Benjamin David Simons

The epidermis is a stratified epithelium comprising proliferative basal cells and differentiated suprabasal cells that are continuously renewed throughout life. Conflicting theories have been proposed regarding the heterogeneity of the basal cell layer, with some studies proposing that all proliferative cells have self-renewal potential during homeostasis ^1–5 . Meanwhile, others posit a model in which stem cell differentiation occurs through an intermediate transit-amplifying-like progenitor state with strictly limited proliferative potential ^6–10 . Here, to determine the heterogeneity and pattern of cell fate, we performed intravital time-lapse imaging to track the fate of lineage-labelled cells in the skin interfollicular epidermis. Results from quantitative analyses of clonal dynamics support a two-compartment model of epidermal homeostasis, whereby basal cells are organized in a stem cell–transit-amplifying cell hierarchy. Critical to these conclusions was the discovery of sexual dimorphism in epidermal fate, with female mice showing a highly synchronized pattern of cell division, differentiation and delamination that contributes to accelerated wound healing. Single-cell RNA sequencing, transcriptional profiling and clonal analyses of castrated mice revealed that male sex hormones promote stem cell differentiation and delamination. In summary, our study provides insight into the functional identity of the stem cell population and identifies sex-specific differences in the regulation of epidermal stem cells and transit-amplifying cell fate.

Chromatin mobility, the exploration of 3D conformations by the chromatin fibre, is implicated in transcription and DNA repair, and is thought to decrease progressively as cells differentiate. Here, combining live-cell 3D single-nucleosome tracking with genomic approaches, we show that chromatin mobility instead changes in a cell-type-specific manner, increasing as pluripotent cells acquire posterior fates. This is accompanied by 3D genome reorganisation, including loss of Polycomb-associated interactions and emergence of CDX2-associated interactions. Withdrawal of posteriorising WNT/β-catenin signalling rapidly reduces chromatin mobility and partially restores Polycomb-associated chromatin interactions, while Cdx1/2/4 -null cells fail to acquire these WNT/β-catenin-dependent changes. Together, our findings establish chromatin mobility as a regulated property of cell state, with WNT/β-catenin signalling and CDX factors acting as mediators that fine-tune the sampling of 3D chromatin interactions during cell state transitions.

By generalizing a class of models recently introduced to account for protracted transients in biological systems, we identify a novel mechanism for hyperuniformity. In this model, competition of individuals over a shared resource serves as feedback that can asymptotically guide the population toward a critical steady state with a divergent individual lifetime. We show that, in its spatially extended form, this many-body model exhibits hyperuniform density fluctuations. Through explicit coarse-graining, we develop a hydrodynamic theory that conforms closely with the results of stochastic simulations. Unlike previous models for nonequilibrium hyperuniform states, our model does not exhibit conservation laws, even in the asymptotic regime. Instead, hyperuniformity arises from the divergence of the range of the resource-mediated interactions. These findings may find applications in engineering, cellular population dynamics, and ecology.

Pathologic transformation represents a critical yet poorly defined window during which mutant epithelial stem cells actively construct the microenvironment that enables tumour initiation ^1,2 . Here using integrated single-cell, spatial and functional analyses, we define the earliest multicellular events that licence this transition following oncogenic activation in the lung. Kras ^G12D -mutant alveolar type II cells rapidly adopt regenerative-like states that act as signalling hubs, orchestrating coordinated stromal and immune reprogramming while enhancing epithelial plasticity. Through secretion of amphiregulin, mutant epithelial cells activate EGFR signalling in adjacent fibroblasts, inducing a fibrotic, injury-like programme. Reprogrammed fibroblasts, in turn, expand and reprogramme alveolar macrophages, amplifying inflammatory signalling and reinforcing epithelial plasticity. These reciprocal interactions establish a self-sustaining epithelial–stromal–immune circuit that generates a tumour-permissive niche before malignant outgrowth. Disruption of the amphiregulin–EGFR axis prevents early niche formation and abrogates tumour initiation. Conservation of this programme in KRAS ^G12D -inducible human alveolar organoids and early-stage lung adenocarcinoma tissues identifies epithelial–microenvironment communication as a therapeutically actionable vulnerability and suggests that intercepting niche formation may prevent progression to treatment-resistant disease.

Cellular identity and fate transitions are governed by continuous molecular processes that form dynamic trajectories within a high-dimensional transcriptomic landscape. Existing methods attempt to model these dynamics from two complementary perspectives: trajectory inference and velocity modeling. Ideally, velocity and trajectory are dual aspects of transcriptomic dynamics where velocity is tangent to trajectory everywhere. This inherent connection between velocity and trajectory is currently absent in transcriptomic analysis. Splicing velocity are precision-limited to inadequately-sequenced genes, while trajectory inference prioritizes the modeling of global trends while omitting local dynamics. This divergence breaks the geometric continuity between local velocities and global trajectories, hindering the reliable interpretation of developmental dynamics. To reconcile trajectory inference and RNA velocity, we introduce VeloTrace, a framework that unifies them through Neural Ordinary Differential Equations (NeuralODEs). VeloTrace learns a continuous-time velocity field whose integral curves constitute the trajectory itself, while ensuring that velocities are tangent to integral paths everywhere. Leveraging a splicing quality score, VeloTrace incorporates high-quality splicing velocity as partial supervision for velocity orientation and grounding. During optimization, VeloTrace incorporates a Monte Carlo multi–time-frame supervision strategy to ensure coherence between local and global trajectorys and suppress sequencing-induced stochastic diffusion. Through refining the velocity field and cell-specific parameters for pseudo-time, expression, and velocity, VeloTrace reconstructs a smooth, local-and-global-coherent velocity-vector-guided flow in the transcriptomic latent space. This strategy ensures a complementary integration of velocity and trajectory, imputing the transcriptional kinetics for genes of insufficient strength, whose kinetics cannot be accurately portrayed by splicing velocity. In simulation benchmarks, VeloTrace captured the transcriptional dynamics of all expressed genes, even those with inadequate sequencing coverage, producing velocity directions that were most consistent with the true direction and every-where tangential across the entire process, outperforming state-of-the-art methods, including scVelo, UniTVelo, VeloVI and scTour. VeloTrace uniquely reconciles RNA velocity and trajectory inference, creating a velocity field where each cell can infer past and future transitions from its current state. Moreover, VeloTrace extends reliable velocity estimation to a broader set of genes. When applied to mouse neural stem cell differentiation data, it successfully recovers dynamics of driver genes for two developmental lineages, including those with low expression, shedding light on their regulatory roles during differentiation. This unified framework lays the foundation for more accurate modeling of gene regulation and cell fate decisions in complex biological systems.

Interactions between mutant cells and their environment have a key role in determining cancer susceptibility ^1–3 . However, understanding of how the precancerous microenvironment contributes to early tumorigenesis remains limited. Here we show that newly emerging tumours at their most incipient stages shape their microenvironment in a critical process that determines their survival. Analysis of nascent squamous tumours in the upper gastrointestinal tract of the mouse reveals that the stress response of early tumour cells instructs the underlying mesenchyme to form a supportive ‘precancerous niche’, which dictates the long-term outcome of epithelial lesions. Stimulated fibroblasts beneath emerging tumours activate a wound-healing response that triggers a marked remodelling of the underlying extracellular matrix, resulting in the formation of a fibronectin-rich stromal scaffold that promotes tumour growth. Functional heterotypic 3D culture assays and in vivo grafting experiments, combining carcinogen-free healthy epithelium and tumour-derived stroma, demonstrate that the precancerous niche alone is sufficient to confer tumour properties to normal epithelial cells. We propose a model in which both mutations and the stromal response to genetic stress together define the likelihood of early tumours to persist and progress towards more advanced disease stages.

Crypt fixation is an early and essential event of colorectal cancer initiation 1–6. It is an evolutionary process where precancerous stem cells compete with their healthy neighbors for monoclonal fixation of intestinal crypts 1–6. This competition involves stochastic interaction between three cell populations, oncogene mutant clones, competing wild-type clones and the cells populating their microenvironment. Interactions between competing WT and mutant clones are well studied; however, the impact of the microenvironment cells on crypt fixation, has been challenging to quantify 1–6. This is due to the absence of suitable experimental and theoretical models to study three-way stochastic interactions between competing clones and cells in their microenvironment. To overcome this challenge, we integrated established mosaic clonal selection models1,7,8 and immune depletion models9 with the theoretical framework provided by Stochastic Conveyor Belt model 10,11. Using this approach, we quantified the selective advantage of colonic stem cells with the BRAFV600E mutation, one of the most prevalent human oncogenes. We showed that BRAFV600E confers a selective advantage to colonic stem cells but approximately half of the clones are destined for clonal extinction. We show mechanistically that clonal extinction of these precancerous cells is actively mediated by adaptive immune system. Specific depletion of regulatory T cells or targeted deletion of Major Histocompatibility Complex Class II (MHCII) or interleukin 10 receptor A (IL10RA) on the BRAFV600E mutated clones, prevented their extinction. Overall, these findings reveal a new immune sensing mechanism whereby Tregs recognize and eliminate precancerous stem cells before malignant transformation. Understanding this interplay between microenvironment and stem cell dynamics at tumor initiation will enable the development of immunoprevention strategies against colon cancer initiation, rather than treat existing tumors. *Birgit Tschiatschek & Miraal Maqsood contributed equally.

The ability of adult epithelial cells to rewire their cell fate programme in response to injury has emerged as a new paradigm in stem cell biology. This plasticity supersedes the concept of strict stem cell hierarchies, granting cells access to a wider repertoire of fate choices. Yet, in order to prevent a disordered cellular response, this process must be finely regulated. Here we investigate the little-known regulatory processes that restrict fate permissibility in adult cells, and keep plasticity in check. Using a 3D regenerative culture system, that enables co-culturing epithelium and stroma of different origins, we demonstrate that oesophageal cells exposed to the ectopic signals of the dermis are capable of switching their identity towards skin. Lineage tracing experiments and histological analysis, however, reveal that the oesophageal-to-skin lineage conversion process is highly inefficient, pointing to the existence of barriers limiting cell fate re-specification. Single-cell RNA sequencing capturing the temporality of this process shows that cells transitioning towards skin identity resist the natural progression towards tissue maturation by remaining in a persistent regenerative state marked by a particularly strong hypoxic signature. Gain and loss of function experiments demonstrate that the HIF1a-SOX9 axis acts as a key modulator of epithelial cell fate plasticity, restricting changes in identity during tissue regeneration. Taken together, our results reveal the existence of lineage conversion barriers that must be resolved for cells to respond to signals instructing alternative fate choices, shedding light on the principles underlying the full regenerative capacity of adult epithelial cells.