Antiviral efficacy against and replicative fitness of an XBB.1.9.1 clinical isolate.

The emergence and spread of new SARS-CoV-2 variants with mutations in the spike protein, such as the XBB.1.5 and XBB.1.9.1 sublineages, raise concerns about the efficacy of current COVID-19 vaccines and therapeutic monoclonal antibodies (mAbs). In this study, none of the mAbs we tested neutralized XBB.1.9.1 or XBB.1.5, even at the highest concentration used. We also found that the bivalent mRNA vaccine could enhance humoral immunity against XBB.1.9.1, but that XBB.1.9.1 and XBB.1.5 still evaded humoral immunity induced by vaccination or infection. Moreover, the susceptibility of XBB.1.9.1 to remdesivir, molnupiravir, nirmatrelvir, and ensitrelvir was similar to that of the ancestral strain and the XBB.1.5 isolate in vitro. Finally, we found the replicative fitness of XBB.1.9.1 to be similar to that of XBB.1.5 in hamsters. Our results suggest that XBB.1.9.1 and XBB.1.5 have similar antigenicity and replicative ability, and that the currently available COVID-19 antivirals remain effective against XBB.1.9.1.

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.

Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level.

Chronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.

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.

Zonisamide promotes survival of human-induced pluripotent stem cell-derived dopaminergic neurons in the striatum of female rats.

The transplantation of dopaminergic (DA) progenitors derived from pluripotent stem cells improves the behavior of Parkinson's disease model animals. However, the survival of DA progenitors is low, and the final yield of DA neurons is only approximately 0.3%-2% the number of transplanted cells. Zonisamide (ZNS) increases the number of survived DA neurons upon the transplantation of mouse-induced pluripotent stem (iPS) cell-derived DA progenitors in the rat striatum. In this study, we induced DA progenitors from human iPS cells and transplanted them into the striatum of female rats with daily administration of ZNS. The number of survived DA neurons was evaluated 1 and 4 months after transplantation by immunohistochemistry, which revealed that the number of survived DA neurons was significantly increased with the administration of ZNS. To assess the mechanism of action of ZNS, we performed a gene expression analysis to compare the gene expression profiles in striatum treated with or without ZNS. The analysis revealed that the expression of SLIT-and NTRK-like protein 6 (SLITRK6) was upregulated in rat striatum treated with ZNS. In conclusion, ZNS promotes the survival of DA neurons after the transplantation of human-iPS cell-derived DA progenitors in the rat striatum. SLITRK6 is suggested to be involved in this supportive effect of ZNS by modulating the environment of the host brain.

Foxo in T Cells Regulates Thermogenic Program through Ccr4/Ccl22 Axis.

Crosstalk between immunity and the thermogenic program has provided insight into metabolic energy regulation. Here, we generated thermogenic program-accelerating mice (T-QKO), in which Foxo1 is knockout and Foxo3 is hetero-knockout in CD4+ T cells. T-QKO exhibit lean phenotype under HFD due to increased energy expenditure. Cold exposure significantly increased expression of the thermogenic genes (Ppargc1a and Ucp1), Th2 cytokines (Il4 and Il13), and Th2 marker gene (Gata3) in subcutaneous adipose tissue (SC) of T-QKO. Furthermore, Ccr4 expression was significantly increased in Th2 cells of T-QKO, and cold exposure induced Ccl22 expression in SC, leading to increased accumulation of Th2 cell population in SC of T-QKO. These data reveal a mechanism by which cold exposure induces selective recruitment of Th2 cells into SC, leading to regulation of energy expenditure by generating beige adipocyte and suggest that inhibition of Foxo in T cells may support a strategy to prevent and treat obesity.

Therapeutic effects of combined cell transplantation and locomotor training in rats with brain injury.

Cell-based therapies are attracting attention as alternative therapeutic options for brain damage. In this study, we investigated the therapeutic effect of a combined therapy of cell transplantation and locomotor training by evaluating the neuronal connectivity. We transplanted neural cells derived from the frontal cortex of E14.5 GFP-expressing mice into the frontal lobe of 3-week-old rats with brain injury, followed by treadmill training (TMT) for 14 days. In the TMT(-) group, graft-derived neurites were observed only in the striatum and internal capsule. In contrast, in the TMT(+) group, they were observed in the striatum, internal capsule, and the cerebral peduncle and spinal cord. The length of the longest neurite was significantly longer in the TMT(+) group than in the TMT(-) group. In the TMT(+) group, Synaptophysin+ vesicles on the neuronal fibers around the ipsilateral red nucleus were found, suggesting that neuronal fibers from the grafted cells formed synapses with the host neurons. A functional analysis of motor recovery using the foot fault test showed that, 1 week after the transplantation, the recovery was significantly better in the cell transplantation and TMT group than the cell transplantation only group. The percentage of cells expressing C-FOS was increased in the grafts in the TMT(+) group. In conclusion, TMT promoted neurite extensions from the grafted neural cells, and the combined therapy of cell transplantation and locomotor training might have the potential to promote the functional recovery of rats with brain injury compared to cell transplantation alone.

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.

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.

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.

Colonic Pro-inflammatory Macrophages Cause Insulin Resistance in an Intestinal Ccl2/Ccr2-Dependent Manner.

High-fat diet (HFD) induces low-grade chronic inflammation and insulin resistance. However, little is known about the mechanism underlying HFD-induced chronic inflammation in peripheral insulin-responsive tissues. Here, we show that colonic pro-inflammatory macrophages regulate insulin sensitivity under HFD conditions. To investigate the pathophysiological role of colonic macrophages, we generated macrophage-specific chemokine (C-C Motif) receptor 2 (Ccr2) knockout (M-Ccr2KO) and intestinal epithelial cell-specific tamoxifen-inducible Ccl2 knockout (Vil-Ccl2KO) mice. Both strains exhibited similar body weight to control under HFD. However, they exhibited decreased infiltration of colonic pro-inflammatory macrophages, decreased intestinal permeability, and inactivation of the colonic inflammasome. Interestingly, they showed significantly improved glucose tolerance and insulin sensitivity with decreased chronic inflammation of adipose tissue. Therefore, inhibition of pro-inflammatory macrophage infiltration prevents HFD-induced insulin resistance and could be a novel therapeutic approach for type 2 diabetes.

X-linked severe combined immunodeficiency (X-SCID) rats for xeno-transplantation and behavioral evaluation.

BACKGROUND: To evaluate the in vivo function of human dopaminergic (DA) neurons, Parkinson's disease (PD) model rats made by the hemi-lateral injection of 6-hydroxydopamine (6-OHDA) are widely used as host animals. In the case of such xeno-transplantation, however, immunosuppression is needed for good survival of the grafted cells. NEW METHODS: In order to determine whether human mature neurons can survive in X-linked severe combined immunodeficiency (X-SCID) rats without immunosuppression, we grafted human embryonic stem cell (ESC)-derived DA neurons into the striatum of X-SCID rats. We next treated the X-SCID rats with 6-OHDA and grafted mouse fetal DA neurons or human induced pluripotent stem cell (iPSC)-derived DA neurons to examine whether these rats can be used as PD model rats. RESULTS: X-SCID rats did not elicit immune responses against human ESC-derived DA neurons and consequently resulted in good survival of the cells without immunosuppression. Furthermore, 6-OHDA-lesioned X-SCID rats exhibited rotational behavior, which was recovered by grafting mouse fetal DA neurons or human iPSC-derived DA neurons. COMPARISON WITH EXISTING METHODS: Immunosuppression by drugs such as Cyclosporine A requires daily injection, which is stressful for rats and moreover may cause renal or hepatic failure. Furthermore, blood levels of the drug may not be stable, which weakens the reliability of the data. CONCLUSIONS: Our results provide a more accessible and reliable method to evaluate the in vivo function of human DA neurons, potentially offering a pre-clinical study for the application of pluripotent stem cells.

The BED finger domain protein MIG-39 halts migration of distal tip cells in Caenorhabditis elegans.

Organs are often formed by the extension and branching of epithelial tubes. An appropriate termination of epithelial tube extension is important for generating organs of the proper size and morphology. However, the mechanism by which epithelial tubes terminate their extension is mostly unknown. Here we show that the BED-finger domain protein MIG-39 acts to stop epithelial tube extension in Caenorhabditis elegans. The gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern during larval development and stop migrating at the young adult stage, generating a gonad with anterior and posterior U-shaped arms. In mig-39 mutants, however, DTCs overshot their normal stopping position. MIG-39 promoted the deceleration of DTCs, leading to the proper timing and positioning of the cessation of DTC migration. Among three Rac GTPase genes, mutations in ced-10 and rac-2 enhanced the overshoot of anterior DTCs, while they suppressed that of posterior DTCs of mig-39 mutants. On the other hand, the mutation in mig-2 suppressed both the anterior and posterior DTC defects of mig-39. Genetic analyses suggested that MIG-39 acts in parallel with Rac GTPases in stopping DTC migration. We propose a model in which the anterior and posterior DTCs respond in an opposite manner to the levels of Rac activities in the cessation of DTC migration.