Biology subjects

Onorati, M.

Publications and source records attributed to Onorati, M..

4 recordsLinked to original sources

bioRxiv2024

Long-term mouse spinal cord organotypic slice culture as a platform for validating cell transplantation in spinal cord injury

Spinal cord injury (SCI) is an extremely invalidating condition with a severe physical and psychological impact. Resolutive cures are still lacking, due to its complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCI. Standard SC organotypic cultures are maintained for up to 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance for up to three months (90 days). The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a GFP reporter were transplanted into mouse SC-slices. 30 days after the transplant, cells still show GFP expression, and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments. SUMMARYIn this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

neuroscience↗

bioRxiv2024

Generation and benchmarking of a collection of hiPSC lines from Schizophrenia Patients with Diverse Clinical Profiles

Limited therapeutic advancements in Schizophrenia (SCZ) depend on the heterogeneous nature of the disorder, impacting drug development and clinical trials that assume uniform therapy response, neglecting individual genetic and epigenomic variability. Disease modeling using human induced pluripotent stem cells (hiPSCs) is ideally suited for precision medicine, enabling individualized treatment approaches. Here, we describe the generation of patient-specific lines from somatic cells of SCZ individuals with well-defined diverse clinical trajectories using a Sendai virus-based reprogramming system. Karyotypically and CGH-array validated, the generated hiPSCs expressed diagnostic markers and demonstrated functional pluripotency. Converting these hiPSCs into neural progenitor cells enables the identification of aberrant cellular phenotypes associated with specific pathologically relevant neural phenotypes. This collection of hiPSC lines serves as a platform for developing therapeutic compounds targeting neural populations, potentially addressing early-stage disease alterations.

neuroscience↗

bioRxiv2022

Low forces push the maturation of neural precursors into neurons

Mechanical stimulation modulates neural development and neuronal activity. In a previous study, we proposed magnetic "nano-pulling" as a tool to generate active forces. By loading neural cells with magnetic nanoparticles (MNPs), a precise force vector is remotely generated through static magnetic fields. In the present study, human neural stem cells (NSCs) were subjected to a standard differentiation protocol, in the presence or absence of nano-pulling. Under mechanical stimulation, we found an increase in the length of the neural processes which showed an enrichment in microtubules, endoplasmic reticulum, and mitochondria. A stimulation lasting up to 52 days induced a strong remodelling at the level of synapse density and a re-organization of the neuronal network, halving the time required for the maturation of neural precursors into neurons. We then injected the MNP-loaded NSCs into mouse spinal cord slices, demonstrating that nano-pulling stimulates the elongation of the NPC processes and modulates their orientation even in an ex vivo model system. To the best of our knowledge, this is the first evidence showing that active mechanical stimuli can guide the outgrowth of NSCs transplanted into the spinal cord tissue. Our findings suggest that MNPs play an important role in neuronal maturation which could be applied in regenerative medicine.

neuroscience↗

bioRxiv2022

Microcephaly-associated WDR62 mutations hamper Golgi apparatus-to-spindle pole shuttling in human neural progenitors

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions during corticogenesis. Recessive mutations in WDR62 are associated with structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated a C-terminal truncating mutation (D955AfsX112) in WDR62 using induced pluripotent stem cells (iPSCs) obtained from a patient with MCPH2. We generated neuroepithelial stem (NES) cells and cerebro-cortical progenitors and neurons from patient-derived and isogenic retro-mutated iPSC lines. We found that WDR62 dysfunction resulted in impaired cell cycle progression and alterations of the neurogenic trajectories of iPSC neuroderivatives. Moreover, we report WDR62 localization at the Golgi apparatus during interphase, both in human neural progenitors in vitro and in human fetal brain tissue. WDR62 shuttling from the Golgi apparatus to spindle poles is dynamic and microtubule-dependent. Impairment of WDR62 function and localization results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in MCPH etiology.

neuroscience↗