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Formation of the fibrotic reticulum. Representative images of the IPF and HLF grafts localized to the head and trunk regions of the 1 and 2 dpf host embryos. Left panel: Fluorescence: High cell motility is characteristic of the IPF grafts in 2 dpf embryos. Arrows pointing at the processes, wedge indicating the graft margin, * marking the foci. Yolk autofluorescence (background) and no signal in head and trunk regions in embryos without grafts (CON). Right panel: Brightfield images showing graft-related malformations in the head (unilateral anophthalmia and microphthalmia) and trunk (lump, axial deformities) regions in the graft-bearing embryo, and no deformities in embryos without grafts (CON); e, eye; ov, otic vesicle; p, pericardium; Y, yolk sack. Lateral view. Scale bar: 250 lm.
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Fibroblasts have a central role in the maintenance of tissue homeostasis and repair after injury. Currently, there are no tractable, cost-effective model systems for studying the biology of human fibroblasts in vivo. Here we demonstrate that primary human fibroblasts survive transplantation into zebrafish embryos. Transplanted cells migrate and pro...
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... pushed towards the blastula apex mechanically by host cells. According to the zebrafish fate map, 9 the apical position predetermines cell restriction to the head region of the developing embryo. Indeed, out of 115 grafts we inspected, the majority of grafts (80%) were ob- served in the head and only 20% in the trunk region of the host embryos (Fig. 2, left panel). All grafts remained visible in live embryos for 3 ...
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... a variety of deformities. As evidenced by fluorescence microscopy, grafts localized to the posterior head and trunk regions caused trunk and tail un- derdevelopment and malformations, while grafts positioned in the anterior and mid-head regions predominantly hindered brain development, causing microphthalmia, bilateral or unilateral anophthalmia (Fig. 2, right panel). Of note, phe- notype screening for malformations greatly facilitated selec- tion of graft-bearing embryos at 2 days post fertilization (dpf). Despite these abnormalities, the majority of host embryos survived for 5-6 ...
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... for 3 days. During this period, there were major changes in graft shape and fluorescence intensity. At day 1, grafts were compact, show- ing strong adhesion of the CSFE-positive cells to each other. Grafts were spheroid in shape, with indistinct outer edges and a few (2 to 5) short protrusions of approximately 0.1 to 0.5 graft diameters in length (Fig. 2, upper panel). By day 2, graft Margin size width (lm) Foci, N Processes, N outgrowth increased significantly and graft shape became irregular. The central highly fluorescent area was surrounded by an uneven fine mesh-like margin with diminished fluo- rescence due to partitioning of the CFSE dye between daughter cells as engrafted cells proliferated. ...
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... margin with diminished fluo- rescence due to partitioning of the CFSE dye between daughter cells as engrafted cells proliferated. In other regions there were highly fluorescent processes resulting from cell migration. The processes varied in number, length, and tra- jectory, with occasional focal swellings at the outer surface of the host embryos (Fig. 2, mid-and lower panels). Of note, these reticular features were independent of graft location (head vs. trunk) or type/severity of host embryo malforma- tion (Fig. 2, mid-panels). By day 3, graft fluorescence was diminished with multiple areas of reduced fluorescence ob- served in the embryos (data not ...
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... fluorescent processes resulting from cell migration. The processes varied in number, length, and tra- jectory, with occasional focal swellings at the outer surface of the host embryos (Fig. 2, mid-and lower panels). Of note, these reticular features were independent of graft location (head vs. trunk) or type/severity of host embryo malforma- tion (Fig. 2, mid-panels). By day 3, graft fluorescence was diminished with multiple areas of reduced fluorescence ob- served in the embryos (data not ...
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Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progeni...
Citations
... Therefore, the murine model has limitations in translating therapeutic treatments to humans, which might be overcome with the utilization of preclinical models that include human lung tissue with established fibrosis or IPF tissue-derived cells. Accordingly, IPF lung-derived fibroblasts have previously been xenografted in immunodeficient mice and zebrafish models to study biological processes and drug response (15,16), whereas human lung tissues have been cultivated ex vivo as precision-cut lung slices (PCLS), in vitro explants, and pulmospheres (17)(18)(19), which lack interaction with a whole organism. ...
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with no curative pharmacological treatment. Current preclinical models fail to accurately reproduce human pathophysiology and are therefore poor predictors of clinical outcomes. Here, we investigated whether the chick embryo chorioallantoic membrane (CAM) assay supports the implantation of xenografts derived from IPF lung tissue and primary IPF lung fibroblasts and can be used to evaluate the efficacy of antifibrotic drugs. We demonstrate that IPF xenografts maintain their integrity and are perfused with chick embryo blood. Size measurements indicate that the xenografts amplify on the CAM, and Ki67 and pro-collagen type I immunohistochemical staining highlight the presence of proliferative and functional cells in the xenografts. Moreover, the IPF phenotype and immune microenvironment of lung tissues are retained when cultivated on the CAM and the fibroblast xenografts mimic invasive IPF fibroblastic foci. Daily treatments of the xenografts with nintedanib and PBI-4050 significantly reduce their size, fibrosis-associated gene expression, and collagen deposition. Similar effects are found with GLPG1205 and fenofibric acid, two drugs that target the immune microenvironment. Our CAM-IPF model represents the first in vivo model of IPF that uses human lung tissue. This rapid and cost-effective assay could become a valuable tool for predicting the efficacy of antifibrotic drug candidates for IPF.
... In our study, we were able to demonstrate that the zebrafish provides human cells with an appropriate niche for them to survive. We and others have proved that not only cancer cell lines can be detected into embryos, but also primary human cancer and healthy cells exhibited the ability to establish and migrate (24,(60)(61)(62). Moreover, other investigations have demonstrated that zebrafish maintain the cancerous phenotype of leukemic cell lines (63,64). ...
Acute leukemia is a heterogeneous set of diseases affecting children and adults. Current prognostic factors are not accurate predictors of the clinical outcome of adult patients and the stratification of risk groups remains insufficient. For that reason, this study proposes a multifactorial analysis which integrates clinical parameters, ex vivo tumor characterization and behavioral in vivo analysis in zebrafish. This model represents a new approach to understand leukemic primary cells behavior and features associated with aggressiveness and metastatic potential. Xenotransplantation of primary samples from patients newly diagnosed with acute leukemia in zebrafish embryos at 48 hpf was used to asses survival rate, dissemination pattern, and metastatic potential. Seven samples from young adults classified in adverse, favorable or intermediate risk group were characterized. Tumor heterogeneity defined by Leukemic stem cell (LSC) proportion, was performed by metabolic and cell membrane biomarkers characterization. Thus, our work combines all these parameters with a robust quantification strategy that provides important information about leukemia biology, their relationship with specific niches and the existent inter and intra-tumor heterogeneity in acute leukemia. In regard to prognostic factors, leukemic stem cell proportion and Patient-derived xenografts (PDX) migration into zebrafish were the variables with highest weights for the prediction analysis. Higher ALDH activity, less differentiated cells and a broader and random migration pattern are related with worse clinical outcome after induction chemotherapy. This model also recapitulates multiple aspects of human acute leukemia and therefore is a promising tool to be employed not only for preclinical studies but also supposes a new tool with a higher resolution compared to traditional methods for an accurate stratification of patients into worse or favorable clinical outcome.
... The transparency of the zebrafish identifies it as a good model for the investigation of vascular development. Moreover, zebrafish mutually share many structural, functional, and molecular features with other vertebrates and are perfect models for xenotransplantation due to their inability to reject graft within 48hpf ( Benyumov et al, 2012;Jung et al, 2017;Mulligan and Weinstein, 2014). In vertebrates, lymphangiogenesis begins from hematopoietic stem cells (HSC) fate determination, through several differentiation steps, to develop arterial and venous progenitors ( Nicenboim et al, 2015). ...
Prostaglandin (PG)-E2 is essential for growth and development of vertebrates. PGE2 binds to G-coupled receptors to regulate embryonic stem cell differentiation and maintains tissue homeostasis. Overproduction of PGE2 by breast tumor cells promotes aggressive breast cancer phenotypes and tumor-associated lymphangiogenesis. In this study, we investigated novel roles of PGE2 in early embryonic vascular development and maturation with the microinjection of PGE2 in fertilized zebrafish (Danio rerio) eggs. We injected Texas Red dextran to trace vascular development. Embryos injected with the solvent of PGE2 served as vehicle. Distinct developmental changes were noted from 28–96 h post fertilization (hpf), showing an increase in embryonic tail flicks, pigmentation, growth, hatching and larval movement post-hatching in the PGE2-injected group compared to the vehicle. We recorded a significant increase in trunk vascular fluorescence and maturation of vascular anatomy, embryo heartbeat and blood vessel formation in the PGE2 injected group. At 96 hpf, all larvae were euthanized to measure vascular marker mRNA expression. We observed a significant increase in the expression of stem cell markers efnb2a, ephb4a, angiogenesis markers vegfa, kdrl, etv2 and lymphangiogenesis marker prox1 in the PGE2-group compared to the vehicle. This study shows the novel roles of PGE2 in promoting embryonic vascular maturation and angiogenesis in zebrafish.
This article has an associated First Person interview with the first author of the paper.
... In parallel with our increased understanding of fibrosis initiation, we have learned that fibrosis progression involves both cell-intrinsic/autonomous and ECM-driven mechanisms. Cellautonomous fibrogenicity was initially identified in studies using primary mesenchymal cells from fibrotic tissue and organs (15)(16)(17)(18)(19)(20), corroborated in zebrafish and mouse xenograft models (21,22), and verified in mouse lineage tracing studies (23). The discovery of fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of patients with IPF provided definitive proof in humans (9,24). ...
The extracellular matrix (ECM) is dynamically tuned to optimize physiological function. Its major properties, including composition and mechanics, profoundly influence cell biology. Cell-ECM interactions operate through an integrated set of sensor and effector circuits that use several classes of receptors and signal transduction pathways. At the single-cell level, the ECM governs differentiation, metabolism, motility, orientation, proliferation, and survival. At the cell population level, the ECM provides higher-order guidance that is essential for physiological function. When pathological changes in the ECM lead to impairment of organ function, we use the term "fibrosis." In this Review, we differentiate fibrosis initiation from progression and focus primarily on progressive lung fibrosis impairing organ function. We present a working model to explain how the altered ECM is not only a consequence but also a driver of fibrosis. Additionally, we advance the concept that fibrosis progression occurs in a fibrogenic niche that is composed of a fibrogenic ECM that nurtures fibrogenic mesenchymal progenitor cells and their fibrogenic progeny.
... Typical readouts allowing for quantification of the behavior of transplanted tumor cells in the fish host are proliferation, migration, and neovascularization. Proliferation of transplanted tumor cells can be investigated in a straightforward way by using available human-specific anti-ki67 antibodies (88,103). Neovascularization can be visualized easily by performing xenotransplantation into transgenic zebrafish strains with fluorescently labeled vasculature (104,105). ...
Over the last decade, zebrafish has proven to be a powerful model in cancer research. Zebrafish form tumors that histologically and genetically resemble human cancers. The live imaging and cost-effective compound screening possible with zebrafish especially complement classic mouse cancer models. Here, we report recent progress in the field, including genetically engineered zebrafish cancer models, xenotransplantation of human cancer cells into zebrafish, promising approaches toward live investigation of the tumor microenvironment, and identification of therapeutic strategies by performing compound screens on zebrafish cancer models. Given the recent advances in genome editing, personalized zebrafish cancer models are now a realistic possibility. In addition, ongoing automation will soon allow high-throughput compound screening using zebrafish cancer models to be part of preclinical precision medicine approaches.
... Three reports describe transplantation of adipocyte-derived and mesenchymal stem cells and show cell survival [92][93][94]. Melanocytes and fibroblasts have also been transplanted into blastoderm embryos and were shown to survive for several days [71,[94][95][96]. The possibility to integrate human iPS cells in the zebrafish could hold great promise. ...
The goal of personalised medicine is to develop tailor-made therapies for patients in whom currently available therapeutics fail. This approach requires correlating individual patient genotype data to specific disease phenotype data and using these stratified data sets to identify bespoke therapeutics. Applications for personalised medicine include common complex diseases which may have multiple targets, as well as rare monogenic disorders, for which the target may be unknown. In both cases, whole genome sequence analysis (WGS) is discovering large numbers of disease associated mutations in new candidate genes and potential modifier genes. Currently, the main limiting factor is the determination of which mutated genes are important for disease progression and therefore represent potential targets for drug discovery. Zebrafish have gained popularity as a model organism for understanding developmental processes, disease mechanisms and more recently for drug discovery and toxicity testing. In this chapter, we will examine the diverse roles that zebrafish can make in the expanding field of personalised medicine, from generating humanised disease models to xenograft screening of different cancer cell lines, through to finding new drugs via in vivo phenotypic screens. We will discuss the tools available for zebrafish research and recent advances in techniques, highlighting the advantages and potential of using zebrafish for high throughput disease modeling and precision drug discovery.
... In addition, with the help of automated image-based phenotypic analysis in zebrafish, this swim bladder platform is amenable to automation necessary for high-throughput drug screening. 16 Zebrafish models have been utilized to investigate the pathogeneses of lung diseases, including idiopathic pulmonary fibrosis modeled by embryo xenotransplantation, 17 the host-pathogen interactions that occur after bacterial infections 18 and tailfin injury-induced neutrophilic inflammation. 19 Nevertheless, these studies do not provide direct and definitive evidence for the feasibility and effectiveness of zebrafish as an in vivo model for lung diseases. ...
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment. To date, in vivo functional approaches have been limited by the inaccessibility to the alveolar sacs, which are located at the anatomical terminal of the respiratory duct in mammals. We are the first to characterize the swim bladder of the zebrafish larva, which is similar to the mammalian lung, as a real-time in vivo model for examining pulmonary neutrophil infiltration during ALI. We observed that the delivery of exogenous materials, including lipopolysaccharide (LPS), Poly IC and silica nanoparticles, by microinjection triggered significant time- and dose-dependent neutrophil recruitment into the swim bladder. Neutrophils infiltrated the LPS-injected swim bladder through the blood capillaries around the pneumatic duct or a site near the pronephric duct. An increase in the post-LPS inflammatory cytokine mRNA levels coincided with the in vivo neutrophil aggregation in the swim bladder. Microscopic examinations of the LPS-injected swim bladders further revealed in situ injuries, including epithelial distortion, endoplasmic reticulum swelling and mitochondrial injuries. Inhibitor screening assays with this model showed a reduction in neutrophil migration into the LPS-injected swim bladder in response to Shp2 inhibition. Moreover, the pharmacological suppression and targeted disruption of Shp2 in myeloid cells alleviated pulmonary inflammation in the LPS-induced ALI mouse model. Additionally, we used this model to assess pneumonia-induced neutrophil recruitment by microinjecting bronchoalveolar lavage fluid from patients into swim bladders; this injection enhanced neutrophil aggregation relative to the control. In conclusion, our findings highlight the swim bladder as a promising and powerful model for mechanistic and drug screening studies of alveolar injuries.
... In addition, with the help of automated image-based phenotypic analysis in zebrafish, this swim bladder platform is amenable to automation necessary for high-throughput drug screening. 16 Zebrafish models have been utilized to investigate the pathogeneses of lung diseases, including idiopathic pulmonary fibrosis modeled by embryo xenotransplantation, 17 the host-pathogen interactions that occur after bacterial infections 18 and tailfin injury-induced neutrophilic inflammation. 19 Nevertheless, these studies do not provide direct and definitive evidence for the feasibility and effectiveness of zebrafish as an in vivo model for lung diseases. ...
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment.
... 通讯作者：严继舟。Email: jyan2@shou.edu.cn 扩散并形成囊块，并且在囊块内部斑马鱼的血管内皮细胞会被诱导生成 [7] ；人类和鼠类的肿 瘤细胞在移植到受精后 48 小时的斑马鱼的肠下静脉附近时，其所表达生血管成纤维细胞生 长因子和血管内皮生长因子会诱导新的微脉管系统的快速形成 [8] ；乳腺癌细胞系移植到斑马 鱼胚胎后，迁移到胚胎的尾部形成囊块 [9] ；人类胶质瘤细胞在移植到斑马鱼胚胎后，能增殖 生成肿瘤，且这些肿瘤能诱导血管新生 [10] ；向发育中的斑马鱼移植具有扩散性的黑色素瘤 细胞，后者会分泌一种强力的胚胎类似物，导致胚胎体轴的发育异常 [11] 。斑马鱼异种移植 模型也被用来代替鼠类模型，快速高效得评估人类多能干细胞来源的内皮细胞的功能 [12] Fig.5 The fluorescence observation of AB donor cell signals in the brain of AF9 larval recipients 3 讨论 本实验的目的是想利用斑马鱼为模型研究干细胞移植治疗脑神经细胞退化的可行性。 为 此首先检验了干细胞移植后的致癌性，其次是移植细胞增生分化和细胞替代治疗的能力。 许多报道显示人类肿瘤细胞移植入斑马鱼胚胎、 仔鱼或肝脏都表现出侵润和微转移现象 [20] ；斑马鱼胚胎移植人类恶性胶质瘤细胞和其中的胶质瘤干细胞后，在胶质瘤干细胞的促进 下，胶质瘤细胞沿斑马鱼胚胎的血管快速扩散，并表现出强烈的侵润性 [21] ；胶质瘤细胞可以 在斑马鱼胚胎中存活、增殖并诱导血管分支的形成 [22] 。 MLL-AF9 是一种白血病基因，MLL-AF9 在人类 CD34+细胞中表达会引起严重的淋巴 系白血病、髓系白血病或混合型白血病。本次实验将转人类 MLL-AF9-GFP 基因的斑马鱼的 胚胎细胞移植到正常 AB 品系斑马鱼仔鱼脑部，同时也将正常 AB 品系斑马鱼的胚胎细胞移 植到 MLL-AF9-GFP 转基因的斑马鱼仔鱼的脑部， 以此观察带有白血病基因的细胞在正常斑 马鱼体内的致病性和正常的斑马鱼细胞治疗白血病基因表达细胞的可能性。 细胞移植结果显 示带有白血病的细胞在正常斑马鱼仔鱼的脑部没有增殖的迹象，移植细胞数量逐渐减少，无 法像癌细胞一样形成囊块； 相反移植到 MLL-AF9-GFP 转基因斑马鱼仔鱼的脑部的正常胚胎 细胞表现出有限增殖。虽然移植细胞团逐渐增大，但远离转基因细胞团。这些结果不仅证实 微环境对于白血病干细胞的影响较大 [23] ，也提示移植的胚胎细胞和转基因细胞分属两个不 同微环境，而斑马鱼体内微环境更适合于正常细胞移植和生成。 MLL-AF9 转基因鱼细胞移植实验说明斑马鱼有很强抗癌变和抗外源细胞整合的能力。 许多报道也发现正常细胞和多种干细胞及少部分肿瘤细胞， 移植到斑马鱼胚胎和仔鱼后只能 增殖和扩散，无法形成囊块，也很少整合到斑马鱼组织。人类成纤维细胞移植到受精后 2 天斑马鱼的卵黄囊时，会发生迁移，但不会增殖扩散并形成囊块 [7] ；人类脂肪干细胞在移植 到斑马鱼胚胎时，不仅能够存活和增殖，而且在短时间内保持其未分化的状态 [24] ；人类严 重髓性白血病细胞和胚细胞移植到受精后 48 小时的斑马鱼体内时，会停留在循环系统内数 日，且不影响斑马鱼的发育 [25] ；人类黑色素瘤细胞在移植到斑马鱼囊胚期胚胎时，存活并 扩散，但黑色素瘤细胞并没有形成肿瘤或整合入宿主器官，而是分散在细胞间隙中，倾向于 分布在正常的皮肤微环境 [26] ；人类胚胎干细胞来源的内皮细胞异种移植到斑马鱼胚胎后， 只能形成不稳定的管脉系统，不能整合入斑马鱼的血管 [27] ；人类纤维细胞移植入斑马鱼胚 胎后，迁移并增殖，但没有整合入宿主的组织 [28] ...
To establish a brain cell transplant model,here Danio rerio embryo cells were labeled with red fluorescent dye and separately transplanted into the larval brains of AB line and two different transgenic D. rerio. Fluorescence microscopic and histological observations showed the transplanted cells were migrated along interstitial / intercellular space instead of blood vessels,revealing little proliferation and differentiation. 24 hours after transplantation,the brain histological structure looked similar to the control. The transplanted embryo cells from the leukemia transgenic fish did not display any neoplasm proliferation and metastasis in AB larvae. In comparison,the transplant cells from AB lines showed a bit more proliferation and migration at the region far from leukemia cell aggregate in the brain of leukemia transgenic fish. These results suggested that the brain microenvironment was not suitable for the growth of the cell transplants,thereby cell transplant cannot replace the existing cells but fill a few cell sparsities.
... These authors defined a new model for in vivo study of inflammation resolution and their link with apoptosis (Renshaw et al., 2007). By other way, Benyumov et al. (2012) developed an in vivo zebrafish model to test phenotypic differences between human fibroblasts that participate in physiological and pathological process. ...
Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host-pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host-pathogen interactions.
