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Mice 3D testicular organoid system as a novel tool to study Zika virus pathogenesis.
Zika virus (ZIKV) poses a serious threat to global public health due to its close relationship with neurological and male reproductive damage. However, deficiency of human testicular samples hinders the in-depth research on ZIKV-induced male reproductive system injury. Organoids are relatively simple in vitro models, which could mimic the pathological changes of corresponding organs. In this study, we constructed a 3D testicular organoid model using primary testicular cells from adult BALB/c mice. Similar to the testis, this organoid system has a blood-testis barrier (BTB)-like structure and could synthesize testosterone. ZIKV tropism of testicular cells and ZIKV-induced pathological changes in testicular organoid was also similar to that in mammalian testis. Therefore, our results provide a simple and reproducible in vitro testicular model for the investigations of ZIKV-induced testicular injury.
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Retinal Organoid Technology: Where Are We Now?
It is difficult to regenerate mammalian retinal cells once the adult retina is damaged, and current clinical approaches to retinal damages are very limited. The introduction of the retinal organoid technique empowers researchers to study the molecular mechanisms controlling retinal development, explore the pathogenesis of retinal diseases, develop novel treatment options, and pursue cell/tissue transplantation under a certain genetic background. Here, we revisit the historical background of retinal organoid technology, categorize current methods of organoid induction, and outline the obstacles and potential solutions to next-generation retinal organoids. Meanwhile, we recapitulate recent research progress in cell/tissue transplantation to treat retinal diseases, and discuss the pros and cons of transplanting single-cell suspension versus retinal organoid sheet for cell therapies.
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Identification of new AAV vectors with enhanced blood-brain barrier penetration efficiency via organ-on-a-chip.
To overcome limitations in the generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by the insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of the human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, which showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to a 5 times higher titer, while AAV-M8 reached a 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. The transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high-throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell-type specificity of virus vectors.
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Functional bioengineered tissue models of neurodegenerative diseases.
Aging-associated neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases remain poorly understood and no disease-modifying treatments exist despite decades of investigation. Predominant in vitro (e.g., 2D cell culture, organoids) and in vivo (e.g., mouse) models of these diseases are insufficient mimics of human brain tissue structure and function and of human neurodegenerative pathobiology, and have thus contributed to this collective translational failure. This has been a longstanding challenge in the field, and new strategies are required to address both fundamental and translational needs. Bioengineered tissue culture models constitute a class of promising alternatives, as they can overcome the low cell density, poor nutrient exchange, and long term culturability limitations of existing in vitro models. Further, they can reconstruct the structural, mechanical, and biochemical cues of native brain tissue, providing a better mimic of human brain tissues for in vitro pathobiological investigation and drug development. We discuss bioengineering techniques for the generation of these neurodegenerative tissue models, including biomaterials-, organoid-, and microfluidics-based approaches, and design considerations for their construction. To aid the development of the next generation of functional neurodegenerative disease models, we discuss approaches to incorporate greater cellular diversity and simulate aging processes within bioengineered brain tissues.
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Methadone interrupts neural growth and function in human cortical organoids.
Prenatal opioids exposure can lead to both neonatal abstinence syndrome in newborns and neurological deficits later in life. Although opioids have been well studied in general, the cellular and molecular mechanisms by which opioids affect human fetal brain development has not been well understood. In this work, we have taken advantage of a human 3D-brain cortical organoid (hCO) that facilitated enormously the investigation of early human brain development. Using imaging, immunofluorescence, multi-electrode array (MEA) and patch clamp recording techniques, we have investigated the effect of methadone, a frequently used opioid during pregnancy, on early neural development, including neuronal growth, neural network activity and synaptic transmission in hCOs. Our results demonstrated that methadone dose-dependently halted the growth of hCOs and induced organoid disintegration after a prolonged exposure. In addition, methadone dose-dependently suppressed the firing of spontaneous action potentials in hCOs and this suppression could be reversed upon methadone withdrawal in hCOs treated with lower dosages. Further investigation using patch clamp whole cell configuration revealed that, at clinically relevant concentrations, methadone decreased the frequency and amplitude of excitatory postsynaptic currents in neurons, indicating a critical role of methadone in weakening synaptic transmission in neural networks in hCOs. In addition, methadone significantly attenuated the voltage-dependent Na+ current in hCOs. We conclude that methadone interrupts neural growth and function in early brain development.
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Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus.
The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.
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An approach to measuring protein turnover in human induced pluripotent stem cell organoids by mass spectrometry.
Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson's disease (PD) - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain essential as organoid models evolve. To solve this problem, we have designed a workflow to reproducibly extract proteins from brain organoids, measure global turnover using mass spectrometry, and statistically investigate turnover differences between genotypes. We also provide robust methodology for data filtering and statistical treatment of turnover data. Using human midbrain organoids (hMO) as a model system, our method accurately characterized the half-lives of 773 midbrain proteins. We compared these half-lives both to Parkin knockout hMOs and to previously reported data from primary cell cultures and in vivo models. Overall, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.
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A brief history of testicular organoids: from theory to the wards.
In recent years, researchers have successfully generated many human and mammalian organoid models, including organoids representing the intestine, prostate, ovary, bladder, liver, and brain. Therefore, organoids have become an important research model in the fields of regenerative medicine, drug research, and gene therapy, acting as a bridge between in vivo and in vitro experiments. In addition, testicular organoids (TOs) represent the highest level of in vitro culture of spermatogenic cells in a simulated testicular environment. However, the generation of TOs is still in the early stages, and there is still much room for improvement in dealing with the many challenges and prospects to achieve the goal of TOs that simulate the testicular microenvironment in vitro or even reconstruct the process of spermatogenesis without the need to reconstruct seminiferous tubules. We review the brief history of TO generation and some major strategies for functional applications of TOs, which are the main concerns of our clinicians and laboratory researchers.
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Development of a deep learning based image processing tool for enhanced organoid analysis.
Contrary to 2D cells, 3D organoid structures are composed of diverse cell types and exhibit morphologies of various sizes. Although researchers frequently monitor morphological changes, analyzing every structure with the naked eye is difficult. Given that deep learning (DL) has been used for 2D cell image segmentation, a trained DL model may assist researchers in organoid image recognition and analysis. In this study, we developed OrgaExtractor, an easy-to-use DL model based on multi-scale U-Net, to perform accurate segmentation of organoids of various sizes. OrgaExtractor achieved an average dice similarity coefficient of 0.853 from a post-processed output, which was finalized with noise removal. Correlation between CellTiter-Glo assay results and daily measured organoid images shows that OrgaExtractor can reflect the actual organoid culture conditions. The OrgaExtractor data can be used to determine the best time point for organoid subculture on the bench and to maintain organoids in the long term.
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The application progress of organoid technology in the toxicity assessment of environmental pollutants.
Human production activities and daily life are the main sources of environmental pollutants, and their potential health impacts have attracted growing attention. Currently, the assessment of environmental pollutant toxicity depends heavily on animal models and some cell culture models. However, these approaches have inherent limitations, such as their inability to recapitulate the in vivo microenvironment and the presence of interspecies differences. Organoids not only recapitulate cell types and tissue structures of human organs, but also exhibit key physiological functions, rendering them ideal alternative models for assessing the health risks of environmental pollutants. In this review, we briefly introduce the development history of organoids as models for environmental pollutant toxicity assessment. Additionally, organoids are categorized by cell sources, and current mainstream organoid culture techniques are systematically summarized. Next, we highlight the application value of typical organoids including lung, brain, cardiac, intestine, liver and kidney organoids in environmental pollutant toxicity assessment. Finally, we thoroughly analyze the strengths and limitations of organoid models for such toxicity testing. Therefore, this review aims to advance organoid technology, and provide new insights into the toxicity assessment of environmental pollutants and the exploration of relevant toxic mechanisms.