Biology subjects

Treutlein, B.

Publications and source records attributed to Treutlein, B..

At least 37 records · Page 2Linked to original sources

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↗

bioRxiv2023

Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells

Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.

genomics↗

bioRxiv2022

Single-cell brain organoid screening identifies developmental defects in autism

Development of the human brain involves processes that are not seen in many other species, but can contribute to neurodevelopmental disorders (1-4). Cerebral organoids can be used to investigate neurodevelopmental disorders in a human context but are limited by variability and low throughput. We have developed the CRISPR-human organoids-scRNA-seq (CHOOSE) system that utilizes verified pairs of gRNAs, inducible CRISPR/Cas9-based genetic disruption, and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Genetic perturbations of 36 high-risk autism spectrum disorder (ASD) genes related to transcriptional regulation allowed us to identify their effects on cell fate determination and discover developmental stages susceptible to ASD gene perturbations. We construct a developmental gene regulatory network (GRN) of cerebral organoids from single-cell multiomic data including transcriptome and chromatin modalities and identify ASD-associated and perturbation-enriched regulatory modules. We show that perturbing members of the BAF chromatin remodeling complex leads to an expanded population of ventral telencephalon progenitors. Specifically, the BAF subunit ARID1B controls the fate transitions of progenitors to oligodendrocyte precursor cells and interneurons, which we confirmed in patient-specific induced pluripotent stem cell (iPSC) derived organoids. Our study paves the way for phenotypically characterizing disease susceptibility genes in human organoid models with cell type, developmental trajectory, and gene regulatory network readouts.

developmental biology↗

bioRxiv2022

Fate and state transitions during human blood vessel organoid development

Blood vessel organoids (BVOs) derived from human pluripotent stem cells have emerged as a novel system to understand human vascular development, model disorders, and develop regenerative therapies. However, it is unclear which molecular states constitute BVOs and how cells differentiate and self-organize within BVOs in vitro and after transplantation. Here we reconstruct BVO development over a time course using single-cell transcriptomics. We observe progenitor states that bifurcate into endothelial and mural fates, and find that BVOs do not acquire definitive arterio-venous endothelial identities in vitro. Chromatin accessibility profiling identifies gene regulatory network (GRN) features associated with endothelial and mural fate decisions, and transcriptome-coupled lineage recording reveals multipotent progenitor states within BVOs. We perform single-cell genetic perturbations within mosaic BVOs to dissect the impact of transcription factor (TF) and receptor depletion on cell differentiation, and highlight multiple TFs including MECOM and ETV2 as strong-effect regulators of human BVO development. We show that manipulation of VEGF and Notch signaling pathways alters BVO morphogenesis and endothelial GRNs, and induces arteriovenous-like state differentiation. We analyze matured BVOs after transplantation using scRNA-seq, and observe matured endothelium with clear arteriovenous specification. We also observe off-target cell fates with bone and adipocyte features, suggesting multipotent states reside within the BVOs in vitro that expand and diversify in less restrictive conditions. Finally, we map vascular disease associated genes to BVO cell states to highlight the potential of BVOs for disease modeling. Altogether, our data and analyses provide the first comprehensive cell state atlas of BVO development and illuminate both the power and limitation of BVOs for translational research.

developmental biology↗

bioRxiv2022

Single-cell analyses of axolotl forebrain organization, neurogenesis, and regeneration

Salamanders are important tetrapod models to study brain organization and regeneration, however the identity and evolutionary conservation of brain cell types is largely unknown. Here, we delineate cell populations in the axolotl telencephalon during homeostasis and regeneration, representing the first single-cell genomic and spatial profiling of an anamniote tetrapod brain. We identify glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved GABAergic neuron classes. We infer transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific direct and indirect neurogenesis, and unravel conserved signatures. Following brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield key insights into the organization, evolution, and regeneration of a tetrapod nervous system.

developmental biology↗

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↗

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

Maturation of human intestinal epithelium from pluripotency in vitro

Methods to generate human intestinal tissue from pluripotent stem cells (PSCs) open new inroads into modeling intestine development and disease. However, current protocols require organoid transplantation into an immunocompromised mouse to achieve matured and differentiated epithelial cell states. Inspired by developmental reconstructions from primary tissues, we establish a regimen of inductive cues that enable stem cell maturation and epithelial differentiation entirely in vitro. We show that the niche factor Neuregulin1 (NRG1) promotes morphological change from proliferative epithelial cysts to matured epithelial tissue in three-dimensional cultures. Single-cell transcriptome analyses reveal differentiated epithelial cell populations, including diverse secretory and absorptive lineages. Comparison to multi-organ developmental and adult intestinal cell atlases confirm the specificity and maturation state of cell populations. Altogether, this work opens a new direction to use in vitro matured epithelium from human PSCs to study human intestinal epithelium development, disease, and evolution in controlled culture environments.

cell 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↗

bioRxiv2021

Characterization of RNA content in individual phase-separated coacervate microdroplets

Liquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

genomics↗

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

An organoid and multi-organ developmental cell atlas reveals multilineage fate specification in the human intestine

Human intestinal organoids (HIOs) generated from pluripotent stem cells provide extraordinary opportunities to explore development and disease. Here, we generate a single-cell transcriptome reference atlas from HIOs and from multiple developing human organs to quantify the specificity of HIO cell fate acquisition, and to explore alternative fates. We identify epithelium-mesenchyme interactions, transcriptional regulators involved in cell fate specification, and stem cell maturation features in the primary tissue that are recapitulated in HIOs. We use an HIO time course to reconstruct the molecular dynamics of intestinal stem cell emergence, as well as the specification of multiple mesenchyme subtypes. We find that the intestinal master regulator CDX2 correlates with distinct phases of epithelial and mesenchymal development, and CDX2 deletion perturbs the differentiation of both intestinal epithelium and mesenchyme. Collectively our data provides a comprehensive and quantitative assessment of HIO development, and illuminates the molecular machinery underlying endodermal and mesodermal cell fate specification.

developmental biology↗

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↗

bioRxiv2020

Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

We generated induced excitatory sensory neurons (iNeurons, iNs) from chimpanzee, bonobo and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single cell-RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee than the human cells. In accordance, during the first two weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs and this difference in timing likely depends on functions downstream of NGN2.

evolutionary biology↗

bioRxiv2020

Cluster similarity spectrum integration of single-cell genomics data

Technologies to sequence the transcriptome, genome or epigenome from thousands of single cells in an experiment provide extraordinary resolution into the molecular states present within a complex biological system at any given moment. However, it is a major challenge to integrate single-cell sequencing data across experiments, conditions, batches, timepoints and other technical considerations. New computational methods are required that can integrate samples while simultaneously preserving biological information. Here, we propose an unsupervised reference-free data representation, Cluster Similarity Spectrum (CSS), where each cell is represented by its similarities to clusters independently identified across samples. We show that CSS can be used to assess cellular heterogeneity and enable reconstruction of differentiation trajectories from cerebral organoid single-cell transcriptomic data, and to integrate data across experimental conditions and human individuals. We compare CSS to other integration algorithms and show that it can outperform other methods in certain integration scenarios. We also show that CSS allows projection of single-cell genomic data of different modalities to the CSS-represented reference atlas for visualization and cell type identity prediction. In summary, CSS provides a straightforward and powerful approach to understand and integrate challenging single-cell multi-omic data.

bioinformatics↗

bioRxiv2020

In vitro and in vivo development of the human intestine at single cell resolution

The human intestinal stem cell (ISC) niche supports ISC self-renewal and epithelial function, yet little is known about the development of the human ISC niche. We used single-cell mRNA sequencing (scRNA-seq) to interrogate the human intestine across 7-21 weeks of gestation. Using these data coupled with marker validation in situ, molecular identities and spatial locations were assigned to several cell populations that comprise the epithelial niche, and the cellular origins of many niche factors were determined. The major source of WNT and RSPONDIN ligands were ACTA2+ cells of the muscularis mucosa. EGF was predominantly expressed in the villus epithelium and the EGF-family member NEUREGULIN1 (NRG1) was expressed by subepithelial mesenchymal cells. Functional data from enteroid cultures showed that NRG1 improved cellular diversity, enhanced the stem cell gene signature, and increased enteroid forming efficiency, whereas EGF supported a secretory gene expression profile and stimulated rapid proliferation. This work highlights unappreciated complexities of intestinal EGF/ERBB signaling and identifies NRG1 as a stem cell niche factor.

developmental biology↗