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Directed differentiation and direct reprogramming: Applying stem cell technologies to hearing research.
Hearing loss is the most widely spread sensory disorder in our society. In the majority of cases, it is caused by the loss or malfunctioning of cells in the cochlea: the mechanosensory hair cells, which act as primary sound receptors, and the connecting auditory neurons of the spiral ganglion, which relay the signal to upper brain centers. In contrast to other vertebrates, where damage to the hearing organ can be repaired through the activity of resident cells, acting as tissue progenitors, in mammals, sensory cell damage or loss is irreversible. The understanding of gene and cellular functions, through analysis of different animal models, has helped to identify causes of disease and possible targets for hearing restoration. Translation of these findings to novel therapeutics is, however, hindered by the lack of cellular assays, based on human sensory cells, to evaluate the conservation of molecular pathways across species and the efficacy of novel therapeutic strategies. In the last decade, stem cell technologies enabled to generate human sensory cell types in vitro, providing novel tools to study human inner ear biology, model disease, and validate therapeutics. This review focuses specifically on two technologies: directed differentiation of pluripotent stem cells and direct reprogramming of somatic cell types to sensory hair cells and neurons. Recent development in the field are discussed as well as how these tools could be implemented to become routinely adopted experimental models for hearing research.
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Harnessing three-dimensional (3D) cell culture models for pulmonary infections: State of the art and future directions.
Pulmonary infections have been a leading etiology of morbidity and mortality worldwide. Upper and lower respiratory tract infections have multifactorial causes, which include bacterial, viral, and rarely, fungal infections. Moreover, the recent emergence of SARS-CoV-2 has created havoc and imposes a huge healthcare burden. Drug and vaccine development against these pulmonary pathogens like respiratory syncytial virus, SARS-CoV-2, Mycobacteria, etc., requires a systematic set of tools for research and investigation. Currently, in vitro 2D cell culture models are widely used to emulate the in vivo physiologic environment. Although this approach holds a reasonable promise over pre-clinical animal models, it lacks the much-needed correlation to the in vivo tissue architecture, cellular organization, cell-to-cell interactions, downstream processes, and the biomechanical milieu. In view of these inadequacies, 3D cell culture models have recently acquired interest. Mammalian embryonic and induced pluripotent stem cells may display their remarkable self-organizing abilities in 3D culture, and the resulting organoids replicate important structural and functional characteristics of organs such the kidney, lung, gut, brain, and retina. 3D models range from scaffold-free systems to scaffold-based and hybrid models as well. Upsurge in organs-on-chip models for pulmonary conditions has anticipated encouraging results. Complexity and dexterity of developing 3D culture models and the lack of standardized working procedures are a few of the setbacks, which are expected to be overcome in the coming times. Herein, we have elaborated the significance and types of 3D cell culture models for scrutinizing pulmonary infections, along with the in vitro techniques, their applications, and additional systems under investigation.
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Polygenic risk score of Alzheimer's disease is associated with cognitive trajectories and phenotypes of cerebral organoids.
INTRODUCTION: Polygenic risk score (PRS) identifies individuals at high genetic risk for Alzheimer's disease (AD), but its utility in predicting cognitive trajectories and AD pathologies remains unclear. We optimized PRS (optPRS) for AD, investigated its association with cognitive trajectories and AD phenotypes of cerebral organoids. METHODS: Using genome-wide association study (GWAS) summary statistics from a European population, we developed optPRS to predict AD in Korean individuals (n = 1634). We analyzed the association between optPRS and cognitive trajectories (n = 771). We generated induced pluripotent stem cell-derived cerebral organoids from patients with high (n = 3) and low (n = 4) optPRS to evaluate amyloid beta (Aβ) and phosphorylated tau (p-tau) levels. RESULTS: OptPRS predicted AD dementia and Aβ positivity, independent of apolipoprotein E (APOE). Higher optPRSs correlated with rapid cognitive decline. Cerebral organoids from the high optPRS group exhibited increased Aβ insolubility and p-tau levels. CONCLUSION: OptPRS predicted cognitive decline and AD phenotypes of cerebral organoids, supporting its use in risk assessments and drug-screening platform. HIGHLIGHTS: Optimized polygenic risk scores (optPRSs) improve the prediction of Alzheimer's disease (AD) dementia and amyloid beta positivity (Aβ+). High optPRS is associated with faster cognitive decline, particularly in Aβ+. Induced pluripotent stem cell (iPSC)-derived cerebral organoids from high optPRSs show high Aβ insolubility and phosphorylated tau (p-tau). PRS genetic risk stratification provides insight into AD progression and pathology.
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A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.
Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening. iPSC-derived neural spheroids or organoids can be developed to include complex neuronal and glial cell populations and display spontaneous, synchronous activity, which is a hallmark of in vivo neural communication. In this report we present a proof-of-concept study evaluating 3D iPSC-derived cortical neural spheroids as a physiologically- and pharmacologically-relevant high-throughput screening (HTS) platform and investigate their potential for use for therapeutic development. To this end, a library of 687 neuroactive compounds were tested in a phenotypic screening paradigm which measured calcium activity as a functional biomarker for neural modulation through fluctuations in calcium fluorescence. Pharmacological responses of cortical neural spheroids were analyzed using a multi-parametric approach, whereby seven peak characteristics from the calcium activity in each well were quantified and incorporated into principal component analysis and Sammon mapping to measure compound response. Here, we describe the implementation of the 687-compound library screen and data analysis demonstrating that iPSC-derived cortical spheroids are a robust and information-rich assay platform for HTS.
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Retinal Ganglion Cells With a Glaucoma OPTN(E50K) Mutation Exhibit Neurodegenerative Phenotypes when Derived from Three-Dimensional Retinal Organoids.
Retinal ganglion cells (RGCs) serve as the connection between the eye and the brain, with this connection disrupted in glaucoma. Numerous cellular mechanisms have been associated with glaucomatous neurodegeneration, and useful cellular models of glaucoma allow for the precise analysis of degenerative phenotypes. Human pluripotent stem cells (hPSCs) serve as powerful tools for studying human disease, particularly cellular mechanisms underlying neurodegeneration. Thus, efforts focused upon hPSCs with an E50K mutation in the Optineurin (OPTN) gene, a leading cause of inherited forms of glaucoma. CRISPR/Cas9 gene editing introduced the OPTN(E50K) mutation into existing lines of hPSCs, as well as generating isogenic controls from patient-derived lines. RGCs differentiated from OPTN(E50K) hPSCs exhibited numerous neurodegenerative deficits, including neurite retraction, autophagy dysfunction, apoptosis, and increased excitability. These results demonstrate the utility of OPTN(E50K) RGCs as an in vitro model of neurodegeneration, with the opportunity to develop novel therapeutic approaches for glaucoma.
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Accumulation of axoplasmic organelles in swollen nerve fibers.
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Tubulin assembly sites and the organization of cytoplasmic microtubules in cultured mammalian cells.
The number, distribution, and nucleating capacity of microtubule-organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells. Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material. MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm. The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCs was assayed by incubating tubulin-depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 65 brain tubulin and microtubule assembly buffer. Initiation and assembly of 65 tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific. Our results are consistent with the notion that the specification of microtubule length, number, and spatial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits.
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Neuronal alteration and reinnervation following axonal regeneration and sprouting in mammalian spinal cord.
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Young adult-form of dementia with neurofibrillary changes and Lewy bodies.
Alzheimer's neurofibrillary tangles, Lewy bodies and chromatolytic neurons were found in the brain at autopsy of a 28-year-old male with pyramidal and extrapyramidal signs, and severe dementia of 7-year duration prior to his death. Review of histological material showed generalized changes involving both cortical and subcortical structures. These changes were characterized by the presence of neurofibrillary myelin in long tracts and in subcortical regions. The neurofibrillary tangles were mostly composed of Alzheimer's paired helical filaments (PHF), PHF were immunostained with both polyclonal and monoclonal antibodies to PHF and the microtubule-associated protein tau. Some Lewy bodies were immunolabelled with monoclonal antibodies to PHF. To the best of our knowledge it is the first reported case of a young adult-form of dementia with extensive formation of neurofibrillary changes and Lewy bodies.
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Cathepsin D precursors in clathrin-coated organelles from human fibroblasts.
Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphate. Material adsorbed to S. aureus cells coated with control immunoglobulins was nearly free of clathrin and contained a small amount of the cathepsin D precursor (less than 20% of that adsorbed with anti-clathrin antibodies). The extraction of this cathepsin D precursor was independent of mannose 6-phosphate and was complete after a brief exposure to saponin. The amount of cathepsin D precursor in coated membranes varied between 0.4 and 2.5% of total precursor. Analysis of pulse chase-labeled fibroblasts revealed that cathepsin D was only transiently associated with coated membranes. The mean residence time of cathepsin D precursor in coated membranes was estimated to be 2 min. These observations support the view that coated membranes participate in the transfer of precursor forms of endogenous lysosomal enzymes to lysosomes.