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Author(s): Tetsuhiro Kikuchi [1]; Asuka Morizane [1]; Daisuke Doi [1]; Hiroaki Magotani [1]; Hirotaka Onoe [2]; Takuya Hayashi [2]; Hiroshi Mizuma [2]; Sayuki Takara [2]; Ryosuke Takahashi [3]; Haruhisa Inoue [...]

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. [ABSTRACT FROM AUTHOR]

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, MHCmatching 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. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)