Biology subjects

Lepine, P.

Publications and source records attributed to Lepine, P..

8 recordsLinked to original sources

bioRxiv2026

Applications of adeno-associated virus for 3D single-cell morphometric analysis in iPSC-derived midbrain organoids.

Human midbrain organoids (hMBOs) are emerging in vitro models to mirror the cellular diversity and the structural complexity of the developing human brain. However, the dense neural network, limits the investigation of individual cells morphology or cell-cell connectivity, which is mostly restricted to fixed organoids following extensive optical clearing techniques. To better resolve individual cells within a brain organoid and for longitudinal tracking of its growth and development, we turned to adeno-associated virus (AAVs) for targeted gene delivery. In particular, we applied AAVs for expressing specific markers that provide the foundation to image individual cells within 3D hMBOs. Thus, we developed a phenotypic platform to specifically inspect the neuronal and astrocytic cytoarchitecture and to examine their connectivity in living hMBOs derived from two genetically unrelated control iPSC lines. We demonstrate that through AAV transduction, we could capture and reconstruct the 3D architecture of both neurons and astrocytes within the hMBO as a whole. Transduced cells exhibited an intrinsic heterogeneity in term of soma volume, arbor complexity and territory covered, regardless of both genetic background, age, and cell-type. Yet, these cellular morphometrics remained equivalent between the two cell lines, indicative of homogeneity in hMBO cellular development. We were able to establish longitudinal profiling of transduced cells, demonstrating how neurons and astrocytes could expand their network over time. Lastly, we describe time-lapse studies to track cellular motility and morphology fluctuations in neurons and astrocytes over time, highlighting the dynamic nature of these cells within the ramified architecture of the neural network in the developing hMBOs. Overall, our platform underscores the versatility of AAVs in studying single cell-morphometrics and cellular connectivity for longitudinal monitoring of cellular dynamics in live 3D hMBOs instead of a static snapshot.

neuroscience↗

bioRxiv2026

Integration of iPSC-derived microglia into human midbrain organoids enhances microglial maturation and inflammatory signaling

Microglia are the resident immune cells of the central nervous system and play key roles in the healthy brain during development and adulthood, as well as during neurodegenerative diseases - including Parkinsons disease (PD). Yet the role of microglia in PD pathogenesis has not been fully elucidated. Limitations of 2D cell culture and animal models in simulating human microglia in the brain parenchyma have contributed to this knowledge gap. Human midbrain organoids (hMOs) provide a promising model that can recapitulate elements of PD pathology but lack microglial cells. Here we adapt protocols for the differentiation of hMOs and human iPSC-derived microglia (iMG) to generate iMG-hMO assembloids. Within assembloids, integrated iMG (intMG) express canonical microglia markers and induce the release of cytokines and chemokines. Transcriptomic profiling by single cell RNA sequencing reveals that intMG adopt a more mature and inflammation-responsive state compared to 2D iMG. The integration of microglia results in increased signaling through inflammatory and trophic pathways that drive altered transcriptional signatures of dopaminergic neurons and astrocytes within assembloids. Overall, iMG-hMO assembloids have the potential to more faithfully model the role of microglia and neuroinflammation in PD pathogenesis.

neuroscience↗

bioRxiv2026

Stress-adaptive biomaterials with tunable yielding architectures regulate organoid morphogenesis

The yield stress at which biomaterials undergo plastic deformation limits the stresses that can be developed in encapsulated growing tissues. While mechanical properties of the matrix such as stiffness and viscoelasticity have a profound effect on cells, the role of yield stress has remained challenging to define. Here we design a self-healing granular hydrogel platform with supramolecular host-guest dynamic crosslinkers to precisely and quantitatively tune the stress at which the matrix repeatedly yields and reconfigures around tissues as they grow. Designed to provide similar mechanical constraints as a mesh stress ball, matrix yield stresses can be tuned between 12 and 370 Pa, while maintaining a storage modulus below [~]0.1kPa. We show that this range of yield stress is sufficient to promote or limit peripheral shedding in a model of non-adhesive cancer migration; and that early development of midbrain organoids is exquisitely sensitive to yield stress. Optimal yield stresses of only 25 Pa promoted budlike protrusions and large, lumenized neural rosettes, while variations as small as 10 Pa limited these phenotypes. These studies demonstrate that morphogenesis and tissue organization are exquisitely sensitive to yield stress, suggesting a new material property to target in designing biomaterials for disease modeling and regenerative medicine.

biophysics↗

bioRxiv2022

CelltypeR: A framework to identify and characterize cell types in human midbrain organoids using flow cytometry

Motivated by the growing number of single cell RNA sequencing datasets (scRNAseq) revealing the cellular heterogeneity in complex tissues, particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models, we developed a high-throughput, standardized approach for reproducibly characterizing cell types in complex neuronal tissues based on protein expression levels. Our approach combines a flow cytometry (FC) antibody panel targeting brain cells with a computational pipeline called CelltypeR, with functions for aligning and transforming datasets, optimizing unsupervised clustering, annotating and quantifying cell types, and statistical comparisons. We applied this workflow to human iPSC-derived midbrain organoids and identified the expected brain cell types, including neurons, astrocytes, radial glia, and oligodendrocytes. Defining gates based on the expression levels of our protein markers, we performed Fluorescence-Activated Cell Sorting of astrocytes, radial glia, and neurons, cell types were then confirmed by scRNAseq. Among the sorted neurons, we identified three subgroups of dopamine (DA) neurons; one reminiscent of substantia nigra DA neurons, the cell type most vulnerable in Parkinsons disease. Finally, we use our workflow to track cell types across a time course of organoid differentiation. Overall, our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets.

neuroscience↗

bioRxiv2021

Myosin-1b interacts with UNC45A and controls intestinal epithelial morphogenesis

Vesicle trafficking and the establishment of apico-basal polarity are essential processes in epithelium morphogenesis. Myosin-1b, an actin-motor able to bind membranes, regulates membrane shaping and vesicle trafficking. Here, we investigate Myosin-1b function in gut morphogenesis and congenital disorders using cell line and zebrafish larvae as well as patient biopsies. In a 3D Caco-2 cyst model, lumen formation is impaired in absence of Myosin-1b. In zebrafish, both Morpholino knock-down and genetic mutation of myo1b result in intestinal bulb epithelium folding defects associated with vesicle accumulation, reminiscent of a villous atrophy phenotype. We show that Myosin-1b interacts with the chaperone UNC45A, genetic deletion of which also results in gut folding defects in zebrafish. Loss of function mutations in UNC45A have been reported in complex hereditary syndromes, notably exhibiting intestinal disorders associated with villous atrophy. In UNC45A-depleted cells and in patient biopsies, Myosin-1b protein level is strikingly decreased. The appearance of Myosin-1b aggregates upon proteasome inhibition in cells points at a degradation mechanism of misfolded Myosin-1b in the absence of its chaperone. In conclusion, Myosin-1b plays an unexpected role in the development of the intestinal epithelium folds or villi downstream UNC45A, establishing its role in the gut defects reported in UNC45A patients. Summary statementMyosin-1b is important for intestinal epithelium folding during zebrafish development and participates in the villous atrophy clinical manifestation downstream UNC45A loss of function.

developmental biology↗

bioRxiv2021

Microfabricated disk technology: rapid scale up in midbrain organoid generation

By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinsons disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.

neuroscience↗

bioRxiv2021

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy

SNCA, the first gene associated with Parkinsons disease, encodes the -synuclein (-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinsons disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of -syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of -syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of -syn and exhibit an age-dependent increase in -syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of -syn. These phosphorylated -syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinsons disease and provide a powerful system to study the pathogenesis of synucleinopathies.

neuroscience↗

bioRxiv2019

RNA-binding protein altered expression and mislocalization in multiple sclerosis

ObjectiveNuclear depletion and mislocalization of RNA-binding proteins (RBPs) trans-activation response DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS) are thought to contribute to the pathogenesis of a number of disorders, including amyotrophic lateral sclerosis (ALS). We recently found that TDP-43 as well as polypyrimidine tract binding protein (PTB) have decreased expression and mislocalization in oligodendrocytes in demyelinated lesions in an experimental mouse model of multiple sclerosis (MS) caused by Theilers murine encephalomyelitis virus infection.\n\nMethodsThe latter finding prompted us to investigate TDP-43, FUS, and PTB in the demyelinated lesions of MS and in in vitro cultured human brain-derived oligodendrocytes.\n\nResultsWe found: i) mislocalized TDP-43 in oligodendrocytes in active lesions in some MS patients; ii) decreased PTB1 expression in oligodendrocytes in mixed active/inactive demyelinating lesions; iii) decreased nuclear expression of PTB2 in neurons in cortical demyelinating lesions; iv) nuclear depletion of TDP-43 in oligodendrocytes under metabolic stress induced by low glucose/low nutrient conditions compared to optimal culture conditions.\n\nConclusionTDP-43 has been found to have a key role in oligodendrocyte function and viability, while PTB is important in neuronal differentiation, suggesting that altered expression and mislocalization of these RBPs in MS lesions may contribute to the pathogenesis of demyelination and neurodegeneration. Our findings also identify nucleocytoplasmic transport as a target for treatment.

neuroscience↗