Biology subjects

Lutolf, M. P.

Publications and source records attributed to Lutolf, M. P..

At least 19 recordsLinked to original sources

bioRxiv2026

Growth factor-free synthetic matrices reveal intrinsic skeletal progenitor competence

Clinical translation of human skeletal progenitor cells has been limited by the inability to reliably predict their regenerative performance, as current characterization methods rely on phenotypic markers or instructive differentiation assays that obscure intrinsic cell function. Here we demonstrate that human skeletal progenitor competence can only be revealed within a precisely defined and growth factor-free synthetic microenvironment. By independently tuning matrix mechanics, degradability, adhesion, and cell density within a fully synthetic poly(ethylene glycol) hydrogel, we create a permissive niche that enables human bone marrow stromal cells (hBMSCs) to undergo spontaneous bone formation in vivo without osteoinductive supplementation. These exogenous growth factor-free conditions expose donor-specific differences in bone forming capacity that are otherwise masked. Strikingly, supplementation with even low concentrations of BMP-2 abolished inter-donor variability, directly demonstrating that osteoinductive factors override intrinsic progenitor competence. Single-cell transcriptomics, proteomic profiling, and longitudinal in vivo analyses converge to show that high performing donors are defined by chondrocyte-primed transcriptional states and coordinated extracellular matrix (ECM) remodeling programs that establish a pro-osteochondral niche and drive bone formation via endochondral ossification. In contrast, low performing donors fail to initiate this cascade despite comparable phenotypic profiles and proliferation rates. Together, these findings implicate matrix context as a critical regulator of stem cell competence. Within this permissive synthetic niche, intrinsic ECM remodeling capacity emerges as a defining feature of donor-specific osteogenic potential, one that remains invisible to conventional assays. These matrices thus provide a functional platform for prospective stratification of skeletal progenitor cells, with direct implications for donor selection in regenerative medicine. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731042v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@6fa9ceorg.highwire.dtl.DTLVardef@f51dddorg.highwire.dtl.DTLVardef@7bb040org.highwire.dtl.DTLVardef@706a36_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

High-resolution single-cell atlas of the human B cell compartment and immune microenvironment across tissues

The immune system is a dynamic network of diverse cell types distributed across tissues. While mouse studies have highlighted the importance of tissue-localized B-cell responses in infection and tissue repair, research on human B cells has remained largely confined to peripheral blood. Here, we specifically investigated the human B-cell compartment and its interactions with the immune microenvironment across 10 tissues, using single-cell RNA sequencing and paired B-cell receptor sequencing. We mapped the full spectrum of B-cell populations and revealed diverse differentiation trajectories spanning naive, memory, and plasma cell types. Germinal center B cells and plasma cells showed tissue-specific adaptations in transcriptional states, isotype usage, and functional profiles. Plasma cell isotypes influenced both effector functions and predicted interactions with T cells. Finally, we defined tissue-specific residency gene modules that outperformed existing memory B-cell signatures. Together, this dataset serves as a foundation for systematically studying tissue-localized B cells and reveals how local microenvironments shape humoral immunity.

immunology↗

bioRxiv2025

An integrated platform for high-throughput phenospace learning of 3D multilineage organoid systems

Complex multilineage organoid systems lack quantitative phenotyping methods preserving spatial architecture at high throughput. Current approaches compromise biological complexity, spatial resolution, or robust homogeneous multilineage assembly. We establish an integrated experimental-computational platform for high-throughput spatial phenotyping of multilineage organoids through developing a modular tumoroid culture system incorporating pancreatic ductal adenocarcinoma (PDAC) cells and cancer-associated fibroblasts (CAFs) in 384-well format with multiplexed whole-mount imaging. We developed Phenocoder, a machine learning framework combining conditional variational autoencoders with spatial graph analysis to extract multiscale organoid features. Rigorous validation demonstrates robust performance in PDAC tumoroids. The platform identifies pathway modulators that disrupt the fibrotic microenvironment and discovers stroma-dependent vulnerabilities, undetectable in monocultures. Extending to immuno-competent tumoroids, we assess fibrosis modulators in combination with T cell bispecific antibodies, identifying treatments that enhance immune cell proliferation and infiltration inducing cancer cell death, validated in patient-derived explants. This platform establishes a generalizable framework for multilineage organoid phenotyping.

cancer 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

Regulation of mRNA polyadenylation governs mammalian body plan formation in gastruloids

The establishment of the body plan during gastrulation represents a hallmark of animal life. It emerges from the interplay of gene-regulatory programs and positional cues, yet how these signals are integrated post-transcriptionally remains largely unexplored. Here, we combine the scalability of mouse gastruloids with a single-cell CRISPR screening platform to functionally dissect germ layer specification at single-cell transcriptomic resolution. Focusing on post-transcriptional regulation, we systematically map drivers of mesodermal and endodermal fate and identify the deadenylase Cnot8. Loss of Cnot8 leads to widespread poly(A) tail elongation and transcript stabilization, shifting mesoderm differentiation toward ectopic notochord fate, thereby profoundly impacting axial patterning. Collectively, our findings identify mRNA deadenylation as a fundamental mechanism linking cellular identity with morphogenetic signaling during mammalian body plan formation.

developmental biology↗

bioRxiv2025

Modeling host-microbe interactions in immunocompetent engineered human gut tissues

The intestinal mucosal barrier contains microbial organisms within the lumen while preserving the ability to absorb nutrients. Dietary, microbial, and other exposures shaped human barrier evolution and continue to impact disease susceptibility. Here, we established engineered barrier models of the human small intestine and colon composed of a multilineage epithelium, mucus layer, accessible microbial compartment and autologous tissue-resident immune cells. The epithelium has crypt- and villus-like topological domains, with stem cells differentiating into absorptive and secretory lineages with region-specific identities. Secreted mucins accumulate apically, forming a dense mucus layer separating the epithelium from colonizing commensal and pathogenic bacteria. Intestinal memory T cells integrate into and interact with the epithelium. We use the engineered intestinal tissues to identify an epithelial gene regulatory network underlying response to Salmonella Typhimurium infection, and uncover epithelial-immune-pathogen crosstalk coordinating cytokine release and epithelial damage. Overall, this work allows for the modular integration of epithelial, microbial, and immune compartments providing a versatile system for studying human intestinal physiology and pathologies.

bioengineering↗

bioRxiv2024

Watch-breaker: establishment of a microwell array-based miniaturized thymic organoid model suitable for high throughput applications.

T-cell development depends critically on the thymic stroma, in particular the diverse array of functionally distinct thymic epithelial cell (TEC) types. However, a robust in vitro thymus model mimicking the native thymus and compatible with medium/high-throughput analyses is currently lacking. Here, we demonstrate a novel high-density microwell array-based miniaturized thymus organoid (mTO) model, that supports T-cell commitment and development, possesses key organizational characteristics of the native thymus and is compatible with live-imaging and medium/high-throughput applications. We establish the minimum cellular input required for functional mTO and show that mTO TEC phenotype and complexity closely mirrors the native thymus. Finally, we use mTO to probe the role of fetal thymic mesenchyme, revealing a requirement beyond maintenance of Foxn1 in differentiation/maintenance of mature TEC subpopulations. Collectively, mTO present a new in vitro model of the native thymus adaptable to medium/high-throughput applications and validated for exploration of thymus- and thymus organoid-biology.

immunology↗

bioRxiv2024

Specialized signaling centers direct cell fate and spatial organization in a limb organoid model

Specialized signaling centers orchestrate robust development and regeneration. Limb morphogenesis, for instance, requires interactions between the mesoderm and the signaling center apical-ectodermal ridge (AER), whose properties and role in cell fate decisions have remained challenging to dissect. To tackle this, we developed mouse embryonic stem cells (mESCs)-based heterogeneous cultures and a limb organoid model, termed budoids, comprising cells with AER, surface ectoderm, and mesoderm properties. mESCs were first induced into heterogeneous cultures that self-organized into domes in 2D. Aggregating these cultures resulted in formation of limb bud-like structures in 3D, exhibiting chondrogenesis-based symmetry breaking and elongation. Using our organoids and quantitative in situ expression profiling, we uncovered that AER-like cells support nearby limb mesoderm and fibroblast identities while enhancing tissue polarization that permits distant cartilage formation. Together, our findings provide a powerful model to study aspects of limb morphogenesis, and reveal the ability of signaling center AER cells to concurrently modulate cell fate and spatial organization.

developmental biology↗

bioRxiv2024

A Bioengineered Platform for Enhanced Observability of Patterned Gastrointestinal Organoid Monolayers with Bilateral Access

Studying the physiology and pathology of gastrointestinal (GI) tissues requires tools that can accurately mimic their complex architecture and functionality in vitro. Organoids have emerged as one such promising tool, though their closed structures with poorly accessible lumen and limited observability makes readouts challenging. In this study, we introduce a bioengineered organoid platform that generates bilaterally accessible 3D tissue models, allowing independent manipulation of both the apical and basal sides of patterned epithelial monolayers. We successfully constructed gastric, small intestinal, caecal, and colonic epithelial models that faithfully reproduce tissue-respective geometries and exhibit high physiological relevance, evidenced by the regionalization of stem cells and transcriptional resemblance to real epithelia. The gained observability allowed single-cell tracking over time and studies into the motility of cells in immersion and air-liquid interface cultures. Additionally, this model recapitulated Trichuris muris infection of the caecum epithelium, allowing the first live imaging of syncytial tunnel formation. Overall, this platform offers accessible organoids with improved observability, making it a valuable tool for investigating the dynamics of GI epithelial cells and their interactions with pathogens.

bioengineering↗

bioRxiv2023

Dissection and reconstruction of the colorectal cancer tumor microenvironment

Patient-derived organoids (PDOs) are the reference in vitro human disease models. However, the utility of colorectal cancer (CRC) PDOs is hindered by the lack of a tumor microenvironment (TME). To address this limitation, we built a living biobank of CRC PDOs with autologous stromal and immune TME. We characterized the original tumors and traditional monocultures using single-cell RNA-seq (scRNA-seq) and whole exome sequencing (WES) to obtain insights into cell type selection and phenotypic drift in culture. Subsequently, we developed culture conditions supporting all cell types to recapitulate the CRC-TME around PDOs. From the transcriptomes of >180k cells obtained from 260 such co-cultures, we illuminated the mutual influence of cells within CRC tumors. Based on original tumor data, atlases of predicted interactions and transcriptional networks elucidated why monocultures were altered and suggested that TME reconstruction more accurately reflected original tumor behavior. We found that inflammatory signals were absent in vitro and recovered upon co-culture with tumor-infiltrating lymphocytes (TILs). We also functionally confirmed that stromal, not cancer cells, mediated immune evasion. Additionally, stroma induced an invasive phenotype in cancer cells. From this deep dive into CRC-TME interactions, we built the human CRC-TME atlas (https://crc-tme.com/), an online portal for interactive exploration of gene expression data, prediction of cell-cell interactions at the pathway and receptor/ligand levels, transcriptional networks, and more. We anticipate PDO cultures with reconstructed TMEs will be valuable for discovery efforts, preclinical studies, and personalized medicine, with the atlas as a framework and inspiration for future CRC-TME studies.

cancer biology↗

bioRxiv2023

Bioengineered human colon organoids with in vivo-like complexity and function

Organoids and microphysiological systems, such as organs-on-a-chip, have emerged as powerful tools for modeling human gut physiology and disease in vitro. However, although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell-type diversity, and maturity necessary for mimicking adult human intestinal mucosa. To instead generate models closely resembling the in vivo cell-type composition and spatial compartmentalization, we herein integrated organoid and organ-on-a-chip technology to develop a primary human stem-cell-derived organoid model, called mini-colons. The luminal access and flow in human mini-colons removes shed cells to greatly enhance tissue longevity and differentiation over physically inaccessible human intestinal organoids that accumulate trapped cellular debris and waste. By establishing a gradient of growth factors, we replicated and sustained in vivo-like cell fate patterning and concurrent differentiation to secretory cell types and colonocytes. These long-lived human mini-colons contain abundant mucus-producing Goblet cells that lubricate the colonic epithelial lining. The stem and proliferative progenitor cells are also realistically confined to the crypts, facilitating stable homeostatic tissue turnover and preserving tissue integrity for several weeks. Also signifying mini-colon in vivo-like maturation, single-cell RNA sequencing showed emerging mature colonocytes and absorptive BEST4+ colonocytes. This methodology could be expanded to generate microtissues derived from the small intestine and incorporate additional microenvironmental components, thus emulating the intricate complexity of the native gut in an in vitro setting. Our bioengineered human organoids provide a highly accurate, long-lived, functional platform to systematically study human gut physiology and pathology, and for the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2023

OpenSIM: open source microscope add-on for structured illumination microscopy

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

bioengineering↗

bioRxiv2022

COORDINATION BETWEEN EMBRYO GROWTH AND TROPHOBLAST MIGRATION UPON IMPLANTATION DELINEATES MOUSE EMBRYOGENESIS

Implantation marks a key transition in mammalian development. The role of embryo-uterus interaction in periimplantation development is however poorly understood due to inaccessibility in utero. Here, we develop an engineered uterus-like microenvironment to recapitulate mouse development ex vivo up to E5.25 and discover an essential role of integrin-mediated trophoblast adhesion to the uterine matrix. Light-sheet microscopy shows that trophoblast cells undergo Rac1-dependent collective migration upon implantation, displacing Reicherts membrane and generating space for egg cylinder growth. The key role of coordination between trophoblast migration and embryo growth is verified by experimentally manipulating the migration velocity and geometry of the engineered uterus. Modeling the implanting embryo as a wetting droplet links the tissue shape dynamics to underlying changes in trophoblast adhesion and suggests that the corresponding tension release facilitates egg cylinder formation. Together, this study provides mechanisms by which dynamic embryo-uterus interactions play an essential role in peri-implantation development.

developmental biology↗

bioRxiv2021

Primate-specific cis- and trans-regulators shape transcriptional networks during human development

The human genome contains more than 4.5 million inserts derived from transposable elements (TE), the result of recurrent waves of invasion and internal propagation throughout evolution. For new TE copies to be inherited, they must become integrated in the genome of the germline or preimplantation embryo, which requires that their source TE be expressed at these stages. Accordingly, many TEs harbor DNA binding sites for the pluripotency factors OCT4, NANOG, SOX2, KLFs and are transiently expressed during embryonic genome activation. Here, we describe how many primate-restricted TEs have additional binding sites for lineage-specific transcription factors driving their expression during human gastrulation and later steps of fetal development. These TE integrants serve as lineage-specific enhancers fostering the transcription, amongst other targets, of KRAB-zinc finger proteins of similar evolutionary age, which in turn corral the activity of TE-embedded regulatory sequences in an equally lineage-restricted fashion. Thus, TEs and their KZFP controllers play broad roles in shaping transcriptional networks during early human development.

evolutionary biology↗

bioRxiv2021

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such a vascular compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

bioengineering↗

bioRxiv2021

Probing the killing potency of tumor-infiltrating lymphocytes on microarrayed autologous tumoroids

Immunotherapy has shown promise as an approach to fight cancer by harnessing the immune system. However, due to the lack of biomarkers to guide treatment regimens and predict response rates, there is an unmet need for more robust ex vivo and in vitro systems that recapitulate patient-specific tumor biology and enable response prediction for immune therapies in an autologous setting. To address this issue, we developed a high-content screening-compatible assay based on microcavity arrays to study tumor-infiltrating lymphocyte (TIL) functionality on 3D tumoroid models. We validated our system using the pmel-1 activated T cell mouse model to assess both cancer immunogenicity and T cell functionality. To demonstrate the translational potential of the platform, we used it to evaluate the response of patient-derived TILs to autologous human colorectal cancer (CRC) tumoroids. Using a combination of imaging and flow cytometry, we determined several features of the antitumor activity of TILs, including the extent of tumoroid killing and secretion of cytokines. We then used the approach to identify responders to immunotherapy, such as the immune checkpoint blockade (ICB) agent Nivolumab (PD-1 inhibitor) and Ipilimumab (CTLA-4 inhibitor). Our system allows not only the identification of immunogenic tumors, but also the testing of patients for response to immunomodulators, enabling personalized immuno-oncology.

cancer biology↗

bioRxiv2021

Gastruloids as in vitro models of embryonic blood development with spatial and temporal resolution

Gastruloids are three-dimensional embryonic organoids that reproduce key features of early mammalian development in vitro with unique scalability, accessibility, and spatiotemporal similarity to real embryos. Recently, we adapted gastruloid culture conditions to promote cardiovascular development. In this work, we extended these conditions to capture features of embryonic blood development through a combination of immunophenotyping, detailed transcriptomics analysis, and identification of blood stem/progenitor cell potency. We uncovered the emergence of blood progenitor and erythroid-like cell populations in late gastruloids and showed the multipotent clonogenic capacity of these cells, both in vitro and after transplantation into irradiated mice. We also identified the spatial localization near a vessel-like plexus in the anterior of gastruloids with similarities to the emergence of blood stem cells in the embryo. These results highlight the potential and applicability of gastruloids to the in vitro study of complex processes in embryonic blood development with spatiotemporal fidelity.

developmental biology↗

bioRxiv2021

Bioengineered embryoids mimic post-implantation development in vitro

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are not yet optimal for modeling post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. A high-throughput cell aggregation approach was employed to simultaneously coax mouse embryonic stem cells (ESCs) into hundreds of uniform epiblast-like (EPI) aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell (TSC) aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

developmental biology↗