In vivo selection in non-human primates identifies AAV capsids for on-target CSF delivery to spinal cord.

Systemic administration of adeno-associated virus (AAV) vectors for spinal cord gene therapy has challenges including toxicity at high doses and pre-existing immunity that reduces efficacy. Intrathecal (IT) delivery of AAV vectors into cerebral spinal fluid can avoid many issues, although distribution of the vector throughout the spinal cord is limited, and vector entry to the periphery sometimes initiates hepatotoxicity. Here we performed biopanning in non-human primates (NHPs) with an IT injected AAV9 peptide display library. We identified top candidates by sequencing inserts of AAV DNA isolated from whole tissue, nuclei, or nuclei from transgene-expressing cells. These barcoded candidates were pooled with AAV9 and compared for biodistribution and transgene expression in spinal cord and liver of IT injected NHPs. Most candidates displayed increased retention in spinal cord compared with AAV9. Greater spread from the lumbar to the thoracic and cervical regions was observed for several capsids. Furthermore, several capsids displayed decreased biodistribution to the liver compared with AAV9, providing a high on-target/low off-target biodistribution. Finally, we tested top candidates in human spinal cord organoids and found them to outperform AAV9 in efficiency of transgene expression in neurons and astrocytes. These capsids have potential to serve as leading-edge delivery vehicles for spinal cord-directed gene therapies.

Circadian coordination: understanding interplay between circadian clock and mitochondria.

Biological rhythms play a crucial role in temporally regulating behavioral, physiological, and cellular processes within our bodies. One prominent example is the circadian rhythm, which enables our bodies to anticipate external cues and regulate our internal processes accordingly. The circadian rhythm is controlled by a molecular feedback loop known as the circadian clock, present in nearly all cells. The regulation of genes involved in mitochondrial function is no exception. Key aspects such as oxidative phosphorylation, mitochondrial biogenesis, and mitochondrial morphology are regulated by the circadian clock. Functional changes in mitochondria can retrogradely affect the circadian rhythm. Furthermore, there are also transcriptional circadian clock-independent rhythms within mitochondria. This review discusses mitochondrial rhythms independently or in communication with the circadian clock in the nucleus at the cellular level.

The Differential Developmental Neurotoxicity of Valproic Acid on Anterior and Posterior Neural Induction of Human Pluripotent Stem Cells.

Valproic acid (VPA), widely used as an antiepileptic drug, exhibits developmental neurotoxicity when exposure occurs during early or late pregnancy, resulting in various conditions ranging from neural tube defects to autism spectrum disorders. However, toxicity during the very early stages of neural development has not been addressed. Therefore, we investigated the effects of VPA in a model where human pluripotent stem cells differentiate into anterior or posterior neural tissues. Exposure to VPA during the induction of neural stem cells induced different developmental toxic effects in a dose-dependent manner. For instance, VPA induced cell death more profoundly during anteriorly guided neural progenitor induction, while inhibition of cell proliferation and enhanced differentiation were observed during posteriorly guided neural induction. Furthermore, acute exposure to VPA during the posterior induction step also retarded the subsequent neurulation-like tube morphogenesis process in neural organoid culture. These results suggest that VPA exposure during very early embryonic development might exhibit cytotoxicity and subsequently disrupt neural differentiation and morphogenesis processes.

Polarization of organoids by bioengineered symmetry breaking.

Symmetry breaking leading to axis formation and spatial patterning is crucial for achieving more accurate recapitulation of human development in organoids. While these processes can occur spontaneously by self-organizing capabilities of pluripotent stem cells, they can often result in variation in structure and composition of cell types within organoids. To address this limitation, bioengineering techniques that utilize geometric, topological and stiffness factors are increasingly employed to enhance control and consistency. Here, we review how spontaneous manners and engineering tools such as micropattern, microfluidics, biomaterials, etc. can facilitate the process of symmetry breaking leading to germ layer patterning and the formation of anteroposterior and dorsoventral axes in blastoids, gastruloids, neuruloids and neural organoids. Furthermore, brain assembloids, which are composed of multiple brain regions through fusion processes are discussed. The overview of organoid polarization in terms of patterning tools can offer valuable insights for enhancing the physiological relevance of organoid system.

Establishment and Validation of a Model for Fetal Neural Ischemia Using Necrotic Core-Free Human Spinal Cord Organoids.

Fetal spinal cord ischemia is a serious medical condition that can result in significant neurological damage and adverse outcomes for the fetus. However, the lack of an appropriate experimental model has hindered the understanding of the pathology and the development of effective treatments. In our study, we established a system for screening drugs that affect fetal spinal cord ischemia using spinal cord organoids. Importantly, we produced necrotic core-free human spinal cord organoids (nf-hSCOs) by reducing the organoid size to avoid potential complications of spontaneous necrosis in large organoids. Exposing nf-hSCOs to CoCl2 as a hypoxia mimetic and hypoglycemic conditions resulted in significant neuronal damage, as assessed by multiple assay batteries. By utilizing this model, we tested chemicals that have been reported to exhibit beneficial effects in brain organoid-based ischemia models. Surprisingly, these chemicals did not provide sufficient benefit, and we discovered that rapamycin is a mild neuroprotective reagent for both axon degeneration and neuronal survival. We propose that nf-hSCO is suitable for large-scale screening of fetal neural ischemia due to its scalability, ease of ischemic induction, implementation of quantifiable assay batteries, and the absence of spontaneous necrosis.

Comparing two mucin secretagogues for the treatment of dry eye disease: a prospective randomized crossover trial.

This study aimed to compare the clinical efficacy and investigate patients' preferences for two mucin secretagogues in the treatment of dry eye disease (DED). Thirty patients with DED were randomly treated with either 3% diquafosol or 2% rebamipide ophthalmic solution for 4 weeks, followed by an additional 4-week treatment using the other eye drop after a 2-week washout period. Objective and subjective assessments, including the corneal and conjunctival staining score, tear breakup time (TBUT), Schirmer 1 test, tear osmolarity, tear matrix metalloproteinase-9 (MMP-9), lipid layer thickness (LLT) and ocular surface disease index (OSDI), were performed at baseline, 4 weeks, 6 weeks, and 10 weeks. Patient preferences were assessed based on four categories (comfort, efficacy, convenience, willingness to continue) using a questionnaire and the overall subjective satisfaction score for each drug was obtained at the end of the trial. In total, 28 eyes from 28 patients were included in the analysis. Both diquafosol and rebamipide significantly improved the OSDI (p = 0.033 and 0.034, respectively), TBUT (p < 0.001 and 0.026, respectively), and corneal (p < 0.001 and 0.001, respectively) and conjunctival (p = 0.017 and 0.042, respectively) staining after 4 weeks of treatment. An increase in Schirmer test scores was observed only after rebamipide treatment (p = 0.007). No significant changes were detected in tear osmolarity, MMP-9, and LLT following both treatments. The patients' preference was slightly greater for diquafosol (46.4%) than rebamipide (36.7%), presumably due to rebamipide's bitter taste. The self-efficacy of both drugs and overall satisfaction scores were comparable. These findings indicate that two mucin secretagogues showed comparable effects in ameliorating symptoms and improving signs (TBUT, corneal and conjunctival staining) in patients with DED.

Revolutionizing biomedical research: The imperative need for heart-kidney-connected organoids.

Organoids significantly advanced our comprehension of organ development, function, and disease modeling. This Perspective underscores the potential of heart-kidney-connected organoids in understanding the intricate relationship between these vital organs, notably the cardiorenal syndrome, where dysfunction in one organ can negatively impact the other. Conventional models fall short in replicating this complexity, necessitating an integrated approach. By co-culturing heart and kidney organoids, combined with microfluidic and 3D bioprinting technologies, a more accurate representation of in vivo conditions can be achieved. Such interconnected systems could revolutionize our grasp of multi-organ diseases, drive drug discovery by evaluating therapeutic agents on both organs simultaneously, and reduce the need for animal models. In essence, heart-kidney-connected organoids present a promising avenue to delve deeper into the pathophysiology underlying cardiorenal disorders, bridging existing knowledge gaps, and advancing biomedical research.

Enhancing cartilage regeneration through spheroid culture and hyaluronic acid microparticles: A promising approach for tissue engineering.

Cell therapy using chondrocytes has shown promise for cartilage regeneration, but maintaining functional characteristics during in vitro culture and ensuring survival after transplantation are challenges. Three-dimensional (3D) cell culture methods, such as spheroid culture, and hydrogels can improve cell survival and functionality. In this study, a new method of culturing spheroids using hyaluronic acid (HA) microparticles was developed. The spheroids mixed with HA microparticles effectively maintained the functional characteristics of chondrocytes during in vitro culture, resulting in improved cell survival and successful cartilage formation in vivo following transplantation. This new method has the potential to improve cell therapy production for cartilage regeneration.

Guidelines for Manufacturing and Application of Organoids: Brain.

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

Capturing cell morphology dynamics with high temporal resolution using single-shot quantitative phase gradient imaging.

Significance: Label-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75 frames / s ). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining. Aim: We aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images. Approach: Here, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability. Results: We validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3 lp / mm , and our analysis scheme accurately retrieves the phase with a high correlation coefficient ( ∼ 0.99 ), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55 µ m for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings. Conclusions: Label-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.

Measuring Heat Dissipation and Entropic Potential in Battery Cathodes Made with Conjugated and Conventional Polymer Binders Using Operando Calorimetry.

This study explores the influence of electronic and ionic conductivities on the behavior of conjugated polymer binders through the measurement of entropic potential and heat generation in an operating lithium-ion battery. Specifically, the traditional poly(vinylidene fluoride) (PVDF) binder in LiNi0.8Co0.15Al0.05O2 (NCA) cathode electrodes was replaced with semiconducting polymer binders based on poly(3,4-propylenedioxythiophene). Two conjugated polymers were explored: one is a homopolymer with all aliphatic side chains, and the other is a copolymer with both aliphatic and ethylene oxide side chains. We have shown previously that both polymers have high electronic conductivity in the potential range of NCA redox, but the copolymer has a higher ionic conductivity and a slightly lower electronic conductivity. Entropic potential measurements during battery cycling revealed consistent trends during delithiation for all of the binders, indicating that the binders did not modify the expected NCA solid solution deintercalation process. The entropic signature of polymer doping to form the conductive state could be clearly observed at potentials below NCA oxidation, however. Operando isothermal calorimetric measurements showed that the conductive binders resulted in less Joule heating compared to PVDF and that the net electrical energy was entirely dissipated as heat. In a comparison of the two conjugated polymer binders, the heat dissipation was lower for the homopolymer binder at lower C-rates, suggesting that electronic conductivity rather than ionic conductivity was the most important for reducing Joule heating at lower rates, but that ionic conductivity became more important at higher rates.

Robust and customizable spheroid culture system for regenerative medicine.

Three-dimensional cell spheroids show promise for the reconstruction of native tissues. Herein, we report a sophisticated, uniform, and highly reproducible spheroid culture system for tissue reconstruction. A mesh-integrated culture system was designed to precisely control the uniformity and reproducibility of spheroid formation. Furthermore, we synthesized hexanoyl glycol chitosan, a material with ultralow cell adhesion properties, to further improve spheroid formation efficiency and biological function. Our results demonstrate improved biological function in various types of cells and ability to generate spheroids with complex structures composed of multiple cell types. In conclusion, our spheroid culture system offers a highly effective and widely applicable approach to generating customized spheroids with desired structural and biological features for a variety of biomedical applications.

Re-Esterified Triglyceride ω-3 Fatty Acids in Dry Eye Disease With Meibomian Gland Dysfunction: A Randomized Clinical Trial.

Importance: Taking ω-3 supplements has been associated with a reduction in symptoms of dry eye disease (DED) associated with meibomian gland dysfunction (MGD). However, a recent relatively large clinical trial concluded that treating DED with ω-3 consumption was ineffective, potentially warranting additional investigations. Objectives: To investigate the effect of re-esterified triglyceride (rTG) ω-3 fatty acid supplementation on DED associated with MGD. Design, Setting, and Participants: This double-masked, parallel-group, randomized clinical trial was conducted at 7 institutions from September 2020 to January 2023. Patients with DED associated with MGD were included and randomly assigned to the ω-3 group (received 1680 mg of eicosapentaenoic acid and 560 mg of docosahexaenoic acid), whereas those in the grape-seed group received 3000 mg of grape-seed oil daily. Interventions: rTG ω-3 Fatty acid supplementation vs grape-seed oil. Main Outcome Measures: The primary end point was the Ocular Surface Disease Index (OSDI) from baseline to 6 and 12 weeks. The safety parameters were visual acuity and intraocular pressure change. Results: A total of 132 patients (mean [SD] age, 50.6 [13.8] years; 103 female [78.0%]) were included in this study. The mean (SD) baseline OSDI scores of the ω-3 and grape-seed groups were 43.5 (16.5) and 44.1 (16.6), respectively. A total of 58 patients (87.9%) and 57 patients (86.4%) in the ω-3 and grape-seed groups, respectively, completed 12 weeks of follow-up. There were no differences in compliance with the dietary supplement intake between groups (ω-3, 95.8% and grape-seed, 95.4%). The OSDI (SD) change from baseline to 6 and 12 weeks was -20.5 (16.0) and -22.7 (15.7), respectively, in the ω-3 group and -15.1 (20.2) and -18.8 (21.7), respectively, in the grape-seed control group (difference at 6 weeks = -5.4; 95% CI, -12.15 to 1.33; P = .12 and at 12 weeks = -3.9; 95% CI, -10.90 to 3.13; P = .28). There were no changes in safety parameters or adverse events related to taking the dietary supplement in either group. Conclusions and Relevance: This randomized clinical trial did not show a benefit of the rTG form of ω-3 for ameliorating symptoms of DED associated with MGD, although fewer than 60 participants were evaluated in each group. Any secondary outcomes from this study should be considered for hypothesis generation of future evaluations of the effect of the rTG form of ω-3 on DED associated with MGD. Trial Registration: CRIS Identifier: KCT0004927.