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Genetic lineage tracing has been widely employed to investigate cell lineages and fate. However, conventional reporting systems often label the entire cytoplasm, making it challenging to discern cell boundaries. Additionally, single Cre-loxP recombination systems have limitations in tracing specific cell populations. This study proposes three reporting systems that utilizing Cre, Dre, and Dre + Cre mediated recombination. These systems incorporate tdTomato expression on the cell membrane and PhiYFP expression within the nucleus, allowing for clear observation of the nucleus and membrane. The efficacy of these systems is successfully demonstrated by labeling cardiomyocytes and hepatocytes. The potential for dynamic visualization of the cell membrane is showcased using intravital imaging microscopy or three-dimensional imaging. Furthermore, by combining this dual recombinase system with the ProTracer system, hepatocyte proliferation is traced with enhanced precision. This reporting system holds significant importance for advancing the understanding of cell fate studies in development, homeostasis, and diseases.
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BACKGROUND: Endothelial cell (EC) generation and turnover by self-proliferation contributes to vascular repair and regeneration. The ability to accurately measure the dynamics of EC generation would advance our understanding of cellular mechanisms of vascular homeostasis and diseases. However, it is currently challenging to evaluate the dynamics of EC generation in large vessels such as arteries because of their infrequent proliferation. METHODS: By using dual recombination systems based on Cre-loxP and Dre-rox, we developed a genetic system for temporally seamless recording of EC proliferation in vivo. We combined genetic recording of EC proliferation with single-cell RNA sequencing and gene knockout to uncover cellular and molecular mechanisms underlying EC generation in arteries during homeostasis and disease. RESULTS: Genetic proliferation tracing reveals that ≈3% of aortic ECs undergo proliferation per month in adult mice during homeostasis. The orientation of aortic EC division is generally parallel to blood flow in the aorta, which is regulated by the mechanosensing protein Piezo1. Single-cell RNA sequencing analysis reveals 4 heterogeneous aortic EC subpopulations with distinct proliferative activity. EC cluster 1 exhibits transit-amplifying cell features with preferential proliferative capacity and enriched expression of stem cell markers such as Sca1 and Sox18. EC proliferation increases in hypertension but decreases in type 2 diabetes, coinciding with changes in the extent of EC cluster 1 proliferation. Combined gene knockout and proliferation tracing reveals that Hippo/vascular endothelial growth factor receptor 2 signaling pathways regulate EC proliferation in large vessels. CONCLUSIONS: Genetic proliferation tracing quantitatively delineates the dynamics of EC generation and turnover, as well as EC division orientation, in large vessels during homeostasis and disease. An EC subpopulation in the aorta exhibits more robust cell proliferation during homeostasis and type 2 diabetes, identifying it as a potential therapeutic target for vascular repair and regeneration.
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Diabetes Mellitus Tipo 2 , Fator A de Crescimento do Endotélio Vascular , Animais , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Aorta/metabolismo , Células Endoteliais/metabolismo , Homeostase , Canais Iônicos/metabolismoRESUMO
Various factors contribute to different types of surgical site infections (SSI) in gastric cancer patients undergoing surgery, and the risk factors remain uncertain. This meta-analysis aims to clarify the relationship between various factors and SSI, resolving existing controversies. Thirty-four eligible articles with 66 066 patients were included in the meta-analysis. Significant risk factors for SSI included age ≥65 years, male gender, BMI ≥25 kg/m2, diabetes, hypertension, advanced TNM stage ≥III, pathologic T stage ≥T3, pathologic N stage ≥N1, ASA ≥3, open surgery, blood transfusion, extensive resection, combined resection, splenectomy, D2 or more lymph node dissection, and operative time ≥240 min. Operative time showed a nonlinear relationship with SSI risk. Subgroup analysis revealed significant differences in the effects of risk factors among different infection types. These findings inform the development of targeted preventive measures to reduce SSI rates.
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Neoplasias Gástricas , Infecção da Ferida Cirúrgica , Humanos , Infecção da Ferida Cirúrgica/etiologia , Infecção da Ferida Cirúrgica/epidemiologia , Neoplasias Gástricas/cirurgia , Neoplasias Gástricas/complicações , Fatores de Risco , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Adulto , Idoso de 80 Anos ou maisRESUMO
The platelet-derived growth factor (PDGF) and its receptor, PDGFRα, are critical for tissue development and injury repair. To track PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expresses green fluorescent protein (GFP) under the control of the PDGFRα promoter. This genetic tool enabled us to detect PDGFRα expression in various organs during both neonatal and adult stages. Additionally, we confirmed the correlation between endogenous PDGFRα and transgenic PDGFRα expression using mouse injury models, showing the potential of this genetic reporter for studying PDGFRα-mediated signaling pathways and developing therapeutic strategies. Overall, the PDGFRα-GFP knock-in mouse line serves as a valuable tool for investigating the biology of PDGFRα and its role in normal development and disease.
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Fibroblastos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Camundongos , Animais , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Camundongos Transgênicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismoRESUMO
The maternal liver undergoes dramatic enlargement to adapt to the increased metabolic demands during pregnancy. However, the cellular sources for liver growth during pregnancy remain largely elusive. Here, we employed a proliferation recording system, ProTracer, to examine the spatial-temporal proliferation of hepatocytes during pregnancy. We discovered that during early to late pregnancy, hepatocyte proliferation initiated from zone 1, to zone 2, and lastly to zone 3, with the majority of new hepatocytes being generated in zone 2. Additionally, using single-cell RNA sequencing, we observed that Ccnd1 was highly enriched in zone 2 hepatocytes. We further applied dual-recombinase-mediated genetic lineage tracing to reveal that Ccnd1+ hepatocytes expanded preferentially during pregnancy. Moreover, we demonstrated that estrogen induces liver enlargement during pregnancy, which was abolished in Ccnd1 knockout mice. Our work revealed a unique spatial-temporal hepatocyte proliferation pattern during pregnancy, with Ccnd1+ hepatocytes in zone 2 serving as the major cellular source for hepatic enlargement.
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Hepatócitos , Regeneração Hepática , Camundongos , Animais , Feminino , Gravidez , Hepatócitos/metabolismo , Fígado/metabolismo , Proliferação de Células , Camundongos KnockoutRESUMO
A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.
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Conventional polarization beam splitters (PBSs) suffer energy loss and signal distortion due to backscattering caused by disturbances. Topological photonic crystals provide backscattering immunity and anti-disturbance robustness transmission owing to the topological edge states. Here, we put forward a kind of dual-polarization air hole-type fishnet valley photonic crystal with a common bandgap (CBG). The Dirac points at the K point formed by different neighboring bands for transverse magnetic and transverse electric polarizations are drawn closer via changing the filling ratio of the scatterer. Then the CBG is constructed by lifting the Dirac cones for dual polarizations within a same frequency range. We further design a topological PBS using the proposed CBG via changing the effective refractive index at the interfaces which guide polarization-dependent edge modes. Based on these tunable edge states, the designed topological PBS (TPBS) achieves efficient polarization separation and is robust against sharp bends and defects, verified by simulation results. The TPBS's footprint is approximately 22.4 × 15.2 µ m 2, allowing high-density on-chip integration. Our work has potential application in photonic integrated circuits and optical communication systems.
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Dispositivos Ópticos , Simulação por Computador , Eletricidade , Fótons , Células Fotorreceptoras Retinianas ConesRESUMO
The ability to experimentally measure cell proliferation is the basis for understanding the sources of cells that drive organ development, tissue regeneration and repair. Recently, we generated a genetic approach to detect cell proliferation: we used genetic lineage-tracing technologies to achieve seamless recording of in vivo cell proliferation in a tissue-specific manner. We provide a detailed protocol (generation of mouse lines, characterization of mouse lines, mouse line crossing and cell-proliferation tracing) for using this genetic system to study cell proliferation. This cell-proliferation tracing system, which we term 'ProTracer' (Proliferation Tracer), permits lifelong noninvasive monitoring of cell proliferation of specific cell lineages in live animals. Compared with other short-term strategies that require execution of animals, ProTracer does not require sampling or animal sacrifice for tissue processing. To highlight these features, we used ProTracer to study the proliferation of hepatocytes during liver homeostasis and after tissue injury in mice. We show that the protocol is applicable to study any in vivo cell proliferation, which takes ~9 months to finish from mouse generation to data analysis. This protocol can easily be carried out by researchers skilled in mouse-related experiments.
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Hepatócitos , Fígado , Camundongos , Animais , Diferenciação Celular , Fígado/metabolismo , Hepatócitos/metabolismo , Linhagem da Célula , Proliferação de CélulasRESUMO
Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/ß-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.
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Regeneração Hepática , Fígado , Proliferação de Células/genética , Hepatócitos , Células Epiteliais , Células-Tronco , Diferenciação Celular/genéticaRESUMO
After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.
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Cardiopatias , Fibroblastos/metabolismo , Cardiopatias/genética , Cardiopatias/patologia , Fibrose/genética , Animais , Camundongos , Envelhecimento , Proliferação de Células , Via de Sinalização Wnt , Camundongos TransgênicosRESUMO
Unraveling cell fate plasticity during tissue homeostasis and repair can reveal actionable insights for stem cell biology and regenerative medicine. In the pancreas, it remains controversial whether lineage transdifferentiation among the exocrine cells occur under pathophysiological conditions. Here, to address this question, we used a dual recombinase-mediated genetic system that enables simultaneous tracing of pancreatic acinar and ductal cells using two distinct genetic reporters, avoiding the "ectopic" labeling by Cre-loxP recombination system. We found that acinar-to-ductal transdifferentiation occurs after pancreatic duct ligation or during caerulein-induced pancreatitis, but not during homeostasis or after partial pancreatectomy. On the other hand, pancreatic ductal cells contribute to new acinar cells after significant acinar cell loss. By genetic tracing of cell proliferation, we also quantify the cell proliferation dynamics and deduce the turnover rate of pancreatic exocrine lineages during homeostasis. Together, these results suggest that the lineage transdifferentiation happens between acinar cells and ductal cells in the pancreatic exocrine glands under specific conditions.
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Unraveling cell-cell interaction is fundamental to understanding many biological processes. To date, genetic tools for labeling neighboring cells in mammals are not available. Here, we developed a labeling strategy based on the Cre-induced intercellular labeling protein (CILP). Cre-expressing donor cells release a lipid-soluble and membrane-permeable fluorescent protein that is then taken up by recipient cells, enabling fluorescent labeling of neighboring cells. Using CILP, we specifically labeled endothelial cells surrounding a special population of hepatocytes in adult mice and revealed their distinct gene signatures. Our results highlight the potential of CILP as a platform to reveal cell-cell interactions and communications in vivo.
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Células Endoteliais , Proteínas de Membrana , Animais , Camundongos , Hepatócitos/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Monitoring of cell-cell communication in multicellular organisms is fundamental to understanding diverse biological processes such as embryogenesis and tumorigenesis. To track cell-cell contacts in vivo, we developed an intercellular genetic technology to monitor cell-cell contact and to trace cell contact histories by permanently marking contacts between cells. In mice, we engineered an artificial Notch ligand into one cell (the sender cell) and an artificial receptor into another cell (the receiver cell). Contact between the sender and receiver cells triggered a synthetic Notch signaling that activated downstream transcriptional programs in the receiver cell, thereby transiently or permanently labeling it. In vivo cell-cell contact was observed during development, tissue homeostasis, and tumor growth. This technology may be useful for studying dynamic in vivo cell-cell contacts and cell fate plasticity.
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Comunicação Celular , Perfilação da Expressão Gênica , Receptores Notch , Animais , Camundongos , Carcinogênese , Plasticidade Celular , Transdução de Sinais , Receptores Notch/genética , Perfilação da Expressão Gênica/métodosRESUMO
Cells and tissues are exposed to a wide range of mechanical stimuli during development, tissue homeostasis, repair, and regeneration. Over the past few decades, mechanosensitive ion channels (MSCs), as force-sensing integral membrane proteins, have attracted great attention with regard to their structural dynamics and mechanics at the molecular level and functions in various cells. Piezo-type MSC component 1 (Piezo1) is a newly discovered MSC; it is inherently mechanosensitive. However, which type of cells express Piezo1 in vivo remains unclear. To detect and trace Piezo1-expressing cells, we generated and characterized a novel tamoxifen-inducible Cre knock-in mouse line, Piezo1-CreER, which expresses CreER recombinase under the control of the endogenous Piezo1 promoter. Using this genetic tool, we detected the expression of Piezo1 in various cell types at the embryonic, neonatal, and adult stages. Our data showed that Piezo1 was highly expressed in endothelial cells in all the three stages, while the Piezo1 expression in epithelial cells was dynamic during development and growth. In summary, we established a new genetic tool, Piezo1-CreER, to study Piezo1-expressing cells in vivo during development, injury response, and tissue repair and regeneration.
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Células Endoteliais , Canais Iônicos , Animais , Células Endoteliais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Tamoxifeno/farmacologiaRESUMO
Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages.
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Linhagem da Célula , Coração , Macrófagos , Miocárdio , Animais , Aorta/citologia , Endocárdio/citologia , Coração/embriologia , Hematopoese/genética , Miocárdio/citologia , Saco Vitelino/citologiaRESUMO
Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato+ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.
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Linhagem da Célula , Integrases , Recombinases , Animais , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Recombinases/genética , Recombinases/metabolismo , Recombinação GenéticaRESUMO
Venetoclax has emerged as a breakthrough for treatment of leukemia with a wide interindividual variability in pharmacokinetics. Herein, a rapid, sensitive, and reliable UPLC-MS/MS method for quantification of venetoclax in plasma was developed and validated. The method was operated in the multiple-reaction monitoring (MRM) mode to detect venetoclax at m/z transition 868.5 > 321.0 and IS at 875.5 > 321.0, respectively. Protein precipitation prior to injection into the LC-MS/MS and the analyte was separated on an ACQUITY UPLC BEH C18 column by gradient elution with acetonitrile and 0.1% formic acid in water. Linear calibration curves were obtained in the range of 25−8000 ng/mL. The specificity, recovery, matrix effect, and stability also met the acceptance criteria of FDA guidance. The method was successfully applied to analyze plasma in acute myeloid leukemia (AML) patients. The peak plasma concentration (Cmax) of venetoclax in Chinese AML patient was 2966.0 ± 1595.0 ng/mL while the trough concentration (Cmin) was 1018.0 ± 729.4 ng/mL. Additionally, Cmax and Cmin showed a positive correlation with AST levels. Furthermore, Cmax was significantly higher in the older patients. The present method can be applied for TDM of venetoclax in treatment of hematological cancers.