Biology subjects

Picelli, S.

Publications and source records attributed to Picelli, S..

11 recordsLinked to original sources

bioRxiv2026

Odor preference maps to cohesive transcriptional domains in the olfactory bulb

Animals classify odors along behaviorally relevant dimensions, yet how the olfactory bulb, the first stage of olfactory processing, is organized and how this organization relates to odor classification remains unclear. Here, we identify cohesive transcriptional domains within the larval zebrafish olfactory bulb whose differential functional recruitment reflects odor preference. Using a flow-based two-choice assay, we characterized behavioral preferences across chemically diverse odors. Mapping odor-evoked neuronal activity patterns revealed that similarly preferred odors converge onto shared spatial activation patterns in the olfactory bulb. Whole-mount multiplexed spatial transcriptomics revealed that projection neuron and interneuron subtypes form cohesive spatial domains. Mapping odor-activated neurons onto these domains identified preference-biased recruitment of specific neuronal subtypes. Targeted ablation of a TH+ interneuron population associated with attraction selectively impaired attraction while sparing aversion. Together, these findings reveal that the olfactory bulb is organized into spatially cohesive, transcriptionally defined domains and that selective recruitment of specific subtypes is linked to odor preference. Key findingsO_LIChemically diverse odorants elicit behaviors reflecting olfactory preference. C_LIO_LITranscriptionally defined neuronal subtypes form cohesive spatial domains in the olfactory bulb. C_LIO_LIOdorants of opposing preference recruit distinct transcriptional domains in the olfactory bulb. C_LIO_LIA TH+ interneuron population is selectively required for odor attraction but not aversion. C_LI

neuroscience↗

bioRxiv2026

Proliferative Capacity and Neural Lineage Commitment of Muller Glia in the Adult Human Retina

BackgroundThe mammalian retina lacks meaningful regenerative capacity, and degeneration usually leads to irreversible vision loss. Although lower vertebrates regenerate retinal neurons through Muller glia, this capacity has generally been considered absent in humans. This study investigated whether defined humoral cues alone are sufficient to unlock a latent neurogenic program in human Muller cells. MethodsLong-term organotypic retinal cultures were established from 39 adult donors. Cultures were treated with FGF-2 and GSK-3 inhibition to assess proliferation across the peripheral and central retina. Cellular responses were evaluated using single-cell transcriptomics and immunohistochemistry. ResultsFGF-2 treatment and GSK-3 inhibition induced robust proliferation across both peripheral and central retina, with 79.09 {+/-} 6.32% of dividing cells identified as Muller glia, some completing multiple cell cycles. Single-cell transcriptomics revealed activation of progenitor-like and neuronal differentiation pathways, whereas immunohistochemistry demonstrated expression of early and late neuronal markers spanning all major retinal lineages. Newly generated cells expressed markers of cone, rod, bipolar, horizontal, amacrine, and ganglion cell identities, together with evidence of early synaptogenesis. ConclusionsThese findings reveal an intrinsic regenerative potential in adult human Muller glia and show that defined humoral cues can activate a latent neurogenic program in these cells. This may have implications for future vision-restoration strategies in degenerative retinal disease.

neuroscience↗

bioRxiv2026

Striving towards improved full-length single-cell RNA-sequencing

Full-length single-cell RNA-sequencing (scRNA-seq) methods provide superior transcript coverage and isoform resolution compared to their 3-end counterparts, but are typically limited to short-read platforms. Here, we report efforts to improve the FLASH-seq protocol and adapt it for long-read sequencing on the Oxford Nanopore Technologies (ONT) platform (FLASH-seq-ONT). We developed two plate-barcoding strategies enabling higher multiplexing: a custom PCR-ligation approach (PCR-LIG) and ONT native barcoding (NB-ONT). To support data processing, we built FSNanoporeR, a comprehensive bioinformatics pipeline for barcode demultiplexing, chimeric read detection and splitting, UMI extraction, and transcript quantification. Both barcoding strategies produced high-quality transcriptomic data from HEK293T cells, with notable differences in read length distributions. We further demonstrated that monomeric and trimeric UMIs can be reliably detected in >82% of reads, enabling accurate molecular counting at isoform resolution. However, both multiplexing approaches exhibited also critical limitations, including high chimeric read rates and index-swapping artifacts. Our results highlight both the promise and current technical hurdles of full-length single-cell long-read sequencing, and provide a practical framework for researchers considering ONT-based scRNA-seq workflows.

genomics↗

bioRxiv2025

SEQURNA enhances FLASH-seq gene detection while eliminating DTT dependence

Effective RNase inhibition is critical for single-cell RNA-sequencing, yet commercial recombinant RNase inhibitors (RRIs) require reducing agents for stability and impose substantial costs. Here, we systematically benchmark SEQURNA, a synthetic thermostable RNase inhibitor, against commercial alternatives using FLASH-seq in human retinal organoids and peripheral blood mononuclear cells (PBMCs). SEQURNA at 0.5-1 U/l achieved 14-50% higher gene detection than Takara RRI, with the greatest improvement in low-RNA PBMCs. Surprisingly, DTT supplementation at standard concentrations (5-10 mM) significantly impaired gene detection across all SEQURNA concentrations without improving RNA quality metrics, challenging established reverse transcription protocols. SEQURNA preserved biological heterogeneity, maintained sample stability during one-month storage at -80{degrees}C, and reduced reagent costs by 70%. We recommend SEQURNA at 1 U/l without DTT as an optimized formulation that simultaneously enhances data quality and cost-effectiveness for full-length single-cell RNA sequencing.

genomics↗

bioRxiv2023

Cell type-focused compound screen in human organoids reveals molecules and pathways controlling cone photoreceptor death

Human organoids that mirror their corresponding organs in cell-type diversity present an opportunity to perform large-scale screens for compounds that protect disease-affected or damage healthy cell types. However, such screens have not yet been performed. Here, we generated 20,000 human retinal organoids with GFP-labeled cone photoreceptors. Since degeneration of cones is a leading cause of blindness, we induced cone death and screened 2,707 compounds with known targets, for those that saved cones or those that further damaged cones. We identified inhibitors of CK1 or MAPK11 that protected cones, HSP90 inhibitors that saved cones in the short term but damaged them in the longer term, and broad HDAC inhibition by many compounds that significantly damaged cones. This work provides a database for cone-damaging compounds and describes compounds that can be starting points to develop neuroprotection for cones in diseases such as macular degeneration.

neuroscience↗

bioRxiv2023

High-efficiency base editing for Stargardt disease in mice, non-human primates, and human retina tissue

Stargardt disease is a currently untreatable, inherited neurodegenerative disease that leads to macular degeneration and blindness due to loss-of-function mutations in the ABCA4 gene. We have designed a dual adeno-associated viral vector split-intein adenine base-editing strategy to correct the most common mutation in ABCA4 (c.5882G>A, p.G1961E). We optimized ABCA4 base editing in human models, including retinal organoids, iPSC-derived retinal pigment epithelial (RPE) cells, as well as adult human retinal- and RPE/choroid explants in vitro. The resulting gene therapy vectors achieved high levels of gene correction in mutation-carrying mice and in non-human primates, with an average editing of 37% of photoreceptors and 73% of RPE cells in vivo. The high editing rates in primates make way for precise and efficient gene editing in other neurodegenerative ocular diseases.

neuroscience↗

bioRxiv2022

Embryonic cortical layer 5 pyramidal neurons form an active, transient circuit motif perturbed by autism-associated mutations

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that embryonic layer 5 pyramidal neurons, identified through single cell transcriptomics, display two phases of circuit assembly in vivo. At E14.5, a multi-layered circuit motif, composed of a single layer 5 cell type, forms. This motif is transient, switching to a second circuit motif, involving all three types, by E17.5. In vivo targeted single cell recordings and two-photon calcium imaging of embryonic layer 5 neurons reveal that, in both phases, neurons have active somas and neurites, tetrodotoxin-sensitive voltage-gated conductances, and functional glutamatergic synapses. Embryonic layer 5 neurons strongly express autism-associated genes, and perturbing these genes disrupts the switch between the two motifs. Hence, layer 5 pyramidal neurons form transient active pyramidal-to-pyramidal circuits, at the inception of neocortex, and studying these circuits could yield insights into the etiology of autism.

neuroscience↗

bioRxiv2022

Multimodal spatiotemporal phenotyping of human organoid development

Organoids generated from human pluripotent stem cells (PSCs) provide experimental systems to study development and disease. However, we lack quantitative spatiotemporal descriptions of organoid development that incorporate measurements across different molecular modalities. Here we focus on the retina and use a single-cell multimodal approach to reconstruct human retinal organoid development. We establish an experimental and computational pipeline to generate multiplexed spatial protein maps over a retinal organoid time course and primary adult human retina, registering protein expression features at the population, cellular, and subcellular levels. We develop an analytical toolkit to segment nuclei, identify local and global tissue units, infer morphology trajectories, and analyze cell neighborhoods from multiplexed imaging data. We use this toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components, and global patterning in each organoid and primary tissue. In addition, we generate a single-cell transcriptome and chromatin accessibility time course dataset and infer a gene regulatory network underlying organoid development. We then integrate genomic data with spatially segmented nuclei into a multi-modal atlas enabling virtual exploration of retinal organoid development. We visualize molecular, cellular, and regulatory dynamics during organoid lamination, and identify regulons associated with neuronal differentiation and maintenance. We use the integrated atlas to explore retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death. Finally, we show that mosaic CRISPR/Cas genetic perturbations in retinal organoids provide insight into cell fate regulation. Altogether, our work is a major advance toward a virtual human retinal organoid, and provides new directions for how to approach disorders of the visual system. More broadly, our approaches can be adapted to many organoid systems.

systems biology↗

bioRxiv2021

Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq

In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1-3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs.

genomics↗

bioRxiv2019

Clonally distinct differentiation trajectories shape CD8+ memory T cell heterogeneity after acute viral infections in humans

CD8+ T cells play essential roles in immunity to viral and bacterial infections, and to guard against malignant cells. The CD8+ T cell response to an antigen is composed of many T cell clones with unique T cell receptors, together forming a heterogenous repertoire of phenotypically and functionally distinct effector and memory cells1, 2. How individual T cell clones contribute to this heterogeneity during an immune response is key to understand immunity but remains largely unknown. Here, we longitudinally tracked CD8+ T cell clones expanding in response to yellow fever virus vaccination at the single cell level in humans. We show that only a fraction of the clones detected in the acute response persists as circulating memory T cells, indicative of clonal selection. Clones persisting in the memory phase displayed biased differentiation trajectories along a gradient of stem cell memory (SCM) towards terminally differentiated effector memory (EMRA) fates. Reactivation of single memory CD8+ T cells revealed that they were poised to recapitulate skewed differentiation trajectories in secondary responses, and this was generalizable across individuals for both yellow fever and influenza virus. Together, we show that the sum of distinct clonal differentiation repertoires results in the multifaceted T cell response to acute viral infections in humans.

immunology↗

bioRxiv2017

STRT-seq-2i: dual-index 5′ single cell and nucleus RNA-seq on an addressable microwell array

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but currently no high-throughput platform has been used successfully on archived human brain samples. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform.

molecular biology↗