Phosphatidylinositol-3-phosphate mediates Arc capsid secretion through the multivesicular body pathway.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalize with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knockdown, cells in mouse cortical neurons reduces their surface GlutA1 levels. These results suggest that unlike the HIV Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), the assembly of Arc capsids is mediated by PI3P at endocytic membranes. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Extended depth-of-field wide-field fluorescence microscopy with a micro-mirror array lens system for versatile cellular examination.

We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.

Feasibility of moxifloxacin and proflavine dual fluorescence imaging for detecting gastrointestinal neoplastic lesions: A prospective study.

OBJECTIVES: High-contrast and high-resolution imaging techniques would enable real-time sensitive detection of the gastrointestinal lesions. This study aimed to investigate the feasibility of novel dual fluorescence imaging using moxifloxacin and proflavine in the detection of neoplastic lesions of the human gastrointestinal tract. METHODS: Patients with the colonic and gastric neoplastic lesions were prospectively enrolled. The lesions were biopsied with forceps or endoscopically resected. Dual fluorescence imaging was performed by using custom axially swept wide-field fluorescence microscopy after topical moxifloxacin and proflavine instillation. Imaging results were compared with both confocal imaging with cell labeling and conventional histological examination. RESULTS: Ten colonic samples (one normal mucosa, nine adenomas) from eight patients and six gastric samples (one normal mucosa, five adenomas) from four patients were evaluated. Dual fluorescence imaging visualized detail cellular structures. Regular glandular structures with polarized cell arrangement were observed in normal mucosa. Goblet cells were preserved in normal colonic mucosa. Irregular glandular structures with scanty cytoplasm and dispersed elongated nuclei were observed in adenomas. Goblet cells were scarce or lost in the colonic lesions. Similarity analysis between moxifloxacin and proflavine imaging showed relatively high correlation values in adenoma compared with those in normal mucosa. Dual fluorescence imaging showed good detection accuracies of 82.3% and 86.0% in the colonic and the gastric lesions, respectively. CONCLUSIONS: High-contrast and high-resolution dual fluorescence imaging was feasible for obtaining detail histopathological information in the gastrointestinal neoplastic lesions. Further studies are needed to develop dual fluorescence imaging as an in vivo real-time visual diagnostic method.

Severe headache in a middle-aged woman after device closure of a ventricular septal defect.

Reversible cerebral vasoconstriction syndrome presents with thunderclap headache and represents a group of conditions that show reversible multifocal narrowing of cerebral arteries. Some patients who undergo device closure of an atrial septal defect complain of headache, which are posited as a migraine. Here we report a case of severe headache due not to migraine but reversible cerebral vasoconstriction syndrome after device closure of a ventricular septal defect.

Multi-Focus Image Fusion Using Focal Area Extraction in a Large Quantity of Microscopic Images.

The non-invasive examination of conjunctival goblet cells using a microscope is a novel procedure for the diagnosis of ocular surface diseases. However, it is difficult to generate an all-in-focus image due to the curvature of the eyes and the limited focal depth of the microscope. The microscope acquires multiple images with the axial translation of focus, and the image stack must be processed. Thus, we propose a multi-focus image fusion method to generate an all-in-focus image from multiple microscopic images. First, a bandpass filter is applied to the source images and the focus areas are extracted using Laplacian transformation and thresholding with a morphological operation. Next, a self-adjusting guided filter is applied for the natural connections between local focus images. A window-size-updating method is adopted in the guided filter to reduce the number of parameters. This paper presents a novel algorithm that can operate for a large quantity of images (10 or more) and obtain an all-in-focus image. To quantitatively evaluate the proposed method, two different types of evaluation metrics are used: "full-reference" and "no-reference". The experimental results demonstrate that this algorithm is robust to noise and capable of preserving local focus information through focal area extraction. Additionally, the proposed method outperforms state-of-the-art approaches in terms of both visual effects and image quality assessments.

Changes in movements of neck, trunk, and hip according to height and foot position during sit-to-stand.

[Purpose] The purpose of this study is to analyze changes in movements of the neck, trunk and hip according to foot position while performing sit-to-stand (STS) exercises from a height-fixed chair. [Subjects and Methods] The study subjects consisted of 22 university students (12 males and 10 females). STS was conducted using a height-fixed chair at three positions: symmetric foot position, right foot position, and left foot position. Through three-dimensional motion analyzer, the movements of the neck, trunk, and hip were analyzed. [Results] While performing STS, the height was more influential on changed in angle of the neck, trunk, and hip. Moreover, when the height of the chair and the height of the subject were not matched correctly, more effective STS could be achieved when both of feet were laid symmetrically rather than at the other two positions. [Conclusion] It is necessary to employ an appropriate chair height that is matched with the height of the patients when therapy using STS is performed.

Optimal Atmospheric Correction for Above-Ground Forest Biomass Estimation with the ETM+ Remote Sensor.

The reflectance of the Earth's surface is significantly influenced by atmospheric conditions such as water vapor content and aerosols. Particularly, the absorption and scattering effects become stronger when the target features are non-bright objects, such as in aqueous or vegetated areas. For any remote-sensing approach, atmospheric correction is thus required to minimize those effects and to convert digital number (DN) values to surface reflectance. The main aim of this study was to test the three most popular atmospheric correction models, namely (1) Dark Object Subtraction (DOS); (2) Fast Line-of-sight Atmospheric Analysis of Spectral Hypercubes (FLAASH) and (3) the Second Simulation of Satellite Signal in the Solar Spectrum (6S) and compare them with Top of Atmospheric (TOA) reflectance. By using the k-Nearest Neighbor (kNN) algorithm, a series of experiments were conducted for above-ground forest biomass (AGB) estimations of the Gongju and Sejong region of South Korea, in order to check the effectiveness of atmospheric correction methods for Landsat ETM+. Overall, in the forest biomass estimation, the 6S model showed the bestRMSE's, followed by FLAASH, DOS and TOA. In addition, a significant improvement of RMSE by 6S was found with images when the study site had higher total water vapor and temperature levels. Moreover, we also tested the sensitivity of the atmospheric correction methods to each of the Landsat ETM+ bands. The results confirmed that 6S dominates the other methods, especially in the infrared wavelengths covering the pivotal bands for forest applications. Finally, we suggest that the 6S model, integrating water vapor and aerosol optical depth derived from MODIS products, is better suited for AGB estimation based on optical remote-sensing data, especially when using satellite images acquired in the summer during full canopy development.

Surface tracking integrated extended depth-of-field microscopy for rapid non-contact examination of conjunctival goblet cells in humans.

Conjunctival goblet cells (CGCs) are specialized epithelial cells playing key roles for ocular surface homeostasis, and their examination is important for diagnosing ocular surface diseases. Despite recent advancements in high-contrast CGC imaging for non-invasive examination, significant challenges remain for human applications. High-speed large-area imaging over the curved ocular surface is needed to assess statistically meaningful CGCs in the extensive human conjunctiva. To address this challenge, we developed a novel surface detection method and an integrated microscopy system for human use. With both a long detection range of 2 mm and a high update rate of 50 Hz, the surface detection method enabled real-time surface tracking during large-area imaging. The integrated microscopy could complete 5 × 2 patch imaging in approximately 10 s. CGC density analysis showed significantly reduced uncertainties with large-area imaging. This is the first demonstration of non-contact large-area cellular examination in humans, and this new development holds promise for non-invasive CGC examination and accurate diagnosis of ocular surface diseases.

Analysis of Fatty Acid Compositions and Acid Values of Krill Oil Supplementary Products from the Korean Market.

In order to provide the qualitative data for the 20 commercially available krill oil supplementary products ,the levels of omega-3 polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid ( DHA) and eicosapentaenoic acid (EPA),fatty acid compositions, and chemical indices, including acid values , of the supplements , were determined . The acid values ranged from 7.4 to 43 .7 mg of potassium hydroxide (KOH)/ g of oil. The relative percentages of EPA andDHA in the oils ranged from 14.2 to 34.8 % (w/w).Although all 20 krill oil supplements used 100% krill oil as raw material,the fatty acid composition of 4 samples differed from typical krill oil in terms ofthe content of myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), linoleic acid (C18:2, n-6), and eicosenoic acid (C20:1, n-9). Accordingly, the Ministry of Food and Drug Safety recently standardized linoleic acid (3% orless) and myristic acid (5-13%) as part ofthe fatty acid components of krill oil. This study provides a reference for analyzing the chemical and nutritional properties and evaluating the adulteration of krill oil supplements in theKorean market.

Pituitary Apoplexy After Leuprolide Therapy in a Breast Cancer Patient: A Case Report.

Pituitary apoplexy (PA) is a clinical syndrome resulting from sudden hemorrhage and/or infarction of the pituitary gland. Recent reports documented the development of PA secondary to treatment with gonadotropin-releasing hormone (GnRH) agonists for prostate cancer. A 52-year-old woman visited our emergency room with a severe headache, occurred 1 day prior. She underwent breast-conserving surgery for breast cancer 1 month prior. She was currently undergoing radiation and hormone therapy, consisting of leuprorelin. Brain contrast-enhanced MRI revealed a pituitary adenoma with internal hemorrhage in the sellar and suprasellar areas. Pachymeningeal enhancement was observed along the retroclival and bilateral frontal areas. The patient was diagnosed with PA and aseptic meningitis. The patient underwent total excision via transsphenoidal surgery 8 days after admission. The patient was pathologically diagnosed with a pituitary adenoma with necrosis. On immunochemical staining, the tumor was positive for follicle-stimulating hormone. The follow-up MRI revealed no evidence of residual tumor or an improved pachymeningeal enhancement. She is currently undergoing follow-up at the neurosurgery and endocrinology outpatient departments with no noted complications. In breast cancer patients receiving GnRH agonist therapy, PA may be rare complication.

Phosphatidylinositol 3-phosphate mediates Arc capsids secretion through the multivesicular body pathway.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. The recent discovery that Arc mediates the inter-neuronal RNA transfer implies its role in regulating neuronal functions across long distances. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag). By assembling into high-order, virus-like capsids, Arc mediates the intercellular RNA transfer. However, the exact secretion pathway through which Arc capsids maneuver cargos is unclear. Here, we identified that Arc capsids assemble and secrete through the endosomal-multivesicular body (MVB) pathway. Arc's endosomal entry is likely mediated by phosphatidylinositol-3-phosphate (PI3P). Indeed, reconstituted Arc protein preferably binds to PI3P. In mammalian cells, Arc forms puncta that colocalizes with FYVE, an endosomal PI3P marker, and competitive binding to PI3P via prolonged FYVE expression reduces the average number of Arc puncta per cell. Overexpression of MTMR1, a PI3P phosphatase, significantly reduces Arc capsid secretion. Arc capsids secrete through the endosomal-MVB axis as extracellular vesicles. Live-cell imaging shows that fluorescently labeled Arc primarily colocalizes Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces Arc-mediated RNA transfer efficiency. These results suggest that, unlike the Human Immunodeficiency Virus Gag, which assembles on and bud off from the plasma membrane, Arc capsids assemble at the endocytic membranes of the endosomal-MVB pathway mediated by PI3P. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Dual-energy computed tomography material decomposition improves prediction accuracy of hematoma expansion in traumatic intracranial hemorrhage.

OBJECTIVE: The angiographic spot sign (AS) on CT angiography (CTA) is known to be useful for predicting expansion in intracranial hemorrhage, but its use is limited due to its relatively low sensitivity. Recently, dual-energy computed tomography (DECT) has been shown to be superior in distinguishing between hemorrhage and iodine. This study aimed to evaluate the diagnostic performance of hematoma expansion (HE) using DECT AS in traumatic intracranial hemorrhage. METHODS: We recruited participants with intracranial hemorrhage confirmed via CTA for suspected traumatic cerebrovascular injuries. We evaluated AS on both conventional-like and fusion images of DECT. AS is grouped into three categories: intralesional enhancement without change, delayed enhancement (DE), and growing contrast leakage (GL). HE was evaluated by measuring hematoma size on DECT and follow-up CT. Logistic regression analysis was used to evaluate whether AS on fusion images was a significant risk factor for HE. Diagnostic accuracy was calculated, and the results from conventional-like and fusion images were compared. RESULTS: Thirty-nine hematomas in 24 patients were included in this study. Of these, 18 hematomas in 13 patients showed expansion on follow-up CT. Among the expanders, AS and GL on fusion images were noted in 13 and 5 hematomas, respectively. In non-expanders, 10 and 1 hematoma showed AS and GL, respectively. In the logistic regression model, GL on the fusion image was a significant independent risk factor for predicting HE. However, when AS was used on conventional-like images, no factors significantly predicted HE. In the receiver operating characteristic curve analysis, the area under the curve of AS on the fusion images was 0.71, with a sensitivity and specificity of 66.7% and 76.2%, respectively. CONCLUSIONS: GL on fusion images of DECT in traumatic intracranial hemorrhage is a significant independent radiologic risk factor for predicting HE. The AS of DECT fusion images has improved sensitivity compared to that of conventional-like images.

Noninvasive Imaging of Conjunctival Goblet Cells as a Method for Diagnosing Dry Eye Disease in an Experimental Mouse Model.

Purpose: The purpose of this study was to evaluate a noninvasive conjunctival goblet cell (GC) imaging method for assessing dry eye disease (DED) in an experimental mouse model. Methods: Moxifloxacin-based fluorescence microscopy (MBFM) was used to examine GCs noninvasively in 56 mice. Forty-two (42) DED-induced mice were divided into 2 groups and treated topically for 14 days with cyclosporine (CsA) or normal saline (NS). In vivo MBFM imaging and clinical DED evaluations were performed and goblet cell density (GCD) and goblet cell area (GCA) were obtained and compared with histological GCD using periodic acid-Schiff (PAS) staining. Correlation and receiver operating characteristic (ROC) analyses showed MBFM's high diagnostic value. Results: The GCD and GCA of the DED mice obtained from in vivo MBFM imaging were highly correlated with clinical DED parameters and GCD obtained from PAS histology. The therapeutic effect of CsA, as observed by in vivo MBFM, was significant with respect to that of NS treatment. The ROC curves derived from in vivo MBFM showed high diagnostic value in assessing DED. Conclusions: The proposed noninvasive method has high diagnostic value in assessing the severity of DED and the effect of treatment for this disease. Translational Relevance: A noninvasive imaging method using moxifloxacin-based fluorescence microscopy was evaluated for assessing DED in an experimental mouse model. The method showed high diagnostic value in assessing the severity of DED and the effect of treatment, bridging the gap between basic research and clinical treatment. The study provides a promising tool for diagnosing and monitoring DED.

Deep learning framework for automated goblet cell density analysis in in-vivo rabbit conjunctiva.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells secreting mucins for the mucus layer of protective tear film and playing immune tolerance functions for ocular surface health. Because GC loss is observed in various ocular surface diseases, GC examination is important for precision diagnosis. Moxifloxacin-based fluorescence microscopy (MBFM) was recently developed for non-invasive high-contrast GC visualization. MBFM showed promise for GC examination by high-speed large-area imaging and a robust analysis method is needed to provide GC information. In this study, we developed a deep learning framework for GC image analysis, named dual-channel attention U-Net (DCAU-Net). Dual-channel convolution was used both to extract the overall image texture and to acquire the GC morphological characteristics. A global channel attention module was adopted by combining attention algorithms and channel-wise pooling. DCAU-Net showed 93.1% GC segmentation accuracy and 94.3% GC density estimation accuracy. Further application to both normal and ocular surface damage rabbit models revealed the spatial variations of both GC density and size in normal rabbits and the decreases of both GC density and size in damage rabbit models during recovery after acute damage. The GC analysis results were consistent with histology. Together with the non-invasive high-contrast imaging method, DCAU-Net would provide GC information for the diagnosis of ocular surface diseases.

Multiple De Novo Cerebral Arteriovenous Malformations in a Patient with Alcoholic Liver Cirrhosis.

Cerebral arteriovenous malformations (cAVMs) are traditionally considered congenital anomalies. The literature includes only two reported cases of de novo solitary cAVMs associated with liver cirrhosis (LC). Here, we report a unique case of multiple de novo cAVMs in a patient with alcoholic LC. Teaching point: In patients with potential risks for de novo cAVM, including liver cirrhosis, the possibility of multiple de novo cAVMs should be considered.

Association of cerebral white matter hyperintensities with coronary artery calcium in a healthy population: a cross-sectional study.

In brain magnetic resonance imaging (MRI), white matter hyperintensity (WMH) is a commonly encountered finding and is known to reflect cerebral small vessel disease. The aim of our study was to investigate the association of coronary artery calcium (CAC) with WMH and elucidate the relationship between WMH and atherosclerotic risk factors in a large-scale healthy population. This retrospective study included 1337 individuals who underwent brain MRI and CAC scoring computed tomography at healthcare centers affiliated with a tertiary hospital. Cerebral WMH was defined as Fazekas score greater than 2 on brain MRI. Intracranial artery stenosis (ICAS) was also assessed and determined to be present when stenosis was more than 50% on angiography. The associations of risk factors, CAC score, and ICAS with cerebral WMH were assessed by multivariable regression analysis. In multivariable analysis, categories of higher CAC scores showed increased associations with both periventricular and deep WMHs in a dose-dependent relationship. The presence of ICAS was also significantly related to cerebral WMH, and among the clinical variables, age and hypertension were independent risk factors. In conclusion, CAC showed a significant association with cerebral WMH in a healthy population, which might provide evidence for referring to the CAC score to identify individuals with risk of cerebral WMH.

Moxifloxacin-Based Extended Depth-of-Field Fluorescence Microscopy for Real-Time Conjunctival Goblet Cell Examination.

Conjunctival goblet cells (CGCs) are mucin-secreting cells in the eye and play essential roles for ocular surface homeostasis. Since various ocular surface pathologies are related to CGC dysfunction, CGC examination is important for the evaluation of ocular surface conditions. Recently we introduced moxifloxacin-based fluorescence microscopy (MBFM) for non-invasive CGC imaging. However, the imaging speed was up to 1 frame per second (fps) and needed to be improved for clinical applications. In this study, we developed a high-speed moxifloxacin-based, extended depth-of-field (EDOF) microscopy system that operates at a maximum imaging speed of 15 fps. The system used a deformable mirror for the high-speed axial sweeping of focal plane during single-frame acquisitions. The acquired images contained both in-focus and out-of-focus information, and deconvolution was used to filter the in-focus information. The system had a DOF of 800 [Formula: see text], field-of-view of 1.2 mm ×1.2 mm, and resolution of [Formula: see text]. Its performance was demonstrated by real-time, breathing-motion-insensitive CGC imaging of mouse and rabbit models, in vivo. High-speed EDOF microscopy has potentials for non-invasive, real-time CGC examinations of human subjects.

Brainstem Venous Infarction from Thrombosis on the Jugular Bulb as a Serious Complication of Blunt Head Trauma Injury: A Case Report.

The incidence of dural venous sinus thrombosis (DVST) and the cerebral venous infarct have not exactly known, but DVST is closely related to the skull fracture around the venous sinus. A 56-year-old man experienced massive watery discharge after hitting on his face by a falling machine. He was alert and no cerebrospinal fluid discharge on admission. Air-density was shown on the jugular fossa in the brain computed tomography. On the 3th day of trauma, he suddenly had dyspnea and loss of consciousness and became comatose. Acute edema on medulla, pons and right cerebellar hemisphere and focal infarct on right medulla were visualized on the brain magnetic resonance imaging. And the sigmoid sinus and the jugular vein were occluded and venous circulation on the right posterior fossa was diminished on the cerebral angiography. Air-density on the sinus may be an indicator into developing venous thrombosis and brainstem venous infarct could be followed by the DVST round the jugular bulb.

Evaluation of Reproducibility of Brain Volumetry between Commercial Software, Inbrain and Established Research Purpose Method, FreeSurfer.

BACKGROUND AND PURPOSE: We aimed to determine the intermethod reproducibility between the commercial software Inbrain (MIDAS IT) and the established research-purpose method FreeSurfer, as well as the effect of MRI resolution and the pathological condition of subjects on their intermethod reproducibility. METHODS: This study included 45 healthy volunteers and 85 patients with mild cognitive impairment (MCI). In 43 of the 85 patients with MCI, three-dimensional, T1-weighted MRI data were obtained at an in-plane resolution of 1.2 mm. The data of the remaining 42 patients with MCI and the healthy volunteers were obtained at an in-plane resolution of 1.0 mm. The within-subject coefficient of variation (CoV), intraclass correlation coefficient (ICC), and effect size were calculated, and means were compared using paired t-tests. The parameters obtained at 1.0-mm and 1.2-mm resolutions in patients with MCI were compared to evaluate the effect of the in-plane resolution on the intermethod reproducibility. The parameters obtained at a 1.0-mm in-plane resolution in patients with MCI and healthy volunteers were used to analyze the effect of subject condition on intermethod reproducibility. RESULTS: Overall the two methods showed excellent reproducibility across all regions of the brain (CoV=0.5-3.9, ICC=0.93 to >0.99). In the subgroup of healthy volunteers, the intermethod reliability was only good in some regions (frontal, temporal, cingulate, and insular). The intermethod reproducibility was better in the 1.0-mm group than the 1.2-mm group in all regions other than the nucleus accumbens. CONCLUSIONS: Inbrain and FreeSurfer showed good-to-excellent intermethod reproducibility for volumetric measurements. Nevertheless, some noticeable differences were found based on subject condition, image resolution, and brain region.

Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson's disease: Midbrain organoid-NSCs (Og-NSC) as a stable source for PD treatment.

Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.