Biology subjects

Santel, M.

Publications and source records attributed to Santel, M..

16 recordsLinked to original sources

bioRxiv2026

Live single-molecule imaging reveals global shifts in mRNA mobility during human stem cell differentiation

Spatiotemporal regulation of mRNA localisation is fundamental to cell identity specification and function, yet tracking transcript dynamics in living differentiating cells remains technically challenging. Here, we establish a robust pipeline for MS2 tagging of endogenous transcripts in human induced pluripotent stem cells (iPSCs), coupled with single-particle tracking and Hidden Markov Modelling to map mRNA mobility landscapes during differentiation and cell state transitions. Applying this approach to different cytoskeleton-encoding transcripts in diverse contexts -- neural organoids, directly programmed neurons, and vascular organoids -- we reveal a conserved principle: Both, {beta}-actin and {beta}2b-tubulin particle dynamics progressively shift towards constrained, compartmentalised patterns as cells acquire cell type identity. Perturbation experiments demonstrate that microtubule-dependent tethering is a common, conserved mechanism controlling {beta}-actin mRNA localisation in all cell types studied, whereas translation-dependent anchoring and actin filaments contribute in a context-dependent manner. Analysis of particle dynamics in migrating blood vessel progenitors further showed that {beta}-actin mRNAs accumulating at cell edges are highly diffusive, while those in perinuclear regions show constrained movement. Together, our integrated framework provides a scalable foundation for mechanistic dissection of mRNA targeting in human developmental and disease models.

cell biology↗

bioRxiv2025

Integrated cell atlas and tumoroids chart pancreatic cancer therapeutic targets

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense, fibroblast-rich stroma that actively shapes the tumor microenvironment. Most PDAC cases arise from conserved genetic transformations initiated by oncogenic KRAS mutations, developing into metastatic disease with high mortality rates. To chart universal PDAC cell states and identify therapeutic inroads, we integrated published single-cell transcriptomes from 200 patient samples, and used the atlas to define prevalent cancer cell and cancer-associated fibroblast (CAF) states, gene expression programs, and ligand-receptor interactions. We established modular tumoroids incorporating patient-derived cancer cells and CAFs that recapitulate aspects of ductal architecture and desmoplastic stroma. Single-cell and spatial transcriptomic profiling confirmed preservation of key cellular states and signaling networks in vitro. We identified Syndecan-1 (SDC1) as a CAF-responsive cancer cell receptor correlating with poor patient survival. Functional SDC1 blockade disrupted cancer growth in tumoroids, highlighting therapeutic relevance. This study provides a framework for dissecting cancer-stroma dynamics and identifying actionable targets using patient-derived tumoroid models.

cancer biology↗

bioRxiv2025

Multi-omic human neural organoid cell atlas of the posterior brain

Patterning of the neural tube establishes midbrain and hindbrain structures that coordinate motor movement, process sensory input, and integrate cognitive functions. Cellular impairment within these structures underlie diverse neurological disorders, and in vitro organoid models promise inroads to understand development, model disease, and assess therapeutics. Here, we use paired single-cell transcriptome and accessible chromatin sequencing to map cell composition and regulatory mechanisms in organoid models of midbrain and hindbrain. We find that existing midbrain organoid protocols generate ventral and dorsal cell types, and cover regions including floor plate, dorsal and ventral midbrain, as well as adjacent hindbrain regions, such as cerebellum. Gene regulatory network (GRN) inference and transcription factor perturbation resolve mechanisms underlying neuronal differentiation. A single-cell multiplexed patterning screen identifies morphogen concentration and combinations that expand existing organoid models, including conditions that generate medulla glycinergic neurons and cerebellum glutamatergic subtypes. Differential abundance of cell states across screen conditions enables differentiation trajectory reconstruction from region-specific progenitors towards diverse neuron types of mid- and hindbrain, which reveals morphogen-regulon regulatory relationships underlying neuronal fate specification. Altogether, we present a single-cell multi-omic atlas and morphogen screen of human neural organoid models of the posterior brain, advancing our understanding of the co-developmental dynamics of regions within the developing human brain.

developmental biology↗

bioRxiv2024

Decoding morphogen patterning of human neural organoids with a multiplexed single-cell transcriptomic screen

Morphogens, secreted signalling molecules that direct cell fate and tissue development, are used to direct neuroepithelial progenitors towards discrete regional identities across the central nervous system. Neural tissues derived from pluripotent stem cells in vitro (neural organoids) provide new models for studying neural regionalization, however, we lack a comprehensive survey of how the developing human neuroepithelium responds to morphogen cues. Here, we produce a detailed map of morphogen-induced effects on the axial and regional specification of human neural organoids using a multiplexed single-cell transcriptomics screen. We find that the timing, concentration, and combination of morphogens strongly influence organoid cell type and regional composition, and that cell line and neural induction method strongly impact the response to a given morphogen condition. We apply concentration gradients in microfluidic chips or a range of static concentrations in multi-well plates to explore how human neuroepithelium interprets morphogen concentrations and observe similar dose-dependent induction of patterned domains in both scenarios. Altogether, we provide a detailed resource that supports the development of new regionalized neural organoid protocols and enhances our understanding of human central nervous system patterning.

developmental biology↗

bioRxiv2023

Human neuron subtype programming through combinatorial patterning with scSeq readouts

Human neurons programmed through transcription factor (TF) overexpression model neuronal differentiation and neurological diseases. However, programming specific neuron types remains challenging. Here, we modulate developmental signaling pathways combined with TF overexpression to explore the spectrum of neuron subtypes generated from pluripotent stem cells. We screened 480 morphogen signaling modulations coupled with NGN2 or ASCL1/DLX2 induction using a multiplexed single-cell transcriptomic readout. Analysis of 700,000 cells identified diverse excitatory and inhibitory neurons patterned along the anterior-posterior and dorsal-ventral axes of neural tube development. We inferred signaling and TF interaction networks guiding differentiation of forebrain, midbrain, hindbrain, spinal cord, peripheral sympathetic and sensory neurons. Our approach provides a strategy for cell subtype programming and to investigate how cooperative signaling drives neuronal fate.

bioengineering↗

bioRxiv2023

An integrated transcriptomic cell atlas of human neural organoids

Neural tissues generated from human pluripotent stem cells in vitro (known as neural organoids) are becoming useful tools to study human brain development, evolution and disease. The characterization of neural organoids using single-cell genomic methods has revealed a large diversity of neural cell types with molecular signatures similar to those observed in primary human brain tissue. However, it is unclear which domains of the human nervous system are covered by existing protocols. It is also difficult to quantitatively assess variation between protocols and the specific cell states in organoids as compared to primary counterparts. Single-cell transcriptome data from primary tissue and neural organoids derived with guided or un-guided approaches and under diverse conditions combined with large-scale integrative analyses make it now possible to address these challenges. Recent advances in computational methodology enable the generation of integrated atlases across many data sets. Here, we integrated 36 single-cell transcriptomics data sets spanning 26 protocols into one integrated human neural organoid cell atlas (HNOCA) totaling over 1.7 million cells. We harmonize cell type annotations by incorporating reference data sets from the developing human brain. By mapping to the developing human brain reference, we reveal which primary cell states have been generated in vitro, and which are under-represented. We further compare transcriptomic profiles of neuronal populations in organoids to their counterparts in the developing human brain. To support rapid organoid phenotyping and quantitative assessment of new protocols, we provide a programmatic interface to browse the atlas and query new data sets, and showcase the power of the atlas to annotate new query data sets and evaluate new organoid protocols. Taken together, the HNOCA will be useful to assess the fidelity of organoids, characterize perturbed and diseased states and facilitate protocol development in the future.

developmental biology↗

bioRxiv2023

Single-cell epigenomic reconstruction of developmental trajectories in human neural organoid systems from pluripotency

Human cell type diversity emerges through a highly regulated series of fate restrictions from pluripotent progenitors. Fate restriction is orchestrated in part through epigenetic modifications at genes and regulatory elements, however it has been difficult to study these mechanisms in humans. Here, we use organoid models of the human central nervous system and establish single-cell profiling of histone modifications (H3K27ac, H3K27me3, H3K4me3) in organoid cells over a time course to reconstruct epigenomic trajectories governing cell identity acquisition from human pluripotency. We capture transitions from pluripotency through neuroepithelium, to retinal and brain region specification, as well as differentiation from progenitors to neuronal and glial terminal states. We find that switching of repressive and activating epigenetic modifications can precede and predict decisions at each stage, providing a temporal census of gene regulatory elements and transcription factors that we characterize in a gene regulatory network underlying human cerebral fate acquisition. We use transcriptome and chromatin accessibility measurements in the same cell from a human developing brain to validate this regulatory mode in a primary tissue. We show that abolishing histone 3 lysine 27 trimethylation (H3K27me3) through inhibition of the polycomb group protein Embryonic Ectoderm Development (EED) at the neuroectoderm stage disrupts fate restriction and leads to aberrant cell fate acquisition, ultimately influencing cell type composition in brain organoids. Altogether, our single-cell genome wide map of histone modifications during human neural organoid development serves as a blueprint (https://episcape.ethz.ch) to explore human cell fate decisions in normal physiology and in neurodevelopmental disorders. More broadly, this approach can be used to study human epigenomic trajectory mechanisms in any human organoid system. SummaryUnguided neural organoids reveal widespread and dynamic switching of epigenetic modifications during development and recapitulate fate restriction from pluripotency to terminally differentiated cells of the human central nervous system.

developmental biology↗

bioRxiv2023

Morphodynamics of human early brain organoid development

Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. Here, we establish long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. We provide a novel dual-channel, multi-mosaic and multi-protein labeling strategy combined with a computational demultiplexing approach to enable simultaneous quantification of distinct subcellular features during organoid development. We track Actin, Tubulin, plasma membrane, nucleus, and nuclear envelope dynamics, and quantify cell morphometric and alignment changes during tissue state transitions including neuroepithelial induction, maturation, lumenization, and brain regionalization. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrixinduced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of nontelencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.

developmental biology↗

bioRxiv2022

Reconstructing cell interactions and state trajectories in pancreatic cancer stromal tumoroids

Interlineage communication within a cancer microenvironment can augment cancer cell behaviour and impact response to therapy. Patient-derived cancer organoids provide an opportunity to explore cancer cell biology, however it is a major challenge to generate a complex cancer microenvironment in vitro. Here, we established a stromal tumoroid culture system modeling pancreatic ductal adenocarcinoma (PDAC) that reconstitutes multilineage interactions between cancer, endothelial, and fibroblast cells and recapitulates several aspects of primary tumors. Whole-mount immunohistochemistry on cleared tumoroids reveals organized vessel, desmoplastic fibroblast, and glandular cancer cell phenotypes that emerge over time. Time-course scRNA-seq measurements show that tumoroid formation activates fibroblasts, altering the extracellular matrix (ECM) composition and inducing cancer cell signal-response signatures and metabolic state change. Comparison between tumoroids with normal or cancer associated fibroblasts (CAFs) reveals different ECM compositions, as well as differential effects on cancer cell behaviors and metabolism. We identify Syndecan 1 (SDC1) and Peroxisome proliferator-activated receptor gamma (PPARG) as receptor and metabolic nodes involved in cancer cell response to CAF signals, and blocking SDC1 disrupts cancer cell growth within the tumoroid. Tumoroids from multiple PDAC patients revealed co-existence of subpopulations associated with classical and basal phenotypes, and CAF-induced migration behaviors emerged in certain patient tumoroids. Comparisons between patient tumoroids revealed a multigene migration signature that develops over time reflecting a stress response mechanism that correlates with worse clinical outcome. Altogether, stromal tumoroids can be used to explore dynamic and reciprocal interactions between cancer, CAF and endothelial cell states, and our data provides new inroads into the discovery of personalized pancreatic cancer therapies.

cancer biology↗

bioRxiv2021

Inferring and perturbing cell fate regulomes in human cerebral organoids

Self-organizing cerebral organoids grown from pluripotent stem cells combined with single-cell genomic technologies provide opportunities to explore gene regulatory networks (GRNs) underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin profiling data over a dense time course covering multiple phases of organoid development including neuroepithelial formation, patterning, brain regionalization, and neurogenesis. We identify temporally dynamic and brain region-specific regulatory regions, and cell interaction analysis reveals emergent patterning centers associated with regionalization. We develop Pando, a flexible linear model-based framework that incorporates multi-omic data and transcription binding site predictions to infer a global GRN describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoid. We find that certain factors regulate the abundance of cell fates, whereas other factors impact neuronal cell states after differentiation. We show that the zinc finger protein GLI3 is required for cortical fate establishment in humans, recapitulating previous work performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify a regulome central to the dorsoventral telencephalic fate decision. This regulome suggests that Notch effectors HES4/5 are direct GLI3 targets, which together coordinate cortex and ganglionic eminence diversification. Altogether, we provide a framework for how multi-brain region model systems and single-cell technologies can be leveraged to reconstruct human brain developmental biology.

developmental biology↗

bioRxiv2021

Cell atlas of the regenerating human liver after portal vein embolization

The liver has the remarkable capacity to regenerate. In the clinic, this capacity can be induced by portal vein embolization (PVE), which redirects portal blood flow resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the liver during regeneration. We first establish an atlas of cell subtypes from the healthy human liver using fresh and frozen tissues, and then compare post-PVE samples with their reference counterparts. We find that PVE alters portal-central zonation of hepatocytes and endothelial cells. Embolization upregulates expression programs associated with development, cellular adhesion and inflammation across cell types. Analysis of interlineage crosstalk revealed key roles for immune cells in modulating regenerating tissue responses. Altogether, our data provides a rich resource for understanding homeostatic mechanisms arising during human liver regeneration and degeneration.

genomics↗

bioRxiv2021

Nuclei are mobile processors enabling specialization in a gigantic single-celled syncytium

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleated syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters, and if nuclei within the cell diversify into heterogeneous states. Here we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions, and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile, and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleated cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization, and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

cell biology↗

bioRxiv2020

Ngn2 induces diverse neuronal lineages from human pluripotency

Human neurons engineered from induced pluripotent stem cells (iPSCs) through Neurogenin 2 (Ngn2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here we used single-cell transcriptomics to dissect the cell states that emerge during Ngn2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to Ngn2 expression level and the duration of Ngn2 forced expression. Our data reveals that Ngn2 dosage can regulate neuron fate acquisition, and that Ngn2-iN heterogeneity can confound results that are sensitive to neuron type.

neuroscience↗

bioRxiv2020

Lineage recording reveals dynamics of cerebral organoid regionalization

Diverse regions develop within cerebral organoids generated from human induced pluripotent stem cells (iPSCs), however it has been a challenge to understand the lineage dynamics associated with brain regionalization. Here we establish an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during cerebral organoid development. We infer fate-mapped whole organoid phylogenies over a scarring time course, and reconstruct progenitor-neuron lineage trees within microdissected cerebral organoid regions. We observe increased fate restriction over time, and find that iPSC clones used to initiate organoids tend to accumulate in distinct brain regions. We use lineage-coupled spatial transcriptomics to resolve lineage locations as well as confirm clonal enrichment in distinctly patterned brain regions. Using long term 4-D light sheet microscopy to temporally track nuclei in developing cerebral organoids, we link brain region clone enrichment to positions in the neuroectoderm, followed by local proliferation with limited migration during neuroepithelial formation. Our data sheds light on how lineages are established during brain organoid regionalization, and our techniques can be adapted in any iPSC-derived cell culture system to dissect lineage alterations during perturbation or in patient-specific models of disease.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2019

Single-cell-resolution transcriptome map of human, chimpanzee, bonobo, and macaque brains

Identification of gene expression traits unique to the human brain sheds light on the mechanisms of human cognition. Here we searched for gene expression traits separating humans from other primates by analyzing 88,047 cell nuclei and 422 tissue samples representing 33 brain regions of humans, chimpanzees, bonobos, and macaques. We show that gene expression evolves rapidly within cell types, with more than two-thirds of cell type-specific differences not detected using conventional RNA sequencing of tissue samples. Neurons tend to evolve faster in all hominids, but non-neuronal cell types, such as astrocytes and oligodendrocyte progenitors, show more differences on the human lineage, including alterations of spatial distribution across neocortical layers.

evolutionary biology↗

bioRxiv2019

Single-cell genomic atlas of great ape cerebral organoids uncovers human-specific features of brain development

The human brain has changed dramatically since humans diverged from our closest living relatives, chimpanzees and the other great apes1-5. However, the genetic and developmental programs underlying this divergence are not fully understood6-8. Here, we have analyzed stem cell-derived cerebral organoids using single-cell transcriptomics (scRNA-seq) and accessible chromatin profiling (scATAC-seq) to explore gene regulatory changes that are specific to humans. We first analyze cell composition and reconstruct differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. We find that brain region composition varies in organoids from different iPSC lines, yet regional gene expression patterns are largely reproducible across individuals. We then analyze chimpanzee and macaque cerebral organoids and find that human neuronal development proceeds at a delayed pace relative to the other two primates. Through pseudotemporal alignment of differentiation paths, we identify human-specific gene expression resolved to distinct cell states along progenitor to neuron lineages in the cortex. We find that chromatin accessibility is dynamic during cortex development, and identify instances of accessibility divergence between human and chimpanzee that correlate with human-specific gene expression and genetic change. Finally, we map human-specific expression in adult prefrontal cortex using single-nucleus RNA-seq and find developmental differences that persist into adulthood, as well as cell state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene regulatory features that are unique to humans.

developmental biology↗