The cytoprotective, catabolic process of autophagy is a highly conserved response to conditions of cellular stress and nutrient depletion. Large intracellular substrates, like misfolded or aggregated proteins and organelles, experience degradation due to this mechanism. The process of self-degradation is vital for maintaining protein balance in post-mitotic neurons, demanding meticulous control over its actions. Driven by its homeostatic function and the implications it holds for certain disease states, autophagy research is expanding rapidly. Included in a practical toolkit for examining autophagy-lysosomal flux in human iPSC-derived neurons are two assays. For the assessment of autophagic flux in human iPSC neurons, a western blotting approach is outlined in this chapter, targeting two proteins of interest for quantification. In the concluding section of this chapter, a flow cytometry assay utilizing a pH-sensitive fluorescent reporter for assessing autophagic flux is detailed.
Exosomes, a type of extracellular vesicle (EV), are produced through endocytic processes. Their function in intercellular signaling is significant, and they are implicated in the dispersal of protein aggregates linked to neurological diseases. The plasma membrane is the final destination for multivesicular bodies, also known as late endosomes, to release exosomes into the extracellular environment. A novel application of live-imaging microscopy in exosome research has enabled the simultaneous capture of MVB-PM fusion and exosome release within single cells. Researchers have engineered a construct that merges CD63, a tetraspanin enriched in exosomes, with the pH-sensitive marker pHluorin. The fluorescence of the CD63-pHluorin fusion protein is quenched within the acidic MVB lumen, subsequently fluorescing only upon release into the less acidic extracellular medium. Stereotactic biopsy A method for visualizing MVB-PM fusion/exosome secretion in primary neurons is described here, utilizing a CD63-pHluorin construct in combination with total internal reflection fluorescence (TIRF) microscopy.
Active cellular uptake of particles, known as endocytosis, is a dynamic process. Late endosome-lysosome fusion represents a pivotal step in the degradation pathway for both newly synthesized lysosomal proteins and endocytosed material. Neurological disorders are a consequence of disturbances in this neuronal process. Consequently, examining endosome-lysosome fusion within neurons holds the potential to reveal new understandings of the mechanisms driving these diseases, while simultaneously presenting promising avenues for therapeutic intervention. Yet, the quantification of endosome-lysosome fusion proves to be a problematic and protracted undertaking, which consequently hampers investigations in this specific field of study. We engineered a high-throughput method using the Opera Phenix High Content Screening System and pH-insensitive dye-conjugated dextrans. This method proved effective in segregating endosomes and lysosomes within neurons, and time-lapse imaging documented endosome-lysosome fusion events observed in hundreds of cells. Both assay set-up and analysis processes can be undertaken in a manner that is both swift and effective.
Genotype-to-cell type connections are frequently elucidated via the widespread application of large-scale transcriptomics-based sequencing methods, a consequence of recent technological developments. Employing CRISPR/Cas9-edited mosaic cerebral organoids, we describe a fluorescence-activated cell sorting (FACS) and sequencing method designed to ascertain or validate correlations between genotypes and specific cell types. Comparisons across different antibody markers and experiments are possible due to the quantitative and high-throughput nature of our approach, which utilizes internal controls.
Cell cultures and animal models offer avenues for studying neuropathological diseases. Animal models, unfortunately, often fall short in replicating the intricate nature of brain pathologies. Flat-surface cell cultures, a tried-and-true method, have been used for decades, beginning in the early 1900s, to cultivate cells. Ordinarily, 2D neural culture systems, which lack the intricate three-dimensional architecture of the brain, often provide a flawed representation of the diverse cell types and their interactions during physiological and pathological processes. A donut-shaped sponge, featuring a central window that is optically transparent, contains an NPC-derived biomaterial scaffold. This scaffold is made of silk fibroin interspersed with a hydrogel, and it accurately replicates the mechanical properties of natural brain tissue, enabling sustained neural cell development. This chapter details the process of incorporating iPSC-derived neural progenitor cells (NPCs) within silk-collagen scaffolds and subsequently inducing their maturation into neural cells.
Modeling early brain development is gaining significant traction thanks to the rising utility of region-specific brain organoids, including those of the dorsal forebrain. Of particular importance, these organoids provide a context for investigating the mechanisms that contribute to neurodevelopmental disorders, mimicking the developmental stages of early neocortical structures. Remarkably, the development of neural precursors, their transformation into intermediate cell types, and eventual differentiation into neurons and astrocytes mark significant progress, as do the essential neuronal maturation processes like synapse formation and pruning. Using human pluripotent stem cells (hPSCs), we demonstrate the creation of free-floating dorsal forebrain brain organoids, the method detailed here. The organoids are also validated through cryosectioning and immunostaining techniques. A refined protocol is included for the high-quality dissociation of brain organoid tissues into individual living cells, a necessary first step for subsequent single-cell assays.
In vitro cell culture models are useful for high-resolution and high-throughput investigation of cellular activities. narrative medicine However, in vitro culture procedures frequently fail to fully reproduce intricate cellular processes that depend on harmonious interactions between diverse neural cell populations and the enveloping neural microenvironment. Detailed procedures for the formation of a three-dimensional primary cortical cell culture system, compatible with live confocal microscopy, are presented here.
The brain's key physiological component, the blood-brain barrier (BBB), safeguards it from peripheral processes and pathogens. The BBB's dynamic structure is actively engaged in cerebral blood flow, angiogenesis, and other neural functions. Nevertheless, the BBB functions as a formidable obstacle to the penetration of therapeutics into the brain, obstructing more than 98% of drugs from interacting with the brain. Neurovascular co-morbidities in neurological diseases, such as Alzheimer's and Parkinson's, are indicative of a potential causal involvement of blood-brain barrier impairment in the process of neurodegeneration. However, the underlying methodologies by which the human blood-brain barrier is built, preserved, and declines in the context of illnesses remain largely unclear, as human blood-brain barrier tissue is difficult to obtain. In an effort to alleviate these constraints, we developed an in vitro induced human blood-brain barrier (iBBB), derived from pluripotent stem cells. To advance understanding of disease mechanisms, identify novel drug targets, screen potential drugs, and apply medicinal chemistry to boost the brain penetration of central nervous system treatments, the iBBB model provides a valuable platform. The present chapter elaborates on the techniques to differentiate induced pluripotent stem cells into endothelial cells, pericytes, and astrocytes, as well as methods for their assembly into the iBBB.
Brain microvascular endothelial cells (BMECs) form the blood-brain barrier (BBB), a high-resistance cellular interface that isolates the blood from the brain parenchyma. see more Preservation of brain homeostasis depends upon a healthy blood-brain barrier (BBB), although this barrier can impede the access of neurotherapeutic medications. Nevertheless, there are restricted possibilities when it comes to testing BBB permeability specifically in humans. The use of human pluripotent stem cell models allows for a powerful dissection of this barrier's components in vitro, including the understanding of blood-brain barrier mechanisms and the development of approaches to boost the permeability of molecular and cellular treatments directed at the brain. A comprehensive, step-by-step protocol for differentiating human pluripotent stem cells (hPSCs) into cells displaying key BMEC characteristics, including paracellular and transcellular transport resistance, and transporter function, is presented here for modeling the human blood-brain barrier (BBB).
Human neurological disease modeling has significantly benefited from the innovations in induced pluripotent stem cell (iPSC) techniques. The induction of neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells has been facilitated by several well-established protocols. In spite of their merits, these protocols are still constrained by limitations, including the substantial period of time necessary to isolate the specific cells, or the difficulty of culturing several different cell types simultaneously. The process of developing standardized protocols for addressing multiple cell types within a compressed timeframe remains in progress. We detail a straightforward and dependable co-culture setup for investigating the interplay between neurons and oligodendrocyte precursor cells (OPCs), both in healthy and diseased states.
The generation of oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) is possible through the employment of human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Through the strategic modification of culture parameters, pluripotent cell populations are sequentially guided via intermediary cell types, transforming initially into neural progenitor cells (NPCs) and subsequently into oligodendrocyte progenitor cells (OPCs) before achieving their mature state as central nervous system-specific oligodendrocytes (OLs).