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Pluripotent Stem Cell Platforms for Drug Discovery.
Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis.
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Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin.
Monospecific antibody directed against bovine brain tubulin has been purified by affinity chromatography and tested against soluble tubulin and intact microtubules of brain and mitotic apparatus. Binding of the tubulin antibody to the mitotic spindle of rat kangaroo cells was demonstrated in all stages of mitosis by indirect immunofluorescence.
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Midline assembloids reveal regulators of human axon guidance.
Organizers orchestrate cell patterning and axon guidance in the developing nervous system. Although nonhuman models have led to fundamental discoveries about floor plate (FP)-mediated midline organization, an experimental model of the human FP would enable insights into human neurodevelopment and midline connectivity. Here, we developed organoids resembling human FP (hFpOs) and assembled them with human spinal cord organoids (hSpOs) to generate midline assembloids (hMAs). We demonstrate that hFpOs promote ventral patterning, commissural axon guidance, and bilateral connectivity. To investigate midline regulators, we profiled the hFpO secretome, identifying 27 human-enriched genes compared with mouse. In an arrayed CRISPR screen of hMAs, we discovered that loss of GALNT2 and PLD3 impaired FP-mediated guidance of axons. This platform holds promise for revealing aspects of human-specific neurobiology and disease.
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Modeling Down syndrome in cells: From stem cells to organoids.
Down Syndrome (DS) is a complex chromosomal disorder, with neurological issues, featuring among the symptoms. Primary neuronal cells and tissues are extremely useful, but limited both in supply and experimental manipulability. To better understand the cellular, molecular and pathological mechanisms involved in DS neurodevelopment and neurodegeneration, a range of different cellular models have been developed over the years including human: mouse hybrid cells, transchromosomic mouse embryonic stem cells (ESCs) and human ESC and induced pluripotent stem cells derived from different sources. All of these model systems have provided useful information in the study of DS. Furthermore, different technologies to genetically modify or correct trisomy of either single genes or the whole chromosome have been developed using these cellular models. New techniques and protocols to allow better modeling of cellular mechanisms and disease processes are being developed and the use of cerebral organoids offers great promise for future research into the neural phenotypes seen in DS.
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Magnetic particles in the liver: a probe for intracellular movement.
Previous studies have used magnetic particles to estimate the viscosity of cell cytoplasm in vitro 1-4. Here we describe how magnetic Fe2O3 particles can be used to estimate non-invasively the motion of organelles in hepatic macrophages in intact animals. We report that when these particles are injected intravenously (i.v.), most are phagocytosed by hepatic macrophages (Fig. 1)5. When an external magnetic field is applied to the rabbit, these particles become magnetized and aligned. After removal of the field, the particles collectively produce a remanent magnetic field which can be measured at the body surface. This field decreases with time due to particle rotation (relaxation) 6,7. As the particles are contained in phagosomes or secondary lysosomes, we conclude that motions of these organelles are responsible for the particle rotation and relaxation.
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PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.
Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.
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Immunofluorescent localization of 100K coated vesicle proteins.
A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.
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Organoids as a model of status epilepticus.
Status epilepticus (SE) is a neurological emergency that can be studied in animal models, particularly mice. However, these models are labour-intensive and require large numbers of animals, which raises ethical and logistical challenges. Additionally, rodent-based models could lack direct relevance to human physiology. While reduced models offer some insights, they fail to replicate the full complexity of brain connectivity and interactions with other organs. To address this, human forebrain assembloids, formed by both cortical excitatory and subpallial inhibitory neurons, could be an alternative SE model. Assembloids offer a middle ground, enabling high-throughput screening of potential treatments while maintaining relevant human cell biology. This approach could serve as an intermediate step before transitioning to animal models, ultimately reducing the time and number of animals required for SE research. This paper is based on a presentation made at the 9thLondon-Innsbruck Colloquium on Status Epilepticus and Acute Seizures in April 2024.
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Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephaly.
Actins are cytoskeletal proteins that are essential for multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma-cytoskeletal actin isoforms, respectively, cause a broad spectrum of neurodevelopmental disorders, with microcephaly as the most frequent one. To investigate the pathogenesis underlying this cortical malformation, we studied patient-derived cerebral organoids from induced pluripotent stem cells of individuals with the Baraitser-Winter-CerebroFrontoFacial syndrome (BWCFF-S) carrying an ACTB/ACTG1 missense mutation. These organoids were reduced in size, showing a thinner ventricular zone (VZ) due to reduced VZ progenitor abundance. Strikingly, VZ progenitors in BWCFF-S cerebral organoids displayed a shift in the orientation of their cleavage plane from a predominantly vertical to a majoritarian horizontal orientation. The latter cleavage plane orientation is incompatible with increasing VZ progenitor abundance and instead promotes basal progenitor generation. Various cytoskeletal and morphological irregularities of BWCFF-S VZ progenitors, notably in the apical region, seemingly contribute to this change in cleavage plane orientation. Our results provide insight into the cell biological basis of the microcephaly associated with BWCFF-S caused by actin mutations.
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Human cerebral organoids reveal PFOA-induced axonal injury as a conserved mechanism of neurodevelopmental disruption.
Perfluorooctanoic acid (PFOA), a persistent environmental contaminant of growing public health concern, has been epidemiologically linked to neurodevelopmental deficits, yet its direct pathogenic mechanisms remain poorly defined. Our study addresses this critical gap by employing a unique cross-species framework that integrates zebrafish behavior with human neural organoids-a innovative approach that bridges in vivo physiology with human-specific molecular insights. We demonstrate that embryonic PFOA exposure selectively impairs social behavior in zebrafish while sparing basic motor function, a deficit paralleled by disrupted network activity in human cortical organoids. Mechanistically, we identify a precise developmental disruption: PFOA blocks neuronal differentiation without altering progenitor proliferation, leading to transient growth impairment and lasting neuronal structural deficits. Cross-model transcriptomics revealed conserved axonal and synaptic pathway disruption, centered on downregulation of the structural gene NEFL, which we validated at the protein level. Our findings not only establish a new paradigm for PFOA neurotoxicity but also highlight the value of human neural organoids in environmental risk assessment. By directly linking molecular pathogenesis to functional impairment across species, this work provides crucial mechanistic evidence for regulatory decision-making and underscores the unique vulnerability of the developing human nervous system to environmental contaminants.