Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies

Cell culture is an invaluable tool for investigation of basic biological processes. However, technical hurdles including low cell yield, poor cell differentiation and poor attachment to the growth substrate have limited the use of this tool for studies of the genetic model organism Caenorhabditis elegans. This protocol describes a method for the large-scale culture of C. elegans embryo cells. We also describe methods for in vitro RNA interference, fluorescence-activated cell sorting of embryo cells and imaging of cultured cells for patch-clamp electrophysiology studies. Developing embryos are isolated from gravid adult worms. After eggshell removal by enzymatic digestion, embryo cells are dissociated and plated onto glass substrates. Isolated cells terminally differentiate within 24 h. Analysis of gene expression patterns and cell-type frequency suggests that in vitro embryo cell cultures recapitulate the developmental characteristics of L1 larvae. Cultured embryo cells are well suited for physiological analysis as well as molecular and cell biological studies. The embryo cell isolation protocol can be completed in 5-6 h.     

Isolation and expansion of high yield of pure mesenchymal stromal cells from fresh and cryopreserved placental tissues

The injection of placental stromal cells isolated from fetal human tissues (f-hPSC) was reported to indirectly induce tissue regeneration in different animal models. A procedure of f-hPSC isolation from fragments of both selected fresh or cryopreserved bulk placental neonate tissues is proposed, based on their high migratory potential,. The fragments of the desired fetal placental tissues are adhered to a culture dish by traces of diluted fibrin and covered with culture medium. Spontaneous migration of pure f-hPSC from the tissue fragments to the cell culture dishes is followed by their rapid expansion by numerous passages. The isolated f-hPSC express typical mesenchymal surface antigens, including CD29, CD105, CD166 and CD146, with negative expression of white blood cell lineage and endothelial cells markers. Optimal yields of f-hPSC cultures can also be obtained from tissue samples cryopreserved in medium composed of 10% dimethyl sulfoxide (M₂SO) and 50% fetal calf serum. Slightly better yields are obtained with media supplemented with 1% human albumin. Medium with 5% M₂SO and/or 0.25 mg/ml PEG yielded inferior results. The f-hPSC from fresh or cryopreserved tissues express similar cell markers and growth kinetics. The proposed isolation protocol may also be applied for high yield isolation of stromal cells from fresh and cryopreserved tissue of other organs.     

Establishment and characterization of an immortalized epicardial cell line

Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40-LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.     

Adoptive transfer of acute lung injury

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.     

Heart on a Plate: Histological and Functional Assessment of Isolated Adult Zebrafish Hearts Maintained in Culture

The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min⁻¹) declined significantly between day 0 and day 1 in culture (96.7±19.5 to 45.2±8.2 min⁻¹, mean±SD, p = 0.001), and thereafter declined more slowly to 27.6±7.2 min⁻¹ on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7±13.0 µm vs day 4, 16.9±1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6±14.8 vs 15.2±5.3 µms⁻¹, respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5±11.9 vs day 1, 29.5±5.8 µms⁻¹, p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival.     

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure¹. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury¹. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized^1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules^1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types³. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely^1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals^4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish⁶. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish^7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.     

A primary culture technique of adult retina for regeneration studies on adult CNS neurons

This protocol details a tissue culture technique that allows for quantified regeneration studies on adult retinal ganglion cells (RGCs), that is, CNS neurons. The method may also allow for elucidation of molecular cues, for example of signals relevant in neuronal survival and axon regeneration. The procedure relies on fractioned stripe culture of previously injured retina in defined culture media. Naive dendritic cell contacts of RGCs are preserved, and the system is independent of growth factors. In contrast to other techniques, the protocol is based on tissue grown from adult animals; it dispenses immature co-cultures and evaluates the outgrowth of unmyelinated neurites in a milieu lacking CNS myelin. The technique is suitable for rodent retina from mouse or rat. A growth-conditioning injury of the optic nerve is set 10 days before retinal explantation. Explants are cultured for 5-7 days. Mere preparation of a single retina should be completed within 20 min.     

Treatment of Glucocorticoids Inhibited Early Immune Responses and Impaired Cardiac Repair in Adult Zebrafish

Myocardial injury, such as myocardial infarction (MI), can lead to drastic heart damage. Zebrafish have the extraordinary ability to regenerate their heart after a severe injury. Upon ventricle resection, fibrin clots seal the wound and serve as a matrix for recruiting myeloid-derived phagocytes. Accumulated neutrophils and macrophages not only reduce the risk of infection but also secrete cytokines and growth factors to promote tissue repair. However, the underlying cellular and molecular mechanisms for how immune responses are regulated during the early stages of cardiac repair are still unclear. We investigated the role and programming of early immune responses during zebrafish heart regeneration. We found that zebrafish treated with an anti-inflammatory glucocorticoid had significantly reduced heart regenerative capacities, consistent with findings in other higher vertebrates. Moreover, inhibiting the inflammatory response led to excessive collagen deposition. A microarray approach was used to assess the differential expression profiles between zebrafish hearts with normal or impaired healing. Combining cytokine profiling and immune-staining, our data revealed that impaired heart regeneration could be due to reduced phagocyte recruitment, leading to diminished angiogenesis and cell proliferation post-cardiac injury. Despite their robust regenerative ability, our study revealed that glucocorticoid treatment could effectively hinder cardiac repair in adult zebrafish by interfering with the inflammatory response. Our findings may help to clarify the initiation of cardiac repair, which could be used to develop a therapeutic intervention that may enhance cardiac repair in humans to compensate for the loss of cardiomyocytes after an MI.     

Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies

Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chemical and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, we provide a step-by-step protocol for a hybrid chemical-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the reduction of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR(+) cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.     

Fibronectin is deposited by injury-activated epicardial cells and is necessary for zebrafish heart regeneration

Unlike adult mammals, adult zebrafish vigorously regenerate lost heart muscle in response to injury. The epicardium, a mesothelial cell layer enveloping the myocardium, is activated to proliferate after cardiac injury and can contribute vascular support cells or provide mitogens to regenerating muscle. Here, we applied proteomics to identify secreted proteins that are associated with heart regeneration. We found that Fibronectin, a main component of the extracellular matrix, is induced and deposited after cardiac damage. In situ hybridization and transgenic reporter analyses indicated that expression of two fibronectin paralogues, fn1 and fn1b, are induced by injury in epicardial cells, while the itgb3 receptor is induced in cardiomyocytes near the injury site. fn1, the more dynamic of these paralogs, is induced chamber-wide within one day of injury before localizing epicardial Fn1 synthesis to the injury site. fn1 loss-of-function mutations disrupted zebrafish heart regeneration, as did induced expression of a dominant-negative Fibronectin cassette, defects that were not attributable to direct inhibition of cardiomyocyte proliferation. These findings reveal a new role for the epicardium in establishing an extracellular environment that supports heart regeneration.     

Role of the cytoskeleton during injury-induced cell migration in corneal endothelium

The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming reestablished. When injured corneas are placed in media containing 5 x 10(-7) M cytochalasin B, endothelial cell migration occurs; but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10(-8) M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 micrograms/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.     

Cell tracking using a photoconvertible fluorescent protein

The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS-Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two-photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2-8 d.     

A Surgery Protocol for Adult Zebrafish Spinal Cord Injury

Adult zebrafish has a remarkable capability to recover from spinal cord injury,providing an excellent model for studying neuro-regeneration. Here we list equipment and reagents,and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol,potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed.In addition,two assessments are introduced to monitor the success of the surgery and functional recovery:one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing.In the swimming behavior test,successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury,followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury.As a morphometric correlate,anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury,confirming functional recovery.This zebrafish model provides a paradigm for recovery from spinal cord injury,enabling the identification of pathways and components of neuroregeneration.     

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.     

Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish

The heart maintains structural and functional integrity during years of continual contraction, but the extent to which new cell creation participates in cardiac homeostasis is unclear. Here, we assessed cellular and molecular mechanisms of cardiac homeostasis in zebrafish, which display indeterminate growth and possess an unusual capacity to regenerate after acute cardiac injury. Lowering fish density in the aquarium triggered rapid animal growth and robust cardiomyocyte proliferation throughout the adult ventricle, greater than that observed during slow animal growth or size maintenance. Rapid animal growth also induced strong expression of the embryonic epicardial markers raldh2 (aldh1a2) and tbx18 in adult epicardial tissue. Pulse-chase dye labeling experiments revealed that the epicardium recurrently contributes cells to the ventricular wall, indicating an active homeostatic process. Inhibition of signaling by Fibroblast growth factors (Fgfs) decreased this epicardial supplementation of the ventricular wall in growing zebrafish, and led to spontaneous ventricular scarring in animals maintaining cardiac size. Our results demonstrate that the adult zebrafish ventricle grows and is maintained by cardiomyocyte hyperplasia, and that epicardial cells are added to the ventricle in an Fgf-dependent fashion to support homeostasis.     

Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM epsilon-aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen-depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.