The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease, the second most common human neurodegenerative disease. Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear, the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy. The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons, which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies. The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells. The benefits of induced pluripotent stem cell-based research are highlighted. Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared. The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated. Finally, limitations, challenges, and future directions of induced pluripotent stem cell-based approaches are analyzed and proposed, which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.

Neurodegenerative diseases are characterized by selective loss of neurons. Cell replacement therapies are the most promising therapeutic strategies to restore the neuronal functions lost during these neurodegenerative processes. However, cell replacement-based clinical trials for Huntington's (HD) and Parkinson's diseases (PD) failed due to the large heterogeneity of the samples. Here, we identify CD200 as a cell surface marker for human striatal neuroblasts (NBs) using massively parallel single-cell RNA sequencing. Next, we set up a CD200-based immunomagnetic sorting pipeline that allows high-yield enrichment of human striatal NBs from in vitro differentiation of human pluripotent stem cells (hPSCs). We also show that sorted CD200-positive cells are striatal projection neuron (SPN)-committed NBs which survive upon intra-striatal transplantation in adult mice with no evidence of graft overgrowth in vivo. In conclusion, we implemented a new CD200 cell selection strategy that reduces the heterogeneity and batch-to-batch variation and potentially decreases the teratogenic risk of hPSC-based cell therapy for neurodegenerative diseases.

Induced pluripotent stem cells (iPSCs), generated through somatic cell reprogramming, exhibit self-renewal capacity and multilineage differentiation potential. In recent years, iPSC-derived neurons have emerged as a significant platform for researching mechanisms and developing therapies for neurological diseases. This paper reviews the targeted differentiation strategies of iPSCs into dopaminergic neurons, motor neurons, cholinergic neurons and medium spinal neurons, providing detailed insights into the differentiation processes. Additionally, this paper discusses the challenges associated with the future application of iPSCs-derived neurons in the treatment of nervous system diseases are also discussed in this paper, aiming to provide references for the application of iPSCs in cellular therapies for neurodegenerative disorders.

Many neurological diseases involving tissue damage cannot be treated with drug-based approaches, and the inaccessibility of human brain samples further hampers the study of these diseases. Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an excellent model for studying neural development and function. PSCs can be differentiated into various neural cell types, providing a renewal source of functional human brain cells. Therefore, PSC-derived neural cells are increasingly used for multiple applications, including neurodevelopmental and neurotoxicological studies, neurological disease modeling, drug screening, and regenerative medicine. In addition, the neural cells generated from patient iPSCs can be used to study patient-specific disease signatures and progression. With the recent advances in genome editing technologies, it is possible to remove the disease-related mutations in the patient iPSCs to generate corrected iPSCs. The corrected iPSCs can differentiate into neural cells with normal physiological functions, which can be used for autologous transplantation. This review highlights the current progress in using PSCs to understand the fundamental principles of human neurodevelopment and dissect the molecular mechanisms of neurological diseases. This knowledge can be applied to develop better drugs and explore cell therapy options. We also discuss the basic requirements for developing cell transplantation therapies for neurological disorders and the current status of the ongoing clinical trials.

Parkinson's disease is caused by the loss of dopamine neurons, causing motor symptoms. Initial cell therapies using fetal tissues showed promise but had complications and ethical concerns1-5. Pluripotent stem (PS) cells emerged as a promising alternative for developing safe and effective treatments6. In this phase I/II trial at Kyoto University Hospital, seven patients (ages 50-69) received bilateral transplantation of dopaminergic progenitors derived from induced PS (iPS) cells. Primary outcomes focused on safety and adverse events, while secondary outcomes assessed motor symptom changes and dopamine production for 24 months. There were no serious adverse events, with 73 mild to moderate events. Patients' anti-parkinsonian medication doses were maintained unless therapeutic adjustments were required, resulting in increased dyskinesia. Magnetic resonance imaging showed no graft overgrowth. Among six patients subjected to efficacy evaluation, four showed improvements in the Movement Disorder Society Unified Parkinson's Disease Rating Scale part III OFF score, and five showed improvements in the ON scores. The average changes of all six patients were 9.5 (20.4%) and 4.3 points (35.7%) for the OFF and ON scores, respectively. Hoehn-Yahr stages improved in four patients. Fluorine-18-L-dihydroxyphenylalanine (18F-DOPA) influx rate constant (Ki) values in the putamen increased by 44.7%, with higher increases in the high-dose group. Other measures showed minimal changes. This trial (jRCT2090220384) demonstrated that allogeneic iPS-cell-derived dopaminergic progenitors survived, produced dopamine and did not form tumours, therefore suggesting safety and potential clinical benefits for Parkinson's disease.

Transplantation therapy using induced pluripotent stem cell (iPS) cell-derived dopamine (DA) progenitors for Parkinson's disease (PD) has attracted attention as an innovative treatment to restore DA neurons in PD, which leads to the improvement of motor disturbance. iPS cells are multipotent stem cells with very high proliferation activity, created by reprogramming mature somatic cells through the transduction of four transcription factors. Relative to fetal midbrain DA neurons, iPS cells have advantages in terms of ethical aspects and availability. On the other hand, the most serious concern associated with therapies with ES/iPS cells is the risk of tumor growth that is caused by the proliferation of undifferentiated ES/iPS cells. Human ES cells that differentiate into DA neurons have been shown to form teratomas. Another concern is graft-induced dyskinesia (GID). GID, which is likely caused by several factors including contamination with serotonergic neurons, developed in the recipients of fetal ventral midbrain (VM) in randomized controlled trials. To enrich the DA progenitor cells and eliminate unwanted cells, a protocol for sorting midbrain DA neurons with antibodies against CORIN, a marker for floor plates, was developed. CORIN-sorted dopamine progenitors were transplanted into the bilateral putamina of MPTP-treated Parkinson models of cynomolgus monkeys, resulting in 18F-DOPA PET-positive graft formation and motor improvement without tumor formation two years after the surgeries. Very recently, a phase I/II trial of iPSC-derived, CORIN-sorted dopaminergic cells for Parkinson's disease was completed (The Kyoto Trial) (Takahashi, 2020; Sawamoto et al., 2025) [1,2]. Based on the results of the trial, we would like to discuss the current status and future perspectives of iPS cell therapy for PD.

Although pluripotent stem cell (PSC) properties, such as differentiation and infinite proliferation, have been well documented within the frameworks of transcription factor networks, epigenomes, and signal transduction, they remain unclear and fragmented. Directing attention toward translational regulation as a bridge between these events can yield additional insights into previously unexplained mechanisms. Our functional CRISPR interference screen-based approach revealed that EIF3D, a translation initiation factor, is crucial for maintaining primed pluripotency. Loss of EIF3D disrupted the balance of pluripotency-associated signaling pathways, thereby compromising primed pluripotency. Moreover, EIF3D ensured robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, EIF3D-mediated translation contributes to tuning the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. This study provides further insights into the translation network in maintaining pluripotency.

Advancements in cell separation are essential for understanding cellular phenotypes and functions, with implications for both research and therapeutic applications. This review examines the evolution of cell separation techniques, categorizing them into physical and affinity-based methods, with a primary focus on the latter due to its high specificity. Among affinity techniques, DNA nanomaterials have emerged as powerful tools for biomolecular recognition owing to their unique properties and diverse range of nanostructures. We discuss various DNA nanomaterials, including linear aptamers, multivalent DNA constructs, DNA origami, and DNA hydrogels and their roles in cell recognition and separation. Each section highlights the distinctive characteristics of these DNA nanostructures, providing examples from recent studies that demonstrate their applications in cell isolation and release. We also compare the four DNA nanomaterials, outlining their individual contributions and identifying the remaining challenges and opportunities for further development. We conclude that DNA nanotechnology holds great promise as a transformative solution for cell separation, particularly in the context of personalized therapeutics.

Cell replacement therapies using human pluripotent stem cell-derived ventral midbrain (VM) dopaminergic (DA) progenitors are currently in clinical trials for treatment of Parkinson's disease (PD). Recapitulating developmental patterning cues, such as fibroblast growth factor 8 (FGF8), secreted at the midbrain-hindbrain boundary (MHB), is critical for the in vitro production of authentic VM DA progenitors. Here, we explored the application of alternative MHB-secreted FGF-family members, FGF17 and FGF18, for VM DA progenitor patterning. We show that while FGF17 and FGF18 both recapitulated VM DA progenitor patterning events, FGF17 induced expression of key VM DA progenitor markers at higher levels than FGF8 and transplanted FGF17-patterned progenitors fully reversed motor deficits in a rat PD model. Early activation of the cAMP pathway mimicked FGF17-induced patterning, although strong cAMP activation came at the expense of EN1 expression. In summary, we identified FGF17 as a promising alternative FGF candidate for robust VM DA progenitor patterning.

Parkinson's disease (PD) is a neurodegenerative disease primarily affecting the central nervous system and impacting both the motor system and non-motor systems. Although administration of L-DOPA is effective, it is not a fundamental treatment and has side effects such as diurnal fluctuation and dyskinesia, highlighting the need for new treatment methods. There is a growing interest in dopaminergic neuron transplantation as a potential treatment. Dopaminergic neurons derived from pluripotent stem (iPS) cells provide a valuable source for transplantation therapies. Developing an efficient method to differentiate iPS cells into dopaminergic cells is essential for cell transplantation therapy. While Cell differentiation is typically controlled by the addition of specific reagents, the physical characteristics of culture substrate, especially in the charge and stiffness, are also crucial factors in regulating differentiation. In this research, we show that two newly developed electrically charged polymeric hydrogels composed of cationic (C) and anionic (A) monomers inratio of 1-9 and 2 to 8 can significantly promote Dopaminergic neuron differentiation. Our findings emphasize the importance of culture substrates in effective dopaminergic cell differentiation.

The advancement of induced pluripotent stem cell (iPSC) technology has revolutionized regenerative medicine, enabling breakthroughs in disease modeling, drug discovery, and cell replacement therapies. This review examines the progression of iPSC-based regenerative medicine, focusing on cell replacement therapy and mechanisms like the Replacement Effect, which is crucial for long-term tissue regeneration. Using Parkinson's disease as a key example, it discusses the induction of midbrain dopaminergic neurons from iPSCs and the importance of precise signaling for safety and efficacy. By demonstrating the integration and safety of these cells, animal studies have paved the way for clinical trials. This review highlights the need for strategic collaboration among stakeholders-regulatory authorities, research and medical staff, and industry-to ensure successful clinical applications. iPSC technology's ongoing evolution holds significant promise for broader therapeutic applications and improved patient outcomes.

Brain development may be influenced by both genetic and environmental factors, with potential consequences that may last through the lifespan. Alterations during neurogenesis are linked to neurodevelopmental cognitive disorders. Many neurotransmitters and their systems play a vital role in brain development, as most are present prior to synaptogenesis, and they are involved in the aetiology of many neurodevelopmental disorders. For instance, dopamine (DA) receptor expression begins at the early stages of development and matures at adolescence. The long maturation period suggests how important it is for the stabilisation and integration of neural circuits. DA and dopaminergic (DAergic) system perturbations have been implicated in the pathogenesis of several neurological and neuropsychiatric disorders. The DAergic system controls key cognitive and behavioural skills including emotional and motivated behaviour through DA as a neurotransmitter and through the DA neuron projections to major parts of the brain. In this review, we summarise the current understanding of the DAergic system's influence on neurodevelopment and its involvement in the aetiology and progression of major disorders of the developing brain including autism, schizophrenia, attention deficit hyperactivity disorder, down syndrome, and fragile X syndrome.

While clinical trials are ongoing using human pluripotent stem cell-derived midbrain dopamine (mDA) neuron precursor grafts in Parkinson's disease (PD), current protocols to derive mDA neurons remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of some mDA neuron and subtype-specific markers can be further improved. For example, characterization of mDA grafts by single cell transcriptomics has yielded only a small proportion of mDA neurons and a considerable fraction of contaminating cell populations. Here we present an optimized mDA neuron differentiation strategy that builds on our clinical grade ("Boost") protocol but includes the addition of FGF18 and IWP2 treatment ("Boost+") at the mDA neurogenesis stage. We demonstrate that Boost+ mDA neurons show higher expression of EN1, PITX3 and ALDH1A1. Improvements in both mDA neurons yield and transcriptional similarity to primary mDA neurons is observed both in vitro and in grafts. Furthermore, grafts are enriched in authentic A9 mDA neurons by single nucSeq. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo analyses show increased percentages of TH+ mDA neurons resulting in efficient rescue of amphetamine induced rotation behavior in the 6-OHDA rat model and rescue of some motor deficits in non-drug induced assays, including the ladder rung assay that is not improved by Boost mDA neurons. The Boost+ conditions present an optimized protocol with advantages for disease modeling and mDA neuron grafting paradigms.

Parkinson's disease (PD) is a neurodegenerative disease characterized by degeneration of dopamine neurons in the substantia nigra pars compacta. The patient exhibits a series of motor symptoms, such as static tremors, which impair their capacity to take care for themselves in daily life. In the late stage, the patient is unable to walk independently and is bedridden for an extended period of time, reducing their quality of life significantly. So far, treatment methods for PD mainly include drug therapy and deep brain stimulation. Pharmacotherapy is aimed at increasing dopamine (DA) levels; however, the treatment effect is more pronounced in the short term, and there is no benefit in improvement in the overall progression of the disease. In recent years, novel therapeutic strategies have been developed, such as cell reprogramming, trying to generate more DA in PD treatment. This review mainly discusses the advantages, methodology, cell origin, transformation efficiency, and practical application shortcomings of cell reprogramming therapy in PD strategy.

Parkinson's disease (PD), characterized by progressive degeneration of dopaminergic neurons in substantia nigra, has no disease-modifying therapy. Mesenchymal stem cell (MSC) therapy has shown great promise as a disease-modifying solution for PD. Induced pluripotent stem cell-derived MSC (iMSC) not only has stronger neural repair function, but also helps solve the problem of MSC heterogeneity. So we evaluated the therapeutic effects of iMSCs on PD. iMSCs were administered by tail vein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD models of C57BL/6 mice. The results showed iMSCs increased body weights, inhibited the prolongation of latencies to descend in pole tests, the decrease of grip strength in grip strength tests and increase of open arm entries in elevated plus maze test, and showed a trend to alleviate striatal dopamine loss. They indicate iMSCs might improve functions partially by preserving striatal dopamine in PD. We for the first time (1) found that iMSC has therapeutic effects on PD; (2) tested specifically muscle strength in cell therapy for PD and found it increases muscle strength; (3) found cell therapy alleviated the increase of entries into the open arms in PD. It suggests iMSC is a promising candidate for clinical investigations and drug development for PD.

Non-invasive cell culture monitoring technology is crucial to improve the manufacturing efficiency of cell products. We have found that extracellular vesicles (EVs) are secreted into the culture supernatants in the differentiation process from human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells, and that the composition of EVs changes in accordance with the differentiation processes. In this study, we hypothesized that it is possible to evaluate the cultured cellular states by detecting compositional changes of EVs secreted from cultured cells with label-free Raman spectroscopy in a non-invasive manner. Therefore, Raman signal analysis derived from EV fractions isolated from culture supernatants throughout the differentiation process was conducted. iPSCs cultures were simultaneously implemented under a standard condition (control) and an artificial deviation condition inducing reductions in pluripotency by depleting FGF2 in culture medium (-FGF2), which is indispensable for maintaining the pluripotency. Subsequently, the differentiation step was conducted for each iPSCs culture under the same condition. As a result, it was found that under -FGF2, the expression level of the pluripotency marker NANOG decreased compared to that of the control and correlated with the identification results based on Raman signals with a correlation coefficient of 0.77. Lipid-derived Raman signals were extracted as identification factors, suggesting that changes in the lipid component of EV occur depending on the cellular states. From the above, we have found that the change in composition of EVs in the culture supernatant by detecting Raman signals would be a monitoring index of the cellular state of differentiation and pluripotency.

Parkinson's disease (PD) is characterized by the loss of the dopaminergic nigrostriatal pathway which has led to the successful development of drug therapies that replace or stimulate this network pharmacologically. Although these drugs work well in the early stages of the disease, over time they produce side effects along with less consistent clinical benefits to the person with Parkinson's (PwP). As such there has been much interest in repairing this pathway using transplants of dopamine neurons. This work which began 50 years ago this September is still ongoing and has now moved to first in human trials using human pluripotent stem cell-derived dopaminergic neurons. The results of these trials are eagerly awaited although proof of principle data has already come from trials using human fetal midbrain dopamine cell transplants. This data has shown that developing dopamine cells when transplanted in the brain of a PwP can survive long term with clinical benefits lasting decades and with restoration of normal dopaminergic innervation in the grafted striatum. In this article, we discuss the history of this field and how this has now led us to the recent stem cell trials for PwP.

The transplantation of injury/ischemia-induced stem cells (iSCs) extracted from post-stroke human brains can improve the neurological functions of mice after stroke. However, the usefulness of iSCs as an alternative stem cell source remains unclear. The current study aimed to assess the efficacy of iSC and mesenchymal stem cell (MSC) transplantation. In this experiment, equal numbers of human brain-derived iSCs (h-iSCs) (5.0 × 104 cells/μL) and human bone marrow-derived MSCs (h-MSCs) (5.0 × 104 cells/μL) were intracranially transplanted into post-stroke mouse brains after middle cerebral artery occlusion. Results showed that not only h-iSC transplantation but also h-MSC transplantation activated endogenous neural stem/progenitor cells (NSPCs) around the grafted sites and promoted neurological functional improvement. However, mice that received h-iSC transplantation experienced improvement in a higher number of behavioral tasks compared with those that received h-MSC transplantation. To investigate the underlying mechanism, NSPCs extracted from the ischemic areas of post-stroke mouse brains were cocultured with h-iSCs or h-MSCs. After coincubation, NSPCs, h-iSCs, and h-MSCs were selectively collected via fluorescence-activated cell sorting. Next, their traits were analyzed via microarray analysis. The genes related to various neuronal lineages in NSPCs after coincubation with h-iSCs were enriched compared with those in NSPCs after coincubation with h-MSCs. In addition, the gene expression patterns of h-iSCs relative to those of h-MSCs showed that the expression of genes related to synapse formation and neurotransmitter-producing neurons increased more after coincubation with NSPCs. Hence, cell-cell interactions with NSPCs promoted transdifferentiation toward functional neurons predominantly in h-iSCs. In accordance with these findings, immunohistochemistry showed that the number of neuronal networks between NSPCs and h-iSCs was higher than that between NSPCs and h-MSCs. Therefore, compared with h-MSC transplantation, h-iSC transplantation is associated with a higher neurological functional improvement, presumably by more effectively modulating the fates of endogenous NSPCs and grafted h-iSCs themselves.

Parkinson's disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson's disease. The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson's disease, which could substantially alleviate the symptoms of Parkinson's disease in clinical practice. However, ethical issues and tumor formation were limitations of its clinical application. Induced pluripotent stem cells can be acquired without sacrificing human embryos, which eliminates the huge ethical barriers of human stem cell therapy. Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons, without the need for intermediate proliferation states, thus avoiding issues of immune rejection and tumor formation. Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson's disease. However, there are also ethical concerns and the risk of tumor formation that need to be addressed. This review highlights the current application status of cell reprogramming in the treatment of Parkinson's disease, focusing on the use of induced pluripotent stem cells in cell replacement therapy, including preclinical animal models and progress in clinical research. The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson's disease, as well as the controversy surrounding in vivo reprogramming. These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder resulting from the loss of dopamine-producing neurons in the brain, causing motor symptoms like tremors and stiffness. Although current treatments like medication and deep brain stimulation can alleviate symptoms, they don't address the root cause of neuron loss. Therefore, cell replacement therapy emerges as a promising treatment strategy. However, the generation of engraftable dopaminergic (DA) cells in clinically relevant quantities is still a challenge. Recent advances in cell reprogramming technologies open up vast possibilities to produce patient-specific cells of a desired type in therapeutic quantities. The main cell reprogramming strategies involve the enforced expression of individual or sets of genes through viral transduction or transfection, or through small molecules, known as the chemical approach, which is a much easier and safer method. In our previous studies, using a small molecule approach (combinations of epigenetic modifiers and SMAD inhibitors such asDorsomorphin and SB431542), we have been able to generate DA progenitors from human mesenchymal stem cells (hMSCs). The aim of this study was to further improve the method for the generation of DA progenitors and to test their therapeutic effect in an animal model of Parkinson's. The results showed that the addition of an autophagy enhancer (AE) to our DA cell induction protocol further increased the yield of DA progenitor cells. The results also showed that DA progenitors transplanted into the mouse model of PD survived, integrated, and improved PD motor symptoms. These data suggest that chemically-produced DA cells can be very promising and safe cellular therapeutics for PD.

A breadth of preclinical studies now support the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, most cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such a format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with cryopreserved single-cell DA progenitors. Here, we used a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabling the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods, including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner using a Parkinsonian rat model. The survival of the cells was confirmed by post-mortem histological analysis, characterised by the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson's disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks after transplantation using cryopreserved 3D format.

Recent advances in human pluripotent stem cell (hPSC) technologies have prompted the emergence of new research fields and applications for human neurons and brain organoids. Brain organoids have gained attention as an in vitro model system that recapitulates the higher structure, cellular diversity and function of the brain to explore brain development, disease modeling, drug screening, and regenerative medicine. This progress has been accelerated by abundant interactions of brain organoid technology with various research fields. A cross-disciplinary approach with human brain organoid technology offers a higher-ordered advance for more accurately understanding the human brain. In this review, we summarize the status of neural induction in two- and three-dimensional culture systems from hPSCs and the modeling of neurodegenerative diseases using brain organoids. We also highlight the latest bioengineered technologies for the assembly of spatially higher-ordered neural tissues and prospects of brain organoid technology toward the understanding of the potential and abilities of the human brain.

Parkinson's disease (PD) is one of the most devastating neurological diseases; however, there is no effective cure yet. The availability of human induced pluripotent stem cells (iPSCs) provides unprecedented opportunities to understand the pathogenic mechanism and identification of new therapy for PD. Here a new model system of PD, including 2D human iPSC-derived midbrain dopaminergic (mDA) neurons, 3D iPSC-derived midbrain organoids (MOs) with cellular complexity, and more advanced microphysiological systems (MPS) with 3D organoids, is introduced. It is believed that successful integrations and applications of iPSC, organoid, and MPS technologies can bring new insight on PD's pathogenesis that will lead to more effective treatments for this debilitating disease.

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.

Parkinson's disease (PD) stands as the second most common neurodegenerative disorder after Alzheimer's disease, and its prevalence continues to rise with the aging global population. Central to the pathophysiology of PD is the specific degeneration of midbrain dopamine neurons (mDANs) in the substantia nigra. Consequently, cell replacement therapy (CRT) has emerged as a promising treatment approach, initially supported by various open-label clinical studies employing fetal ventral mesencephalic (fVM) cells. Despite the initial favorable results, fVM cell therapy has intrinsic and logistical limitations that hinder its transition to a standard treatment for PD. Recent efforts in the field of cell therapy have shifted its focus towards the utilization of human pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, to surmount existing challenges. However, regardless of the transplantable cell sources (e.g., xenogeneic, allogeneic, or autologous), the poor and variable survival of implanted dopamine cells remains a major obstacle. Emerging evidence highlights the pivotal role of host immune responses following transplantation in influencing the survival of implanted mDANs, underscoring an important area for further research. In this comprehensive review, building upon insights derived from previous fVM transplantation studies, we delve into the functional ramifications of host immune responses on the survival and efficacy of grafted dopamine cells. Furthermore, we explore potential strategic approaches to modulate the host immune response, ultimately aiming for optimal outcomes in future clinical applications of CRT for PD.

Depression poses a significant threat to global physical and mental health, impacting around 3.8% of the population with a rising incidence. Current treatment options primarily involve medication and psychological support, yet their effectiveness remains limited, contributing to high relapse rates. There is an urgent need for innovative and more efficacious treatment modalities. Stem cell therapy, a promising avenue in regenerative medicine for a spectrum of neurodegenerative conditions, has recently garnered attention for its potential application in depression. While much of this work remains preclinical, it has demonstrated considerable promise. Identified mechanisms underlying the antidepressant effects of stem cell therapy encompass the stimulation of neurotrophic factors, immune function modulation, and augmented monoamine levels. Nonetheless, these pathways and other undiscovered mechanisms necessitate further investigation. Depression fundamentally manifests as a neurodegenerative disorder. Given stem cell therapy's success in addressing a range of neurodegenerative pathologies, it opens the door to explore its application in depression treatment. This exploration may include repairing damaged nerves directly or indirectly and inhibiting neurotoxicity. Nevertheless, significant challenges must be overcome before stem cell therapies can be applied clinically. Successful resolution of these issues will ultimately determine the feasibility of incorporating stem cell therapies into the clinical landscape. This narrative review provides insights into the progress of research, potential avenues for exploration, and the prevailing challenges in the implementation of stem cell therapy for treatment of depression.

Human cellular models and their neuronal derivatives have afforded unprecedented advances in elucidating pathogenic mechanisms of neuropsychiatric diseases. Notwithstanding their indispensable contribution, animal models remain the benchmark in neurobiological research. In an attempt to harness the best of both worlds, researchers have increasingly relied on human/animal chimeras by xenografting human cells into the animal brain. Despite the unparalleled potential of xenografting approaches in the study of the human brain, literature resources that systematically examine their significance and advantages are surprisingly lacking. We fill this gap by providing a comprehensive account of brain diseases that were thus far subjected to all three modeling approaches (transgenic rodents, in vitro human lineages, human-animal xenografting) and provide a critical appraisal of the impact of xenografting approaches for advancing our understanding of those diseases and brain development. Next, we give our perspective on integrating xenografting modeling pipeline with recent cutting-edge technological advancements.

The translational stem cell research field has progressed immensely in the past decade. Development and refinement of differentiation protocols now allows the generation of a range of cell types, such as pancreatic β-cells and dopaminergic neurons, from human pluripotent stem cells (hPSCs) in an efficient and good manufacturing practice-compliant fashion. This has led to the initiation of several clinical trials using hPSC-derived cells to replace lost or dysfunctional cells, demonstrating evidence of both safety and efficacy. Here, we highlight successes from some of the hPSC-based trials reporting early signs of efficacy and discuss common challenges in clinical translation of cell therapies.

The Wnt/β-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of β-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/β-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

Induced pluripotent stem cells (iPSCs) hold a promising potential for rescuing dopaminergic neurons in therapy for Parkinson's disease (PD). This study clarifies a TREM2-dependent mechanism explaining the function of iPSC differentiation in neuronal repair of PD.

PD-related differentially expressed genes were screened by bioinformatics analyses and their expression was verified using RT-qPCR in nigral tissues of 6-OHDA-lesioned mice. Following ectopic expression and depletion experiments in iPSCs, cell differentiation into dopaminergic neurons as well as the expression of dopaminergic neuronal markers TH and DAT was measured. Stereotaxic injection of 6-OHDA was used to develop a mouse model of PD, which was injected with iPSC suspension overexpressing TREM2 to verify the effect of TREM2 on neuronal repair.

TREM2 was poorly expressed in the nigral tissues of 6-OHDA-lesioned mice. In the presence of TREM2 overexpression, the iPSCs showed increased expression of dopaminergic neuronal markers TH and DAT, which facilitated the differentiation of iPSCs into dopaminergic neurons. Mechanistic investigations indicated that TREM2 activated the TGF-β pathway and induced iPSC differentiation into dopaminergic neurons. In vivo data showed that iPSCs overexpressing TREM2 enhanced neuronal repair in 6-OHDA-lesioned mice.

This work identifies a mechanistic insight for TREM2-mediated TGF-β activation in the regulation of neuronal repair in PD and suggests novel strategies for neurodegenerative disorders.

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson's disease. Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson's disease. However, transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche. Here, we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells. These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion, effectively maintaining axonal integrity in vitro. Importantly, midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts. Overall, our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and loss of DA transmission in the striatum, thus making cell transplantation an effective treatment strategy. Here, we develop a cellular therapy based on induced pluripotent stem cell (iPSC)-derived midbrain organoids. By transplanting midbrain organoid cells into the striatum region of a 6-OHDA-lesioned PD mouse model, we found that the transplanted cells survived and highly efficiently differentiated into DA neurons. Further, using a dopamine sensor, we observed that the differentiated human DA neurons could efficiently release dopamine and were integrated into the neural network of the PD mice. Moreover, starting from four weeks after transplantation, the motor function of the transplanted mice could be significantly improved. Therefore, cell therapy based on iPSC-derived midbrain organoids can be a potential strategy for the clinical treatment of PD.

Parkinson's disease is a progressive neurodegenerative disorder, which is mainly characterized by unintended or uncontrollable body movements. Pathophysiologically, disturbances in the neurotransmission system of the brain like dopaminergic system and synaptic dysfunction are classified as top-rated causes of the onset of Parkinson's disease, which symptoms can be different according to the involvement of neurotransmission system type and the effect of the disease on the motor and non-motor systems. Although some pharmacological and non-pharmacological approaches have been applied to control and slow down the progression of the disease, a definitive cure has not yet been discovered. One of the factors involved in this issue is the lack of appropriate laboratory models to investigate the pathological mechanisms involved in the disease as well as various aspects of candidate drugs, which ultimately leads to the failure of drug discovery and development pipelines. To deal with these challenges, the application of stem cells, especially cellular reprogramming of somatic cells to human pluripotent stem cells, also known as induced pluripotent stem cells, has been able to promise a new chapter in the modeling of Parkinson's disease. Induced pluripotent stem cells have the stemness capability; therefore, they can differentiate into any type of cell such as nerve cells. Also, since these cells are obtained from the reprogramming of somatic cells in the patient's body, they maintain the patient's genetic content, which can play an important role in increasing the quality of disease modeling and the validity of the results of laboratory studies. Therefore, the procedure for modeling induced pluripotent stem cells for Parkinson's disease is explained in this chapter.

Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits, however, relies on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson's disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9-assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single-cell RNA sequencing of iPSC-derived neural cultures confirm developmental recapitulation of the human fetal midbrain and high-quality midbrain cells. By modelling Parkinson's disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP+), we showed a preferential reduction of BFP+ cells, a finding demonstrated independently by cell death assays and single-cell transcriptomic analysis of MPP+ treated neural cultures. Together, these results highlight the importance of disease-relevant cell types in stem cell modelling.

Ventral midbrain (VM) dopaminergic progenitor cells derived from human pluripotent stem cells have the potential to replace endogenously lost dopamine neurons and are currently in preclinical and clinical development for treatment of Parkinson's Disease (PD). However, one main challenge in the quality control of the cells is that rostral and caudal VM progenitors are extremely similar transcriptionally though only the caudal VM cells give rise to dopaminergic (DA) neurons with functionality relevant for cell replacement in PD. Therefore, it is critical to develop assays which can rapidly and reliably discriminate rostral from caudal VM cells during clinical manufacturing.

We performed shotgun proteomics on cell culture supernatants from rostral and caudal VM progenitor cells to search for novel secreted biomarkers specific to DA progenitors from the caudal VM. Key hits were validated by qRT-PCR and ELISA.

We identified and validated novel secreted markers enriched in caudal VM progenitor cultures (CPE, LGI1 and PDGFC), and found these markers to correlate strongly with the expression of EN1, which is a predictive marker for successful graft outcome in DA cell transplantation products. Other markers (CNTN2 and CORIN) were found to conversely be enriched in the non-dopaminergic rostral VM cultures. Key novel ELISA markers were further validated on supernatant samples from GMP-manufactured caudal VM batches.

As a non-invasive in-process quality control test for predicting correctly patterned batches of caudal VM DA cells during clinical manufacturing, we propose a dual ELISA panel measuring LGI1/CORIN ratios around day 16 of differentiation.

The induced pluripotent stem cells (iPSCs) are able to differentiate into dopaminergic neurons and execute the therapeutic effects for Parkinson's disease (PD). Here, we established a animal model of PD in Lanyu pigs by injecting 5 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). Next, the porcine iPSC-like cells (piPSC-like cells) were differentiated into D18 neuronal progenitors (D18 NPs) that were transplanted into the striatum to evaluate their therapeutic effects of PD. We showed that after 8 weeks of cell transplantation, the behavior score was significantly ameliorated and fully recovered at the 14th week of cell transplantation. The number of dopaminergic neurons was also significantly improved at the end of the experiment although the number was still about 50% lower than that in the control group. Our findings suggest that piPSC-like cell-derived D18 NPs exhibit a potential for the treatment of PD in the Lanyu pig model.

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.

Here, we present a protocol for the generation of functional midbrain dopaminergic (mDA) neurons from human embryonic stem cells (hESCs), which mimics the development of the human ventral midbrain. We describe steps for hESC proliferation, induction of mDA progenitors, freezing stocks of mDA progenitors as an intermediate starting point to reduce the time to make mDA neurons, and maturation of mDA neurons. The entire protocol is feeder-free and uses chemically defined materials. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2023).1.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.