Dopaminergic Cell Replacement for Parkinson's Disease: Addressing the Intracranial Delivery Hurdle.

Parkinson's disease (PD) is an increasingly prevalent neurological disorder, affecting more than 8.5 million individuals worldwide. α-Synucleinopathy in PD is considered to cause dopaminergic neuronal loss in the substantia nigra, resulting in characteristic motor dysfunction that is the target for current medical and surgical therapies. Standard treatment for PD has remained unchanged for several decades and does not alter disease progression. Furthermore, symptomatic therapies for PD are limited by issues surrounding long-term efficacy and side effects. Cell replacement therapy (CRT) presents an alternative approach that has the potential to restore striatal dopaminergic input and ameliorate debilitating motor symptoms in PD. Despite promising pre-clinical data, CRT has demonstrated mixed success clinically. Recent advances in graft biology have renewed interest in the field, resulting in several worldwide ongoing clinical trials. However, factors surrounding the effective neurosurgical delivery of cell grafts have remained under-studied, despite their significant potential to influence therapeutic outcomes. Here, we focus on the key neurosurgical factors to consider for the clinical translation of CRT. We review the instruments that have been used for cell graft delivery, highlighting current features and limitations, while discussing how future devices could address these challenges. Finally, we review other novel developments that may enhance graft accessibility, delivery, and efficacy. Challenges surrounding neurosurgical delivery may critically contribute to the success of CRT, so it is crucial that we address these issues to ensure that CRT does not falter at the final hurdle.

Cell therapy for Parkinson's disease with induced pluripotent stem cells.

Parkinson's disease (PD) is the second most common neurodegenerative disease and a prime target of cell therapies. In fact, aborted fetal tissue has been used as donor material for such therapies since the 1980s. These cell therapies, however, suffer from several problems, such as a short supply of donor materials, quality instability of the tissues, and ethical restrictions. The advancement of stem cell technologies has enabled the production of donor cells from pluripotent stem cells with unlimited scale, stable quality, and less ethical problems. Several research groups have established protocols to induce dopamine neural progenitors from pluripotent stem cells in a clinically compatible manner and confirmed efficacy and safety in non-clinical studies. Based on the results from these non-clinical studies, several clinical trials of pluripotent stem cell-based therapies for PD have begun. In the context of immune rejection, there are several modes of stem cell-based therapies: autologous transplantation, allogeneic transplantation without human leukocyte antigen-matching, and allogeneic transplantation with matching. In this mini-review, several practical points of stem cell-based therapies for PD are discussed.

Pretreatment with Perlecan-Conjugated Laminin-E8 Fragment Enhances Maturation of Grafted Dopaminergic Progenitors in Parkinson's Disease Model.

The therapeutic effect of a cell replacement therapy for Parkinson's disease (PD) depends on the proper maturation of grafted dopaminergic (DA) neurons and their functional innervation in the host brain. In the brain, laminin, an extracellular matrix protein, regulates signaling pathways for the survival and development of neurons by interacting with integrins. The heparan sulfate (HS) chain binds mildly to various neurotrophic factors and regulates their intracellular signaling. Perlecan-conjugated laminin 511/521-E8 fragments (p511/p521) were designed to contain an integrin-binding site and HS chains. Here we examined the effect of treating DA progenitors with p511/p521 prior to transplantation in rodent PD models. In vitro and in vivo experiments showed that p511/p521 treatment enhanced the maturation and neurite extension of the grafted DA progenitors by activating RAS-ERK1/2 signaling. This strategy will contribute to an efficient cell replacement therapy for PD in the future.

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application.

BACKGROUND: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson's disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. OBJECTIVE: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. METHODS: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. RESULTS: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. CONCLUSION: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Evading the Immune System: Immune Modulation and Immune Matching in Cell Replacement Therapies for Parkinson's Disease.

Stem cell-based therapies for Parkinson's disease are now being applied clinically. Notably, studies have shown that controlling the graft-induced immune response improves the results. In this mini-review, we concisely summarize current approaches used for this control. We focus on four modes of stem cell-based therapies: autologous transplantation, allogeneic transplantation with human leukocyte antigen-matching and allogeneic transplantation without, and finally the application of "universal" pluripotent stem cells. We also discuss immuno-suppressive treatments and the monitoring of immune reactions in the brain.

Axonal Extensions along Corticospinal Tracts from Transplanted Human Cerebral Organoids.

The reconstruction of lost neural circuits by cell replacement is a possible treatment for neurological deficits after cerebral cortex injury. Cerebral organoids can be a novel source for cell transplantation, but because the cellular composition of the organoids changes along the time course of the development, it remains unclear which developmental stage of the organoids is most suitable for reconstructing the corticospinal tract. Here, we transplanted human embryonic stem cell-derived cerebral organoids at 6 or 10 weeks after differentiation (6w- or 10w-organoids) into mouse cerebral cortices. 6w-organoids extended more axons along the corticospinal tract but caused graft overgrowth with a higher percentage of proliferative cells. Axonal extensions from 10w-organoids were smaller in number but were enhanced when the organoids were grafted 1 week after brain injury. Finally, 10w-organoids extended axons in cynomolgus monkey brains. These results contribute to the development of a cell-replacement therapy for brain injury and stroke.

Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson's disease.

Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.

Exercise Promotes Neurite Extensions from Grafted Dopaminergic Neurons in the Direction of the Dorsolateral Striatum in Parkinson's Disease Model Rats.

BACKGROUND: Cell transplantation is expected to be a promising treatment for Parkinson's disease (PD), in which re-innervation of the host striatum by grafted dopamine (DA) neurons is essential. In particular, the dorsolateral part of the striatum is important because it is the target of midbrain A9 DA neurons, which are degenerated in PD pathology. The effect of exercise on the survival and maturation of grafted neurons has been reported in several neurological disease models, but never in PD models. OBJECTIVE: We investigated how exercise influences cell transplantation for PD, especially from the viewpoint of cell survival and neurite extensions. METHODS: Ventral mesencephalic neurons from embryonic (E12.5) rats were transplanted into the striatum of adult 6-OHDA-lesioned rats. The host rats then underwent treadmill training as exercise after the transplantation. Six weeks after the transplantation, they were sacrificed, and the grafts in the striatum were analyzed. RESULTS: The addition of exercise post-transplantation significantly increased the number of surviving DA neurons. Moreover, it promoted neurite extensions from the graft toward the dorsolateral part of the striatum. CONCLUSIONS: This study indicates a beneficial effect of exercise after cell transplantation in PD.

Induction of the germ cell fate from pluripotent stem cells in cynomolgus monkeys†.

In vitro reconstitution of germ-cell development from pluripotent stem cells (PSCs) has created key opportunities to explore the fundamental mechanisms underlying germ-cell development, particularly in mice and humans. Importantly, such investigations have clarified critical species differences in the mechanisms regulating mouse and human germ-cell development, highlighting the necessity of establishing an in vitro germ-cell development system in other mammals, such as non-human primates. Here, we show that multiple lines of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in cynomolgus monkeys (Macaca fascicularis; cy) can be maintained stably in an undifferentiated state under a defined condition with an inhibitor for WNT signaling, and such PSCs are induced efficiently into primordial germ cell-like cells (PGCLCs) bearing a transcriptome similar to early cyPGCs. Interestingly, the induction kinetics of cyPGCLCs from cyPSCs is faster than that of human (h) PGCLCs from hPSCs, and while the transcriptome dynamics during cyPGCLC induction is relatively similar to that during hPGCLC induction, it is substantially divergent from that during mouse (m) PGCLC induction. Our findings delineate common as well as species-specific traits for PGC specification, creating a foundation for parallel investigations into the mechanism for germ-cell development in mice, monkeys, and humans.

MicroRNA-Based Separation of Cortico-Fugal Projection Neuron-Like Cells Derived From Embryonic Stem Cells.

The purification of pluripotent stem cell-derived cortico-fugal projection neurons (PSC-CFuPNs) is useful for disease modeling and cell therapies related to the dysfunction of cortical motor neurons, such as amyotrophic lateral sclerosis (ALS) or stroke. However, no CFuPN-specific surface markers for the purification are known. Recently, microRNAs (miRNAs) have been reported as alternatives to surface markers. Here, we investigated this possibility by applying the miRNA switch, an mRNA technology, to enrich PSC-CFuPNs. An array study of miRNAs in mouse fetal brain tissue revealed that CFuPNs highly express miRNA-124-3p at E14.5 and E16.5. In response, we designed a miRNA switched that responds to miRNA-124-3p and applied it to mouse embryonic stem cell (ESC)-derived cortical neurons. Flow cytometry and quantitative polymerase chain reaction (qPCR) analyses showed the miRNA-124-3p switch enriched CFuPN-like cells from this population. Immunocytechemical analysis confirmed vGlut1/Emx1/Bcl11b triple positive CFuPN-like cells were increased from 6.5 to 42%. Thus, our miRNA-124-3p switch can uniquely enrich live CFuPN-like cells from mouse ESC-derived cortical neurons.

[Cell therapy for Parkinson's disease with induced pluripotent stem cells].

Cell therapy for Parkinson's disease has a history of being applied clinically with aborted embryos as donor source. Efficacy of the therapy under the appropriate condition has been reported. Based on this experience and the advancement of stem cell technology, clinical trials of cell therapy with embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are going to start soon in several countries. In Japan a physician-initiated clinical trial of iPSC-based therapy for Parkinson's disease has launched since 2018. This trial adopts allogeneic transplantation with a cell line from iPSC stock. This article discusses patient selection, procedure, and risk of the therapy. It also introduces the world's current situation of the cell therapy for Parkinson's disease.

Efficacy of constant long-term delivery of YM-58483 for the treatment of rheumatoid arthritis.

The aim of this study was to investigate the efficacy and safety of YM-58483, a small molecular antagonist of Ca2+ release-activated Ca2+ (CRAC) channels, for the treatment of rheumatoid arthritis (RA), in vivo and ex vivo. YM-58483 was continuously injected subcutaneously in a collagen-induced arthritis (CIA) mouS.E.M.odel using an implanted osmotic pump. The severity of CIA was evaluated using the following parameters: body weight, hind paw volume, clinical score, histological analysis, cytokine levels, Ca2+ influx, and specific IgG production. The efficacy of long-term application of YM-58483 was also verified ex vivo in RA patient-derived peripheral blood monocytes. Assessment of the clinical severity of CIA, cytokine profile in serum and joint protein extracts, and specific IgG production showed that continuous application of YM-58483 suppressed synovial inflammation by inhibiting immune cell activity. Chemical screening and hepatography indicated that long-term subcutaneous delivery of YM-58483 was safer than oral administration for systemic application. Moreover, constant preincubation with YM-58483 at an IC50 of 0.1-1 nM altered proinflammatory cytokine production ex vivo in peripheral T cells derived from RA patients. Our findings suggest that continuous long-term application of appropriate CRAC inhibitors such as YM-58483 is a potential therapeutic strategy for global immunosuppression in RA.

Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

MHC matching improves engraftment of iPSC-derived neurons in non-human primates.

The banking of human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cells (iPSCs) is considered a future clinical strategy for HLA-matched cell transplantation to reduce immunological graft rejection. Here we show the efficacy of major histocompatibility complex (MHC)-matched allogeneic neural cell grafting in the brain, which is considered a less immune-responsive tissue, using iPSCs derived from an MHC homozygous cynomolgus macaque. Positron emission tomography imaging reveals neuroinflammation associated with an immune response against MHC-mismatched grafted cells. Immunohistological analyses reveal that MHC-matching reduces the immune response by suppressing the accumulation of microglia (Iba-1+) and lymphocytes (CD45+) into the grafts. Consequently, MHC-matching increases the survival of grafted dopamine neurons (tyrosine hydroxylase: TH+). The effect of an immunosuppressant, Tacrolimus, is also confirmed in the same experimental setting. Our results demonstrate the rationale for MHC-matching in neural cell grafting to the brain and its feasibility in a clinical setting.Major histocompatibility complex (MHC) matching improves graft survival rates after organ transplantation. Here the authors show that in macaques, MHC-matched iPSC-derived neurons provide better engraftment in the brain, with a lower immune response and higher survival of the transplanted neurons.

Enhanced Axonal Extension of Subcortical Projection Neurons Isolated from Murine Embryonic Cortex using Neuropilin-1.

The cerebral cortical tissue of murine embryo and pluripotent stem cell (PSC)-derived neurons can survive in the brain and extend axons to the spinal cord. For efficient cell integration to the corticospinal tract (CST) after transplantation, the induction or selection of cortical motor neurons is important. However, precise information about the appropriate cell population remains unclear. To address this issue, we isolated cells expressing Neuropilin-1 (NRP1), a major axon guidance molecule receptor during the early developmental stage, from E14.5 mouse embryonic frontal cortex by fluorescence-activated cell sorting. Aggregates of NRP1+ cells gradually expressed subcortical projection neuron markers, Ctip2 and VGluT1, and axon guidance molecule receptors, Robo1 and deleted in colorectal calcinoma (Dcc), in vitro, suggesting that they contained early-stage subcortical projection neurons. We transplanted NRP1+ cells into the frontal cortex of P2 neonatal mice. Compared with grafts derived from NRP1- or unsorted cells, those derived from NRP1+ cells extended a larger number of axons to the spinal cord along the CST. Our data suggest that sorting NRP1+ cells from the embryonic cerebral cortex enriches subcortical projection neurons to reconstruct the CST.

Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multi-system disease caused by expanded CTG repeats in dystrophia myotonica protein kinase (DMPK). The expanded CTG repeats are unstable and can increase the length of the gene with age, which worsens the symptoms. In order to establish a human stem cell system suitable for the investigation of repeat instability, DM1 patient-derived iPSCs were generated and differentiated into three cell types commonly affected in DM1, namely cardiomyocytes, neurons and myocytes. Then we precisely analysed the CTG repeat lengths in these cells. Our DM1-iPSCs showed a gradual lengthening of CTG repeats with unchanged repeat distribution in all cell lines depending on the passage numbers of undifferentiated cells. However, the average CTG repeat length did not change significantly after differentiation into different somatic cell types. We also evaluated the chromatin accessibility in DM1-iPSCs using ATAC-seq. The chromatin status in DM1 cardiomyocytes was closed at the DMPK locus as well as at SIX5 and its promoter region, whereas it was open in control, suggesting that the epigenetic modifications may be related to the CTG repeat expansion in DM1. These findings may help clarify the role of repeat instability in the CTG repeat expansion in DM1.

Idiopathic Parkinson's disease patient-derived induced pluripotent stem cells function as midbrain dopaminergic neurons in rodent brains.

Patient-specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinson's disease (PD) patients, however, their vulnerability and the transmission of pathological α-Synuclein are possible drawbacks that may prevent PD-specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC-derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α-Synuclein transgenic mice, PDiPSC-derived DA neurons did not cause pathological α-Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD. © 2017 Wiley Periodicals, Inc.

MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells.

The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.

Cell Therapy for Parkinson's Disease.

In Parkinson's disease (PD), dopamine neurons in the substantia nigra are degenerated and lost. Cell therapy for PD replaces the lost dopamine neurons by transplanting donor dopamine neural progenitor cells. Cell therapy for PD has been performed in the clinic since the 1980s and uses donor cells from the mesencephalon of aborted embryos. Regenerative medicine for PD using induced pluripotent stem (iPS) cell technology is drawing attention, because it offers a limitless and more advantageous source of donor cells than aborted embryos.