Viral delivery of L1CAM promotes axonal extensions by embryonic cerebral grafts in mouse brain.

Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy.

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.

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.

Serum CA 125 Level after Neoadjuvant Chemotherapy is Predictive of Prognosis and Debulking Surgery Outcomes in Advanced Epithelial Ovarian Cancer.

Recently, neoadjuvant chemotherapy followed by interval debulking surgery (NACT-IDS) has been recommended for selected patients with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV disease and bulky tumors. The aim of this study was to evaluate associations between post-NACT serum CA 125 levels, surgical outcomes, and clinical outcomes in patients with advanced epithelial ovarian cancer. We retrospectively analyzed 107 patients with FIGO stage III or IV ovarian cancer who were treated with NACT-IDS at the Gynecology Department of Kanagawa Cancer Center between January 2001 and December 2012. Serum CA 125 levels after NACT were significantly lower in the complete/optimal IDS group compared to the suboptimal IDS group (mean±standard deviation: 48.1±27.6 vs. 346.5±295.2 U/mL, p<0.01). Patients with low preoperative CA 125 levels (<35 U/mL) had a higher probability of optimal IDS (78.1±41.9% vs. 33.3±19.2%, p<0.01) and longer progression-free survival (mean±standard deviation: 30.4±14.3 months vs. 21.3±7.3 months, p<0.05) than patients with high CA 125 levels (>100 U/mL). Patients with low CA 125 levels (<35 U/mL) had a higher probability of complete/optimal IDS and longer progression-free survival compared to patients with high CA 125 levels (>100 U/mL).

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.

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.

Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1.

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.

Fail-Safe Therapy by Gamma-Ray Irradiation Against Tumor Formation by Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors.

Cell replacement therapy holds great promise for Parkinson's disease (PD), but residual undifferentiated cells and immature neural progenitors in the therapy may cause tumor formation. Although cell sorting could effectively exclude these proliferative cells, from the viewpoint of clinical application, there exists no adequate coping strategy in the case of their contamination. In this study, we analyzed a component of proliferative cells in the grafts of human-induced pluripotent stem cell-derived neural progenitors and investigated the effect of radiation therapy on tumor formation. In our differentiating protocol, analyses of neural progenitors (day 19) revealed that the proliferating cells expressed early neural markers (SOX1, PAX6) or a dopaminergic neuron progenitor marker (FOXA2). When grafted into the rat striatum, these immature neurons gradually became postmitotic in the brain, and the rosette structures disappeared at 14 weeks. However, at 4-8 weeks, the SOX1(+)PAX6(+) cells formed rosette structures in the grafts, suggesting their tumorigenic potential. Therefore, to develop a fail-safe therapy against tumor formation, we investigated the effect of radiation therapy. At 4 weeks posttransplantation, when KI67(+) cells comprised the highest ratio, radiation therapy with (137)Cs Gammacell Exactor for tumor-bearing immunodeficient rats showed a significant decrease in graft volume and percentage of SOX1(+)KI67(+) cells in the graft, thus demonstrating the preventive effect of gamma-ray irradiation against tumorigenicity. These results give us critical criteria for the safety of future cell replacement therapy for PD.

Estradiol Facilitates Functional Integration of iPSC-Derived Dopaminergic Neurons into Striatal Neuronal Circuits via Activation of Integrin α5ß1.

For cell transplantation therapy for Parkinson's disease (PD) to be realized, the grafted neurons should be integrated into the host neuronal circuit to restore the lost neuronal function. Here, using wheat-germ agglutinin-based transsynaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. In addition, we found that integrin α5ß1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5ß1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administered rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.

Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities.

BACKGROUND: In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. METHODS: We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. RESULTS: Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. CONCLUSIONS: The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading.

A phase II study of paclitaxel and carboplatin with a biweekly schedule in patients with epithelial ovarian cancer: Gynecologic Cancer Network trial.

AIM: The objective of this multicenter phase II study was to evaluate the effects of biweekly paclitaxel and carboplatin combination chemotherapy on response rate and toxicities in patients with epithelial ovarian cancer. PATIENTS AND METHODS: Patients with International Federation of Gynecology and Obstetrics stage II to IV ovarian cancer received paclitaxel at a dose of 120 mg/m(2) and carboplatin at an area under the curve of 3 mg/mL per minute every 2 weeks for 8 or more cycles. Inclusion criteria included an Eastern Cooperative Oncology Group performance status of 0 to 2 and no previous chemotherapy. Informed consent was obtained from each patient before the start of treatment. RESULTS: From March 2003 through July 2009, 42 patients from 5 institutions were eligible to be evaluated for response and toxicity. The median age was 60.5 years (age range, 34-81 years). The International Federation of Gynecology and Obstetrics stage was stage II in 3 patients, stage III in 31 patients, and stage IV in 8 patients. The response rate was 66.7% (95% confidence interval: 50.5%-80.4%). Sixty-nine percent (29 of 42) of patients received 8 or more cycles of chemotherapy. The median progression-free survival was 18.5 months, and overall survival was 59.1 months. The most common grade 3 or 4 hematological toxicity was neutropenia (61.0%). No patients had grade 3 or 4 thrombocytopenia. The most common grade 3 nonhematological toxicities were neuropathy (4.9%) and nausea (2.4%). CONCLUSION: Paclitaxel combined with carboplatin using a biweekly schedule is a safe and effective chemotherapy regimen for patients with epithelial ovarian cancer. Our results suggest that a biweekly schedule is well tolerated and is less toxic than a triweekly schedule.

Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation.

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson's disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN(+) cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN(+) cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN(+) cells in a NURR1(+) cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.

In order to apply human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Since the conventional culture system has some issues that need to be addressed to achieve this goal, we developed a novel culture system. We found that recombinant laminin-511 E8 fragments are useful matrices for maintaining hESCs and hiPSCs when used in combination with a completely xeno-free (Xf) medium, StemFit™. Using this system, hESCs and hiPSCs can be easily and stably passaged by dissociating the cells into single cells for long periods, without any karyotype abnormalities. Human iPSCs could be generated under feeder-free (Ff) and Xf culture systems from human primary fibroblasts and blood cells, and they possessed differentiation abilities. These results indicate that hiPSCs can be generated and maintained under this novel Ff and Xf culture system.

Direct comparison of autologous and allogeneic transplantation of iPSC-derived neural cells in the brain of a non-human primate.

Induced pluripotent stem cells (iPSCs) provide the potential for autologous transplantation using cells derived from a patient's own cells. However, the immunogenicity of iPSCs or their derivatives has been a matter of controversy, and up to now there has been no direct comparison of autologous and allogeneic transplantation in the brains of humans or nonhuman primates. Here, using nonhuman primates, we found that the autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain. In contrast, the allografts caused an acquired immune response with the activation of microglia (IBA-1(+)/MHC class II(+)) and the infiltration of leukocytes (CD45(+)/CD3(+)). Consequently, a higher number of dopaminergic neurons survived in the autografts. Our results suggest that the autologous transplantation of iPSC-derived neural cells is advantageous for minimizing the immune response in the brain compared with allogeneic grafts.

Neural induction with a dopaminergic phenotype from human pluripotent stem cells through a feeder-free floating aggregation culture.

Pluripotent stem cells are promising potential sources for cell replacement therapy and are useful research tools for exploring disease mechanisms. Neural cells are one of the cell types that have been most efficiently differentiated through several established protocols. This chapter describes the feeder-free floating aggregation culture system for the induction of dopaminergic neurons. This method is simple and highly efficient for the production of dopaminergic neurons. It has several advantages for application in clinical usage in comparison to the other protocols using either feeder cells or Matrigel.

Functional recovery of the murine brain ischemia model using human induced pluripotent stem cell-derived telencephalic progenitors.

Induced pluripotent stem (iPS) cells possess the properties of self-renewal and pluripotency, similar to embryonic stem cells. They are a good candidate as a source of suitable cells for cell replacement therapy. In this study, we transplanted human iPS cell-derived neural progenitors into an ischemic mouse brain. Human iPS cells were differentiated into neuronal progenitors by serum-free culture of embryoid body-like aggregates (SFEBs). Focal cerebral ischemia was induced by occluding the middle cerebral artery using the intraluminal filament technique. Donor cells were transplanted into the ischemic lateral striatum 1 week after ischemia induction. Cells survived at the transplantation site, with migration of a proportion of cells along the external capsule and corpus callosum. Cells that were positive for the basal telencephalon marker, Nkx2.1, migrated into the basal part of the telencephalon. The pallial telencephalon marker, Emx1, was detected in cells that had migrated into the pallial part of the telencephalon. SFEBs differentiated into various types of neurons, and a retrograde tracer labeling study showed that differentiated cells integrated into host neural circuitry. Behavioral recovery was significantly enhanced in the transplanted group. Our results suggest that human iPS cell-derived neuronal progenitors survive and migrate in the ischemic brain, and contribute toward functional recovery via neural circuit reconstitution.

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.

Survival of human induced pluripotent stem cell-derived midbrain dopaminergic neurons in the brain of a primate model of Parkinson's disease.

Before induced pluripotent stem cells (iPSCs) can be used to treat neurologic diseases, human iPSC-derived neural cells must be analyzed in the primate brain. In fact, although mouse and human iPSCs have been used to generate dopaminergic (DA) neurons that are beneficial in rat models of Parkinson's disease (PD), human iPSC-derived neural progenitor cells (NPCs) have not been examined in primate brains. Here, we generated NPCs at different stages of predifferentiation using a feeder-free culture method, and grafted them into the brains of a monkey PD model and NOD-SCID mice. Magnetic resonance imaging (MRI), positron emission tomography (PET), immunocytochemistry, and behavioral analyses revealed that NPCs pretreated with Sonic hedgehog and fibroblast growth factor-8 followed by glial cell-derived neurotrophic factor, brain-derived neurotrophic factor, ascorbic acid, and dibutyryl cyclic AMP resulted in smaller grafts than those without these treatments, and survived as DA neurons in a monkey brain as long as six months. Thus, for the first time, we describe a feeder-free neural differentiation method from human iPSCs and an evaluation system that can be used to assess monkey PD models.

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.