Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

Development of pial collaterals by extension of pre-existing artery tips.

Pial collaterals provide protection from ischemic damage and improve the prognosis of stroke patients. The origin or precise sequence of events underlying pial collateral development is unclear and has prevented clinicians from adapting new vascularization and regeneration therapies. We use genetic lineage tracing and intravital imaging of mouse brains at cellular resolution to show that during embryogenesis, pial collateral arteries develop from extension and anastomoses of pre-existing artery tips in a VegfR2-dependent manner. This process of artery tip extension occurs on pre-determined microvascular tracks. Our data demonstrate that an arterial receptor, Cxcr4, earlier shown to drive artery cell migration and coronary collateral development, is dispensable for the formation and maintenance of pial collateral arteries. Our study shows that collateral arteries of the brain are built by a mechanism distinct from that of the heart.

3D model for human glia conversion into subtype-specific neurons, including dopamine neurons.

Two-dimensional neuronal cultures have a limited ability to recapitulate the in vivo environment of the brain. Here, we introduce a three-dimensional in vitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2 weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for in vitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain.

Morphomolecular pathology and Genomic Insights into the Cells of Origin of Cholangiocarcinoma and combined Hepatocellular-Cholangiocarcinoma.

Cholangiocarcinoma is a highly heterogenous group of malignancies that, despite recent progress in the understanding of its molecular pathogenesis and clinical management, continue to pose a major challenge to public health. The traditional view posits that cholangiocarcinomas derive from the neoplastic transformation of cholangiocytes lining the biliary tree. However, increasing genetic and experimental evidence has recently pointed to a more complex - and nuanced - scenario for the potential cell of origin of cholangiocarcinomas, with hepatocytes as well as hepatic stem/progenitor cells being considered as additional potential sources, depending on microenvironmental contexts including liver injury. The hypothesis of potentially diverse cells of origin for CCA, albeit controversial, is certainly not surprising given the plasticity of the cells populating the liver as well as the existence of liver cancer subtypes with mixed histological and molecular features. This review carefully looks at the current pathological, genomic and experimental evidence supporting the existence of multiple cells of origin of liver and biliary tract cancers, with particular focus on cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma.

Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Deciphering the spatial distribution of Gli1-lineage cells in dental, oral, and craniofacial regions.

The craniofacial bone, crucial for protecting brain tissue and supporting facial structure, undergoes continuous remodeling through mesenchymal (MSCs) or skeletal stem cells (SSCs) in their niches. Gli1 is an ideal marker for labeling MSCs and osteoprogenitors in this region, and Gli1-lineage cells are identified as pivotal for bone growth, development, repair, and regeneration. Despite its significance, the distribution of Gli1-lineage cells across the dental, oral, and craniofacial (DOC) regions remains to be systematically explored. Utilizing tissue-clearing and light sheet fluorescence microscopy (LSFM) with a Gli1CreER; tdTomatoAi14 mouse model, we mapped the spatial distribution of Gli1-lineage cells throughout the skull, focusing on calvarial bones, sutures, bone marrow, teeth, periodontium, jaw bones, and the temporomandibular joint (TMJ). We found Gli1-lineage cells widespread in these areas, underscoring their significance in DOC regions. Additionally, we observed their role in repairing calvarial bone defects, providing novel insights into craniofacial biology and stem cell niches and enhancing our understanding of stem cells and their progeny's behavior in vivo.

This study investigates the presence and role of a specific stem cell population, known as Gli1-lineage cells, in various parts of the skull and facial bones. Using advanced imaging techniques, we found that these cells are widely distributed across the dental, oral, and craniofacial regions, especially in the cranial sutures, teeth, and jaw. Notably, Gli1-lineage cells migrate to the injury site, which is essential in bone repair and regeneration. These findings enhance our understanding of how stem cells contribute to healing and development in the craniofacial region.

Hematopoietic Stem Cell Fates and the Cellular Hierarchy of Mammalian Hematopoiesis: from Transplantation Models to New Insights from in Situ Analyses.

Hematopoiesis is the process that generates the cells of the blood and immune system from hematopoietic stem and progenitor cells (HSPCs) and represents the system with the most rapid cell turnover in a mammalian organism. HSPC differentiation trajectories, their underlying molecular mechanisms, and their dysfunctions in hematologic disorders are the focal research questions of experimental hematology. While HSPC transplantations in murine models are the traditional tool in this research field, recent advances in genome editing and next generation sequencing resulted in the development of many fundamentally new approaches for the analyses of mammalian hematopoiesis in situ and at single cell resolution. The current review will cover many recent developments in this field in murine models, from the bulk lineage tracing studies of HSPC differentiation to the barcoding of individual HSPCs with Cre-recombinase, Sleeping Beauty transposase, or CRISPR/Cas9 tools, to map hematopoietic cell fates, together with their transcriptional and epigenetic states. We also address studies of the clonal dynamics of human hematopoiesis, from the tracing of HSPC clonal behaviours based on viral integration sites in gene therapy patients to the recent analyses of unperturbed human hematopoiesis based on naturally accrued mutations in either nuclear or mitochondrial genomes. Such studies are revolutionizing our understanding of HSPC biology and hematopoiesis both under homeostatic conditions and in the response to various forms of physiological stress, reveal the mechanisms responsible for the decline of hematopoietic function with age, and in the future may advance the understanding and management of the diverse disorders of hematopoiesis.

Smooth muscle cells clonally expand in a murine carotid allograft model complicated by immune reactions to reporter transgenes.

BACKGROUND AND AIMS: Most experimental studies of allograft vasculopathy (AV) have relied on transplantation between major histocompatibility complex-mismatched inbred mouse strains, but this leads to the complete eradication of donor smooth muscle cells (SMCs) and lesions formed by recipient cells. This is unlike human AV which is thought to form mainly by donor SMCs. Here, we studied sources of neointimal cells in a minor histocompatibility antigen-mismatched AV model by combining male-to-female orthotopic carotid transplantations and lineage tracing by SMC-specific expression of fluorescent proteins. METHODS: To track SMC-derived cells in allograft vasculopathy, we used male donor mice with SMC-restricted Cre recombination of the mT/mG reporter transgene, which switches expression of membrane-bound red fluorescent protein (RFP) to green fluorescent protein (GFP), or the stochastically recombining Confetti reporter transgene, which yields a mosaic expression of four fluorescent proteins. Donor carotid segments were harvested and orthotopically allografted to female recipients that were wildtype or had non-recombined reporter transgenes. Inhibition of T cell responses by CTLA4Ig was used in some experiments. Sections of lesions harvested after 4 weeks were analyzed by fluorescence microscopy. RESULTS: Donor-derived SMCs survived and gave rise to part of the neointimal cells in experiments where carotid segments from recombined mT/mG male mice were transplanted into wild-type or non-recombined mT/mG female mice. Sex-mismatched transplants developed significant lesions, increasing the intimal and medial area 4.6-fold (p = 0.038) and 2.0-fold (p = 0.024) compared to sex- and fluorescence-matched controls, respectively. Interestingly, sex-matched fluorescence-positive transplants developed intimal lesions in 50% of fluorescence-naïve recipient controls. To study the clonal structure of the neointimal donor-derived SMC lineage cells, we then transplanted male carotids with heterozygous or homozygous recombined Confetti transgenes into female recipients. These transplants developed lesions with few surviving donor SMCs, indicating that expression of the Confetti reporter increased rejection and donor-specific SMC death. Some of the few remaining donor SMCs underwent clonal expansion. CTLA4Ig administration at the time of surgery did not improve SMC survival in mT/mG or Confetti transplants. CONCLUSION: Male-to-female transplant models feature donor-derived SMCs, some of which undergo clonal expansion, but immune rejection to fluorescence reporters appears to bias results in lineage tracing models. Overcoming these challenges with alternative reporter transgenes or tolerant recipients is necessary to study the mechanisms by which donor SMCs contribute to allograft vasculopathy.

Enteric glial cell diversification is influenced by spatiotemporal factors and source of neural progenitors in mice.

Previously focused primarily on enteric neurons, studies of the enteric nervous system (ENS) in both health and disease are now broadening to recognize the equally significant role played by enteric glial cells (EGCs). Commensurate to the vast array of gastrointestinal functions they influence, EGCs exhibit considerable diversity in terms of location, morphology, molecular profiles, and functional attributes. However, the mechanisms underlying this diversification of EGCs remain largely unexplored. To begin unraveling the mechanistic complexities of EGC diversity, the current study aimed to examine its spatiotemporal aspects in greater detail, and to assess whether the various sources of enteric neural progenitors contribute differentially to this diversity. Based on established topo-morphological criteria for categorizing EGCs into four main subtypes, our detailed immunofluorescence analyses first revealed that these subtypes emerge sequentially during early postnatal development, in a coordinated manner with the structural changes that occur in the ENS. When combined with genetic cell lineage tracing experiments, our analyses then uncovered a strongly biased contribution by Schwann cell-derived enteric neural progenitors to particular topo-morphological subtypes of EGCs. Taken together, these findings provide a robust foundation for further investigations into the molecular and cellular mechanisms governing EGC diversity.

Alveolar regeneration by airway secretory-cell-derived p63+ progenitors.

Lung injury activates epithelial stem or progenitor cells for alveolar repair and regeneration. Unraveling the origin and fate of injury-induced progenitors is crucial for elucidating lung repair mechanisms. Here, we report that p63-expressing progenitors emerge upon bleomycin-induced mouse lung injury. Single-cell RNA sequencing and clonal analysis reveal that these p63+ progenitors proliferate rapidly and differentiate into alveolar type 1 and type 2 cells through different trajectories. Dual recombinase-mediated sequential genetic-lineage tracing demonstrates that p63+ progenitors originate from airway secretory cells and subsequently generate alveolar cells. Functionally, p63 activation is essential for efficient alveolar regeneration from secretory cells post injury. Our study identifies secretory-cell-derived p63+ progenitors as contributors to alveolar repair, suggesting a potential therapeutic avenue for lung regeneration following injury.

Combining mutation and recombination statistics to infer clonal families in antibody repertoires.

B-cell repertoires are characterized by a diverse set of receptors of distinct specificities generated through two processes of somatic diversification: V(D)J recombination and somatic hypermutations. B-cell clonal families stem from the same V(D)J recombination event, but differ in their hypermutations. Clonal families identification is key to understanding B-cell repertoire function, evolution, and dynamics. We present HILARy (high-precision inference of lineages in antibody repertoires), an efficient, fast, and precise method to identify clonal families from single- or paired-chain repertoire sequencing datasets. HILARy combines probabilistic models that capture the receptor generation and selection statistics with adapted clustering methods to achieve consistently high inference accuracy. It automatically leverages the phylogenetic signal of shared mutations in difficult repertoire subsets. Exploiting the high sensitivity of the method, we find the statistics of evolutionary properties such as the site frequency spectrum and dN/dS ratio do not depend on the junction length. We also identify a broad range of selection pressures spanning two orders of magnitude.

The cell fates of intermediate cell population in prostate development.

Organ development, regeneration and cancer initiation are typically influenced by the proliferation and lineage plasticity of tissue-specific stem cells. Prostate intermediate cells, which exhibit characteristics of both basal and luminal cells, are prevalent in pathological states and during organ development. However, the identity, fate and function of these intermediate cells in prostate development are not well understood. Through single-cell RNA-seq analysis on neonatal urogenital sinus tissue, we identified intermediate cells exhibiting stem cell potential. A notable decline in the population of intermediate cells was observed during prostate development. Prostate intermediate cells were specifically labeled in early and late postnatal development by the enhanced dual-recombinase-mediated genetic tracing systems. Our findings revealed that these cells possess significant stem cell capabilities as demonstrated in organoid formation and cell fate mapping assays. These intermediate cells also exhibited intrinsic bipotential properties, enabling them to differentiate into both basal and luminal cells. Additionally, we discovered a novel transition from intermediate cell expressing neuroendocrine markers to neuroendocrine cell during prostate development. This study highlights intermediate cells as a crucial stem cell population and enhances our understanding of their role in prostate development and the plasticity of prostate cancer lineage.

Identification of Meibomian gland stem cell populations and mechanisms of aging.

Meibomian glands secrete lipid-rich meibum, which prevents tear evaporation. Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease, a common condition lacking effective treatment. The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined. Using snRNA-seq, in vivo lineage tracing, ex vivo live imaging, and genetic studies in mice, we identified markers for stem cell populations that maintain distinct regions of the gland and uncovered Hh signaling as a key regulator of stem cell proliferation. Consistent with this, human Meibomian gland carcinoma exhibited increased Hh signaling. Aged glands displayed decreased Hh and EGF signaling, deficient innervation, and loss of collagen I in niche fibroblasts, indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration. These findings suggest new approaches to treat aging-associated Meibomian gland loss.

Hepatic stellate cells in zone 1 engage in capillarization rather than myofibroblast formation in murine liver fibrosis.

The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRß-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

LineageVAE: Reconstructing Historical Cell States and Transcriptomes toward Unobserved Progenitors.

MOTIVATION: Single-cell RNA sequencing (scRNA-seq) enables comprehensive characterization of the cell state. However, its destructive nature prohibits measuring gene expression changes during dynamic processes such as embryogenesis. Although recent studies integrating scRNA-seq with lineage tracing have provided clonal insights between progenitor and mature cells, challenges remain. Because of their experimental nature, observations are sparse, and cells observed in the early state are not the exact progenitors of cells observed at later time points. To overcome these limitations, we developed LineageVAE, a novel computational methodology that utilizes deep learning based on the property that cells sharing barcodes have identical progenitors. RESULTS: LineageVAE is a deep generative model that transforms scRNA-seq observations with identical lineage barcodes into sequential trajectories toward a common progenitor in a latent cell state space. This method enables the reconstruction of unobservable cell state transitions, historical transcriptomes, and regulatory dynamics at a single-cell resolution. Applied to hematopoiesis and reprogrammed fibroblast datasets, LineageVAE demonstrated its ability to restore backward cell state transitions and infer progenitor heterogeneity and transcription factor activity along differentiation trajectories. AVAILABILITY AND IMPLEMENTATION: The LineageVAE model was implemented in Python using the PyTorch deep learning library. The code is available on GitHub at https://github.com/LzrRacer/LineageVAE/. SUPPLEMENTARY INFORMATION: Available at Bioinformatics online.

Regulation of Ptbp1-controlled alternative splicing of pyruvate kinase muscle by Liver kinase b1 governs vascular smooth muscle cell plasticity in vivo.

AIMS: Vascular smooth muscle cell (VSMC) plasticity is a state in which VSMCs undergo phenotypic switching from a quiescent contractile phenotype into other functionally distinct phenotypes. Although emerging evidence suggest that VSMC plasticity plays critical roles in the development of vascular diseases, little is known about the key determinant for controlling VSMC plasticity and fate. METHODS AND RESULTS: We found that smooth muscle cell-specific deletion of Lkb1 in tamoxifen-inducible Lkb1flox/flox; Myh11-Cre/ERT2 mice spontaneously and progressively induced aortic/arterial dilation, aneurysm, rupture, and premature death. Single-cell RNA sequencing and imaging-based lineage tracing showed that Lkb1-deficient VSMCs transdifferentiated gradually from early modulated VSMCs to fibroblast-like and chondrocyte-like cells, leading to ossification and blood-vessel rupture. Mechanistically, Lkb1 regulates polypyrimidine tract binding protein 1 (Ptbp1) expression and controls alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2. Lkb1 loss in VSMC results in an increased PKM2/PKM1 ratio and alters the metabolic profile by promoting aerobic glycolysis. Treatment with PKM2 activator TEPP-46 rescues VSMC transformation and aortic dilation in Lkb1flox/flox; Myh11-Cre/ERT2 mice. Furthermore, we found that Lkb1 expression decreased in human aortic aneurysm tissue compared to control tissue, along with changes in markers of VSMC fate. CONCLUSIONS: Lkb1, via its regulation of Ptbp1-dependent alterative splicing of PKM, maintains VSMC in contractile states by suppressing VSMC plasticity.

Bone or Tooth dentin: The TGF-ß signaling is the key.

To investigate the cell linkage between tooth dentin and bones, we studied TGF-ß roles during postnatal dentin development using TGF-ß receptor 2 (Tgfßr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfßr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfßr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-ß signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

An efficient inducible model for the control of gene expression in renin cells.

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/-;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells.NEW & NOTEWORTHY Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy.