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The epicardium is the important outermost mesothelial/epithelial layer of the heart that serves as a signaling center for cardiac development and repair. During heart development, epicardial cells undergo a process known as epithelial-to-mesenchymal transition to form diverse mesenchymal cell lineages, such as fibroblasts, coronary vascular smooth muscle cells, and pericytes. However, it is not clear whether the reverse process, mesenchymal-to-epithelial transition (MET), takes place in the mammalian heart. In this study, we performed apical resection on neonatal hearts and used Fap-CreER;Ai9 labeling to track activated fibroblasts in the injured cardiac regions. We found that these fibroblasts underwent MET to generate epicardial cells during heart regeneration. To our knowledge, this is the first report of MET occurring in vivo during heart development and regeneration. Our findings suggest that it is feasible to directly convert fibroblasts into epicardial cells, providing a novel approach to generate epicardial cells. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease with a poor prognosis. Chronic microinjuries, mainly caused by environmental factors to an aging alveolar epithelium, would lead to the aberrant differentiation and accumulation of aberrant mesenchymal cells with a contractile phenotype, known as fibrosis-associated myofibroblasts, which trigger abnormal extracellular matrix accumulation and fibrosis. The origin of those pathological myofibroblasts in pulmonary fibrosis is not fully understood to date. Lineage tracing methods using mouse models have opened new avenues for studying cell fate in a pathological context. This review aims to present a nonexhaustive list of different potential sources of those harmful myofibroblasts during lung fibrosis, based on these in vivo approaches, and considering the normal and fibrotic lung cellular atlas recently established by single-cell RNA sequencing. [ABSTRACT FROM AUTHOR]

Genetic variation in the mitochondrial genome is linked to important biological functions and various human diseases. Recent progress in single-cell genomics has established single-cell RNA sequencing (scRNAseq) as a popular and powerful technique to profile transcriptomics at the cellular level. While most studies focus on deciphering gene expression, polymorphisms including mitochondrial variants can also be readily inferred from scRNAseq. However, limited attention has been paid to investigate the single-cell landscape of mitochondrial variants, despite the rapid accumulation of scRNAseq data in the community. In addition, a diploid context is assumed for most variant calling tools, which is not appropriate for mitochondrial heteroplasmies. Here, we introduce MitoTrace, an R package for the analysis of mitochondrial genetic variation in bulk and scRNAseq data. We applied MitoTrace to several publicly accessible data sets and demonstrated its ability to robustly recover genetic variants from scRNAseq data. We also validated the applicability of MitoTrace to scRNAseq data from diverse platforms. Overall, MitoTrace is a powerful and user-friendly tool to investigate mitochondrial variants from scRNAseq data. [ABSTRACT FROM AUTHOR]

It has been over three decades since Drs. Herzenberg and Herzenberg proposed the layered immune system hypothesis, suggesting that different types of stem cells with distinct hematopoietic potential produce specific immune cells. This layering of immune system development is now supported by recent studies showing the presence of fetal-derived immune cells that function in adults. It has been shown that various immune cells arise at different embryonic ages via multiple waves of hematopoiesis from special endothelial cells (ECs), referred to as hemogenic ECs. However, it remains unknown whether these fetal-derived immune cells are produced by hematopoietic stem cells (HSCs) during the fetal to neonatal period. To address this question, many advanced tools have been used, including lineage-tracing mouse models, cellular barcoding techniques, clonal assays, and transplantation assays at the single-cell level. In this review, we will review the history of the search for the origins of HSCs, B-1a progenitors, and mast cells in the mouse embryo. HSCs can produce both B-1a and mast cells within a very limited time window, and this ability declines after embryonic day (E) 14.5. Furthermore, the latest data have revealed that HSC-independent adaptive immune cells exist in adult mice, which implies more complicated developmental pathways of immune cells. We propose revised road maps of immune cell development. [ABSTRACT FROM AUTHOR]

Aims Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality. Methods and results We investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (<0.1%) of clonal VSMC proliferation is also observed in vitro. Single-cell RNA-sequencing revealed progressive cell state changes across a contiguous VSMC population at onset of injury-induced proliferation. Proliferating VSMCs mapped selectively to one of two distinct trajectories and were associated with cells showing extensive phenotypic switching. A proliferation-associated transitory state shared pronounced similarities with atypical SCA1+ VSMCs from uninjured mouse arteries and VSMCs in healthy human aorta. We show functionally that clonal expansion of SCA1+ VSMCs from healthy arteries occurs at higher rate and frequency compared with SCA1− cells. Conclusion Our data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion. [ABSTRACT FROM AUTHOR]

Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail. We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/β-catenin signalling and related metabolomic disturbance. We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci. Understanding the source of cells which regenerate the liver is crucial to harness their potential to regrow injured livers. Herein, we show that cells which were previously thought to repopulate the liver play only a limited role in physiological regeneration. Our data helps to reconcile differing conclusions drawn from results from a number of prior studies and highlights methodological challenges which are relevant to preclinical models more generally. [Display omitted] • Pericentral hepatocytes do not play a dominant role in physiological hepatocyte regeneration. • The Wnt pathway is suppressed by Axin2 haploinsufficiency. • Mouse genetics and environmental conditions should be rigorously controlled in preclinical models. [ABSTRACT FROM AUTHOR]

The lung is the most critical organ of the respiratory system supporting gas exchange. Constant interaction with the external environment makes the lung vulnerable to injury. Thus, a deeper understanding of cellular and molecular processes underlying lung development programs and evaluation of progenitor status within the lung is an essential part of lung regenerative medicine. In this review, we aim to discuss the current understanding of lung development process and regenerative capability. We highlight the advances brought by multi-omics approaches, single-cell transcriptome, in particular, that can help us further dissect the cellular player and molecular signaling underlying those processes. [ABSTRACT FROM AUTHOR]

Agonists of trained immunity induce epigenetic changes in hematopoietic stem and progenitor cells (HSPCs) to generate long-lasting immune protection. Although trained HSPCs generate myeloid cells with increased responsiveness to secondary challenges, whether their differentiation kinetics is affected by prior exposure to inducers of trained immunity remains elusive. Here, we used lineage tracing to examine the cell fates of endothelial protein C receptor-positive hematopoietic stem cells (EPCR+ HSCs) and fms-like tyrosine kinase 3-positive multipotent progenitor cells (Flt3+ MPPs) in β-glucan-induced trained immunity. We found that although β-glucan triggered the expected expansion of myeloid progenitors, the differentiation behaviors of EPCR+ HSCs and Flt3+ MPPs in multiple cycles of hematopoietic regeneration were hardly affected. Thus, our results rule out changed kinetics in cell differentiation by EPCR+ HSC and Flt3+ MPP as the cause of enhanced myelopoiesis upon secondary immune challenges. [ABSTRACT FROM AUTHOR]

Abstract Background Colorectal cancer (CRC) ranks as the second-leading cause of cancer-related death worldwide with metastases being the main cause of cancer-related death. Here, we investigated the genomic and transcriptomic alterations in matching adjacent normal tissues, primary tumors, and metastatic tumors of CRC patients. Methods We performed whole genome sequencing (WGS), multi-region whole exome sequencing (WES), simultaneous single-cell RNA-Seq, and single-cell targeted cDNA Sanger sequencing on matching adjacent normal tissues, primary tumors, and metastatic tumors from 12 metastatic colorectal cancer patients (n=84 for genomes, n=81 for exomes, n=9120 for single cells). Patient-derived tumor organoids were used to estimate the anti-tumor effects of a PPAR inhibitor, and self-renewal and differentiation ability of stem cell-like tumor cells. Results We found that the PPAR signaling pathway was prevalently and aberrantly activated in CRC tumors. Blocking of PPAR pathway both suppressed the growth and promoted the apoptosis of CRC organoids in vitro, indicating that aberrant activation of the PPAR signaling pathway plays a critical role in CRC tumorigenesis. Using matched samples from the same patient, distinct origins of the metastasized tumors between lymph node and liver were revealed, which was further verified by both copy number variation and mitochondrial mutation profiles at single-cell resolution. By combining single-cell RNA-Seq and single-cell point mutation identification by targeted cDNA Sanger sequencing, we revealed important phenotypic differences between cancer cells with and without critical point mutations (KRAS and TP53) in the same patient in vivo at single-cell resolution. Conclusions Our data provides deep insights into how driver mutations interfere with the transcriptomic state of cancer cells in vivo at a single-cell resolution. Our findings offer novel knowledge on metastatic mechanisms as well as potential markers and therapeutic targets for CRC diagnosis and therapy. The high-precision single-cell RNA-seq dataset of matched adjacent normal tissues, primary tumors, and metastases from CRCs may serve as a rich resource for further studies.

Low back pain is one of the most serious public health problems worldwide and the major clinical manifestation of intervertebral disc degeneration (IVDD). The key pathological change during IVDD is dysfunction of the annulus fibrosus (AF). However, due to the lack of an in-depth understanding of AF biology, the methods to reconstruct the AF are very limited. In this study, the mice AF cell atlas were decoded by single-cell RNA sequencing to provide a guide for AF reconstruction. The results first identify a new population of AF cells, fibrochondrocyte-like AF cells, which synthesize both collagen I and collagen II and are potential functional cells for AF reconstruction. According to the dual features of the AF extracellular matrix, a composite hydrogel based on the acylation of methacrylated silk fibroin with methacrylated hyaluronic acid was produced. To obtain the ability to stimulate differentiation, the composite hydrogels were combined with a fibrochondrocyte-inducing supplement. Finally, reconstruction of the AF defects, by the novel AF stem cell-loaded composite hydrogel, could be observed, its amount of chondroid matrices recovered to 31.7% of AF aera which is significantly higher than that in other control groups. In summary, this study decodes the AF cell atlas, based on which a novel strategy for AF reconstruction is proposed.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumour thought to arise from ductal cells via pancreatic intraepithelial neoplasia (PanIN) precursor lesions. Modelling of different genetic events in mice suggests both ductal and acinar cells can give rise to PDAC. However, the impact of cellular context alone on tumour development and phenotype is unknown. DESIGN: We examined the contribution of cellular origin to PDAC development by inducing PDAC-associated mutations, KrasG12D expression and Trp53 loss, specifically in ductal cells (Sox9CreER;KrasLSL-G12D;Trp53flox/flox (Duct:KPcKO )) or acinar cells (Ptf1aCreER;KrasLSL-G12D;Trp53flox/flox (Acinar:KPcKO )) in mice. We then performed a thorough analysis of the resulting histopathological changes. RESULTS: Both mouse models developed PDAC, but Duct:KPcKO mice developed PDAC earlier than Acinar:KPcKO mice. Tumour development was more rapid and associated with high-grade murine PanIN (mPanIN) lesions in Duct:KPcKO mice. In contrast, Acinar:KPcKO mice exhibited widespread metaplasia and low-grade as well as high-grade mPanINs with delayed progression to PDAC. Acinar-cell-derived tumours also had a higher prevalence of mucinous glandular features reminiscent of early mPanIN lesions. CONCLUSION: These findings indicate that ductal cells are primed to form carcinoma in situ that become invasive PDAC in the presence of oncogenic Kras and Trp53 deletion, while acinar cells with the same mutations appear to require a prolonged period of transition or reprogramming to initiate PDAC. Our findings illustrate that PDAC can develop in multiple ways and the cellular context in which mutations are acquired has significant impact on precursor lesion initiation, disease progression and tumour phenotype.

Abstract The application of hematopoietic stem cells (HSCs) has been restricted due to limited cell sources and conventional methods for generating these cells by cell expansion and pluripotent stem cell differentiation have not been clinically achieved. Cell reprogramming technique provides a new hope for generating desirable cells. We previously reported that mouse differentiated hematopoietic cell reprogramming could be induced by small molecule compounds to generate hematopoietic stem/progenitor-like cells, whether the human hematopoietic cells could also be reprogrammed into HSCs by chemical compounds remains elusive. Here, we demonstrated for the first time that human committed hematopoietic progenitors could be reprogrammed into multipotent progenitors by spliceosome inhibitor. Combination of single cell RNA-sequencing and genetic lineage tracing including exogenous barcodes and endogenous mitochondrial DNA mutations confirmed the reprogramming procession. Although the small chemical compound inhibiting spliceosome function only induces the differentiated hematopoietic progenitors to acquire plasticity and reprograms them into multipotent progenitors but not stem cells so far, this study still provides a proof-of-concept strategy for generating HSCs based on combining two independent steps together in future, first differentiating rare HSCs into large number of progenitors then reprogramming these progenitors into huge number of HSCs. Further dissecting the mechanism underlying spliceosome inhibitor-induced human hematopoietic cell reprogramming in future will help us comprehensively understanding not only the chemical reprogramming to generate desirable human cells for clinical translation but also hematopoiesis under physiological and pathological conditions.

During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation-stage human embryos. Human-induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single-cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single-cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.

We recently discovered a novel subset of beta cells that resemble immature beta cells during pancreas development. We named these "virgin" beta cells as they do not stem from existing mature beta cells. Virgin beta cells are found exclusively at the islet periphery in areas that we therefore designated as the "neogenic niche." As beta cells are our only source of insulin, their loss leads to diabetes. Islets also contain glucagon-producing alpha cells and somatostatin-producing delta cells, that are important for glucose homeostasis and form a mantle surrounding the beta cell core. This 3D architecture is important and determines access to blood flow and innervation. We propose that the distinctive islet architecture may also play an important, but hitherto unappreciated role in generation of new endocrine cells, including beta cells. We discuss several predictions to further test the contribution of the neogenic niche to beta cell regeneration.

We developed the Rainbow-seq technology to trace cell division history and reveal single-cell transcriptomes. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA sequencing (RNA-seq) experiment to simultaneously decode the lineage marker genes and read single-cell transcriptomes. We triggered lineage tracking in each blastomere at the 2-cell stage, observed microscopically inequivalent contributions of the progeny to the two embryonic poles at the blastocyst stage, and analyzed every single cell at either 4- or 8-cell stage with deep paired-end sequencing of full-length transcripts. Although lineage difference was not marked unequivocally at a single-gene level, it became clear when the transcriptome was analyzed as a whole. Moreover, several groups of novel transcript isoforms with embedded repeat sequences exhibited lineage difference, suggesting a possible link between DNA demethylation and cell fate decision. Rainbow-seq bridged a critical gap between division history and single-cell RNA-seq assays.

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward. [ABSTRACT FROM AUTHOR]

Background: CD34+ cells have been used to treat the patients with heart failure, but the outcome is variable. It is of great significance to scrutinize the fate and the mechanism of CD34+ cell differentiation in vivo during heart failure and explore its intervention strategy. Methods: We performed single-cell RNA sequencing (scRNA-seq) of the total non-cardiomyocytes and enriched Cd34-tdTomato+ lineage cells in the murine (male Cd34-CreERT2; Rosa26-tdTomato mice) pressure overload model (transverse aortic constriction, TAC), and total non-cardiomyocytes from human adult hearts. Then, in order to determine the origin of CD34+ cell that plays a role in myocardial fibrosis, bone marrow transplantation model was performed. Furthermore, to further clarify the role of CD34 + cells in myocardial remodeling in response to TAC injury, we generated Cd34-CreERT2; Rosa26-eGFP-DTA (Cre/DTA) mice. Results: By analyzing the transcriptomes of 59,505 single cells from the mouse heart and 22,537 single cells from the human heart, we illustrated the dynamics of cell landscape during the progression of heart hypertrophy, including CD34+ cells, fibroblasts, endothelial and immune cells. By combining genetic lineage tracing and bone marrow transplantation models, we demonstrated that non-bone-marrow-derived CD34+ cells give rise to fibroblasts and endothelial cells, while bone-marrow-derived CD34+ cell turned into immune cells only in response to pressure overload. Interestingly, partial depletion of CD34+ cells alleviated the severity of myocardial fibrosis with a significant improvement of cardiac function in Cd34-CreERT2; Rosa26-eGFP-DTA model. Similar changes of non-cardiomyocyte composition and cellular heterogeneity of heart failure were also observed in human patient with heart failure. Furthermore, immunostaining showed a double labeling of CD34 and fibroblast markers in human heart tissue. Mechanistically, our single-cell pseudotime analysis of scRNA-seq data and in vitro cell culture study revealed that Wnt-β-catenin and TGFβ1/Smad pathways are critical in regulating CD34+ cell differentiation toward fibroblasts. Conclusions: Our study provides a cellular landscape of CD34+ cell-derived cells in the hypertrophy heart of human and animal models, indicating that non-bone-marrow-derived CD34+ cells differentiating into fibroblasts largely account for cardiac fibrosis. These findings may provide novel insights for the pathogenesis of cardiac fibrosis and have further potential therapeutic implications for the heart failure. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM), a highly malignant and heterogeneous brain tumor, contains various types of tumor and non-tumor cells. Whether GBM cells can trans-differentiate into non-neural cell types, including mural cells or endothelial cells (ECs), to support tumor growth and invasion remains controversial. Here we generated two genetic GBM models de novo in immunocompetent mouse brains, mimicking essential pathological and molecular features of human GBMs. Lineage-tracing and transplantation studies demonstrated that, although blood vessels in GBM brains underwent drastic remodeling, evidence of trans-differentiation of GBM cells into vascular cells was barely detected. Intriguingly, GBM cells could promiscuously express markers for mural cells during gliomagenesis. Furthermore, single-cell RNA sequencing showed that patterns of copy number variations (CNVs) of mural cells and ECs were distinct from those of GBM cells, indicating discrete origins of GBM cells and vascular components. Importantly, single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages. Rather than expansion owing to trans-differentiation, vascular cell expanded by proliferation during tumorigenesis. Therefore, cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis. Our findings advance understanding of cell lineage dynamics during gliomagenesis, and have implications for targeted treatment of GBMs. [ABSTRACT FROM AUTHOR]

Increasingly, patients with gastrointestinal tumors can benefit from immunotherapy, but not patients with pancreatic cancer. While this lack of benefit has been attributed to lower T-cell infiltration in pancreatic cancer, other studies have demonstrated the presence of numerous T cells in pancreatic cancer, suggesting another mechanism for the poor efficacy of immunotherapy. Single-cell RNA sequencing studies on the pancreatic cancer immune microenvironment have demonstrated the predominance of innate immune cells (e.g., macrophages, dendritic cells, mast cells, and innate immune lymphoid cells). Therefore, in-depth research on the source and function of innate immune lymphocytes in pancreatic cancer could guide pancreatic cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of preexisting versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration. [ABSTRACT FROM AUTHOR]

In male mammals, spermatogonial stem cells (SSCs) are essential for sustaining lifelong spermatogenesis within the testicular open niche, a unique environment that allows SSC migration over an extended niche area. As SSCs undergo continuous mitotic division, mutations accumulate and are transmitted to the descendant SSC clones. Therefore, SSC clonal fate behaviors, in terms of their efficiencies in completing spermatogenesis and undergoing expansion within the niche, influence sperm genomic diversity. We aimed to elucidate the effects of physiological aging on SSC clonal fate behavior within the testicular open niche. We used single-cell RNA sequencing, lineage tracing, and intravital live imaging to investigate SSC behavior in aged mouse testes, where spermatogenesis, although reduced, persists. We found that undifferentiated spermatogonia maintained gene expression heterogeneity during aging. Among these, GFRα1 + cells, which exhibited state heterogeneity, showed accelerated proliferation and persistent motility, continuing to function as SSCs in older mice. In contrast, a subset of SSCs characterized by low Egr4 and Cops5 expression did not contribute to spermatid formation. These non-sperm-forming SSC clones increased in proportion among the total SSC clones and expanded spatially within the testicular open niche in old mice, a phenomenon not observed in young mice. The expansion of non-sperm-forming SSC clones in aged testes suggests that they occupy a niche space, limiting the availability of functional SSCs and potentially reducing sperm production and genetic diversity. These findings highlight age-specific clonal characteristics as hallmarks of stem cell aging within the testicular open niche and provide novel insights into the mechanisms governing reproductive aging., (© 2025 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Cancer cells display wide phenotypic variation even across patients with the same mutations. Differences in the cell of origin provide a potential explanation, but traditional assays lack the resolution to distinguish clonally heterogeneous subsets of stem and progenitor cells. To address this challenge, we developed simultaneous tracking of recombinase activation and clonal kinetics (STRACK), a method to trace clonal dynamics and gene expression before and after the acquisition of cancer mutations. Using mouse models, we studied two leukemic mutations, Dnmt3a-R878H and Npm1c, and found that their effect was highly variable across different stem cell states. Specifically, a subset of differentiation-primed stem cells, which normally becomes outcompeted with time, expands with both mutations. Intriguingly, Npm1c mutations reversed the intrinsic bias of the clone of origin, with differentiation-primed stem cells giving rise to more primitive malignant states. Thus, we highlight the relevance of single-cell lineage tracing to unravel early events in cancer evolution and posit that different cellular histories carry distinct cancer phenotypic potential., Competing Interests: Declaration of interests A.E.R.-F. is an advisor for Retro Biosciences., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The developmental diversity among smooth muscle cells (SMCs) plays a crucial role in segment-specific aortic diseases. However, traditional genetic approaches are inadequate for enabling in vivo analysis of disease susceptibility associated with cellular origin. There is an urgent need to build genetic technologies that target different developmental origins to investigate the mechanisms of aortopathies, thereby facilitating the development of effective therapeutics., Methods: To address this challenge, we developed an advanced dual recombinase-mediated intersectional genetic system, specifically designed to precisely target SMCs from various developmental origins in mice. Specifically, we used Isl1-Dre , Wnt1-Dre , Meox1-DreER , and Upk3b-Dre to target SMC progenitors from the second heart field, cardiac neural crest, somites, and mesothelium, respectively. This system was combined with single-cell RNA sequencing to investigate the impact of TGF-β (transforming growth factor-β) signaling in different segments of the aorta by selectively knocking out Tgfbr2 in the ascending aorta and Smad4 in the aortic arch, respectively., Results: Through intersectional genetic approaches, we use the Myh11-Cre(ER ) driver along with origin-specific Dre drivers to trace cells of diverse developmental origins within the SMC population. We found that a deficiency of Tgfbr2 in SMCs of the ascending aorta leads to aneurysm formation in this specific region. We also demonstrate the critical role of Smad4 in preserving aortic wall integrity and homeostasis in SMCs of the aortic arch., Conclusions: Our approach to genetically targeting SMC subtypes provides a novel platform for exploring origin-dependent or location-specific aortic vascular diseases. This genetic system enables comprehensive analysis of contributions from different cell lineages to SMC behavior and pathology, thereby paving the way for targeted research and therapeutic interventions in the future.

Sexual dimorphism is found in gene expression and polarization of macrophages in mammals but remains unclear in the ontogeny of tissue-resident macrophages. Salivary glands show remarkable gender differences and macrophages here are essential for the maintenance of immune quiescence and tissue repair after damages. Therefore, we compared origins of macrophages in salivary glands between male and female mice using conditional Cx3cr1 and Ccr2 lineage tracing approaches. We found most salivary gland macrophages in adult males are locally maintained and derived from yolk sac progenitors or postnatal monocytes, whereas these cells in adult females are mostly short-lived and continuously replenished by monocytes. Female salivary glands also contain more monocytes and polarized macrophages. Cx3cr1 knockout made the macrophage ontogeny in adult male salivary glands like females. These gender differences appear related to female-dominant salivary gland phenotypes in a common autoimmune disease, Sjogren's syndrome, and need be considered in developing macrophage-targeting therapies of dry mouth caused by autoimmunity or radiation., Competing Interests: Conflict of interest statement: The authors declare no potential conflicts of interest.

Postnatal neocortical development is a complex period wherein radial glial progenitors (RGPs) complete excitatory neurogenesis and transition to the production of glia. Here, we take advantage of a multi-layered lineage tracing tool pbacBarcode, to examine the contributions of individual cortical RGPs to the postnatal cortex. We reveal that some individual cortical RGPs are multipotent and give rise to olfactory bulb interneurons, astrocytes, and oligodendrocytes in a ∼2:1:1 ratio. We provide evidence that differentiation potential into terminal cell types is maintained as late as post-natal day (P)4, suggesting that a population decline model, as opposed to cell fate restriction, underlies postnatal neocortical development. Moreover, a pool of proliferative intermediary cells, which may represent a multipotent postnatal intermediate progenitor cell population, may contribute to the production of the three major cell types. Lastly, we examine RGP postnatal contribution to oligodendrocytes and show that oligodendrocyte progenitor founder cell production by cortical RGPs is largely complete by P3., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pulmonary vascular remodeling (PVR), encompassing microvascular loss and muscularization, contributes to multiple respiratory diseases. c-Kit + cells exhibit differentiation potential into both endothelial cells (ECs) and smooth muscle cells. The potential role of lung c-Kit + cell differentiation in PVR, however, remains unclear. Lung c-Kit + cells increase in pulmonary hypertension patients and in the SU5416/hypoxia (SuHx)-induced PVR mouse model. Employing genetic lineage tracing and single-cell RNA sequencing (scRNA-seq), we elucidate that lung-resident c-Kit + cells display an aerocyte and venular endothelial differentiation in the SuHx model. Ablation of tissue-resident c-Kit + cells exacerbates PVR. We identify an Nr2f2-expressing c-Kit + cell subgroup, which exhibitsvenous EC differentiation and increases during PVR. Notably, the elevation of Nr2f2 in c-Kit + cells via AAV enhances differentiation and mitigates PVR. These findings underscore the protective role of lung tissue-resident c-Kit + cells in PVR, achieved by differentiating into mature ECs. Targeting NR2F2 expression in c-Kit + cells emerges as a promising strategy for reversing PVR., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in lineage-tracing technologies have significantly improved our understanding of pancreatic cell biology, particularly in elucidating the ontogeny and regenerative capacity of pancreatic cells. A deeper appreciation of the mechanisms underlying pancreatic cell identity and plasticity holds the potential to inform the development of new therapeutic modalities for conditions such as diabetes and pancreatitis. With this goal in mind, here we summarize advances, challenges, and future directions in tracing pancreatic cell origins and fates using lineage-tracing technologies. Given their essential role for blood glucose regulation, we pay particular attention on the insights gained from endocrine cells, especially β-cells., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose of Review: Bone marrow adipose tissue is a distinctive fat depot located within the skeleton, with the potential to influence both local and systemic metabolic processes. Although significant strides have been made in understanding bone marrow adipose tissue over the past decade, many questions remain regarding their precise lineage and functional roles., Recent Findings: Recent studies have highlighted bone marrow adipose tissue's involvement in continuous cross-talk with other organs and systems, exerting both endocrine and paracrine functions that play a crucial role in metabolic homeostasis, skeletal remodeling, hematopoiesis, and the progression of bone metastases. The advancement of imaging techniques, particularly cross-sectional imaging, has profoundly expanded our understanding of the complexities beyond the traditional view of bone marrow adipose tissue as an inert depot. Notably, marrow adipocytes are anatomically and functionally distinct from brown, beige, and classic white adipocytes. Emerging evidence suggests that bone marrow adipocytes, bone marrow adipose tissue originate from the differentiation of bone marrow mesenchymal stromal cells; however, they appear to be a heterogeneous population with varying metabolic profiles, lipid compositions, secretory properties, and functional responses depending on their specific location within the bone marrow. This review provides an up-to-date synthesis of current knowledge on bone marrow adipocytes, emphasizing the relationships between bone marrow adipogenesis and factors such as aging, osteoporosis, obesity, and bone marrow tumors or metastases, thereby elucidating the mechanisms underlying musculoskeletal pathophysiology., Competing Interests: Compliance with Ethical Standards. Competing interests: The authors declare no competing interests. Human and Animal Rights and Informed Consent : This article does not contain any studies with human or animal subjects performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The CRISPR-activated repair lineage tracing (CARLIN) mouse line uses DNA barcoding to enable high-resolution tracing of cell lineages in vivo (Bowling et al, Cell 181, 1410-1422.e27, 2020). CARLIN mice contain expressed barcodes that allow simultaneous interrogation of lineage and gene expression information from single cells. Furthermore, barcode editing is fully inducible, resulting in cell lineage labeling that can be performed at any time point in development or adulthood. This chapter details the protocols followed for maintaining CARLIN mice, inducing barcoding, and amplifying the CARLIN barcode from DNA, RNA, and single-cell RNA-sequencing libraries for next-generation sequencing., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enables highly resolved descriptions of cell states within these systems; however, approaches are needed to directly determine the lineage relationship between cells. Here we provide a detailed protocol (Fig. 1) for the application of iTracer (He Z, Maynard A, Jain A, et al., Nat Methods 19:90-99, 2022), a recently published lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. iTracer is used to explore clonality and lineage dynamics during brain organoid development. More broadly, iTracer can be adapted to any iPSC-derived culture system to dissect lineage dynamics during normal or perturbed development., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the Drosophila brain, neuronal diversity originates from approximately 100 neural stem cells, each dividing asymmetrically. Precise mapping of cell lineages at the single-cell resolution is crucial for understanding the mechanisms that direct neuronal specification. However, existing methods for high-resolution lineage tracing are notably time-consuming and labor-intensive. Here, we outline the best practices for lineage tracing using CLADES (cell lineage access driven by an edition sequence), a revolutionary approach to neuronal lineage tracing that addresses the limitations of previous methods. CLADES effectively traces the birth order of neurons using approximately 100 samples. The technique relies on a genetic cascade of reporter activations and deactivations that delineate lineage progression through color-coded markers. This system not only facilitates the detailed mapping of neuronal lineages but also holds the potential to be applied to tracking biological events and producing cell types for therapeutic purposes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

StarTrack is a powerful multicolor genetic tool designed to unravel cellular lineages arising from neural progenitor cells (NPCs). This innovative technique, based on retrospective clonal analysis and built upon the PiggyBac system, creates a unique and inheritable "color code" within NPCs. Through the stochastic integration of 12 distinct plasmids encoding six fluorescent proteins, StarTrack enables precise and comprehensive tracking of cellular fates and progenitor potentials. The versatility of this tool is further enhanced by the potential of combining multiple promoters. Whether through the use of fluorescent integrable constructs or driving the expression of the PiggyBac transposase, StarTrack broadens the horizons for lineage tracing from progenitors of multiple origins.StarTrack revolutionized our understanding of cellular origins and lineages, offering an invaluable resource for researchers in the field of neural development and lineage tracing. This protocol provides a comprehensive overview of the technique's capabilities and applications, shedding light on its significance within the scientific community., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gene expression memory-based lineage inference (GEMLI) is a computational tool allowing to predict cell lineages solely from single-cell RNA-sequencing (scRNA-seq) datasets and is publicly available as an R package on GitHub. GEMLI is based on the occurrence of gene expression memory, i.e., the gene-specific maintenance of expression levels through cell divisions. This represents a shift away from experimental lineage tracing techniques based on genetic marks or physical cell lineage separation and greatly eases and expands lineage annotation. GEMLI allows to study cell lineages during differentiation in development, homeostasis, and regeneration, as well as disease onset and progression in various physiological and pathological contexts. This makes it possible to dissect cell type-specific gene expression memory, to discriminate symmetric and asymmetric cell fate decisions, and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. GEMLI is particularly promising for its ability to identify small lineages in human samples, a context in which no other lineage tracing methods are applicable. In this chapter, we provide a detailed protocol of the GEMLI R package usage on gene expression matrices derived from standard scRNA-seq on various platforms. We cover the use of the main function to predict cell lineages and how to adjust its parameters to different tasks. We also show how lineage information is extracted, visualized, and fine-tuned. Finally, we describe the use of the package's functions for the detailed analysis of the predicted cell lineages. This includes the analysis of gene expression memory, cell type composition of individual large lineages, and identification of lineages at the transition point between two cell types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mosaic analysis with double markers (MADM) is a powerful in vivo lineage tracing technique. It utilizes Cre recombinase-dependent interchromosomal recombination to restore the stable expression of two fluorescent proteins sparsely in individual dividing stem or progenitor cells and their progenies. Here, we describe the application of this technique for quantitative lineage analysis of radial glial progenitors in the developing mouse neocortex at the single-cell resolution., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Measurements of cell phylogeny based on natural or induced mutations, known as lineage barcodes, in conjunction with molecular phenotype have become increasingly feasible for a large number of single cells. In this chapter, we delve into Quantitative Fate Mapping (QFM) and its computational pipeline, which enables the interrogation of the dynamics of progenitor cells and their fate restriction during development. The methods described here include inferring cell phylogeny with the Phylotime model, and reconstructing progenitor state hierarchy, commitment time, population size, and commitment bias with the ICE-FASE algorithm. Evaluation of adequate sampling based on progenitor state coverage statistics is emphasized for interpreting the QFM results. Overall, this chapter describes a general framework for characterizing the dynamics of cell fate changes using lineage barcoding data., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lineage tracing has significantly advanced our comprehension in many areas of biology, such as development or immunity, by precisely measuring cellular processes like migration, division, or differentiation across labeled cells and their progeny. Traditional recombinase-based prospective lineage tracing is limited by the need for a priori cell type information and is constrained in the numbers of clones it can simultaneously track. In this sense, clonal lineage tracing with integrated random barcodes offers a robust alternative, enabling researchers to label and track a vast array of cells and their progeny over time. Moreover, clonal lineage tracing can be combined with single-cell omics technologies to study cell states and their maintenance over time. Key steps in these protocols include stable barcode integration, cell division to expand clones, and simultaneous capture of cellular properties with barcode information. Here, we comment on those steps and summarize important parameters to take into account during the design of single-cell lineage tracing experiments. Also, we present the main features for various available lentiviral libraries of expressed barcodes than can be captured alongside the transcriptome of individual cells. We cover other crucial aspects of experimental design, such as the optimization of cellular sampling, library diversity, and the minimization of clonal dropouts. Regarding sequencing data analysis, we provide some tips based on our experience, as well as available computational tools for the assignment of clonal identities and the identification of fate determinants. We finally discuss limitations of current methodologies and use an example step-by-step protocol to illustrate key points during the process. In sum, we provide a roadmap for considering and implementing single-cell lineage tracing studies to comprehensively explore fate determinants and their mechanisms., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lineage tracing methods enable the identification of all progeny generated by a single cell. High-throughput lineage tracing in the mammalian brain involves parallel labeling of thousands of progenitor cells with genetic barcodes in vivo followed by single-cell RNA-seq of lineage relations and cell types. Here we describe the generation of barcoded lentivirus, microinjections into the embryonic day 9.5 mouse forebrain, dissociation of 2-week-old mouse brain tissue, single-cell RNA-seq library preparation, and data analysis using a custom software. Compared to traditional methods based on sparse fluorophore labeling of progenitor cells, lineage tracing with genetic barcodes and single-cell RNA-seq has a >100-fold higher throughput while using >10 times fewer mice., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

5-Bromo-2′-deoxyuridine (BrdU) is a halogenated pyrimidine that can be incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU is widely used in fate-mapping studies of embryonic and adult neurogenesis to identify newborn neurons, however side effects on neural stem cells and their progeny have been reported. In vivo astrocyte-to-neuron (AtN) conversion is a new approach for generating newborn neurons by directly converting endogenous astrocytes into neurons. The BrdU-labeling strategy has been used to trace astrocyte-converted neurons, but whether BrdU has any effect on the AtN conversion is unknown. Here, while conducting a NeuroD1-mediated AtN conversion study using BrdU to label dividing reactive astrocytes following ischemic injury, we accidentally discovered that BrdU inhibited AtN conversion. We initially found a gradual reduction in BrdU-labeled astrocytes during NeuroD1-mediated AtN conversion in the mouse cortex. Although most NeuroD1-infected astrocytes were converted into neurons, the number of BrdU-labeled neurons was surprisingly low. To exclude the possibility that this BrdU inhibition was caused by the ischemic injury, we conducted an in vitro AtN conversion study by overexpressing NeuroD1 in cultured cortical astrocytes in the presence or absence of BrdU. Surprisingly, we also found a significantly lower conversion rate and a smaller number of converted neurons in the BrdU-treated group compared with the untreated group. These results revealed an unexpected inhibitory effect of BrdU on AtN conversion, suggesting more caution is needed when using BrdU in AtN conversion studies and in data interpretation.

Background Interstitial and perivascular fibrosis may contribute to diabetes‐associated heart failure. Pericytes can convert to fibroblasts under conditions of stress and have been implicated in the pathogenesis of fibrotic diseases. We hypothesized that in diabetic hearts, pericytes may convert to fibroblasts, contributing to fibrosis and to the development of diastolic dysfunction. Methods and Results Using pericyte:fibroblast dual reporter (NG2Dsred [neuron‐glial antigen 2 red fluorescent protein variant]; PDGFRαEGFP [platelet‐derived growth factor receptor alpha enhanced green fluorescent protein]) mice in a type 2 diabetic db/db background, we found that diabetes does not significantly affect pericyte density but reduces the myocardial pericyte:fibroblast ratio. Lineage tracing using the inducible NG2CreER driver, along with reliable labeling of fibroblasts with the PDGFRα reporter system, showed no significant pericyte to fibroblast conversion in lean and db/db hearts. In addition, db/db mouse cardiac fibroblasts did not undergo myofibroblast conversion and had no significant induction of structural collagens but exhibited a matrix‐preserving phenotype, associated with increased expression of antiproteases, matricellular genes, matrix cross‐linking enzymes, and the fibrogenic transcription factor cMyc. In contrast, db/db mouse cardiac pericytes had increased expression of Timp3, without any changes in expression of other fibrosis‐associated genes. The matrix‐preserving phenotype of diabetic fibroblasts was associated with induction of genes encoding oxidative (Ptgs2/cycloxygenase‐2, and Fmo2) and antioxidant proteins (Hmox1, Sod1). In vitro, high glucose partially recapitulated the in vivo changes in diabetic fibroblasts. Conclusions Diabetic fibrosis is not mediated through pericyte to fibroblast conversion but involves acquisition of a matrix‐preserving fibroblast program, which is independent of myofibroblast conversion and is only partially explained by the effects of the hyperglycemic environment.

Summary: Clustering cells based on their high-dimensional profiles is an important data reduction process by which researchers infer distinct cellular states. The advent of cellular barcoding, however, provides an alternative means by which to group cells: by their clonal origin. We developed ClonoCluster, a computational method that combines both clone and transcriptome information to create hybrid clusters that weight both kinds of data with a tunable parameter. We generated hybrid clusters across six independent datasets and found that ClonoCluster generated qualitatively different clusters in all cases. The markers of these hybrid clusters were different but had equivalent fidelity to transcriptome-only clusters. The genes most strongly associated with the rearrangements in hybrid clusters were ribosomal function and extracellular matrix genes. We also developed the complementary tool Warp Factor that incorporates clone information in popular 2D visualization techniques like UMAP. Integrating ClonoCluster and Warp Factor revealed biologically relevant markers of cell identity.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

Tissue stem cells are vital for organ homeostasis and regeneration owing to their ability to self‐renew and differentiate into the various cell types that constitute organ tissue. These stem cells are formed during complex and dynamic organ development, necessitating spatial–temporal coordination of morphogenetic events and cell fate specification during this process. In recent years, technological advances have enabled the tracing of the cellular dynamics, states, and lineages of individual cells over time in relation to tissue morphological changes. These dynamic data have not only revealed the origin of tissue stem cells in various organs but have also led to an understanding of the molecular, cellular, and biophysical bases of tissue stem cell formation. Herein, we summarize recent findings on the developmental origin of tissue stem cells in the hair follicles, intestines, brain, skeletal muscles, and hematopoietic system, and further discuss how stem cell fate specification is coordinated with tissue topology. [ABSTRACT FROM AUTHOR]

Objectives: The antimalarial drug artemether is suggested to effect pancreatic islet cell transdifferentiation, presumably through activation γ-aminobutyric acid receptors, but this biological action is contested. Methods: We have investigated changes in α-cell lineage in response to 10-days treatment with artemether (100 mg/kg oral, once daily) on a background of β-cell stress induced by multiple low-dose streptozotocin (STZ) injection in GluCreERT2; ROSA26-eYFP transgenic mice. Key findings: Artemether intervention did not affect the actions of STZ on body weight, food and fluid intake or blood glucose. Circulating insulin and glucagon were reduced by STZ treatment, with a corresponding decline in pancreatic insulin content, which were not altered by artemether. The detrimental changes to pancreatic islet morphology induced by STZ were also evident in artemether-treated mice. Tracing of α-cell lineage, through co-staining for glucagon and yellow fluorescent protein (YFP), revealed a significant decrease of the proportion of glucagon+YFP− cells in STZ-diabetic mice, which was reversed by artemether. However, artemether had no effect on transdifferentiation of α-cells into β-cells and failed to augment the number of bi-hormonal, insulin+glucagon+, islet cells. Conclusions: Our observations confirm that artemisinin derivatives do not impart meaningful benefits on islet cell lineage transition events or pancreatic islet morphology. [ABSTRACT FROM AUTHOR]

Epidural fat contains a population of mesenchymal progenitor cells (MPCs), and this study explores the behavior of these cells on the adjacent dura mater during growth and in response to injury in a p21 knockout mouse model. p21−/− mice are known to have increased cell proliferation and enhanced tissue regeneration post‐injury. Therefore, it is hypothesized that the process by which epidural fat MPCs maintain the dura mater can be accelerated in p21−/− mice. Using a Prx1 lineage tracing mouse model, the epidural fat MPCs are found to increase in the dura mater over time in both C57BL/6 (p21+/+) and p21−/− mice; however, by 3 weeks post‐tamoxifen induction, few MPCs are observed in p21−/− mice. These endogenous MPCs also localize to dural injuries in both mouse strains, with MPCs in p21−/− mice demonstrating increased proliferation. When epidural fat MPCs derived from p21−/− mice are transplanted into dural injuries in C57BL/6 mice, these MPCs are found in the injury site. It is demonstrated that epidural fat MPCs play a role in dural tissue maintenance and are able to directly contribute to dural injury repair. This suggests that these MPCs have the potential to treat injuries and/or pathologies in tissues surrounding the spinal cord. [ABSTRACT FROM AUTHOR]

Tongue epithelium is one of the most proliferative and regenerative epithelia in our body. However, tongue stem cell research is hampered partly by the lack of optimal animal models to study tongue injury, repair, and regeneration. Here, we establish a novel chemically induced tongue injury-recovery mouse model. Focal application of sodium hydroxide for a limited time led to the denudation of suprabasal layers, leaving the basal layer. Time course study revealed that tongue epithelial cells robustly proliferate over one week after the tongue injury. Importantly, we demonstrated that our novel mouse model could be employed in the lineage tracing of the tongue stem cells under the injury and repair process and further showed that tongue stem cells proliferate faster and generate larger clones in the injury condition than in the steady state condition. Our data indicate the development of a novel chemically induced tongue injury-recovery mouse model for tongue stem cell research, which will significantly facilitate the preclinical study for the pathogenesis and treatment of caustic ingestion. • We developed a novel chemical tongue injury-recovery mouse model. • Tongue regeneration after chemical injury was robust over one week. • The chemical tongue injury-recovery mouse model can be employed in the lineage tracing of the tongue stem cells. • Tongue stem cells proliferate faster and generate larger clone size upon the injury than in the steady state. [ABSTRACT FROM AUTHOR]

The development of organisms and tissues is dictated by an elaborate balance between cell division, apoptosis and differentiation: the cell population dynamics. To quantify these dynamics, we propose a phylodynamic inference approach based on single-cell lineage recorder data. We developed a Bayesian phylogenetic framework—time-scaled developmental trees (TiDeTree)—that uses lineage recorder data to estimate time-scaled single-cell trees. By implementing TiDeTree within BEAST 2, we enable joint inference of the time-scaled trees and the cell population dynamics. We validated TiDeTree using simulations and showed that performance further improves when including multiple independent sources of information into the inference, such as frequencies of editing outcomes or experimental replicates. We benchmarked TiDeTree against state-of-the-art methods and show comparable performance in terms of tree topology, plus direct assessment of uncertainty and co-estimation of additional parameters. To demonstrate TiDeTree's use in practice, we analysed a public dataset containing lineage data from approximately 100 stem cell colonies. We estimated a time-scaled phylogeny for each colony; as well as the cell division and apoptosis rates underlying the growth dynamics of all colonies. We envision that TiDeTree will find broad application in the analysis of single-cell lineage tracing data, which will improve our understanding of cellular processes during development. [ABSTRACT FROM AUTHOR]