responses, may result in psychiatric issues.
Currently, the above-mentioned therapies may improve PD symptoms, but a fundamental cure is not yet available.Consequently, diverse treatment methods are being researched.One of these, gene therapy, aims to fix genetic mutations in familial PD, and numerous therapeutic techniques have been proposed, including introducing a target gene vector into an adenoassociated virus or lentivirus and delivering this to the patient (Axelsen and Woldbye, 2018).To actually apply this in humans, however, raises safety concerns, since it employs a virus.Cell therapy has emerged to compensate for the limitations of other treatments.In 1989, Olson's group performed the first cell transplantation for PD patients (Lindvall et al., 1989).Ventral mesencephalic tissues derived from aborted human fetuses were transplanted into the striata of two patients, leading to improvement of certain motor symptoms.However, ethical problems may arise regarding the acquisition of fetal tissue, and even if ventral mesencephalic tissue is acquired, problems regarding purity control remain (Spenger et al., 1996).
Since then, a method for differentiating and transplanting cells derived from human embryonic stem cells (hESC) has been developed, and attempts have been made to overcome the limits of previous therapeutic agents.Particularly, after the discovery of dual-SMAD inhibition method in 2009 (Chambers et al., 2009), diverse and efficient differentiation protocols for DA neurons have been established.Once the techniques for generating human induced pluripotent stem cells (hiPSCs), another subset of hPSCs, from somatic cells were established (Takahashi et al., 2007), these hPSCs were subsequently directed towards differentiation into specific target cells, serving their purpose in cell therapy.Using the patient's own cells (patient-derived hiPSCs) do not face ethical issues and do not result in immune reactions, as compared to those using embryonic-derived hESCs.Several groups have attempted using hPSC-based cell therapy for the fundamental treatment of PD.Various groups are attempting to generate ventral midbrain DA (vmDA) neurons derived from hPSCs, and confirming whether these neurons are indeed the intended vmDA neurons is crucial.Transplanting hPSCderived vmDA progenitor cells without sufficient cell lineage quality control (QC) may result in the development of neoplastic tumor masses (Roy et al., 2006), as these cells could be contaminated with undifferentiated hPSCs and neural progenitor cells (NPCs) that have proliferative and differentiation capabilities.Therefore, precise QC is essential before transplantation.Ensuring a thorough QC process is crucial to minimize potential side effects that may occur after transplantation and to achieve appropriate clinical outcomes through cell therapy in PD.It is important to note that the current cell lineage QC methods fall short compared to the vmDA neuron differentiation protocol currently available.In this review, we intend to encompass the various types of PD therapy developed to date and analyze PD cell therapy research, along with an overview of vmDA neuron differentiation protocols and transplantation methods.Additionally, we assess the outcomes of in vivo experiments where differentiated cells are transplanted into a PD animal model, and we evaluate the resultant behavioral effects.

Conventional approaches to PD treatment 2.1 Medication
In the early 1960s, Birkmayer and Hornykiewicz were the first to report that levodopa, the precursor to dopamine, was an effective drug for treating PD (Birkmayer andHornykiewicz, 1962, Birkmayer andHornykiewicz, 1964).Since then, several medications have been developed through research aimed at understanding and addressing the neurodegenerative processes and symptoms of PD (Cheong et al., 2019).Levodopa, MAO-B inhibitors, etc., are used to treat motor symptoms, which are the most apparent PD symptoms.Levodopa is a DA medication that helps to increase dopamine levels.When levodopa was administered to PD patients with low dopamine levels, their impaired motor function improved (Steiger et al., 1996).MAO-B inhibitors are non-DA drugs that may be substituted for powerful treatments, such as levodopa, in the early stages of PD (Ives et al., 2004).Psychosis, one of the non-motor symptoms of PD, is treated with pimavanserin, clozapine, and quetiapine (Seppi et al., 2019).Among the disadvantages of these various drug treatments are the decrease of drug effects over time, the possibility of drug-induced complications, and the occurrence of severe side effects, including motor fluctuations and dyskinesia, impulse-control disorder, sleepiness or sudden-onset sleep, and dopamine-dysregulation syndrome (Moore et al., 2014;Bastide et al., 2015).These medications can improve the symptoms of PD, but they cannot provide a fundamental cure.

Deep brain stimulation
In 1870, Fritsch and Hitzig demonstrated that they could generate movement by electrically stimulating the cerebral cortex in dogs (Fritsch and Hitzig, 2009).Afterwards, attempts were made to verify the function of the cerebral cortex and its inner structures via electrical stimulation.In 1960, it was reported for the first time that tremor could be reduced by stimulating the ventrolateral thalamus with a high frequency (Hassler et al., 1960).In 1987, stimulation of the ventral intermedius nucleus (VIM) improved tremor in drug-resistant PD patients (Benabid et al., 1987).DBS was approved by the U.S. Food and Drug Administration in 2002 and continues to be used to treat PD patients.Initially, many attempts were made to improve PD symptoms by stimulating the VIM of the thalamus.This method was able to reduce PD tremor but had no effect on other motor symptoms.Many PD patients are currently treated with electrodes implanted in the STN or the GPi.Both regions can reduce Parkinsonian tremor, but depending on the severity of nonmotor symptoms, one region may be preferable over the other (Miocinovic et al., 2013).DBS may effectively improve symptoms in drug-resistant patients (Herrington et al., 2016), but surgical complications, such as hemorrhage or infection, and cost issues, such as expensive surgery costs and battery maintenance costs, may hamper its use (Bronstein et al., 2011).

Cell therapy using human fetal midbrain tissues
Transplantation of rodent ventral mesencephalic tissue into the 6-OHDA PD model marked the beginning of cell therapy for PD, as it was confirmed that this treatment improved symptoms in the apomorphine-induced rotation test (Bjorklund and Stenevi, 1979;Perlow et al., 1979).In the 1980s, clinical trials were initiated to transplant human fetal ventral mesencephalic tissue following the discovery of therapeutic effects in animal studies (Lindvall et al., 1988;Madrazo et al., 1988;Lindvall et al., 1989).Open-label trials demonstrated therapeutic effects in PD patients after fetal ventral mesencephalic tissue transplantation (Widner et al., 1992;Piccini et al., 1999).In the double-blind test, however, no significant impact was observed in the older patient group (Freed et al., 2001).Moreover, during the transplantation of fetal ventral mesencephalon that contained undifferentiated cells, the formation of neural rosettes was observed (Spenger et al., 1996).Additionally, the procedure of obtaining fetal ventral mesencephalon may raise ethical concerns.To overcome the limitations associated with fetal ventral mesencephalon transplantation for PD treatment, cell therapy has emerged by transplanting differentiated hPSCs into patients as DA progenitor cells (Table 1).
3 Advances in hPSC-derived DA progenitor transplantation research for PD cell therapy: A comprehensive review of preclinical studies

Literature search strategy
A comprehensive literature search was conducted using PubMed to identify relevant papers published until 31 October 2021.The search terms used were as follows: (Parkinson) AND (hiPSC OR hESC) AND (transplantation).In total, 329 articles were retrieved from the search.To assess the relevance of each publication, the titles, keywords, and abstracts were evaluated.From the initial pool, 2 duplicate articles, 2 non-English articles, 170 articles categorized as reviews, correspondences, or editorials, and 91 articles that did not involve the actual use of hESC or hiPSC in transplantation were excluded, leaving 55 articles eligible for analysis.These 55 papers were selected and reviewed, focusing on their reports regarding the outcomes of cell transplantation using hPSC-derived neuronal cells.

Evolution and specialization of hPSCderived DA progenitors for PD cell therapy
DA neurons derived from hESCs are believed to have the potential to replace the degenerated DA neurons in the PD brain (Ben-Hur et al., 2004).These neurons utilize dopamine as a neurotransmitter and express tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis (Kosaka et al., 1987).While TH-expressing DA neurons are found in multiple brain regions, those primarily associated with PD are located in the ventral midbrain.Specifically, vmDA neurons are distributed across three regions: the SNpc (A9), the ventral tegmental area (VTA, A10), and the retrorubral field (RrF, A8) (Hokfelt et al., 1974).vmDA neurons originating from each of these regions project to distinct locations (Lammel et al., 2008).Agerelated loss of vmDA neurons in the SNpc, affecting the nigrostriatal pathway from A9 to the dorsal striatum, is the underlying cause of PD (Kish et al., 1988;Fearnley and Lees, 1991).To address this vmDA neuronal loss in the SNpc, several research groups have developed hESC-based differentiation protocols for generating DA neurons, aiming to utilize them in cell therapy approaches (Table 1).Initially, experiments using mouse ESCs were conducted (Kawasaki et al., 2000), followed by hESC experiments focusing on the differentiation of vmDA neurons (Perrier et al., 2004).More recently, with the advancement of iPSCs (Takahashi and Yamanaka, 2006;Takahashi et al., 2007), differentiation experiments using patient-derived iPSCs to generate vmDA neurons have been gradually progressing.The vmDA neurons differentiated from hPSCs originate from the ventral midbrain floor plate (vmFP), which is the most ventral region of the neural tube.Co-expression of the floor plate marker Forkhead box protein A2 (FOXA2) and the roof plate marker LIM homeobox transcription factor 1 alpha (LMX1A) generally identifies the progenitor cells in this region, known as vmDA progenitors (Andersson et al., 2006;Bonilla et al., 2008).

Advances and strategies in direct differentiation of PSCs into DA neurons
When differentiating hPSCs into vmDA neurons, one of the first considerations is choosing between adherent culture or suspension culture methods.Historically, vmDA progenitors were primarily differentiated either by co-culturing with feeder cells such as MS5, S2, and PA6 stromal cell lines in adherent culture (Ben-Hur et al., 2004;Perrier et al., 2004;Brederlau et al., 2006;Sonntag et al., 2007), or through the formation of embryoid bodies (EBs) in suspension culture (Cho et al., 2008;Geeta et al., 2008;Cai et al., 2010).Adherent culture involves co-culturing hPSCs with feeder cells or using a specially-coated dish to support the adhesion and subsequent differentiation of hPSCs into vmDA neurons in a two-dimensional (2D) cell culture format (Figure 1A).In contrast, the suspension culture method employs the culturing of hPSCs in a suspended state, leading to the differentiation into vmDA neurons via the formation of EBs (Figure 1B).Recently, a threedimensional (3D) method involving the formation of neurospheres for vmDA neuron differentiation has also been introduced (Figure 1C).For ease of description, these culture methods can conveniently be categorized as 2D-based, EB-based, or neurospherebased differentiation protocols (Figure 1).Currently, the aforementioned methods are extensively employed in various protocols for differentiating vmDA neurons (Table 1).Numerous protocols have been continuously developed over the years to differentiate cells into vmDA neurons by manipulating specific signaling pathways.These protocols use various cell culture techniques and adjust the duration and concentration of a range of small molecules (Figure 2).One notable protocol is the dual-SMAD inhibition developed by the Studer group, which uses Noggin and SB431542 to inhibit BMP and TGF-beta signaling, respectively (Chambers et al., 2009).Additionally, protocols employing small molecules that modulate cell signaling pathways, such as WNT, SHH, and FGF, are also being developed (Castelo-Branco et al., 2004;Joksimovic et al., 2009;Xi et al., 2012).A method combining dual-SMAD inhibition with other small molecules aims to activate these major signaling pathways at varying concentrations and durations.CHIR99021 (CHIR) is used to activate canonical WNT signaling (Lyashenko et al., 2011), while recombinant SHH protein and SHH agonists like purmorphamine are used for SHH signaling.FGF signaling is triggered using the recombinant protein FGF8b (Figure 2).This combination results in a high yield of vmDA progenitor cells capable of differentiating into vmDA neurons (Cai et al., 2010;Kriks et al., 2011;Doi et al., 2012;Sanchez-Danes et al., 2012;Doi et al., 2014;Grealish et al., 2014;Grealish et al., 2015;Samata et al., 2015;Steinbeck et al., 2015;Samata et al., 2016;Adil et al., 2017;Niclis et al., 2017;Nolbrant et al., 2017;Wakeman et al., 2017;Xia et al., 2017;Fathi et al., 2018;Adler et al., 2019;Chang et al., 2019;Zygogianni et al., 2019;Mousavinejad et al., 2020;Schweitzer et al., 2020;Song et al., 2020;Kim T. W. et al., 2021;Xiong et al., 2021).Researchers like Perrier and his colleagues have focused on differentiation using SHH and FGF8 and found that approximately 60%-80% of beta tubulin III (TUJ1)-positive neurons express TH (Perrier et al., 2004).The Studer and Parmar teams have also successfully differentiated vmDA progenitors by modulating similar signaling pathways (Kriks et al., 2011;Kirkeby et al., 2012).More recently, advanced protocols have been introduced to enhance differentiation efficacy through specific 'boosting methods'.The first method involves using 0.7 μM CHIR from day 0, elevating it to 7.5 μM from day 4, and then reducing it to 3 μM from day 7 (Kim T. W. et al., 2021).The second method administers 0.8 μM CHIR from day 2 and doubles the concentration to 1.6 μM from day 11 (Kim S. W. et al., 2021).Due to variations in cell culture methods, small molecule combinations, and timing, different groups have reported varying rates of vmDA neuronal differentiation (Table 1).
challenges in transplanting fetal ventral mesencephalon cells without proper verification.Thus, it's crucial to validate the lineage of hPSC-derived vmDA progenitors before their transplantation.Numerous markers, such as FOXA2, LMX1A, OTX2, CORIN, EN1, NURR1, PITX3, TH, VMAT2 as well as GIRK2 and DAT-which are co-expressed in DA neurons in the A9 region-are employed for lineage confirmation (Figure 2).A number of research groups employ the co-expression of FOXA2 and LMX1A as indicators of vmDA progenitors (Table 1).Alongside these two established vmDA progenitor markers, numerous others are in use, including OTX2, CORIN, a precursor marker for vmFP, and EN1 (Kriks et al., 2011;Xi et al., 2012) (Table 1).To confirm that vmDA progenitor cells have differentiated into functional vmDA neurons of the SNpc suitable for cell therapy, a neuron maturation process was carried out for validation.As part of this validation, the following markers were employed: NURR1, which is essential for the generation and maintenance of vmDA neurons; PITX3, which plays a pivotal role in the survival of vmDA neurons; the vmDA neuronspecific marker TH; and the pan-neuronal marker TUJ1 (Table 1).Additionally, several markers, such as GIRK2 and DAT, which are co-expressed in DA neurons within the A9 region, were utilized to confirm the vmDA neuronal lineage in this area (Table 1).The identified neurons were further confirmed to possess the same electrophysiological properties as the vmDA neurons in the A9 region (Schulz et al., 2004;Brederlau et al., 2006;Cho et al., 2008;Cho et al., 2011;Kriks et al., 2011;Rhee et al., 2011;Sanchez-Danes et al., 2012;Doi et al., 2014;Steinbeck et al., 2015;Samata et al., 2016;Kikuchi et al., 2017a;Kikuchi et al., 2017b;Niclis et al., 2017;Wakeman et al., 2017;Leitner et al., 2019;Zygogianni et al., 2019;Song et al., 2020).This collective evidence establishes that the progenitor cells have been successfully directed to differentiate into cells exhibiting characteristics consistent with the vmDA neuronal lineage.
However, when generating mature vmDA progenitor cells through various differentiation protocols, a small fraction may diverge into other neuronal types such as noradrenergic, adrenergic, GABAergic, glutamatergic, and serotonergic neurons, or even into glial cells (Ben-Hur et al., 2004;Cho et al., 2008;Geeta et al., 2008;Cai et al., 2010;Sanchez-Danes et al., 2012;Samata et al., 2016;Adil et al., 2017;Wakeman et al., 2017;Fathi et al., 2018;Chang et al., 2019;Zygogianni et al., 2019).Contaminants like undifferentiated stem cells and proliferating NPCs can also be present, posing a risk of neoplastic mass formation upon transplantation (Roy et al., 2006).Most research groups adopt their own QC measures, often focusing on confirming successful differentiation and functional potential post-transplantation. Animal-based transplantation experiments also serve as a form of QC prior to human clinical trials (Table 2, Table 3).Currently, there is a gap in the availability of standardized or rapid methods for 'pass/ fail' assessments to eliminate inappropriate cells immediately prior to transplantation.This 'Last-Mile QC' represents a crucial safety step that would significantly improve the reliability of cell transplantation procedures.While methods like CORIN-positive cell sorting have shown promise-indeed, a study has demonstrated that CORIN-positive cells, once purified, were successfully transplanted, engrafted without tumor formation, and led to behavioral recovery in PD animal models (Doi et al., 2014)-they fall short of meeting the full requirements of 'Last-Mile QC.' One significant limitation is that CORIN-based sorting generally necessitates an additional 16 days of in vitro culture post-sorting, leaving an unresolved issue in the existing QC process (Doi et al., 2014;Katsukawa et al., 2016;Nishimura et al., 2016;Samata et al., 2016;Kikuchi et al., 2017a;Kikuchi et al., 2017b).Therefore, rigorous QC measures should be in place at the vmDA progenitor cell stage.To ensure more predictable and effective PD cell therapy outcomes, it is imperative to either develop a new protocol for lineage verification using specific markers or invent new technologies capable of real-time tracking of cell differentiation.By implementing such stringent QC, we can effectively isolate the correct vmDA progenitor cells for  transplantation, thereby mitigating risks associated with cell therapy.

Impact of cell sorting on the efficacy and safety of vmDA progenitor cell transplantation
Before transplanting vmDA progenitor cells into animal models of PD, research practices have varied.Some groups sort the vmDA progenitor cells using specific markers, while others do not.Importantly, both groups of researchers used the same markers for in vitro identification to confirm the differentiation of the transplanted cells into vmDA neurons in vivo (Chang et al., 2019;Gantner et al., 2020;Goggi et al., 2020;Song et al., 2020;Xiong et al., 2021).When vmDA progenitor cells are not sorted prior to transplantation, undesirable lineage differentiation may occur post-transplantation (Cai et al., 2010;Rhee et al., 2011;Wakeman et al., 2017;Kim T. W. et al., 2021).Additionally, unsorted donor cell populations may contain proliferating neural stem cells (NSCs) and hPSCs, raising the risk of teratoma formation (Brederlau et al., 2006;Cai et al., 2010).To minimize these risks, some research groups have used cell sorting with specific markers such as CORIN + , LRTM1 + , CNTN2 + , LMX1A + , and PSA-NCAM + (Table 2).Among these, CORIN, LRTM1, and CNTN2 are specifically used as vmDA progenitor markers, with CORIN being most frequently employed (Doi et al., 2014;Samata et al., 2015;Nishimura et al., 2016;Kikuchi et al., 2017a;Kikuchi et al., 2017b;Miyawaki et al., 2020).Experiments involving CORIN + -sorted cells have shown significant improvements in outcomes.Jun Takahashi's group demonstrated a 96% increase in the number of TH + cells per graft when using CORIN + -sorted cells compared to unsorted cells.In addition, CORIN + -sorted cells also showed an 18% increase in the proportion of TH + cells among NEUN + cells and led to a smaller average graft size (unsorted, 35.0 ± 37.5 mm 3 ; sorted, 3.4 ± 2.9 mm 3 ), thereby indicating improved precision and efficacy of the transplantation procedure.Moreover, the number of proliferating cells and the number of serotonin + cells were decreased in CORIN + -sorted donor cells (Doi et al., 2014).Post-transplantation functionality of the engrafted cells was assessed using [ 18 F]DOPA-PET imaging.Results indicate a substantial increase in dopamine synthesis in the putamen of the striatum over time, affirming the functionality of transplanted cells (Kikuchi et al., 2017a).
When cells were sorted using LRTM1, a distinct vmDA surface marker, there was an increase of 50 ± 3% in FOXA2+ cells, 31 ± 0.8% in NURR1+ cells, and 289% in TH + cells after transplantation.Importantly, the graft size was also reduced in these cases (Samata et al., 2016).When the sorting process was conducted using CNTN2 as the marker, the proportions of both TH+ and DAT + cells saw a rise of approximately 4% following transplantation (Fathi et al., 2018).The maturation of the engrafted cells into vmDA neurons was further confirmed through the observation of mature neuron morphology, including attributes such as neurite extension, arborization, and branching (Nishimura et al., 2016;Kikuchi et al., 2017a;Kikuchi et al., 2017b;Adler et al., 2019;Gantner et al., 2020;Xiong et al., 2021).
4 Progress and challenges in the clinical application of stem cell therapies for PD Advancements in the field of stem cell therapy for PD have come a long way, from initial in vitro generation of vmDA progenitor cells to successful transplantation in animal models, and finally to clinical trials in human patients.A 2019 case-series study spearheaded by Madrazo et al. demonstrated the potential for using human fetal NPCs as a treatment for PD (Madrazo et al., 2019).The study involved injecting NPCs into the dorsal putamina of patients while administering cyclosporine A to mitigate the risk of immune rejection.Longitudinal evaluations over a 4-year period, encompassing neurological, neuropsychological, and brain imaging analyses, revealed motor improvements in all but one of the seven patients followed, with PET scans indicating a trend of increased dopamine activity in the midbrain.
Another milestone was achieved by Jun Takahashi's research group, pioneers in hiPSC-based therapy for PD (Takahashi, 2020).Beginning their clinical trials in August 2018, they have effectively differentiated DA neurons from hiPSCs.To improve transplantation success rates, they developed a method for selecting DA progenitor cells using the specific vmDA floor plate marker, CORIN (Table 1, Table 2).Confirmations were secured that these CORIN + -sorted cells not only survived both in vitro and in vivo conditions but also functionally differentiated into vmDA neurons, as evidenced in various animal models of PD (Doi et al., 2014;Kikuchi et al., 2017a;Doi et al., 2020).The team also validated the safety and effectiveness of their specialized hiPSC line, QHJI-01, focusing on FOXA2 + TUJ1 + cells as the final product (Doi et al., 2020).The threshold for the final cell population designated for transplantation was established at 80%, with the remainder of the population comprised of midbrain glial cells, which play a supportive role for DA neurons.To minimize potential adverse effects, rigorous QC ensured the absence of hiPSCs expressing OCT3/4 and TRA-2-49/6E markers, as well as NPCs expressing SOX1 and PAX6.The study, designed as a single-arm, non-randomized, open-label Phase I/II trial, initiated patient recruitment in August 2018.The first patient intake session took place at Kyoto University Hospital in October of the same year.To mitigate the risk of immune rejection post-transplantation, patients were administered tacrolimus for a period of 1 year.
When utilizing cells obtained from a different individual for transplantation, there arises the issue of immunocompatibility (Kordower et al., 2008).Immunocompatibility issues stemming from HLA mismatches can lead to the transplanted cells being targeted by the recipient's immune system (Kordower et al., 1997).Consequently, transplant recipients must employ immunosuppressive agents to suppress immune responses (Morizane and Takahashi, 2021).Prolonged use of immunosuppressive agents can give rise to infections and other immunological complications (Lopez et al., 2006).
Another notable contribution came from a study by Schweitzer et al., in which autologous transplantation of hiPSC-derived DA neurons was performed without immunosuppression (Schweitzer et al., 2020).These cells survived for up to 2 years posttransplantation, and clinical measures showed that PD symptoms had either stabilized or improved during an 18-24-month follow-up period.Internationally, both China and Australia are also conducting PD cell transplantation trials using hESCs, as ClinicalTrials.govnumbers NCT03119636 and NCT02452723 indicate.In addition to the aforementioned studies, various clinical trials utilizing hPSCderived dopaminergic cells are currently underway (Barker et al., 2017).
Despite these advances, challenges still loom large, such as safety concerns associated with viral vectors used in reprogramming hiPSCs (Ma et al., 2014;Kang et al., 2016).However, recent developments like the protocol by Guan et al., which replaces viruses with chemicals for generating clinical-grade hiPSCs, mark a promising step towards overcoming these obstacles (Guan et al., 2022).In summary, although hPSC-based cell therapies are advancing toward clinical applications, several challenges persist, such as safety concerns and potential complications arising from the use of adult or embryonic cells, as well as immunocompatibility issues.Nevertheless, the momentum gained from these groundbreaking endeavors holds promise for a more effective and hopeful future in the treatment of PD.

Discussion
The potential of human pluripotent stem cells (hPSCs) to differentiate into ventral midbrain dopaminergic (vmDA) progenitors presents an exciting avenue for the treatment of Parkinson's Disease (PD).Various protocols have been developed to guide this differentiation, often achieving high yields of vmDA progenitors and neurons.However, the challenge lies in the concomitant generation of cells from other lineages (Table 1), which risks the introduction of undesired cell types during transplantation.Current QC methods for ensuring the lineagespecificity of the generated cells are not yet fully reliable.Jun Takahashi's group sought to overcome this hurdle by using the surface marker CORIN to sort vmDA progenitors (Table 2, Table 3).Yet, subsequent research indicated that CORIN expression is not confined to the caudal vmDA progenitor region, raising concerns about the specificity of this sorting mechanism (Kirkeby et al., 2017).The clinical implications of these challenges are significant.Transplanting cells with unconfirmed lineage can lead to complications such as tumorigenicity and dyskinesias, emphasizing the need for rigorous QC procedures.While current differentiation protocols show promise, the lack of precise lineageverification methods raises questions about their readiness for clinical applications.Emerging technologies like nanoprobe-based lineage verification may offer a solution to these challenges.The development of more specific surface markers or advanced detection techniques could enable the selective harvesting of vmDA progenitors, reducing the risk of adverse effects and potentially enhancing therapeutic efficacy.
In conclusion, while hPSC-derived vmDA progenitors present an encouraging path for PD treatment, a robust framework for lineage verification is imperative for ensuring the safety and effectiveness of this promising therapeutic approach.Bridging the gap between preclinical research and clinical practice remains a crucial task for the field, demanding continued interdisciplinary efforts to overcome these limitations.

TABLE 1
Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.

TABLE 1 (
Continued) Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.

TABLE 1 (
Continued) Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.

TABLE 1 (
Continued) Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.

TABLE 1 (
Continued) Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.

TABLE 1 (
Continued) Validation summary of dopaminergic neuronal differentiation from human pluripotent stem cells for transplantation.
* Papers with IDs a1 to a39 assessed the impact of cell transplantation on motor function recovery, while papers with IDs b40 to b55 did not assess the effects of motor function recovery after cell transplantation.** 2D, monolayer differentiation.DA, dopamine; DIV, days in vitro; ELISA, confirmed by enzyme-linked immunosorbent assay; EB, embryonic body differentiation; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; HPLC, confirmed by high performance liquid chromatography; hpSC, human parthenogenetic stem cell; ICC, confirmed by immunocytochemistry. n/a, not available.neurosphere, neurosphere differentiation.PD, Parkinson's disease.qRT-PCR, confirmed by quantitative real-time PCR.RNA-seq, confirmed by RNA, sequencing; RP-HPLC, confirmed by reverse-phase high performance liquid chromatography; RT-PCR, confirmed by reverse transcription PCR., scRNA-seq, confirmed by single cell RNA, sequencing; vmDA, ventral midbrain dopaminergic; WB, confirmed by western blotting.

TABLE 2
Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 2 (
Continued) Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 2 (
Continued) Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 2 (
Continued) Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 2 (
Continued) Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 2 (
Continued) Summary of graft verification following human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 3
Summary of behavioral recovery after human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 3 (
Continued) Summary of behavioral recovery after human pluripotent stem cell-derived dopaminergic neuronal transplantation.

TABLE 3 (
Continued) Summary of behavioral recovery after human pluripotent stem cell-derived dopaminergic neuronal transplantation.