Biology subjects

Fiorenzano, A.

Publications and source records attributed to Fiorenzano, A..

10 recordsLinked to original sources

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 Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published2,8-25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials.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↗

bioRxiv2026

Rare variants alter mitochondrial lipid homeostasis and neuronal excitability in PD patient-derived dopaminergic neurons

Parkinsons disease (PD) exhibits substantial genetic heterogeneity, yet how combinations of rare variants converge on disease-relevant cellular mechanisms remains unclear. Here, we generated human induced pluripotent stem cell-derived dopaminergic neurons from PD patients carrying rare variants in recently implicated genes and performed integrated electrophysiological, proteomic, lipidomic, and genetic analyses. Patient-derived neurons showed reduced membrane capacitance and altered action potential firing, indicating impaired intrinsic excitability and synaptic dysfunction, with marked variability across genetic backgrounds. Multi-omics profiling revealed dysregulation of mitochondrial function, glycolysis, and oxidative phosphorylation, accompanied by extensive lipid remodeling, including increased fatty acids, acylcarnitines, and sphingolipids, and reduced gangliosides. These alterations were more pronounced in neurons harboring specific variant combinations in KIF21B, SLC6A3, HMOX2, TMEM175, and AIMP2. Integrative analyses uncovered coordinated protein-lipid changes linking mitochondrial dysfunction and membrane homeostasis. Notably, Calpastatin and CXCR4 were consistently dysregulated across PD neurons. Genetic association analyses in independent cohorts identified PD-associated variants in genes encoding dysregulated proteins, supporting the functional relevance of these pathways. Overall, our results define convergent and variant-specific mechanisms underlying PD and highlight candidate biomarkers and therapeutic targets.

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↗

bioRxiv2025

Non-neuronal, TGF-β- extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

Progressive supranuclear palsy-Richardson syndrome (PSP-RS) is a rapidly progressive tauopathy lacking effective therapies. Although tau aggregation is a defining feature, the initiating mechanisms remain elusive. Here we used patient-derived induced pluripotent stem cell midbrain organoids, integrating single-cell transcriptomics, bulk RNA profiling, and quantitative proteomics, to dissect early pathogenic events. We identified vascular leptomeningeal-like cells (VLMCs) as the first altered population, exhibiting TGF-{beta}-driven extracellular matrix (ECM) remodeling enriched in collagens, integrins, and TGFBI. The resulting pathological ECM increased stiffness, induced integrin clustering, and activated RhoA-ROCK-mediated cytoskeletal disorganization. These changes sustained PI3K-AKT and MAPK-ERK signaling, suppressed PP2A, hyperactivated mTOR, and impaired autophagy, culminating in tau hyperphosphorylation and mislocalization. Pharmacological inhibition of TGF-{beta}, AKT, ERK, or mTORC1 restored autophagic flux, reduced tau burden, and rescued neuronal architecture. Our findings establish non-neuronal, matrix-producing niche cells as upstream drivers of tauopathy and reveal TGF-{beta}-mediated ECM restructuring as a mechanochemical trigger of neurodegeneration, opening multiple therapeutic avenues for PSP-RS and related tauopathies.

neuroscience↗

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↗

bioRxiv2024

Mosaic midbrain organoids: a new tool to study Progressive Supranuclear Palsy and advancing clinical neurology research

Progressive supranuclear palsy (PSP) is a severe neurodegenerative disease pathologically characterized by intracellular tangles of hyperphosphorylated tau protein, widely distributed across the neocortex, basal ganglia, and midbrain. Developing effective drugs for PSP presents challenges due to its complex underpinning mechanism and the absence of robust human models that accurately recapitulate biochemical and pathological features of the disease phenotype. Brain organoids have recently emerged as a three-dimensional tissue culture platform to study brain development and pathology. Here, we present a novel induced pluripotent stem cell (iPSC)-derived mosaic midbrain organoid (mMOs) system from four patients with progressive supranuclear palsy-Richardson syndrome (PSP-RS), aimed at reproducing key molecular disease features while reducing variability across organoids derived from different iPSC donors. The PSP-RS 3D model exhibited accumulation of hyperphosphorylated tau protein, predominance of 4R-tau, increased GFAP-positive cells, and PSP-associated histological alterations compared to organoids derived from healthy donors. Pathologically, diseased mMOs showed typical neurofibrillary tangles and tufted-shaped astrocytes, and poorly branched processes of Tyrosine Hydroxylase-immunoreactive cells with thin terminal branches. Our results suggest that mMOs represent a valuable experimental model for PSP research and hold great promise for future identification of new therapeutic targets for progressive supranuclear palsy. O_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/597136v1_unfig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@8b2942org.highwire.dtl.DTLVardef@41e3baorg.highwire.dtl.DTLVardef@1cddaeeorg.highwire.dtl.DTLVardef@1e0716c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Injectable 3D microcultures enable intracerebral transplantation of mature neurons directly reprogrammed from patient fibroblasts

Direct reprogramming of somatic cells into induced neurons (iNs) has become an attractive strategy for the generation of patient-specific neurons for disease modeling and regenerative neuroscience. To this end, adult human dermal fibroblasts (hDFs) present one of the most relevant cell sources. However, iNs generated from adult hDFs using two-dimensional (2D) cultures poorly survive transplantation into the adult brain in part due to the need for enzymatic or mechanical cellular dissociation before transplantation. Three-dimensional (3D) culturing methodologies have the potential to overcome these issues but have largely been unexplored for the purposes of direct neuronal reprogramming. Here we report a strategy for direct in vitro reprogramming of adult hDFs inside suspension 3D microculture arrays into induced DA neurospheroids (iDANoids). We show that iDANoids express neuronal and DA markers and are capable of firing mature action potentials and releasing dopamine. Importantly, they can be gently harvested and transplanted into the brain of a Parkinsons disease rat model to reproducibly generate functionally integrated neuron-rich grafts. The 3D culturing approach presented here thus eliminates a major bottleneck in direct neuronal reprogramming field and, due to its simplicity and versatility, could readily be adapted as a culturing platform used for a broad range of transplantation studies as well as disease modeling.

neuroscience↗

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↗

bioRxiv2019

Single Cell Gene Expression Analysis Reveals Human Stem Cell-Derived Graft Composition in a Cell Therapy Model of Parkinson’s Disease

Since the pioneering studies using fetal cell transplants in Parkinsons disease (PD), brain repair by cell replacement has remained a long-standing and realistic goal for the treatment of neurodegenerative disorders including PD. Authentic and functional midbrain dopamine (DA) neurons can now be generated from human pluripotent stem cells (hPSCs) via a floor plate intermediate1,2, and these cell preparations are both safe and functional when transplanted to animal models of PD3. However, although resulting grafts from fetal brain tissue and hPSCs contain large numbers of desired DA neurons, these therapeutic cells are a minor component of the grafts. Moreover, the cellular composition of the graft has remained difficult to assess due to limitations in histological methods that rely on pre-conceived notions concerning cell types. Here, we used single cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from ventral midbrain (VM)-patterned human embryonic stem cells (hESCs) and VM fetal tissue after long-term survival and functional maturation in a pre-clinical rat model of PD. The analyses revealed that while both cell preparations gave rise to neurons and astrocytes, oligodendrocytes were only detected in grafts of fetal tissue. On the other hand, a cell type closely resembling a class of newly identified perivascular-like cells was identified as a unique component of hESC-derived grafts. The presence of these cells was confirmed in transplants from three different hESC lines, as well as from iPSCs. Thus, these experiments have addressed one of the major outstanding questions in the field of cell replacement in neurological disease by revealing graft composition and differences between hESC- and fetal cell-derived grafts, which can have important implications for clinical trials.

neuroscience↗