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Klein, D.

Publications and source records attributed to Klein, D..

At least 19 recordsLinked to original sources

Generative inverse design steers human hematopoietic cell fate in vitro

Inverse design has advanced the discovery of molecules and materials. Yet it remains largely unexplored for steering cell fate, where inputs are continuous, combinatorial culture conditions and outputs are distributions of cell states. Human hematopoiesis, in which combinations and doses of growth factors steer stem cells toward each blood lineage, is a demanding test case. While forward models can evaluate discrete conditions, the vast combinatorial space renders searching for a specific, target-producing design an intractable inverse problem. Here, we introduce LabCompass, an inverse-modeling framework that optimizes experimental designs toward candidates expected to produce specified target cell states, using a flow-matching generative model guided by cellular phenotypes predicted by a forward surrogate. We applied LabCompass to primary human CD34+ in vitro cultures, where continuous cytokine and small-molecule parameters govern hematopoietic cell fate. In a lab-in-the-loop setting, we experimentally validated model-driven designs and used these measurements to update the model across four successive loops. LabCompass recovered established lineage-inducing designs, improved predictions with new data, and enriched target populations across lineages. Incorporating new parameters expanded the design space and enriched rare populations beyond initial reach. Across loops, we generated a 78-million-cell spectral flow cytometry dataset and identified distinct culture conditions converging on the same target cell states. Our results establish generative inverse modeling as a compelling paradigm for navigating high-dimensional, continuous experimental spaces, and demonstrate that virtual cell models should be judged not only by how accurately they predict cellular responses, but also by how effectively they guide experiments toward desired biological outcomes.

cell biology↗

A Label-Free Multi-Metric Pipeline for Benchmarking Single-Cell RNA-Sequencing Clustering and Testing the Reproducibility of Cell-Type Heterogeneity

A discovered sub-population from single-cell transcriptomic data is only meaningful if it is reproducible, yet clustering is usually done with one method on one embedding and rarely tested. We present a label- free, multi-metric pipeline that reframes clustering as an auditable, methods-blind decision and separates two notions of stability that are commonly conflated: reproducibility under cell resampling (bootstrap) and reproducibility under re-embedding (retraining the representation). The pipeline evaluates seven clustering configurations across cluster counts using five non-redundant quality metrics. As a whole- dataset control on a mouse retinal atlas, it recovers an eight-cell-type annotation at 96.3% accuracy (adjusted Rand index, ARI = 0.91) without labels. We then validate the discovery mode on two cell types with opposite ground truth. On bipolar cells, which have well-established subtypes, the pipeline accepts the sub-structure: across-embedding reproducibility rises with cluster number to a high plateau (mean pairwise ARI [~]0.93 near the [~]15 known bipolar subtypes), with quality metrics improving in parallel. On rod photoreceptors, treated as homogeneous, it rejects over-clustering: the metric-selected partition passes a bootstrap-stability check but is not reproducible when the embedding is retrained (mean pairwise ARI = 0.69), and the metrics do not improve with cluster number. On synthetic data, the test recovers real structure down to a 5% subpopulation while rejecting null data (high sensitivity and specificity). Bootstrap stability alone is therefore insufficient evidence for sub-population; the across-embedding test discriminates real sub-structure from over-clustering and applies to any cell type as a reproducible alternative to single-method, single-embedding clustering.

bioinformatics↗

Benchmarking gene expression reconstruction from single-cell latent representations

Single-cell transcriptomics is typically modeled in low-dimensional latent representations that improve the signal-to-noise ratio of the data. Such representations underpin data integration, cell state discovery, and perturbation prediction, with applications ranging from large-scale organ atlases to latent trajectory modeling. Recent virtual cell approaches further leverage these representations to predict cellular responses as distributional shifts in latent space. Each of these applications ultimately requires faithful gene expression reconstruction from latent spaces for biological interpretation, enabling gene-level analysis of predicted perturbed or batch-corrected cells. Yet representation choice is typically treated as an implementation detail rather than a primary modeling decision, with no systematic evaluation of how well latent representations support gene expression reconstruction. Here, we introduce ReconEval, a benchmark for evaluating gene expression reconstruction from single-cell latent spaces. We benchmark two classes of latent representations: end-to-end trained models such as PCA, autoencoders, and variational autoencoders, and pretrained single-cell foundation model embeddings coupled to newly trained decoders. Reconstruction is evaluated both directly and after latent-space perturbation prediction. Across perturbational and observational datasets totaling over 100 million cells, our metric suite quantifies statistical fidelity; biological signal preservation, including differential expression, coexpression, cell-cycle structure, cytokine response and pathway activity; and perturbation-specific effects. We find that autoencoders achieve the strongest stand-alone reconstruction at low dimensionality, while variational regularization does not improve generalization in reconstruction. Frozen foundation model embeddings retain recoverable gene-level information, with reconstruction quality depending strongly on decoder architecture and pretraining objective. In latent perturbation modeling, high-dimensional PCA matches foundation model embeddings, while low-dimensional AE embeddings are optimal for flow-based generative models. Overall, reconstruction depends critically on the interplay between representation and downstream model, and simpler representations can outperform complex alternatives given appropriate capacity. Our benchmark establishes reconstruction as a critical evaluation axis for single-cell foundation models. We envision it improving the biological interpretability of latent-space modeling, a prerequisite for future virtual cell models to be validated by domain experts and grounded in biology.

bioinformatics↗

Representations of semantic relations in the human cerebral cortex

An essential aspect of human cognition is the ability to explicitly think about semantic relations between concepts. Neuroimaging studies have found that individual concepts are encoded by distributed patterns of cortical activity, but relatively little is known about how semantic relations between concepts are encoded in the brain. Some theoretical models suggest that relation representations are embedded within concept representations, while others suggest that relation representations are independent of any specific concept pair. We designed a study to compare how semantic relations and concepts are encoded across the cerebral cortex. To characterize how relations are encoded across cortex, fMRI was used to record brain activity while six participants each answered over one thousand questions about different semantic relations. We find that relations are encoded independently of the specific concepts that are connected in any particular instance of the relation. Our results further suggest that relations and concepts are represented in the same set of cortical regions, and that, within these regions, each location is preferentially selective for specific relations. Overall, these results suggest that in the human cerebral cortex, relations and concepts may have the same type of functional representation.

neuroscience↗

Mapping the latent CRBN-molecular glue degrader interactome

Molecular glue degraders (MGDs) are a transformative modality in drug discovery. MGDs that work in concert with the E3 ligase CRL4CRBN can degrade a range of substrates through tailored MGDs. To explore CRL4CRBN reprogrammability, we tested whether reported CRBN-MGD substrates are part of a network of latent CRBN interactors, detectable with generic CRBN-MGDs. Leveraging highly parallel interaction measurement (GluePCA) between CRL4CRBN and human zinc-fingers (ZFs), we identified [~]210 ZFs bound to CRBN-pomalidomide, where top binders are already reported as degraded by dedicated MGDs. To map latent CRBN-MGDs interactions proteome-wide, and thus define the immediately accessible CRBN target space, we combined AI-derived protein surface queries (MaSIF-mimicry) with GluePCA. This pipeline identified 6 known and 43 novel CRBN-pomalidomide binders, thereby providing privileged starting points for MGD development. We expect this binding-focused, highly parallel workflow to be readily applicable to other MGD/E3 ligase systems, extending the target landscape of this emerging drug class.

molecular biology↗

The genetic architecture of the human bZIP family

Generative biology holds the promise to transform our ability to design and understand living systems by creating novel proteins, pathways, and organisms with tailored functions that address challenges in medicine, sustainability, and technology. However, training generative models requires large quantities of data that captures the genetic architecture of protein function in all its complexity, but these are currently scarce. Here, we systematically mutagenized all 54 human basic-leucine zipper (bZIP) domains and quantified their interactions with each other using bindingPCA, a quantitative deep mutational scanning assay. This resulted in [~]2 million interaction measurements, capturing the effect of all single amino acid substitutions at each of the 35 interfacial positions. We found that mutation effects are largely additive in the vicinity of each wild-type bZIPs, but diverge across the family, indicating strong context dependency. A global additive thermodynamic model provided moderate prediction of mutation effects, while individual models per bZIP achieved higher performance, supporting a model of local simplicity and global complexity. Our results therefore suggest that the genetic architecture of protein function is more complex than previously anticipated, which could hinder predictability. However, a convolutional neural network trained on this dataset could accurately predict binding scores from sequence alone. Furthermore, the model enabled the design of synthetic bZIPs with high target specificity, demonstrating practical applicability for bioengineering purposes. Our study shows that capturing family-wide diversity is essential to reveal context dependencies and train accurate quantitative models of protein-protein interactions.

biophysics↗

Reciprocal Regulation Between the SCFFBXO24 Ubiquitin E3 Ligase and FoxP1 Protein

Forkhead Box Protein P1 (FoxP1) is a crucial transcriptional repressor essential for the development of the brain and heart. In adults, FoxP1 protein levels are dysregulated in a variety of disorders, including chronic obstructive pulmonary disease (COPD), atherosclerosis, and heart failure, where they causally contribute to disease pathogenesis. Although independent investigators have reported that FoxP1 protein is ubiquitinated, and E3 ligases have been identified for other FoxP family proteins, the identity of the E3 ligase that controls FoxP1 protein stability has remained unknown. Here, we identify FBXO24, a subunit of the Skp-Cullin-F-box (SCF) ubiquitin E3 ligase complex, as the regulator of FoxP1 ubiquitination and stability. Specifically, FBXO24 regulates K48 and K63 ubiquitination, complexes with, and co-localizes to the nucleus with FoxP1 protein in lung epithelial cells. Depleting FBXO24 reverses the unfolded protein response and cell death triggered by loss of FoxP1 protein in lung epithelium, suggesting a protective role. Additionally, FBXO24 knockout mice exhibit elevated FoxP1 levels in the lung and heart and reduced unfolded protein response activity after short-term cigarette smoke exposure. Intriguingly, we also uncovered bidirectional regulation, whereby FoxP1 protein binds to the FBXO24 promoter to suppress FBXO24 transcription. To our knowledge, this is the first evidence that a substrate for an E3 ligase can also regulate the E3 ligase and, therefore, control levels of other substrates, revealing new regulatory networks. Targeting FBXO24 may offer a therapeutic strategy for COPD, atherosclerosis, and heart failure by stabilizing FoxP1 levels in the heart and lungs and mitigating harmful downstream effects.

molecular biology↗

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↗

Measuring Longitudinal Genome-wide Clonal Evolution of Pediatric Acute Lymphoblastic Leukemia at Single-Cell Resolution

Approximately 15-20% of children with acute lymphoblastic leukemia (pALL) are not cured despite years of treatment with more than ten chemotherapeutic agents, yet bulk sequencing reveals minimal somatic variation that could explain genetic treatment resistance. Using error-corrected bulk sequencing, and primary template-directed amplification (PTA)-enabled single-cell exome and whole-genome sequencing, we discovered much more genetic diversity in pALL. Individual leukemic cells harbor 3.7-fold more mutations (mean 3,553) than detected in bulk samples (mean 965). Many ETV6-RUNX1 patients harbor multiple independent ras-mutated clones, and we also identified convergent evolution of JAK2 mutations as well as copy number alterations. PTA-based phylogenetic analysis of over 150 single-cell genomes linked heritable phenotypes to specific genetic alterations and identified low-frequency clones harboring mutations in treatment resistance-associated genes (ITGA9, NTRK3, SULF2) that increased in frequency during treatment. These findings reveal extensive hidden genetic diversity in pALL while providing a framework for the early detection of high-risk leukemic populations.

cancer biology↗

Learning sequence-based regulatory dynamics in single-cell genomics

Epigenomics assays, such as chromatin accessibility, can identify DNA-sequence-specific regulatory factors. Models that predict read counts from sequence features can explain cell-based readouts using specific DNA patterns (genomic motifs) but do not encode the changes in genomic regulation over time, which is crucial for understanding biological events during cell transitions. To bridge this gap, we present muBind, a deep learning model that accurately predicts genomic counts of single-cell datasets based on DNA sequence features, their cell-based activities, and cell relationships (graphs) in a single architecture, enhancing the interpretability of cell transitions due to the possibility of inspecting motif activities weighted by nearest neighbors. MuBind shows competitive performance in bulk and single-cell genomics. When complemented with graphs learned from RNA-based dynamical models used as injected priors in our model, muBind enhances through motif-graph interactions the identification of transcriptional regulators explaining cell transition events, including Sox9 in pancreatic endocrinogenesis scATAC-seq, and Gli3/Prdm16 in mouse neurogenesis and human organoids scRNA-seq, both supported by independent evidence, including associations between chromatin and motif activities over pseudotime, TF-gene expression patterns, and biological knowledge of these regulators. muBind advances our understanding of cell transitions by revealing regulatory motifs and their interactions, providing valuable insights for genomic research and gene regulatory network dynamics. It is available at https://github.com/theislab/mubind.

genomics↗

Bilingual language processing relies on shared semantic representations that are modulated by each language

Billions of people throughout the world are bilingual, and they can extract meaning from multiple languages. While some evidence suggests that there is a shared system in the human brain for processing semantic information from native and non-native languages, other evidence suggests that semantic processing is language-specific. We conducted a study to determine how semantic information for different languages is represented in the brains of bilinguals. Functional magnetic resonance imaging (fMRI) was used to record brain responses while participants read several hours of natural narratives in their native (Chinese) and non-native (English) languages. These data were then used to compare semantic representations between the two languages. We find that semantic representations are largely shared between languages, but that there are fine-grained differences in the representation of some semantic categories across languages. These results reconcile current competing theories of bilingual language processing. Significance StatementBilinguals understand the meaning of words in multiple languages. Whether this capacity reflects a shared brain system for processing both native and non-native languages, or whether processing is language-specific is still unclear. Here, we examine whether and how semantic representations in the brain support shared and/or language-specific processing. We recorded brain activity from participants reading narratives in their native (Chinese) and non-native (English) languages, and modeled how their brains represent word meaning in each language. We show that semantic representations are similar between the two language conditions, and that these representations are systematically modulated between native and non-native language comprehension.

neuroscience↗

scMicrobe PTA: Near Complete Genomes From Single Bacterial Cells

Microbial genomes produced by single-cell amplification are largely incomplete. Here, we show that primary template amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard amplification approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

genomics↗

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↗

Enhanced efficiency in the bilingual brain through the inter-hemispheric cortico-cerebellar pathway in early second language acquisition

The bilingual experience has a profound impact on the functional and structural organization of the brain, but it is not yet well known how this experience influences whole-brain functional network connectivity. We examined a well-characterized large sample (151 participants) of monolinguals and bilinguals with varied age of second language acquisition, who underwent resting-state functional magnetic brain imaging. We constructed comprehensive functional brain networks for each participant, encompassing cortical, subcortical, and cerebellar regions of interest. Whole-brain analyses revealed that bilingual individuals exhibit higher global efficiency than monolinguals, indicating enhanced functional integration in the brain. Moreover, the age at which the second language was acquired correlated with this increased efficiency, suggesting that earlier exposure to a second language has lasting positive effects on brain functional organization. Further investigation through the network-based statistics (NBS) approach indicates that this effect is primarily driven by heightened functional connectivity between association networks and the cerebellum. This work shows that early learning enhances global whole-brain efficiency and that the timing of learning of two languages has an impact on functional brain organization. Significance statementLong-term learning impacts brain organization at different spatial scales, and this may be particularly enhanced during early stages of life. Bilingualism offers a unique opportunity to test long-term learning effects in the human brain, given that exposure to a second language can occur from birth or later in life, and can be maintained over long periods of time. We found that second language acquisition in early childhood (before five years of age) enhances brain network efficiency, and that this effect goes beyond the language and cognitive control regions, in fact, the interhemispheric cortico-cerebellar circuit plays a key role. This work shows that the timing of bilingual learning experience alters the brain functional organization at the global and local levels.

neuroscience↗

Optimization of a deep mutational scanning workflow to improve quantification of mutation effects on protein-protein interactions

Deep Mutational Scanning (DMS) assays are powerful tools to study sequence-function relationships by measuring the effects of thousands of sequence variants on protein function. During a DMS experiment, several technical artefacts might distort non-linearly the functional score obtained, potentially biasing the interpretation of the results. We therefore tested several technical parameters in the deepPCA workflow, a DMS assay for protein-protein interactions, in order to identify technical sources of non-linearities. We found that parameters common to many DMS assays such as amount of transformed DNA, timepoint of harvest and library composition can cause non-linearities in the data. Designing experiments in a way to minimize these non-linear effects will improve the quantification and interpretation of mutation effects.

systems biology↗

The genetic architecture of protein interaction affinity and specificity

Proteins function in crowded cellular environments in which they must bind to specific target proteins but also avoid binding to many other off-target proteins. In large protein families this task is particularly challenging because many off-target proteins have very similar structures. How this specificity of physical protein-protein interactions in cellular networks is encoded and evolves is not very well understood. Here we address the question of specificity-encoding by comprehensively quantifying the effects of all mutations in one protein, JUN, on its binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. Fitting a global thermodynamic model to the data reveals that most affinity changing mutations equally affect JUNs propensity to bind to all its interaction partners. Mutations that alter the specificity of binding are much rarer. These specificity-altering mutations are, however, distributed throughout the JUN interaction interface. JUNs interaction specificity is encoded by both positive determinants that promote on-target interactions and negative determinants that prevent off-target interactions. Indeed, about half of the specificity-defining residues in JUN have dual functions and both promote on-target binding and prevent off-target binding. Whereas nearly all mutations that alter specificity are pleiotropic and also alter the affinity of binding to all interaction partners, the converse is not true with mutations outside of the interface able to tune affinity without affecting specificity. Our results provide the first global view of how mutations in a protein affect binding to all its potential interaction partners and reveal the distributed encoding of specificity and affinity in an interaction interface. They also show how the modular architecture of coiled-coils provides an elegant solution to the challenge of optimising specificity and affinity in a large protein family.

systems biology↗

Mapping cells through time and space with moscot

Single-cell genomics technologies enable multimodal profiling of millions of cells across temporal and spatial dimensions. Experimental limitations prevent the measurement of all-encompassing cellular states in their native temporal dynamics or spatial tissue niche. Optimal transport theory has emerged as a powerful tool to overcome such constraints, enabling the recovery of the original cellular context. However, most algorithmic implementations currently available have not kept up the pace with increasing dataset complexity, so that current methods are unable to incorporate multimodal information or scale to single-cell atlases. Here, we introduce multi-omics single-cell optimal transport (moscot), a general and scalable framework for optimal transport applications in single-cell genomics, supporting multimodality across all applications. We demonstrate moscots ability to efficiently reconstruct developmental trajectories of 1.7 million cells of mouse embryos across 20 time points and identify driver genes for first heart field formation. The moscot formulation can be used to transport cells across spatial dimensions as well: To demonstrate this, we enrich spatial transcriptomics datasets by mapping multimodal information from single-cell profiles in a mouse liver sample, and align multiple coronal sections of the mouse brain. We then present moscot.spatiotemporal, a new approach that leverages gene expression across spatial and temporal dimensions to uncover the spatiotemporal dynamics of mouse embryogenesis. Finally, we disentangle lineage relationships in a novel murine, time-resolved pancreas development dataset using paired measurements of gene expression and chromatin accessibility, finding evidence for a shared ancestry between delta and epsilon cells. Moscot is available as an easy-to-use, open-source python package with extensive documentation at https://moscot-tools.org.

bioinformatics↗

Mapping lineage-traced cells across time points with moslin

Simultaneous profiling of single-cell gene expression and lineage history holds enormous potential for studying cellular decision-making beyond simpler pseudotime-based approaches. However, it is currently unclear how lineage and gene expression information across experimental time points can be combined in destructive experiments, which is particularly challenging for in-vivo systems. Here we present moslin, a Fused Gromov-Wasserstein-based model to couple matching cellular profiles across time points. In contrast to existing methods, moslin leverages both intra-individual lineage relations and inter-individual gene expression similarity. We demonstrate on simulated and real data that moslin outperforms state-of-the-art approaches that use either one or both data modalities, even when the lineage information is noisy. On C. elegans embryonic development, we show how moslin, combined with trajectory inference methods, predicts fate probabilities and putative decision driver genes. Finally, we use moslin to delineate lineage relationships among transiently activated fibroblast states during zebrafish heart regeneration. We anticipate moslin to play a crucial role in deciphering complex state change trajectories from lineage-traced single-cell data.

bioinformatics↗