Biology subjects

Treutlein, B.

Publications and source records attributed to Treutlein, B..

At least 19 recordsLinked to original sources

bioRxiv2026

SALL1 and neural microenvironmental contextspecify identity of stem cell derived humanmicroglia-like cells

Microglia, the immune cells of the brain parenchyma, play a pivotal role in neurodevelopment and neuroprotection. Human stem cell-derived microglia-like cells (MGL) offer a valuable, human-relevant system to study microglia in vitro. However, their cell states and intercellular interactions within complex 3-dimensional (3D) tissue contexts in vitro remain underexplored. Here, we analyze the gene expression profiles of MGL generated by two differentiation protocols in conventional 2D cultures. We show that co-culture with neural organoids promotes a more mature, in vivo-like MGL phenotype but this signature is lost during long-term co-culture. By varying media compositions, we modulate 2D MGL states toward more active or more homeostatic identities, which may enhance the similarity to primary microglia identities upon subsequent co-culture. Moreover, forced expression of the microglial lineage regulator SALL1 shifts MGL away from a general myeloid state toward a more defined pre-microglial fate. SALL1 overexpression within a 3D neural organoid environment further amplifies the shift toward a homeostatic developmental microglia population. Collectively, our transcriptomic analysis delineates human MGL states in 2D and 3D contexts, refines existing differentiation protocols toward more authentic MGL, and establishes a framework to investigate the intricate microglia-neural tissue interplay.

developmental biology↗

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↗

bioRxiv2026

Analog intrinsic recoding measures RNA dynamics without chemical conversion

Steady-state RNA abundance measurements mask the synthesis and decay rates that shape gene expression. Analog intrinsic recoding sequencing (AIR-seq) repurposes the base-pairing properties of N4-hydroxycytidine (NHC) to mark newly synthesized RNA with C-to-T and T-to-C mismatches in standard RNA-seq libraries, eliminating chemical conversion and enrichment common to RNA metabolic labeling methods. NHC-AIR-seq resolves bulk and single-cell RNA dynamics, improves RNA velocity inference, reveals hidden regulation, and brings RNA kinetics into routine transcriptomic workflows.

genomics↗

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

Integrin-identity shapes and mechanotemporally encodes the fibroblast transcriptome

Fibroblast transcriptomic states reflect physiological and pathological tissue contexts, yet the upstream determinants that stabilize these states remain poorly defined. Integrins mediate extracellular matrix (ECM) adhesion and biochemical signaling, but whether they encode mechanical constraints into stable transcriptomic programs is unclear. Using engineered mouse fibroblasts, bulk and single-cell transcriptomics, and controlled micromechanical confinement, we show that integrins can shape the transcriptomic landscape. The bulk transcriptome of fibroblasts expressing V- and {beta}1-class indicates a shared mechanosensitive baseline, except when these integrin classes are expressed individually. We also found integrin-specific gene clusters, including {beta}1-class integrin-dependent enrichment of Areg, Epha7, Lhpp and Igf2r, which regulate development, regeneration and disease, and altered YAP1 targeted gene expression. At single-cell resolution under confinement, {beta}1-class integrins sustain a progenitor-associated program, whereas their loss or V-class enrichment promotes a constitutively activated state linked to injury repair and wound healing. Mechanical confinement and confinement duration further reshapes these states in an integrin-identity-dependent manner. Our findings establish integrin-identity as a determinant of how fibroblasts transduce mechanotemporal inputs from the cell surface to the nucleus.

Cell Biology↗

bioRxiv2025

DISCO-seq: 3D single-cell transcriptomics of intact biological systems

Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues comparable to standard samples. We demonstrate its versatility by investigating tumor heterogeneity in a syngeneic glioblastoma mouse model, using 3D imaging to identify spatially distinct microenvironments and characterize their unique transcriptomic signatures. Moreover, DISCO-seq enabled unbiased, whole-body mapping of SARS-CoV-2 S1 protein deposition in mice, followed by transcriptomic profiling of spatially defined niches. By bridgingmesoscale 3D imaging with single-cell transcriptomics, DISCO-seq establishes a paradigm for anatomically contextualized, hypothesis-free tissue interrogation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/693352v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@758ee5org.highwire.dtl.DTLVardef@1f862c6org.highwire.dtl.DTLVardef@1cf3b9org.highwire.dtl.DTLVardef@c5082e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDISCO-seq integrates RNA-preserving tissue-clearing chemistry with whole-organ or organism 3D imaging, enabling anatomically unbiased single-cell transcriptomics. C_LIO_LIDISCO-seq yields RNA quality and transcriptome profiles equivalent to those obtained from matched fresh or fixed tissues. C_LIO_LIDISCO-seq identifies discrete glioblastoma microenvironments and defines their transcriptomic states within the intact brain. C_LIO_LIDISCO-seq enables whole-body mapping of SARS-CoV-2 S1 and uncovers region-specific immune and metabolic responses across anatomical niches. C_LI Supplementary movies can be seen at: http://discotechnologies.org/DISCO-seq/

systems biology↗

bioRxiv2025

Unified Generation of Regionalized Neural Organoids from Single-Lumen Neuroepithelium

Human brain development begins with the formation of the neural tube, a neuroepithelium organized around a single continuous lumen that is patterned to specify distinct brain regions. Human pluripotent stem cell (hPSC)-derived neural organoids offer powerful models to study this process and its disruption in disease. However, most existing protocols rely on stochastic self-organization of hPSC aggregates, leading to high variability in tissue architecture and cell type composition, which limits reproducibility and fidelity to natural development. This variability has also hindered the broader adoption of brain organoid technology in translational and pharmaceutical research, where robustness and standardization are critical. Here, we present a scalable and reproducible platform that uses uniform, single-lumen neuroepithelium (SLN) as a standardized starting point for generating diverse, regionally specified neural organoids. SLNs form with high efficiency and reproducibility, can be patterned along dorso-ventral and anterior-posterior axes, and mature in suspension culture into organoids representing forebrain, midbrain, hindbrain, and neural retina. This unified and scalable approach provides a reproducible foundation for modeling human brain development, offering broad translational potential for mechanistic studies, disease modeling, and drug discovery.

developmental 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

Mapping early patterning events in human neural development usingan in-vitro microfluidic stem cell model

Stem cell models can provide insights into human brain development at embryonic stages which are normally inaccessible. We previously developed the Microfluidic Stem Cell Regionalisation (MiSTR) model, which recapitulates the rostro-caudal patterning of human neural tube through a WNT activation (WNTa) gradient. Through temporal single cell transcriptomics of rostro-caudal and dorso-ventral gradient-patterned MiSTR, we found that rostro-caudal subtypes were regionally specified and fate-determined already during the late epiblast stage, several days before onset of neuralisation at day 3-4. Rostral cells were characterised by expression of HESX1 and SHISA2 during pre-neuralisation and PAX6 during early neuralisation, whereas caudal cells expressed FST and HOXA1 during pre-neuralisation and SOX1 as the dominant neuralising factor. In contrast to the early rostro-caudal specification, response to ventralisation in telencephalic progenitors was developmentally delayed and occurred around day 9. We further uncovered temporal events in human midbrain-hindbrain boundary formation and ventral forebrain patterning, contributing new knowledge on early human neural region-specification.

neuroscience↗

bioRxiv2025

CellFlow enables generative single-cell phenotype modeling with flow matching

High-content phenotypic screens provide a powerful strategy for studying biological systems, but the scale of possible perturbations and cell states makes exhaustive experiments unfeasible. Computational models that are trained on existing data and extrapolate to correctly predict outcomes in unseen contexts have the potential to accelerate biological discovery. Here, we present CellFlow, a flexible framework based on flow matching that can model single cell phenotypes induced by complex perturbations. We apply CellFlow to various phenotypic screens, accurately predicting expression responses to a wide range of perturbations, including cytokine stimulation, drug treatments and gene knockouts. CellFlow successfully modeled developmental perturbations at the whole-embryo scale and guided cell fate and organoid engineering by predicting heterogeneous cell populations arising from combinatorial morphogen treatments and by performing a virtual organoid protocol screen. Taken together, CellFlow has the potential to accelerate discovery from phenotypic screens by learning from existing data and generating phenotypes induced by unseen conditions.

bioinformatics↗

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

Single-cell profiling of penta- and tetradactyl mouse limb buds identifies mesenchymal progenitors controlling digit numbers and identities

The cellular mechanisms controlling digit numbers and identities have remained elusive. Profiling of wild-type (pentadactyl) and Grem1 tetradactyl mouse limb buds identifies cellular changes affecting two limb bud mesenchymal progenitor (LMP) populations. In mutant limb buds, the anteriorly biased distribution of peripheral LMPs (pLMPs) is lost and the population expanded, while the distal-posterior LMP (dLMP) population is reduced from early stages onward. Analysis of LMP signature genes in wildtype and mutant mouse limb buds with digit loss or gain establishes that pLMPs are positively regulated by BMP signaling, while dLMPs require GREM1-mediated BMP antagonism. dLMPs encompass digit progenitors and altering their population size prefigures changes in digit numbers. The anteriorly biased pLMP distribution is linked to digit asymmetry as loss of this bias in tetradactyl mouse and pig limb buds underlies middle digit symmetry and identity loss. This study indicates that variable spatial Grem1 expression in mutant and evolutionary diversified limb buds tunes BMP activity, impacting both LMP populations in a complementary manner.

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↗

bioRxiv2024

Somite-independent regeneration of the axolotl primary body axis

Primary body-axis development is a highly conserved process that proceeds through somitogenesis and subsequent subdivision into dermatome, myotome, and sclerotome. Defects in somitic-clock genes such as Hes7 lead to vertebral-segmentation defects in mice and fish. Here we show that in the axolotl, although Hes7 is necessary for proper embryonic vertebral segmentation, it is-- surprisingly--dispensable during tail regeneration. We investigated the mechanism of vertebral segmentation during regeneration which initially occurs through extension of a cartilage rod ventral to the spinal cord. We find that the regenerating cartilage rod undergoes a periodic wrinkling that provides a template for vertebral segmentation. Via direct mechanical measurements and biophysical perturbations, we show that a model of compression-induced buckling instability can predict vertebral segmentation. The cartilage rod and other somitic derivatives (muscle, cartilage, tendon, fibroblasts) arise from tendon-like, Lfng+ multi-potent mesenchymal progenitors, which display a gene regulatory state distinct from somitic progenitors. In summary, we uncover a mechanism of vertebral segmentation during axolotl tail regeneration that is distinct from the somite-based developmental mechanism.

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 endoderm-derived organoids

Human stem cells can generate complex, multicellular epithelial tissues of endodermal origin in vitro that recapitulate aspects of developing and adult human physiology. These tissues, also called organoids, can be derived from pluripotent stem cells or tissue-resident fetal and adult stem cells. However, it has remained difficult to understand the precision and accuracy of organoid cell states through comparison with primary counterparts, and to comprehensively assess the similarity and differences between organoid protocols. Advances in computational single-cell biology now allow the integration of datasets with high technical variability. Here, we integrate single-cell transcriptomes from 218 samples covering organoids of diverse endoderm-derived tissues including lung, pancreas, intestine, liver, biliary system, stomach, and prostate to establish an initial version of a human endoderm organoid cell atlas (HEOCA). The integration includes nearly one million cells across diverse conditions, data sources and protocols. We align and compare cell types and states between organoid models, and harmonize cell type annotations by mapping the atlas to primary tissue counterparts. To demonstrate utility of the atlas, we focus on intestine and lung, and clarify ontogenic cell states that can be modeled in vitro. We further provide examples of mapping novel data from new organoid protocols to expand the atlas, and showcase how integrating organoid models of disease into the HEOCA identifies altered cell proportions and states between healthy and disease conditions. The atlas makes diverse datasets centrally available, and will be valuable to assess organoid fidelity, characterize perturbed and diseased states, and streamline protocol development.

cell biology↗

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↗