Michael A. Rieger

While a differentiation hierarchy with leukemia-initiating stem cells (LICs) at the apex is well documented for acute myeloid leukemia, the existence of LICs and their trajectories in B-cell acute lymphoblastic leukemia (B-ALL) are debated. This study aims to investigate the hierarchical landscape of two B-ALL cases by combining barcoding and multiome single-cell data to unveil the differentiation architecture and temporal dynamics of leukemic differentiation at clonal and single-cell resolution in vivo. Single-cell transcriptome and AbSeq analysis of lentiviral barcoded and transplanted patient-derived B-ALL cells revealed interconnected subpopulations, which could be prospectively isolated via surface markers and exhibited distinct leukemogenicity. Barcode tracking demonstrated the ability of cells to differentiate from a source cluster into differentiated progeny. Using machine learning, we identified expression patterns predicting the differentiation potential of each barcoded cell within the immature cell compartment. To determine the dynamical properties of clones, we developed a mathematical model that simulates the development of these subpopulations from a clonally diverse stem cell compartment. The model confirmed the most likely cluster of origin, supporting our functional data, and revealed the variability in dynamical properties between clones, while largely excluding plasticity. Hence, our work demonstrates unidirectional differentiation in selected B-ALL cases.

During development, transplantation, and homeostasis, hematopoietic stem cells (HSCs) migrate to niches to (re)constitute and maintain the hematopoietic system. A solid mechanistic understanding of their movement is essential but remains poorly examined. Here, we used long-term single-cell imaging and tracking to quantify how motility changes as immature HSCs differentiate into multipotent progenitors (MPPs), establishing motility as an early indicator of differentiation. HSCs and early MPPs exhibit rapid, non-directional movement, while more differentiated MPPs move more slowly and directed. Interestingly, ex vivo expanded HSCs retain HSC characteristics with more non-directional movement, suggesting it as a defining HSC trait. Moreover, we show that movement phenotypes can identify cells with higher HSC potential in long-term cultures. HSCs feature a dedicated cytoskeletal architecture characterized by elevated expression of microtubule- and actin-regulating proteins, and by 3D imaging we show that this coincides with reduced cytoskeletal dynamics. FSCN1, a cytoskeletal regulator prevalent in immature cells, controls HSC locomotion by modulating cell shape, and influences cell cycle kinetics and HSC attributes. Finally, THPO, but not SCF or CXCL12, triggers HSC motility through PI3K/AKT signaling.

How transfer RNA (tRNA) expression changes as hematopoietic stem cells are specified into human blood cell types remains unknown. tRNAs translate the 64-codon genetic code into the 21-amino acid protein code, and their relative abundance can influence proteomic output. Here, we introduce simultaneous single-cell tRNA and mRNA sequencing (sc-STM-seq), a scalable, high-throughput approach for profiling tRNAs alongside mRNA transcriptomes in individual cells. Applying sc-STM-seq to human bone marrow, we map tRNA expression and splicing across hematopoietic differentiation trajectories and identify tRNAs associated with stemness, differentiation, and specific cell lineages. Our study provides an atlas of the hematopoietic tRNA landscape and establishes tRNA expression as a previously underappreciated layer of cellular heterogeneity during human blood cell development.

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression, and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects BC therapy success and remains a major challenge. Although necroptosis, a lytic, and via damage-associated molecular patterns (DAMP) release, highly immunogenic mode of PCD, might overcome apoptosis resistance, there is an urgent need for physiological and translational human models to model necroptosis and PCD resistance in BC. Here, we apply 3D patient-derived, metastatic human mammary organoids (hMOs) to model apoptosis resistance, necroptosis, and inflammatory signaling using single-cell CITE-sequencing, time-lapse live cell brightfield, and immunofluorescent confocal microscopy, as well as biochemical approaches. Smac mimetic-triggered apoptosis could be confirmed in a panel of BC hMOs. Upon inducing apoptosis resistance with caspase inhibition, BC hMOs rapidly undergo necroptosis with profound MLKL phosphorylation. Necroptotic cell death was preceded by prominent transcriptional upregulation of inflammatory cyto- and chemokines, including interferons, that activate natural killer cells. Finally, necroptosis and the expression and release of inflammatory messengers in metastatic BC hMOs could be attenuated upon the inhibition of linear ubiquitination. We describe a novel experimental platform to model PCD, inflammation, and necroptosis that allows therapeutic screening to overcome chemotherapy resistance in patient-derived metastatic BC hMOs. With this platform, we identified necroptosis-induced interferon signaling, suggesting Smac mimetics and necroptosis as a potential immunotherapy strategy against metastatic BC.

Codons function as translation units in open-reading-frames of genes to encode for proteins. Transfer RNAs (tRNAs) connect every codon to its cognate amino acid. The genetic code defines the correspondence between codons and amino acids, yet whether individuals share a common quantitative relationship between tRNA supply and codon demand remains incompletely understood. Here, we established Marathon-tRNA-sequencing combined with matched mRNA-sequencing to reproducibly quantify tRNA and mRNA abundance from blood cells of ten healthy donors. We apply a precision-biology approach quantitatively integrating transcriptomic codon- and corresponding tRNA abundance across all 61 sense codons to compute the relative tRNA supply per codon. Thereby, we discover that relative tRNA supply varied approximately 1000-fold across codons, with broadly consistent patterns of highly and lowly supplied codons across donors. The extent of interindividual variability differed between codons and was highest for ACG (threonine). Relative tRNA supply also differed among synonymous codons, and genomic preferences did not consistently identify the codons with the highest relative supply. In summary, our findings reveal that pronounced inter-codon heterogeneity in relative tRNA supply constitutes a broadly conserved feature of human blood cells, with codon-specific degrees of interindividual variation. By distinguishing shared codon-specific patterns from human variation, this study establishes a reference for investigating codon-dependent gene regulation and synonymous sequence variation.

Background and Aims: Aging significantly increases the risk of cardiovascular disease, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms underlying EC-dependent cardiac aging and identifies a critical role for zinc finger and BTB domain-containing protein 16 (ZBTB16). Methods: Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses of aged hearts were performed to define age-related regulatory changes. Functional studies using genetic models were performed to assess cardiac aging phenotypes. In vitro assays examined EC senescence, secretory profiles, and effects of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested whether ZBTB16 mitigates aging-associated dysfunction. Results: Aged hearts exhibited decreased chromatin accessibility and reduced ZBTB16 expression in both humans and mice. Zbtb16 deletion in young mice, including Zbtb16-haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. ZBTB16-deficient EC supernatants activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified nuclear receptor-interacting protein 1 as a downstream target suppressed by ZBTB16, thereby limiting fibroblast activation and pro-fibrotic signalling. Conclusions: ZBTB16 preserves endothelial integrity and vascular niche homeostasis, protecting against aging-associated cardiac dysfunction. Its loss promotes EC senescence and fibrosis, whereas restoring its expression may represent a therapeutic strategy to improve cardiac function and reduce cardiovascular disease risk during aging.

Background and aim: Aging significantly increases the risk of cardiovascular diseases, characterized by progressive cardiac dysfunction. The vascular niche is crucial for maintaining cardiac homeostasis, yet endothelial cell (EC) impairment during aging remains poorly understood. This study investigates epigenetically regulated mechanisms mediating EC-dependent cardiac aging and identifies a critical role of Zinc finger and BTB domain-containing protein 16 (ZBTB16). Methods: Chromatin accessibility (snATAC-seq) and transcriptomic (snRNA-seq) analyses were performed on aged hearts to identify age-related regulatory changes. Functional studies using genetic models, assessed cardiac aging phenotypes. In vitro assays examined EC senescence and secretory profiles, while co-culture experiments analyzed the impact of ZBTB16-deficient EC supernatants on fibroblasts, cardiomyocytes, and neurons. Overexpression experiments in vitro and in vivo tested the potential for ZBTB16 to mitigate aging-associated dysfunction. Results: Aged hearts exhibited decreased chromatin accessibility and expression of the transcription factor ZBTB16 in both human and mice. Loss of ZBTB16 in young mice, including Zbtb16 haploinsufficient and endothelial-specific knockout mice, led to premature aging, diastolic dysfunction, and increased secretion of pro-fibrotic and inflammatory factors. Supernatants from ZBTB16-deficient ECs activated fibroblasts, induced cardiomyocyte hypertrophy, and impaired neuronal sprouting. Overexpression of ZBTB16 reversed these effects in senescent ECs and aged mice and reduced diastolic dysfunction. Mechanistic studies identified key downstream targets of ZBTB16, including nuclear receptor-interacting protein 1 (NRIP1). ZBTB16 suppressed NRIP1 expression, limiting fibroblast activation and pro-fibrotic signaling. Conclusions: ZBTB16 is a key regulator of endothelial function, maintaining vascular niche homeostasis and mitigating aging-associated cardiac dysfunction. Its loss promotes EC senescence and pro-fibrotic signaling, contributing to diastolic dysfunction. Overexpression of ZBTB16 presents a potential therapeutic strategy for preserving cardiac function during aging. These findings establish a novel role for ZBTB16 in endothelial aging and cardiovascular disease prevention. Graphical

While a differentiation hierarchy with leukemia-initiating stem cells (LICs) at the apex is well documented for acute myeloid leukemia, the existence of LICs and their trajectories in B cell acute lymphoblastic leukemia (B-ALL) are debated. B-ALL is a malignant disease displaying considerable phenotypical and functional heterogeneity, yet the underlying cellular organization remains largely elusive. This study aims to investigate the hierarchical landscape of B-ALL by combining barcoding and multiome single cell data to unveil the differentiation architecture and temporal dynamics of leukemic differentiation at clonal and single cell resolution in vivo . Single-cell transcriptome and AbSeq analysis of lentiviral barcoded and transplanted patient-derived B-ALL cells revealed interconnected subpopulations, which could be prospectively isolated via surface markers and exhibited distinct leukemogenicity. Barcode tracking demonstrated the ability of cells to differentiate from a source cluster into differentiated progeny. Using machine learning we identified expression patterns predicting their differentiation potential of each barcoded cell within the immature cell compartment. To determine the dynamical properties of clones, we have designed a mathematical model that simulates the development of these subpopulations from a clonally diverse stem cell compartment. The model confirmed the most likely cluster of origin, supporting our functional data, and revealed the variability in dynamical properties between clones, while largely excluding plasticity. Hence, our work demonstrates a unidirectional differentiation in B-ALL with an immature population exhibiting a high leukemogenic potential. Key points: A differentiation hierarchy with distinct leukemogenesis potential in B-ALL is demonstrated via clonal tracing and mathematical modeling. Clonal differentiation behavior, while heterogeneous, is cell intrinsically controlled and predictable, yet independent from expansion.

The existing heterogeneity of the human hematopoietic stem cell (HSC) compartment imposes significant challenges in understanding their physiology and molecular constitution. The hematopoietic system is hierarchically organized, with HSCs at the apex, responsible for maintaining homeostasis by ensuring a life-long supply of blood cells. HSCs are highly potent but rare, making their pure isolation challenging. To address this, flow-cytometry-based methods are commonly used to isolate HSCs, bridging the gap between surface marker expression and understanding their functional and molecular properties. However, detailed methodology papers providing practical guidance for the prospective isolation of distinct human hematopoietic stem and progenitor cell (HSPC) populations are rare, hindering reproducible applications across different research groups. Here, we present a comprehensive protocol for isolating multipotent long-term repopulating HSCs (LT-HSCs) and define multipotent progenitor populations (MPPs) from human mobilized peripheral blood (mPB) after leukapheresis using fluorescence-activated cell sorting (FACS). By highlighting the workflow, outlining critical considerations and emphasizing recent advancements in the field, we provide an extensive overview of FACS-based human HSC isolation. This facilitates the enrichment of these rare cells for downstream analysis and enables researchers to improve our understanding of the heterogeneity within the HSC compartment.

We introduce a novel approach for the control of oligonucleotides through enzymatic activation with β‐galactosidase (β‐gal). We use the well‐known enzymatic capability of β‐gal to hydrolyze the β‐galactosidic bond combined with a self‐immolative linker and present three ways of steric or topological blocking of a DNA oligonucleotide. Through a series of in vitro experiments with β‐gal variants (recombinant or from human cell lysates), we systematically investigate stability, transitory perturbation, and enzymatic activation. Our approach holds significant promise for applications related to senescence‐associated β‐gal activity, including targeted modulation of gene expression and programmable molecular interventions. The combination of enzymatic activation applied to oligonucleotides represents a significant advance for targeted release in affected cells without the need for external triggering.