Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

Intercellular genetic tracing of cardiac endothelium in the developing heart.

Cardiac resident macrophages play vital roles in heart development, homeostasis, repair, and regeneration. Recent studies documented the hematopoietic potential of cardiac endothelium that supports the generation of cardiac macrophages and peripheral blood cells in mice. However, the conclusion was not strongly supported by previous genetic tracing studies, given the non-specific nature of conventional Cre-loxP tracing tools. Here, we develop an intercellular genetic labeling system that can permanently trace heart-specific endothelial cells based on cell-cell interaction in mice. Results from cell-cell contact-mediated genetic fate mapping demonstrate that cardiac endothelial cells do not exhibit hemogenic potential and do not contribute to cardiac macrophages or other circulating blood cells. This Matters Arising paper is in response to Shigeta et al. (2019), published in Developmental Cell. See also the response by Liu and Nakano (2023), published in this issue.

Bipotent transitional liver progenitor cells contribute to liver regeneration.

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.

Monitoring of cell-cell communication and contact history in mammals.

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.

The efficacy and mechanism of thoracic photodynamic therapy mediated by hematoporphyrin injection on disseminated pleural malignancies of Lewis lung carcinoma in mice.

In order to avoid the problems of long exposure time and high incidence of photosensitivity by intravenous injection of photosensitizer, our study explore the safety, efficacy, and possible mechanisms of photodynamic therapy (PDT) by intrathoracic administration of hematoporphyrin injection in the treatment of disseminated pleural malignancies of Lewis lung carcinoma in mice to provide a theoretical basis for thoracic PDT in the clinic. Hematoporphyrin was administered into the thoracic cavity of tumor-bearing mice, and the concentrations of hematoporphyrin in normal and tumor pleural tissues were detected by high-performance liquid chromatography. The tumor-bearing mice were randomly divided into four groups: model control, pure laser irradiation, PDT low-dose, and PDT high-dose groups. Hematoxylin and eosin (H&E) staining was used to observe the histological changes in normal pleural tissue. H&E and DNA in situ nick end-labeling staining were used to detect necrosis and apoptosis in the tumor tissues. The tumor volume in each group from high to low was as follows: model control group > pure laser irradiation group > PDT low-dose group > PDT high-dose group. Inflammatory cells infiltrated the normal pleural tissue of the PDT group. Necrosis was observed to different extents in the tumor tissues of the PDT group. The apoptosis index of each group from high to low was as follows: PDT high-dose group > PDT low-dose group > pure laser irradiation group > model control group. The differences were statistically significant (P<0.05). Hematoporphyrin selectively accumulated in tumor pleural tissues. PDT with intrathoracic administration of hematoporphyrin injection could inhibit the thoracic implant tumors in mice by inducing necrosis and apoptosis.

Lineage tracing clarifies the cellular origin of tissue-resident macrophages in the developing heart.

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.

Genetic fate-mapping reveals surface accumulation but not deep organ invasion of pleural and peritoneal cavity macrophages following injury.

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.

Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.

Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ, but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes.

In Vivo AAV-CRISPR/Cas9-Mediated Gene Editing Ameliorates Atherosclerosis in Familial Hypercholesterolemia.

BACKGROUND: Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. METHODS: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, LdlrE208X, based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. CONCLUSIONS: Our work shows that in vivo AAV-CRISPR/Cas9-mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.

Ubiquitination of RIPK1 suppresses programmed cell death by regulating RIPK1 kinase activation during embryogenesis.

The ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing RIPK1K376R which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1K376R are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1K376R/K376R lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1-/- Ripk1K376R/K376R mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity-dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally.

Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction.

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.

Apj+ Vessels Drive Tumor Growth and Represent a Tractable Therapeutic Target.

Identification of cellular surface markers that distinguish tumorous from normal vasculature is important for the development of tumor vessel-targeted therapy. Here, we show that Apj, a G protein-coupled receptor, is highly enriched in tumor endothelial cells but absent from most endothelial cells of adult tissues in homeostasis. By genetic targeting using Apj-CreER and Apj-DTRGFP-Luciferase, we demonstrated that hypoxia-VEGF signaling drives expansion of Apj+ tumor vessels and that targeting of these vessels, genetically and pharmacologically, remarkably inhibits tumor angiogenesis and restricts tumor growth. These in vivo findings implicate Apj+ vessels as a key driver of pathological angiogenesis and identify Apj+ endothelial cells as an important therapeutic target for the anti-angiogenic treatment of tumors.

Genetic lineage tracing of resident stem cells by DeaLT.

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.

Genetic Targeting of Organ-Specific Blood Vessels.

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.

Genetic Fate Mapping Defines the Vascular Potential of Endocardial Cells in the Adult Heart.

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.

Identification of a hybrid myocardial zone in the mammalian heart after birth.

Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa+ or Hey2+ cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa+ and Hey2+ cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa+ and Hey2+ populations. Inhibition of the fetal Hey2+ cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2+ fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.

Genetic tracing of hepatocytes in liver homeostasis, injury, and regeneration.

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.

Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls.

RATIONALE: There is persistent uncertainty regarding the developmental origins of coronary vessels, with 2 principal sources suggested as ventricular endocardium or sinus venosus (SV). These 2 proposed origins implicate fundamentally distinct mechanisms of vessel formation. Resolution of this controversy is critical for deciphering the programs that result in the formation of coronary vessels and has implications for research on therapeutic angiogenesis. OBJECTIVE: To resolve the controversy over the developmental origin of coronary vessels. METHODS AND RESULTS: We first generated nuclear factor of activated T cells (Nfatc1)-Cre and Nfatc1-Dre lineage tracers for endocardium labeling. We found that Nfatc1 recombinases also label a significant portion of SV endothelial cells in addition to endocardium. Therefore, restricted endocardial lineage tracing requires a specific marker that distinguishes endocardium from SV. By single-cell gene expression analysis, we identified a novel endocardial gene natriuretic peptide receptor 3 (Npr3). Npr3 is expressed in the entirety of the endocardium but not in the SV. Genetic lineage tracing based on Npr3-CreER showed that endocardium contributes to a minority of coronary vessels in the free walls of embryonic heart. Intersectional genetic lineage tracing experiments demonstrated that endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls. CONCLUSIONS: Our study suggested that SV, but not endocardium, is the major origin for coronary endothelium in the embryonic ventricular free walls. This work thus resolves the recent controversy over the developmental origin of coronary endothelium, providing the basis for studying coronary vessel formation and regeneration after injury.