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.

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.

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.

Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells.

We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.

A more efficient method to generate integration-free human iPS cells.

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

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.

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease.

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)-derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC-derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinson's disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42-day culture with brain-derived neurotrophic factor/glial cell line-derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain.

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.

Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells.

The balance of bone morphogenic protein (BMP), transforming growth factor-ß (TGFß)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small-molecule inhibitors of BMP (Dorsomorphin) and TGFß/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder-free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine.

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.

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells.

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.

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.

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.

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.

[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.

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.

Meningeal cells induce dopaminergic neurons from embryonic stem cells.

Neural induction of midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells can be achieved by culturing them on a bone marrow-derived stromal cell line, PA6, which possesses stromal cell-derived inducing activity (SDIA). The mechanism of SDIA is unknown, but clinical application of ES cell transplantation requires the use of defined factors for DA neuron induction. Here, we demonstrate that meningeal cells harvested from the developing dura can induce DA neuron differentiation from mouse and human ES cells, as assessed by midbrain DA marker expression and secretion of DA in response to potassium stimuli. Intriguingly, the inductive strength of meningeal cells depends on their developmental stage, with those harvested from embryonic day 18 embryos showing the highest activity. Among six soluble factors known to be involved in DA neuron differentiation, only Wnt-5a and transforming growth factor-beta3 were expressed by both meningeal and PA6 cells, and the expression of Wnt-5a correlated with the DA neuron induction activity of these cells. Furthermore, the induction of DA neuron differentiation by PA6 cell-conditioned medium was reversed by addition of a Wnt-5a neutralizing antibody, whereas recombinant Wnt-5a promoted DA neuron induction when cells were cultured on Matrigel. These results indicate that meningeal cells can be used as feeders to induce DA neurons from ES cells, and that Wnt-5a plays an important role in DA neuron induction by SDIA.

Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model.

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.