Biology subjects

Gagliano, O.

Publications and source records attributed to Gagliano, O..

6 recordsLinked to original sources

bioRxiv2025

Early Reprogramming Intermediates Enable Direct Neuronal Conversion via NGN2

The generation of engineered neurons via Neurogenin-2 (NGN2) overexpression, starting from human induced pluripotent stem cells (hiPSCs), is a powerful tool for modeling neurological diseases. However, using stabilized hiPSCs as a starting point significantly increases the time required to obtain a valuable human neuronal model in vitro. Here, we demonstrated that as little as 3 days of transient expression of reprogramming factors in human fibroblasts can unlock the ability of these cells to transdifferentiate into neurons upon overexpression of NGN2. We used single-cell transcriptomic data to dissect the distinct cell identities that emerge during reprogramming. We identified three distinct reprogramming intermediate populations responsive to NGN2-mediated neuronal induction and found that partial reprogramming for only 3 days is sufficient to mediate NGN2 neuronal conversion of human fibroblasts. Moreover, we found that the efficiency in neuronal fate acquisition mediated by NGN2 overexpression is strictly correlated with the stage of reprogramming used as a starting point. HighlightsO_LIHuman fibroblasts undergo reprogramming into pluripotent stem cells, passing through defined intermediate populations. C_LIO_LIIntermediate reprogramming stages can undergo neuronal fate programming by NGN2 overexpression. C_LIO_LIThe efficiency of neuronal conversion depends on the intermediate stage employed and increases with reprogramming progression. C_LI

neuroscience↗

bioRxiv2024

Engineering assembloids to mimic graft-host skeletal muscle interaction

Skeletal muscle (SkM) tissue engineering aims to generate in vitro three-dimensional (3D) products that can be implanted in patients to replace or repair damaged muscles. Having a humanized in vitro model able to mimic the interaction between the innervated recipient and the engineered SkMs at a functional level would greatly help in the evaluation of the graft potential. Here we developed a 3D in vitro model that allowed to investigate the function, stability, and adaptability of the human NM system in response to an engineered SkM construct. To achieved this, we used decellularized SkMs (dSkM)-based constructs as engineered SkM and human neuromuscular organoids (NMOs) as the recipient-like NM system to create graft-host SkM assembloids. We observed migration of myogenic cells and invasion of neural axons from the NMO to the engineered SkM construct in the assembloids, with the generation of functional neuromuscular junctions (NMJs). Finally, we showed that assembloids were able to regenerate following acute damage, with SkM regeneration and functional recovery. Despite limited by the absence of immunocompetent cells and vasculature, our data showed that our assembloid represents a useful tool to evaluate in vitro the response of the human innervated SkM to a potential tissue-engineered SkM graft.

cell biology↗

bioRxiv2024

From naïve pluripotency to human neural organoids through a three-dimensional morphogenetic continuum

Human central nervous system (CNS) development involves complex transitions from pluripotency to regionalised neural tissues. The early phases of this process are inaccessible in humans but can potentially be modelled in vitro using brain organoids, including to study neurodevelopmental disorders. However, current methods are based on post-implantation-like human pluripotent stem cells (hPSCs), which exhibit a hypermethylated state and show epigenetic memory retention. Here we developed a 3D model of human CNS development, starting from naive human induced PSCs (hiPSCs), which exhibit a hypomethylated pre-implantation-like state of pluripotency and develop into 3D neuroepithelial cysts in a timely morphogenetic continuum. Upon treatment with appropriate signalling cues, naive-derived neuroepithelial cysts can be specified toward different axial identities. Extended culture of anterior-specified organoids results in forebrain-like structures containing both dorsal and ventral neural precursors as well as mature neurons, exhibiting appropriate cellular diversity and functional properties. We applied this system to model Fragile X Syndrome (FXS), an epigenetically regulated neurodevelopmental disorder. We found that FXS patient-derived naive hiPSCs, initially demethylated at the Fmr1 locus, gradually underwent remethylation during organoid development. In addition, Fmr1 silencing started much earlier than can be detected by pre-natal analysis, and is concomitant with the development of mosaicisms. Our approach provides a new platform for studying human CNS development, including early epigenetic events and regional patterning, demonstrating the potential of naive hiPSC-derived organoids for modelling neurodevelopmental disorders with complex epigenetic regulation. Highlights- single naive hiPSCs differentiate into 3D neuroepithelial cysts in a timely morphogenetic continuum - signalling cues at appropriate developmental transitions can direct naive hiPSC- derived organoids to different regional identities of the human CNS - naive hiPSC-derived forebrain organoids display cellular complexity representing both dorsal and ventral identities - forebrain organoids from Fragile X Syndrome patients recapitulate the genetic instability and epigenetic dysregulation of Fmr1 locus.

cell biology↗

bioRxiv2023

Native extracellular matrix promotes human neuromuscular organoid morphogenesis and function.

Human neuromuscular organoids (NMOs) derived from induced pluripotent stem cells (hiPSCs) hold a great potential to study (dys)functional human skeletal muscle (SkM) in vitro. The three-dimensional (3D) self-assembly of NMOs leads to the generation of spheroids, whose 3D organization cannot be controlled. Indeed, proper development, maturation and function of the innervated SkM require a well-defined multiscale 3D organization of the cells in a tissue-specific extracellular matrix (ECM) context. We hypothesized that extracellular structural imprinting along with hiPSC small-molecule-based differentiation could provide self-assembly guidance driving NMO morphogenesis and promoting the maturation and function of the human neuronal-coupled SkM in vitro models. We found that SkM ECM, provided as decellularized skeletal muscle, is able to reproducibly guide the morphogenesis of differentiating hiPSC toward multiscale structured tissue-like NMOs (t-NMOs). T-NMOs show contractile activity and possess functional neuromuscular junctions (NMJs), with mature neuromuscular system upon 30 days of hiPSC differentiation. We found that t-NMO could mimic altered muscle contraction upon administration of neurotoxins that act at NMJ level. Finally, we used hiPSCs derived from patients affected by Duchenne Muscular Dystrophy (DMD) to produce DMD t-NMOs that, upon neuronal stimulation, were able to mimic the altered SkM contractility and calcium dynamics typical of the disease. Altogether, our data confirm the ability of t-NMO platform to model in vitro human neuromuscular system (patho)physiology.

cell biology↗

bioRxiv2022

Effective NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform

Neurodegenerative diseases represent an increasing health burden, with a worrying lack of models recapitulating the hallmarks of the pathology. Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology. For instance, microfluidics allows to mimic the extracellular accumulation of misfolded proteins in the finely controlled microenvironment, thanks to the intrinsic high surface-area-to-volume ratio. Automated microfluidic platforms offer advantages in implementing high-throughput, standardized and parallelized assays, suitable for drug screenings and developing new therapeutic approaches in a cost-effective way. However, the major challenges in the broad application of automated lab-on-a-chip in biological research are the lack of production robustness and ease of use of the devices. Here, we present an automated microfluidic platform able to host the rapid conversion of human induced pluripotent stem cells (hiPSCs) into neurons via NGN2 viral programming in a user-friendly manner. The design of the platform, built with multilayer soft-lithography techniques, shows easiness in the fabrication and assembly thanks to the simple geometry and experimental reproducibility at the same time. The all operations are automatically managed from the cell seeding, medium change, doxycycline-mediated neuronal induction, and selection of the genetically engineered cells, to the analysis, including immunofluorescence assay. Our results show a high-throughput, efficient and homogenous conversion of hiPSCs in neurons in 10 days showing the expression of mature marker MAP2, and calcium signaling. The neurons-on-chip model here described represents a fully automated loop system able to address the challenges in the field of neurodegenerative diseases and improve current preclinical models.

neuroscience↗

bioRxiv2020

SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global public health emergency. COVID-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (GI) symptoms. This is particularly true in children in whom GI symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. By contrast, fetuses seem to be rarely affected by COVID-19, although the virus has been detected in placentas of affected women. These observations raise the question of whether the virus can infect and replicate within the stomach once ingested. Moreover, it is not yet clear whether active replication of SARS-CoV-2 is possible in the stomach of children or in fetuses at different developmental stages. Here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (PCW) fetuses, and from pediatric biopsies, to be used as an in vitro model for SARS-CoV-2 gastric infection. Gastric organoids recapitulate human stomach with linear increase of gastric mucin 5AC along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin A. In order to investigate SARS-CoV-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (RP-GOs) in suspension. In this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ACE2). The pediatric RP-GOs are fully susceptible to infection with SARS-CoV-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. The RP-GOs derived from pediatric patients show sustained robust viral replication of SARS-CoV-2, compared with organoids derived from fetal stomachs. Transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. However, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of SARS-CoV-2.

cell biology↗