Search

After severe heart injury, fibroblasts are activated and proliferate excessively to form scar, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous in respect of their degree of activation, proliferation, and function during cardiac fibrosis. Here, by dual recombinases-mediated genetic lineage tracing, we found that endocardium-derived fibroblasts (EndoFb) preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, the pattern of which mirrors that of EndoFb distribution in the heart. Specific ablation of EndoFb alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of EndoFb during cardiac remodeling. Our study identified EndoFb as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury, and as a potential therapeutic target against cardiac fibrosis.

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/β-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.

Lung injury activates epithelial stem or progenitor cells for alveolar repair and regeneration. However, the origin and fate of injury-induced progenitors are poorly defined. Here, we report that p63-expressing progenitors emerge upon bleomycin-induced lung injury. These p63+progenitors proliferate rapidly and differentiate into alveolar type 1 (AT1) and type 2 (AT2) cells through distinct trajectories. Dual recombinase-mediated sequential genetic lineage tracing reveals that p63+progenitors originate from airway secretory cells and subsequently generate alveolar cells. Functionally, p63 activation is required for efficient alveolar regeneration from secretory cells. Our study identifies a secretory cell-derived p63+progenitor that contributes to alveolar repair, indicating a potential therapeutic avenue for lung regeneration after injury.

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.

Cardiac regeneration involves the generation of new cardiomyocytes from cycling cardiomyocytes. Understanding cell-cycle activity of pre-existing cardiomyocytes provides valuable information to heart repair and regeneration. However, the anatomical locations and in situ dynamics of cycling cardiomyocytes remain unclear. Here we develop a genetic approach for a temporally seamless recording of cardiomyocyte-specific cell-cycle activity in vivo. We find that the majority of cycling cardiomyocytes are positioned in the subendocardial muscle of the left ventricle, especially in the papillary muscles. Clonal analysis revealed that a subset of cycling cardiomyocytes have undergone cell division. Myocardial infarction and cardiac pressure overload induce regional patterns of cycling cardiomyocytes. Mechanistically, cardiomyocyte cell cycle activity requires the Hippo pathway effector YAP. These genetic fate-mapping studies advance our basic understanding of cardiomyocyte cell cycle activity and generation in cardiac homeostasis, repair, and regeneration., The adult mammalian heart exhibits stubbornly low levels of cardiomyocyte proliferation, leading to high morbidity after injury or heart attack. Here the authors develop an approach for tracking cardiomyocyte cell cycling and show that the majority are located adjacent to the endocardium.

Rationale: The developing heart is composed of cardiomyocytes and noncardiomyocytes since the early stage. It is generally believed that noncardiomyocytes including the cardiac progenitors contribute to new cardiomyocytes of the looping heart. However, it remains unclear what the cellular dynamics of nonmyocyte to cardiomyocyte conversion are and when the lineage segregation occurs during development. It also remains unknown whether nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. Objective: We quantify the lineage conversion of noncardiomyocytes to cardiomyocytes in the embryonic and neonatal hearts and determine when the 2 cell lineages segregate during heart development. Moreover, we directly test if nonmyocyte to cardiomyocyte conversion contributes to neonatal heart regeneration. Methods and Results: We generated a dual genetic lineage tracing strategy in which cardiomyocytes and noncardiomyocytes of the developing heart could be simultaneously labeled by 2 orthogonal recombination systems. Genetic fate mapping showed that nonmyocyte to cardiomyocyte conversion peaks at E8.0 (embryonic day) to E8.5 and gradually declines at E9.5 and E10.5. Noncardiomyocytes do not generate any cardiomyocyte at and beyond E11.5 to E12.5. In the neonatal heart, noncardiomyocytes also do not contribute to any new cardiomyocyte in homeostasis or after injury. Conclusions: Noncardiomyocytes contribute to new cardiomyocytes of the developing heart at early embryonic stage before E11.5. The noncardiomyocyte and cardiomyocyte lineage segregation occurs between E10.5 and E11.5, which is maintained afterward even during neonatal heart regeneration.

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.

During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages., Body cavity macrophages reside on the serous surfaces of organs and believed to participate in organ repair following injury. Here the authors show with a fate-mapping reporter system that these cells, although accumulate at the surfaces of injured liver or lung, don’t penetrate deeply into the tissue.

It has been suggested that new beta cells can arise from specific populations of adult pancreatic progenitors or facultative stem cells. However, their existence remains controversial, and the conditions under which they would contribute to new beta-cell formation are not clear. Here, we use a suite of mouse models enabling dual-recombinase-mediated genetic tracing to simultaneously fate map insulin-positive and insulin-negative cells in the adult pancreas. We find that the insulin-negative cells, of both endocrine and exocrine origin, do not generate new beta cells in the adult pancreas during homeostasis, pregnancy or injury, including partial pancreatectomy, pancreatic duct ligation or beta-cell ablation with streptozotocin. However, non-beta cells can give rise to insulin-positive cells after extreme genetic ablation of beta cells, consistent with transdifferentiation. Together, our data indicate that pancreatic endocrine and exocrine progenitor cells do not contribute to new beta-cell formation in the adult mouse pancreas under physiological conditions. Zhao et al. use genetic lineage tracing to demonstrate that pancreatic endocrine and exocrine progenitor cells do not generate new beta cells, thus arguing against beta-cell neogenesis in the adult mouse pancreas.

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

Characterizing the stem cells responsible for lung repair and regeneration is important for the treatment of pulmonary diseases. Recently, a unique cell population located at the bronchioalveolar-duct junctions has been proposed to comprise endogenous stem cells for lung regeneration. However, the role of bronchioalveolar stem cells (BASCs) in vivo remains debated, and the contribution of such cells to lung regeneration is not known. Here we generated a genetic lineage-tracing system that uses dual recombinases (Cre and Dre) to specifically track BASCs in vivo. Fate-mapping and clonal analysis showed that BASCs became activated and responded distinctly to different lung injuries, and differentiated into multiple cell lineages including club cells, ciliated cells, and alveolar type 1 and type 2 cells for lung regeneration. This study provides in vivo genetic evidence that BASCs are bona fide lung epithelial stem cells with deployment of multipotency and self-renewal during lung repair and regeneration.

This letter investigates traffic-oriented resource allocation for millimeter-wave (mmW) multi-hop backhaul (BH) networks. The problem is formulated to minimize the sum of the distances between the data rate and BH traffic of all the small cell base stations (SBSs) under limited BH resources, considering concurrent transmissions to cater for the huge BH traffic occurred in the aggregated SBSs. The problem is casted as a matching game, since it is a two-sided stable and Pareto optimal matching between sub-channels and SBSs. By analyzing the preference profiles and judge functions of SBSs and sub-channels, respectively, an algorithm based on a matching theory is proposed. Simulations demonstrate that our algorithm can effectively cope with the huge aggregated traffic occurred in the mmW multi-hop BH networks with limited BH resources.

Unraveling the fates of resident stem cells during tissue regeneration is an important objective in clinical and basic research. Genetic lineage tracing based on Cre–loxP recombination provides an effective strategy for inferring cell fate and cell conversion in vivo. However, the determination of the exact fates of resident stem cells or their derivatives in disease states and during tissue regeneration remains controversial in many fields of study, partly because of technical limitations associated with Cre-based lineage tracing, such as, for example, off-target labeling. Recently, we generated a new lineage-tracing platform we named DeaLT (dual-recombinase-activated lineage tracing) that uses the Dre–rox recombination system to enhance the precision of Cre-mediated lineage tracing. Here, we describe as an example a detailed protocol using DeaLT to trace the fate of c-Kit+ cardiac stem cells and their derivatives, in the absence of any interference from nontarget cells such as cardiomyocytes, during organ homeostasis and after tissue injury. This lineage-tracing protocol can also be used to delineate the fate of resident stem cells of other organ systems, and takes ~10 months to complete, from mouse crossing to final tissue analysis. In this protocol, the authors explain a new, more precise genetic-lineage-tracing system based on a dual-recombinase strategy. DeaLT enables specific fate mapping of resident stem cells by using both the Cre–loxP and Dre–rox systems.

Background: Whether the adult mammalian heart harbors cardiac stem cells for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers, such as the cardiosphere-derived cells, or with markers such as Sca1 + , Bmi1 + , Isl1 + , or Abcg2 + cardiac stem cells have been reported. Moreover, it remains unclear whether putative cardiac stem cells with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods: To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all nonmyocyte populations that contain putative cardiac stem cells. Using dual lineage tracing system, we assessed whether nonmyocytes generated any new myocytes during embryonic development, during adult homeostasis, and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocyte generation from nonmyocytes. Results: By this stem cell marker–free and dual recombinases–mediated cell tracking approach, our fate mapping data show that new myocytes arise from nonmyocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from nonmyocytes in the skeletal muscle after injury. Conclusions: This study provides in vivo genetic evidence for nonmyocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

Rationale: Organs of the body require vascular networks to supply oxygen and nutrients and maintain physiological function. The blood vessels of different organs are structurally and functionally heterogeneous in nature. To more precisely dissect their distinct in vivo function in individual organs, without potential interference from off-site targets, it is necessary to genetically target them in an organ-specific manner. Objective: The objective of this study was to generate a genetic system that targets vascular endothelial cells in an organ- or tissue-specific manner and to exemplify the potential application of intersectional genetics for precise, target-specific gene manipulation in vivo. Methods and Results: We took advantage of 2 orthogonal recombination systems, Dre-rox and Cre-loxP, to create a genetic targeting system based on intersectional genetics. Using this approach, Cre activity was only detectable in cells that had expressed both Dre and Cre. Applying this new system, we generated a coronary endothelial cell–specific Cre ( CoEC-Cre ) and a brain endothelial cell–specific Cre ( BEC-Cre ). Through lineage tracing, gene knockout and overexpression experiments, we demonstrated that CoEC-Cre and BEC-Cre efficiently and specifically target blood vessels in the heart and brain, respectively. By deletion of vascular endothelial growth factor receptor 2 using BEC-Cre , we showed that vascular endothelial growth factor signaling regulates angiogenesis in the central nervous system and also controls the integrity of the blood-brain barrier. Conclusions: We provide 2 examples to illustrate the use of intersectional genetics for more precise gene targeting in vivo, namely manipulation of genes in blood vessels of the heart and brain. More broadly, this system provides a valuable strategy for tissue-specific gene manipulation that can be widely applied to other fields of biomedical research.

Rationale: Endocardium is the major source of coronary endothelial cells (ECs) in the fetal and neonatal hearts. It remains unclear whether endocardium in the adult stage is also the main origin of neovascularization after cardiac injury. Objective: To define the vascular potential of adult endocardium in homeostasis and after cardiac injuries by fate-mapping studies. Methods and Results: We generate an inducible adult endocardial Cre line ( Npr3 [natriuretic peptide receptor C]- CreER ) and show that Npr3-CreER efficiently and specifically labels endocardial cells but not coronary blood vessels in the adult heart. The adult endocardial cells do not contribute to any vascular ECs during cardiac homeostasis. To examine the formation of blood vessels from endocardium after injury, we generate 4 cardiac injury models with Npr3-CreER mice: myocardial infarction, myocardial ischemia–reperfusion, cryoinjury, and transverse aortic constriction. Lineage tracing experiments show that adult endocardium minimally contributes to coronary ECs after myocardial infarction. In the myocardial ischemia–reperfusion, cryoinjury, or transverse aortic constriction models, adult endocardial cells do not give rise to any vascular ECs, and they remain on the inner surface of myocardium that connects with lumen circulation. In the myocardial infarction model, very few endocardial cells are trapped in the infarct zone of myocardium shortly after ligation of coronary artery, indicating the involvement of endocardial entrapment during blood vessels formation. When these adult endocardial cells are relocated and trapped in the infarcted myocardium by transplantation or myocardial constriction model, very few endocardial cells survive and gain vascular EC properties, and their contribution to neovascularization in the injured myocardium remains minimal. Conclusions: Unlike its fetal or neonatal counterpart, adult endocardium naturally generates minimal, if any, coronary arteries or vascular ECs during cardiac homeostasis or after injuries.

Magnesium hydroxide sulfate whisker (MHSH) was used as flame-retarded synergistic agent of intumescent flame retardant (IFR), and to prepare flame-retarded PBS composites. The mechanical performance and flame-retardant properties of composites were investigated. Experimental data showed that an appropriate MHSH loading favored the mechanical performance and flame-retardant properties of composites. When IFR and MHSH loadings were 23 and 2 wt%, respectively, the limited oxygen index value of 39.8% and UL-94 V0 rate of composite could be achieved. The tensile strength increased by 33.3% in comparison with that of composite only prepared by 25 wt% IFR. XRD analysis indicated that the addition of MHSH significantly increased the crystallization of composites. FT-IR analysis demonstrated that P–O–Mg–O–P bonds could be generated during pyrolysis process. TG and cone calorimeter analyses both indicated that the incorporation of MHSH and IFR lowered the flammability of composites by limiting heat and mass transfer. Based on the experimental and analyses data, especially the SEM analysis, possible mechanism was proposed. The combustion products of MHSH and IFR provided a flame shield during combustion process. MHSH played a reinforcement effect in the shield. A more stable three-dimensional intumescent charred layer could not only effectually prevent the melt from dripping but also hinder the diffusion of oxygen and heat into the interior substrate.

The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.

The liver possesses a remarkable capacity to regenerate after damage. There is a heated debate on the origin of new hepatocytes after injuries in adult liver. Hepatic stem/progenitor cells have been proposed to produce functional hepatocytes after injury. Recent studies have argued against this model and suggested that pre-existing hepatocytes, rather than stem cells, contribute new hepatocytes. This hepatocyte-to-hepatocyte model is mainly based on labeling of hepatocytes with Cre-recombinase delivered by the adeno-associated virus. However, the impact of virus infection on cell fate determination, consistency of infection efficiency, and duration of Cre-virus in hepatocytes remain confounding factors that interfere with the data interpretation. Here, we generated a new genetic tool Alb-DreER to label almost all hepatocytes (>99.5%) and track their contribution to different cell lineages in the liver. By “pulse-and-chase” strategy, we found that pre-existing hepatocytes labeled by Alb-DreER contribute to almost all hepatocytes during normal homeostasis and after liver injury. Virtually all hepatocytes in the injured liver are descendants of pre-existing hepatocytes through self-expansion. We concluded that stem cell differentiation is unlikely to be responsible for the generation of a substantial number of new hepatocytes in adult liver. Our study also provides a new mouse tool for more precise in vivo genetic study of hepatocytes in the field.

Rapid regeneration of smooth muscle after vascular injury is essential for maintaining arterial function. The existence and putative roles of resident vascular stem cells (VSCs) in artery repair are controversial, and vessel regeneration is thought to be mediated by proliferative expansion of pre-existing smooth muscle cells (SMCs). Here, we performed cell fate mapping and single-cell RNA sequencing to identify Sca1+ VSCs in the adventitial layer of artery walls. After severe injury, Sca1+ VSCs migrate into the medial layer and generate de novo SMCs, which subsequently expand more efficiently compared with pre-existing smooth muscle. Genetic lineage tracing using dual recombinases distinguished a Sca1+PDGFRa+ VSC subpopulation that generates SMCs, and genetic ablation of Sca1+ VSCs or specific knockout of Yap1 in Sca1+ VSCs significantly impaired artery repair. These findings provide genetic evidence of a bona fide Sca1+ VSC population that produces SMCs and delineates their critical role in vessel repair. We thank Shanghai Model Organisms Center, Inc. (SMOC) for mouse generation, Dr. Camargo Fernando for Yap-flox mouse, and Baojin Wu, Guoyuan Chen, Zhonghui Weng, and Aimin Huang for animal husbandry. We also thank Wei Bian, Lin Qiu, and members of National Center for Protein Science Shanghai for technical help and assistance with microscopy. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS; XDA16010507 and XDB19000000), the National Key Research and Development Program of China (2019YFA0110400, 2018YFA0107900, 2018YFA0108100, 2016YFC1300600, and 2017YFC1001303), the Shanghai Zhangjiang Stem Cell Project (ZJ2018-ZD-004), the National Science Foundation of China (31730112, 91639302, 31625019, 91849202, 81761138040, 81872241, 31701292, 31801215, and 31922032), the Youth Innovation Promotion Association of the CAS, the Key Project of Frontier Sciences of the CAS (QYZDB-SSW-SMC003), the Shanghai Science and Technology Commission (19JC1415700, 17ZR1449600, and 17ZR1449800), the Program for Guangdong Introduction Innovative and Entrepreneurial Teams (2017ZT07S347), the Major Program of the Development Fund for the Shanghai Zhangjiang National Innovation Demonstration Zone (Stem Cell Strategic Biobank and Stem Cell Clinical Technology Transformation Platform, ZJ2018-ZD-004), the Shanghai Yangfan Project, the China Postdoctoral Science Foundation, the China Postdoctoral Innovative Talent Support Program, the Young Elite Scientist Sponsorship Program by CAST (2018QNRC001, and 2017QNRC001), Boehringer Ingelheim, a Sanofi-SIBS fellowship, AstraZeneca, and a Royal Society Newton advanced fellowship. Sí