Biology subjects

Sozzi, E.

Publications and source records attributed to Sozzi, E..

7 recordsLinked to original sources

bioRxiv2026

Real-Time Neurotransmitter Sensing in Midbrain Organoids Using 3D Carbon Cup Electrodes

Real time, chemically selective detection of neurotransmitter release in brain organoids remains a challenge due to their complex three dimensional architecture and limited optical accessibility. We introduce 3D printed carbon cup electrodes (CCEs) -pyrolyzed, multi level pillar structures - designed to confine a single organoid, increase electroactive surface area, and enable amperometric recordings of oxidizable neurotransmitters. The CCEs exhibit excellent electrochemical performance and reproducibility, supporting diffusion controlled dopamine redox behavior without interference from culture medium. Using ventral midbrain (vMB) organoids at two maturation stages (Day 30 and Day 90), we demonstrate real time detection of potassium evoked dopamine release directly from organoids. Mature organoids exhibit higher peak currents, correlating with increased tyrosine hydroxylase (TH) expression confirmed by immunocytochemistry. Embedding organoids in Matrigel significantly attenuates dopamine signals, indicating a diffusion barrier that limits stimulant access and/or dopamine transport to the electrode. Repetitive stimulations reveal progressive depletion of dopamine stores, consistent with diminished vesicular release. Chemical perturbations using 6 hydroxydopamine and verapamil further validate the platform by producing substantial reductions in dopamine release accompanied by loss of TH neurons. These results establish CCEs as a powerful tool for label free, real-time monitoring of neurotransmission in human organoids, with potential for multiplexed sensing and integration of enzymatic detection strategies for non electroactive neurotransmitters.

neuroscience↗

bioRxiv2026

A human developmental and adult brain atlas benchmarks dopaminergic stem cell models and cell therapy candidates

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons1. Stem cell-derived mDA neurons hold promise for disease modelling2,3 and are currently in clinical trials for cell replacement therapy4-6. However, systematic benchmarking has been limited by the lack of a unified high-resolution reference and methods that quantify incomplete or mixed lineage specification in vitro7. We establish a single-cell and spatial atlas of the human developing diencephalon-midbrain-hindbrain axis resolving 93 cell subtypes, including 39 lacking prior single-cell characterization and 4 entirely novel populations. Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published2,8-25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials. To classify cells and quantify lineage fidelity, we develop CapybaraBrain, a marker-driven non-negative decomposition framework that assigns each cell continuous identity scores across all 93 developmental programs, enabling systematic discrimination of discrete, transitioning, and cross-lineage hybrid states26. We uncover a pervasive landscape of off-target populations reflecting relaxed transcriptional boundaries in vitro, including a previously unrecognized TH-PITX2 midbrain neuronal population, and we validate atlas-predicted latent lineage plasticity through inducible genetic fate mapping in mouse models. We further define maturation-associated transcriptional programs by harmonizing adult mDA subtype atlases, revealing that dopaminergic identity and maturation are partially decoupled across protocols. Finally, projecting PD patient-derived tri-cultures onto the HDNA uncovers genotype- and cell-type-specific transcriptional dysregulation. Together, these integrated atlases and computational framework establish a unified standard for benchmarking differentiation fidelity, exposing off-target states, and guiding next-generation PD models and cell therapies.

Developmental Biology↗

bioRxiv2025

Reconstruction of the human nigrostriatal pathway in vitro reveals target-dependent dopamine neuron maturation

The human nigrostriatal pathway, comprising dopaminergic neurons in the ventral midbrain (vMB) projecting to the dorsolateral striatum, is essential for motor control and selectively vulnerable in Parkinsons disease (PD). How this circuit assembles during development and how it degenerates under pathological conditions remains poorly understood in a human context and in vitro models capturing its long-range connectivity and spatial organization have been lacking. Here, we introduce the connectoid, a compartmentalized, human stem cell-based model of the nigrostriatal pathway that integrates vMB and striatal organoids within a custom-engineered microfluidic device, confining cell bodies while guiding axonal growth, mimicking the in vivo topography. Functional connectivity was confirmed by retrograde rabies tracing, and optogenetic and pharmacological stimulation, while 6-hydroxydopamine-induced selective degeneration of dopamine neurons, recapitulating a key feature of PD. Additionally, single-cell transcriptomics revealed that interaction with striatal targets enhances dopaminergic neuron maturation and activates transcriptional programs linked to synaptic signaling. Thus, connectoids uniquely allow spatial segregation of regionalized organoids while preserving long-range communication, providing a scalable and physiologically relevant platform for studying human circuit assembly, selective vulnerability, and therapeutic interventions in PD.

neuroscience↗

bioRxiv2025

Cografting strategies uncover cell type dependent regulation of dopamine neuron specification and functional maturation in a pre clinical model of Parkinson`s Disease

Parkinsons disease (PD), the second most common neurodegenerative disorder, is characterized by the progressive loss of A9 dopaminergic neurons in the substantia nigra, leading to dopamine (DA) depletion in the striatum and subsequent motor symptoms. Transplantation of ventral midbrain-patterned DA (vmDA) progenitors derived from human pluripotent stem cells, aimed at restoring DA neurotransmission in the striatum, is being developed and currently explored in ongoing clinical trials. One factor that may improve the maturation and fate determination of DA neurons in vivo is the intercellular communication within the graft environment, ultimately affecting the therapeutic outcome. In this study, we co-transplanted vmDA progenitors with either glial, ventral forebrain or striatal progenitors into a preclinical xenograft PD model to investigate how these interactions shape the development, maturation, and function of therapeutic DA neurons. Our findings show that co-grafts with ventral forebrain progenitors increase the yield of DA neurons and also promote their functional maturation. Furthermore, we demonstrated that co-grafts with striatal neurons promote functional maturation and the acquisition of DA subtype identity. From these data, we identified EBF3 and PBX3 as candidate transcription factors directing DA neuron maturation and subtype specification, and then functionally validated their role in brain organoids. Taken together, our data highlight that the cellular microenvironment, including specific interactions with neighbouring cells, guides in vivo DA neuron specification and maturation. These findings provide a foundation for developing more refined and effective cell preparations for replacement therapy in PD, and define a conceptual framework that could inform stem cell-based strategies for other neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Nuclei Isolation Protocol for Single-Nucleus RNA Sequencing of Human Stem Cell-Derived Grafts

Single-nucleus RNA sequencing enables high-resolution transcriptomic profiling of brain tissue, facilitating detailed analysis of cell identity in models of neurodegeneration and repair. Here, we describe a protocol for isolating nuclei from long-term human stem cell-derived grafts in the rat brain, incorporating vibratome sectioning, graft dissection, nuclear extraction, and fluorescence-activated sorting. This workflow supports analysis of human neurons embedded within host tissue or sensitive to dissociation, offering a powerful approach to assess graft composition, integration, and neuronal identity in living brain. For complete details on the use and execution of this protocol, please refer to Fiorenzano et al.1 Subject areasSingle nucleus RNA sequencing, stem cell biology, neuroscience, transplantation, cell replacement therapy Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/672369v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ac3b90org.highwire.dtl.DTLVardef@7aab65org.highwire.dtl.DTLVardef@18a8fd4org.highwire.dtl.DTLVardef@1e8bfae_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIStep-by-step vibratome sectioning of xenografted rat brain tissue. C_LIO_LIPrecise dissection of human stem cell-derived grafts from host brain sections. C_LIO_LIExtraction of intact nuclei from fragile, grafted neurons. C_LIO_LIIsolation of single nuclei via fluorescence-activated nuclei sorting (FANS) for snRNA-seq sample preparation. C_LI

cell biology↗

bioRxiv2024

Non-cell-autonomous control of gastruloid development by the lncRNA T-UCstem1 through DKK1-dependent modulation of WNT signalling

The role and the mechanisms of long non-coding RNA (lncRNAs) in early mammalian embryogenesis remain unclear; this is mostly due to the complexity of the regulatory mechanism of lncRNAs and the limited availability of early embryo samples. The emergence of stem cell-based models of early mammalian embryogenesis offers new opportunities to address these challenging questions. Here we use mouse gastruloids to investigate the role of an ultraconserved lncRNA, T-UCstem1, in the formation of the mammalian body plan. Combining morphological and immunofluorescence analysis with bulk and single cells transcriptomics, we provide unprecedented evidence that T-UCstem1 is a key regulator of gastruloid development and is required for the extension of the anteroposterior axis. Specifically, knock down of T-UCstem1 results in aberrant gastruloid development, which is characterized by altered spatiotemporal expression of the differentiation markers and persistence of pluripotency genes. Single-cell analysis reveals higher cellular heterogeneity in T-UCstem1 KD gastruloids. Notably, the presence of cell populations characterized by the co-expression of pluripotency and differentiation markers points to a key role of T-UCstem1 in establishment and maintenance of proper cellular identity. Mechanistically, we show that T-UCstem1 acts non-cell autonomously through Dickkopf-related protein 1 (DKK-1)-dependent modulation of WNT pathway. Our findings highlight a previously unexplored role for ultraconserved lncRNAs in gastruloid development and open the way for using gastruloids to dissect the functions of lncRNAs in early mammalian development.

developmental biology↗

bioRxiv2020

Single cell transcriptional and functional analysis of human dopamine neurons in 3D fetal ventral midbrain organoid like cultures

Transplantation of midbrain dopamine (DA) neurons for the treatment of Parkinsons disease (PD) is a strategy that has being extensively explored and clinical trials using fetal and stem cell-derived DA neurons are ongoing. An increased understanding of the mechanisms promoting the generation of distinct subtypes of midbrain DA during normal development will be essential for guiding future efforts to precisely generate molecularly defined and subtype specific DA neurons from pluripotent stem cells. In this study, we used droplet-based scRNA-seq to transcriptionally profile a large number of fetal cells from human embryos at different stages of ventral midbrain (VM) development (6, 8, and 11 weeks post conception). This revealed that the emergence of transcriptionally distinct cellular populations was evident already at these early timepoints. To study late events of human DA differentiation and functional maturation, we established a primary fetal 3D culture system that recapitulates key molecular aspects of late human DA neurogenesis and sustains differentiation and functional maturation of DA neurons in a physiologically relevant cellular context. This approach allowed us to define the molecular identities of distinct human DA progenitors and neurons at single cell resolution and construct developmental trajectories of cell types in the developing fetal VM. Overall these findings provide a unique transcriptional profile of developing fetal VM and functionally mature human DA neurons, which can be used to quality control stem cell-derived DA neurons and guide stem cell-based therapies and disease modeling approaches in PD.

neuroscience↗