Multiple Cell Fate Research Articles

Spatial transcriptomics reveals influence of microenvironment on intrinsic fates in melanoma therapy resistance.

Resistance to cancer therapy is driven by both cell-intrinsic and microenvironmental factors. Previous work has revealed that multiple resistant cell fates emerge in melanoma following treatment with targeted therapy and that, in vitro, these resistant fates are determined by the transcriptional state of individual cells prior to exposure to treatment. What remains unclear is whether these resistant fates are shared across different genetic backgrounds and how, if at all, these resistant fates interact with the tumor microenvironment. Through spatial transcriptomics and single-cell RNA sequencing, we uncovered distinct resistance programs in melanoma cells shaped by both intrinsic cellular states and the tumor microenvironment. Consensus non-negative matrix factorization revealed shared intrinsic resistance programs across different cell lines, highlighting the presence of universal and unique resistance pathways. In patient samples, we demonstrated that these resistance programs coexist within individual tumors and associate with diverse immune signatures, suggesting that the tumor microenvironment and distribution of resistant fates are closely connected. Single-cell resolution spatial transcriptomics in xenograft models revealed both intrinsically determined and extrinsically influenced resistant fates. Overall, this work demonstrates that each therapy resistant fate coexists with a distinct immune microenvironment in tumors and that, in vivo, tissue features, such as regions of necrosis, can influence which resistant fate is adopted.

Angiogenic potential in periodontal stem cells from upper and lower jaw: A pilot study.

Teeth and supporting oral tissues are attractive and accessible sources of stem cells. Periodontal ligament stem cells (PDLSC) are readily isolated from extracted third molars, and exhibit the ability to self-renew and differentiate into multiple mesodermal cell fates. Clinical experience suggests that the exact location of periodontal defects affects the oral bone remodeling and wound healing. Compared to the mandible, the maxilla heals quicker and more efficiently. Angiogenesis is key in tissue regeneration including dental tissues, yet few studies focus on the angiogenic potential of PDLSC, none of which considered the differences between upper and lower jaw PDLSC (u-PDLSC and l-PDLSC, respectively). Here we studied the angiogenic potential of u-PDLSC and l-PDLSC and compared the results to well-established mesenchymal stem cells (MSC). Cells were characterized in terms of surface markers, proliferation, and vascular endothelial growth factor (VEGF) secretion, and angiogenic assays were performed. Newly formed capillaries were stained with CD31, and their expression of platelet endothelial cell adhesion molecule (PECAM-1), angiopoietin 2 (ANGPT2), and vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2) were measured. Periodontal stem cells from the upper jaw showed a higher proliferation capacity, secreted more VEGF, and formed capillary networks faster and denser than l-PDLSC. Gene expression of angiogenesis-related genes was significantly higher in u-PDLSC than in l-PDLSC or MSC, given that culture conditions were suitable. The oral cavity is a valuable source of stem cells, particularly PDLSC, which are promising for oral tissue engineering due to their robust growth, lifelong accessibility, low immunogenicity, and strong differentiation potential. Notably, u-PDLSC exhibit higher VEGF secretion and accelerate capillary formation compared to l-PDLSC or MSC. This study suggests a potential molecular mechanism in capillary formation, emphasizing the significance of precise location isolation of PDLSC.

Time-integrated BMP signaling determines fate in a stem cell model for early human development

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore, signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo, we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically, BMP signaling is integrated by SOX2.

Global quantitative understanding of non-equilibrium cell fate decision-making in response to pheromone

Cell-cycle arrest and polarized growth are commonly used to characterize the response of yeast to pheromone. However, the quantitative decision-making processes underlying time-dependent changes in cell fate remain unclear. In this study, we conducted single-cell level experiments to observe multidimensional responses, uncovering diverse fates of yeast cells. Multiple states are revealed, along with the kinetic switching rates and pathways among them, giving rise to a quantitative landscape of mating response. To quantify the experimentally observed cell fates, we developed a theoretical framework based on non-equilibrium landscape and flux theory. Additionally, we performed stochastic simulations of biochemical reactions to elucidate signal transduction and cell growth. Notably, our experimental findings have provided the first global quantitative evidence of the real-time synchronization between intracellular signaling, physiological growth, and morphological functions. These results validate the proposed underlying mechanism governing the emergence of multiple cell fate states. This study introduces an emerging mechanistic approach to understand non-equilibrium cell fate decision-making in response to pheromone.

PHF23 promotes NSCLC proliferation, metastasis, and chemoresistance via stabilization of ACTN4 and activation of the ERK pathway.

At present, non-small cell lung cancer (NSCLC) is still one of the leading causes of cancer-related deaths. Chemotherapy remains the standard treatment for NSCLC. However, the emergence of chemoresistance is one of the major obstacles to lung cancer treatment. Plant homologous structural domain finger protein 23 (PHF23) plays crucial roles in multiple cell fates. However, the clinical significance and biological role of PHF23 in NSCLC remain elusive. The Cancer Genome Atlas data mining, NCBI/GEO data mining, and western blotting analysis were employed to characterize the expression of PHF23 in NSCLC cell lines and tissues. Statistical analysis of immunohistochemistry and the Kaplan-Meier Plotter database were used to investigate the clinical significance of PHF23. A series of in vivo and in vitro assays, including assays for colony formation, cell viability, 5-ethynyl-2'-deoxyuridine (EDU incorporation) and Transwell migration, flow cytometry, RT-PCR, gene set enrichment analysis, co-immunoprecipitation analysis, and a xenograft tumor model, were performed to demonstrate the effects of PHF23 on the chemosensitivity of NSCLC cells and to clarify the underlying molecular mechanisms. PHF23 is overexpressed in NSCLC cell lines and tissues. High PHF23 levels correlate with short survival times and a poor response to chemotherapy in NSCLC patients. PHF23 overexpression facilitates cell proliferation, migration and sensitizes NSCLC cells to Cisplatin and Docetaxel by promoting DNA damage repair. Alpha-actinin-4 (ACTN4), as a downstream regulator, interacts with PHD domain of PHF23. Moreover, PHF23 is involved in ACTN4 stabilization by inhibiting its ubiquitination level. These results show that PHF23 plays an important role in the development and progression of NSCLC and suggest that PHF23 may serve as a therapeutic target in NSCLC patients.

Shared Gene Targets of the ATF4 and p53 Transcriptional Networks.

The master tumor suppressor p53 regulates multiple cell fate decisions, such as cell cycle arrest and apoptosis, via transcriptional control of a broad gene network. Dysfunction in the p53 network is common in cancer, often through mutations that inactivate p53 or other members of the pathway. Induction of tumor-specific cell death by restoration of p53 activity without off-target effects has gained significant interest in the field. In this study, we explore the gene regulatory mechanisms underlying a putative anticancer strategy involving stimulation of the p53-independent integrated stress response (ISR). Our data demonstrate the p53 and ISR pathways converge to independently regulate common metabolic and proapoptotic genes. We investigated the architecture of multiple gene regulatory elements bound by p53 and the ISR effector ATF4 controlling this shared regulation. We identified additional key transcription factors that control basal and stress-induced regulation of these shared p53 and ATF4 target genes. Thus, our results provide significant new molecular and genetic insight into gene regulatory networks and transcription factors that are the target of numerous antitumor therapies.

Lrrn1 Regulates Medial Boundary Formation in the Developing Mouse Organ of Corti.

One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti (OC), is the presence of precise boundaries between sensory and nonsensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells (IHCs) and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 (Lrrn1), which codes for a single-pass, transmembrane protein, is expressed before the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent nonsensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.SIGNIFICANCE STATEMENT Patterning of the developing mammalian cochlea into distinct sensory and nonsensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein Leucine Rich Repeat Neuronal 1 (LRRN1) is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells (IHCs) and adjacent nonsensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/nonsensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.

Osteogenic microenvironment affects palatal development through glycolysis

Palate development involves various events, including proliferation, osteogenic differentiation, and epithelial-mesenchymal transition. Disruption of these processes can result in the cleft palate (CP). Mouse embryonic palatal mesenchyme (MEPM) cells are commonly used to explore the mechanism of palatal development and CP. However, the role of the microenvironment in the biological properties of MEPM cells, which undergoes dynamic changes during palate development, is rarely reported. In this study, we investigated whether there were differences between the palatal shelf mesenchyme at different developmental stages. Our results found that the palatal shelves facilitate proliferation at the early palate stage at mouse embryonic day (E) 13.5 and the tendency towards osteogenesis at E15.5, the late palate development stage. And the osteogenic microenvironment, which was mimicked by osteogenic differentiation medium (OIM), affected the biological properties of MEPM cells when compared to the routine medium. Specifically, MEPM cells showed slower proliferation, shorter S phase, increased apoptosis, and less migration distance after osteogenesis. E15.5 MEPM cells were more sensitive than E13.5, showing an earlier change. Moreover, E13.5 MEPM cells had weaker osteogenic ability than E15.5, and both MEPM cells exhibited different Lactate dehydrogenase A (LDHA) and Cytochrome c (CytC) expressions in OIM compared to routine medium, suggesting that glycolysis might be associated with the influence of the osteogenic microenvironment on MEPM cells. By comparing the stemness of the two cells, we investigated that the stemness of E13.5 MEPM cells was stronger than that of E15.5 MEPM cells, and E15.5 MEPM cells were more like differentiated cells than stem cells, as their capacity to differentiate into multiple cell fates was reduced. E13.5 MEPM cells might be the precursor cells of E15.5 MEPM cells. Our results enriched the understanding of the effect of the microenvironment on the biological properties of E13.5 and E15.5 MEPM cells, which should be considered when using MEPM cells as a model for palatal studies in the future.

The role of peroxisome proliferator-activated receptors in the tumor microenvironment, tumor cell metabolism, and anticancer therapy.

Peroxisome proliferator-activated receptors (PPARs) have been extensively studied for over 3decades and consist of three isotypes, including PPARα, γ, and β/δ, that were originally considered key metabolic regulators controlling energy homeostasis in the body. Cancer has become a leading cause of human mortality worldwide, and the role of peroxisome proliferator-activated receptors in cancer is increasingly being investigated, especially the deep molecular mechanisms and effective cancer therapies. Peroxisome proliferator-activated receptors are an important class of lipid sensors and are involved in the regulation of multiple metabolic pathways and cell fate. They can regulate cancer progression in different tissues by activating endogenous or synthetic compounds. This review emphasizes the significance and knowledge of peroxisome proliferator-activated receptors in the tumor microenvironment, tumor cell metabolism, and anti-cancer treatment by summarizing recent research on peroxisome proliferator-activated receptors. In general, peroxisome proliferator-activated receptors either promote or suppress cancer in different types of tumor microenvironments. The emergence of this difference depends on various factors, including peroxisome proliferator-activated receptor type, cancer type, and tumor stage. Simultaneously, the effect of anti-cancer therapy based on drug-targeted PPARs differs or even opposes among the three peroxisome proliferator-activated receptor homotypes and different cancer types. Therefore, the current status and challenges of the use of peroxisome proliferator-activated receptors agonists and antagonists in cancer treatment are further explored in this review.

Structure-based prediction of Ras-effector binding affinities and design of “branchegetic” interface mutations

Ras is a central cellular hub protein controlling multiple cell fates. How Ras interacts with a variety of potential effector proteins is relatively unexplored, with only some key effectors characterized in great detail. Here, we have used homology modeling based on X-ray and AlphaFold2 templates to build structural models for 54 Ras-effector complexes. These models were used to estimate binding affinities using a supervised learning regressor. Furthermore, we systematically introduced Ras "branch-pruning" (or branchegetic) mutations to identify 200 interface mutations that affect the binding energy with at least one of the model structures. The impacts of these branchegetic mutants were integrated into a mathematical model to assess the potential for rewiring interactions at the Ras hub on a systems level. These findings have provided a quantitative understanding of Ras-effector interfaces and their impact on systems properties of a key cellular hub.

Dynamic single cell lineage recording of endogenous viral specific CD8 T cells reveals multiple potential cell fate pathways

Abstract In response to viral infection, antigen specific naïve CD8 T cells expand to give rise to a heterogenous pool of effector cells consisting of short-lived effector cells (SLECs) and memory precursor effector cells (MPECs). While these effector populations are phenotypically and functionally well characterized, we still don’t fully understand how they arise from antigen responsive naïve CD8 T cells. We have combined progressive lineage recording with single-cell RNA sequencing and T cell receptor sequencing to delineate the early differentiation of OVA-specific endogenous CD8 T cells in response to acute VSV infection. Transcriptional profiling of CD8 T cells captured at the peak of T cell response confirmed 8 distinct T cell states including a unique interferon responsive cluster. RNA Velocity trajectory analysis supported an asymmetric model of CD8 T cell differentiation where early effector cells gave rise to SLECs while MPECs differentiated into further memory precursors. Moreover, our CRISPR/Cas9-based lineage recorder uncovered T cell clones of various sizes and allowed us to follow up to 5 generations of differentiating CD8 T cells. We observed that expanded clones comprised of memory and effector CD8 T cells while medium size clones preferred memory or effector fate, suggesting that different clones follow different differentiation models. Thus, using RNA-seq based trajectory analyses and a dynamic lineage recorder we uncovered potential differentiation pathways taken by early viral specific CD8 T cells. Our single cell full length TCR-seq will further add to our current models and highlight TCR sequences with better memory potential in response to infection. Supported by grants from NIH (R01 AI089805, R01 CA254042) and a training fellowship from the Burroughs Wellcome Fund.

Probing cell identity hierarchies by fate titration and collision during direct reprogramming

Despite the therapeutic promise of direct reprogramming, basic principles concerning fate erasure and the mechanisms to resolve cell identity conflicts remain unclear. To tackle these fundamental questions, we established a single‐cell protocol for the simultaneous analysis of multiple cell fate conversion events based on combinatorial and traceable reprogramming factor expression: Collide‐seq. Collide‐seq revealed the lack of a common mechanism through which fibroblast‐specific gene expression loss is initiated. Moreover, we found that the transcriptome of converting cells abruptly changes when a critical level of each reprogramming factor is attained, with higher or lower levels not contributing to major changes. By simultaneously inducing multiple competing reprogramming factors, we also found a deterministic system, in which titration of fates against each other yields dominant or colliding fates. By investigating one collision in detail, we show that reprogramming factors can disturb cell identity programs independent of their ability to bind their target genes. Taken together, Collide‐seq has shed light on several fundamental principles of fate conversion that may aid in improving current reprogramming paradigms.

Cell fate-inducing CARs orthogonally control multiple signaling pathways.

While chimeric antigen receptor (CAR) has been clinically applied in T cell-mediated cancer therapy, CARs that combinatorially control general cell fates have yet to be practical. In this study, we aim to control multiple cell fates simultaneously by combinatorial activation of multiple cell fate-inducing CARs (cfiCARs). We construct CARs that transduce two different signals using two antigen-specific CARs. The CARs are co-expressed pairwise on the cell surface to let the two antigens act alone or in combination, thus arbitrarily control multiple cell fates. We utilize signaling domains derived from natural receptors (gp130, receptor activator of nuclear factor κB [RANK], c-Fms, and Fas) and tyrosine motif-engineered artificial signaling domains that preferentially recruit target signaling molecules (Shc and STAT3) for inducing specific cell fates by the CARs. Consequently, the signaling molecules downstream of these receptors are activated orthogonally by the corresponding CAR-specific antigens. Furthermore, two completely different cell fates (proliferation and death) are successfully controlled simultaneously or sequentially over time by adding the two antigens, demonstrating proliferation- and apoptosis-inducing CARs (piCAR and aiCAR). Thus, cfiCARs will be applied for delineating the basic principle of cell fate control and improving the efficacy of cell therapy, which would significantly contribute to cell biology and future medicine.

Targeting the Non-Catalytic Functions: a New Paradigm for Kinase Drug Discovery?

Protein kinases have been highly fruitful targets for cancer drug discovery in the past two decades, while most of these drugs bind to the "adenosine triphosphate (ATP)-site" and inhibit kinase catalytic activity. Recently, accumulated evidence suggests that kinases possess functions beyond catalysis through their scaffolds, and the scaffolding functions could play critical roles in multiple cellular signaling and cell fate controls. Small molecules modulating the noncatalytic functions of kinases are rarely reported but emerge as new promising therapeutic strategies for various diseases. Herein, we summarize the characterized noncatalytic functions of kinases, and highlight the recent progress on developing small-molecule modulators of the noncatalytic functions of kinases. Mechanisms and characteristics of different kinds of modulators are also discussed. It is also speculated that targeting the noncatalytic functions would represent a new direction for kinase-based drug discovery.

Cell signaling pathways controlling an axis organizing center in the zebrafish.

Body axis formation in vertebrate development entails the remarkable feat of patterning a myriad of specialized cell types and organ progenitors from a field of unpatterned, multipotent cells. This feat is achieved largely by secreted cell-cell signaling molecules, enabling cells at different positions within the embryo to adopt distinct fates. During patterning of the vertebrate embryonic axes, a multitude of cell fates is induced by a surprisingly small set of signaling pathways: Wnt, Nodal, Bone Morphogenetic Protein (BMP), and Fibroblast Growth Factor (FGF) signaling. These signals function as morphogens, specifying multiple cell fates in a concentration-dependent mechanism, and must therefore be distributed non-uniformly throughout the embryo. A primary signaling center that sets up spatial asymmetries in these signaling pathways to break the symmetry of the vertebrate embryo is known as the dorsal organizer. Discovered nearly a century ago by Hilde Mangold and Hans Spemann in the newt, the organizer has the remarkable ability to induce a secondary body axis when grafted ectopically into a host embryo. Here, we review the cell-cell signaling pathways that control the establishment of the dorsal organizer and its inductive functions in the zebrafish Danio rerio, a vertebrate model highly amenable to genetic manipulation. The organizer's remarkable inductive abilities continue to provide a fascinating source of scientific inquiry in the field of developmental biology.

Overcoming TGFβ-mediated immune evasion in cancer.

Transforming growth factor-β (TGFβ) signalling controls multiple cell fate decisions during development and tissue homeostasis; hence, dysregulation of this pathway can drive several diseases, including cancer. Here we discuss the influence that TGFβ exerts on the composition and behaviour of different cell populations present in the tumour immune microenvironment, and the context-dependent functions of this cytokine in suppressing or promoting cancer. During homeostasis, TGFβ controls inflammatory responses triggered by exposure to the outside milieu in barrier tissues. Lack of TGFβ exacerbates inflammation, leading to tissue damage and cellular transformation. In contrast, as tumours progress, they leverage TGFβ to drive an unrestrained wound-healing programme in cancer-associated fibroblasts, as well as to suppress the adaptive immune system and the innate immune system. In consonance with this key role in reprogramming the tumour microenvironment, emerging data demonstrate that TGFβ-inhibitory therapies can restore cancer immunity. Indeed, this approach can synergize with other immunotherapies - including immune checkpoint blockade - to unleash robust antitumour immune responses in preclinical cancer models. Despite initial challenges in clinical translation, these findings have sparked the development of multiple therapeutic strategies that inhibit the TGFβ pathway, many of which are currently in clinical evaluation.

606 Connecting signaling dynamics with cell fates in live mice

Many tissues are composed by multiple cell types, which are derived from and maintained by tissue resident stem cells. How stem cells coordinately differentiate into multiple cell fates is largely unclear. By using a novel ERK/MAPK signaling reporter mouse, our recent study showed that during early mouse embryogenesis, stem cells bifurcate their fates by transiently modulating ERK activation, suggesting that different cell fates can be determined by distinct types of ERK activation dynamics in stem cell differentiation. Here, we are using hair follicle as a multi-cell-type system to investigate the ERK signal-mediated cell fate coordination mechanism, as ERK pathway has been shown to regulate multiple cell fates in the hair follicle. By combining two-photon imaging approach and the ERK reporter, we are able to capture the real-time ERK signal dynamics at the single cell level throughout hair follicle regeneration in live mice, along with the cellular dynamics we have characterized before. Time-lapse analyses show that ERK signal changes its activation modes at different stages of hair follicle growth, which is coupled with the progressive cell fate specification and maintenance. Drug treatment assays uncover that distinct upstream signaling pathways contribute to different types of ERK signal activation. Further genetic manipulations are performed to understand the genesis of these ERK dynamics as well as their specific roles in coordinating cell fates and behaviors. This work bridges the gap between the dynamic activation of the key regeneration regulator and the coordinated cell fate decisions and will provide new sights into the fundamental principles of tissue regeneration. Given ERK signal is dysregulated by many oncogenes, this work will also have implications in cancer etiology and treatment.

A systematic study of critical miRNAs on cells proliferation and apoptosis by the shortest path

BackgroundMicroRNAs are a class of important small noncoding RNAs, which have been reported to be involved in the processes of tumorigenesis and development by targeting a few genes. Existing studies show that the imbalance between cell proliferation and apoptosis is closely related to the initiation and development of cancers. However, the impact of miRNAs on this imbalance has not been studied systematically.ResultsIn this study, we first construct a cell fate miRNA-gene regulatory network. Then, we propose a systematical method for calculating the global impact of miRNAs on cell fate genes based on the shortest path. Results on breast cancer and liver cancer datasets show that most of the cell fate genes are perturbed by the differentially expressed miRNAs. Most of the top-identified miRNAs are verified in the Human MicroRNA Disease Database (HMDD) and are related to breast and liver cancers. Function analysis shows that the top 20 miRNAs regulate multiple cell fate related function modules and interact tightly based on their functional similarity. Furthermore, more than half of them can promote sensitivity or induce resistance to some anti-cancer drugs. Besides, survival analysis demonstrates that the top-ranked miRNAs are significantly related to the overall survival time in the breast and liver cancers group.ConclusionIn sum, this study can help to systematically study the important role of miRNAs on proliferation and apoptosis and thereby uncover the key miRNAs during the process of tumorigenesis. Furthermore, the results of this study will contribute to the development of clinical therapy based miRNAs for cancers.

The emergence of the two cell fates and their associated switching for a negative auto-regulating gene

BackgroundDecisions in the cell that lead to its ultimate fate are important for fundamental cellular functions such as proliferation, growth, differentiation, development, and death. These cell fate decisions can be influenced by both the gene regulatory network and also environmental factors and can be modeled using simple gene feedback circuits. Negative auto-regulation is a common feedback motif in the gene circuits. It can act to reduce gene expression noise or induce oscillatory expression and is thought to lead to only one cell fate. Here, we present experimental and modeling data to suggest that a self-repressor circuit can lead to two cell fates under specific conditions.ResultsWe show that the introduction of inducers capable of binding and unbinding to a self-repressing gene product (protein), thus regulating the associated gene, can lead to the emergence of two cell states. We suggest that the inducers can alter the effective regulatory binding and unbinding speed of the self-repressor regulatory protein to its destination DNA without changing the gene itself. The corresponding simulation results are consistent with the experimental findings. We propose physical and quantitative explanations for the origin of the two phenotypic cell fates.ConclusionsOur results suggest a mechanism for the emergence of multiple cell fates. This may explain the heterogeneity often observed among cell states, while illustrating that altering gene regulation strength can influence cell fates and their decision-making processes without genetic changes.

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Macrophages promote both injury and repair following myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2− resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4−LYVE1−MHC-IIhiCCR2− macrophages and fully replaced TIMD4−LYVE1−MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4− resident macrophage abundance within infarcted tissue while recruited, CCR2+ monocyte-derived macrophages adopted multiple cell fates, including those nearly indistinguishable from resident macrophages. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, highlighting a non-redundant, cardioprotective role of resident cardiac macrophages. Lastly, we demonstrate the ability of TIMD4 to be used as a durable lineage marker of a subset of resident cardiac macrophages.