Model For Studying Regeneration Research Articles

An Efficient and Reproducible Method for the Isolation and Culture of Primary Cardiomyocytes from Adult Zebrafish.

Zebrafish is a popular animal model in regeneration studies due to their ability to regenerate the heart. Primary cardiomyocytes could be an alternative tool for studying the intrinsic mechanisms of cardiovascular disease in vitro. Thus, our objective is to develop an efficient protocol to isolate primary cardiomyocytes from zebrafish hearts. Low concentration of digestive enzyme (0.5 mg/mL collagenase type II) was utilized in our protocol to obtain single-cell suspension. The ventricles were fragmented, mechanically pipetted, and constantly shaken to ensure adequate contact between the tissues and the enzyme. Preplating the cell suspension onto culture plates for 2 h helped remove cardiac fibroblasts. The purity of isolated cells was validated by flow cytometry analysis of transgenic zebrafish with cardiomyocyte-specific expression of enhanced green fluorescent protein (EGFP) or endothelial cell-specific expression of mCherry. Quantitative real-time PCR analysis revealed a high level of the purity, with cardiac fibroblasts, endothelial cells, and epicardial cell markers scarcely detected in the purified cells. Altogether, this study established a reproducible protocol for isolating primary cardiomyocytes with high purity and activity from adult zebrafish hearts that can be cultured in vitro for up to 4 weeks. This protocol provides a valuable tool for studying the intrinsic mechanisms of cardiovascular disease in vitro using primary cardiomyocytes.

The Role of the Microbiota in Regeneration-Associated Processes.

The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Among these, is the relationship between the microbiota and regenerative processes in various organisms. Here we introduce the concept of the microbiota and its involvement in regeneration-related cellular events. We then review the role of the microbiota in regenerative models that extend from the repair of tissue layers to the regeneration of complete organs or animals. We highlight the role of the microbiota in the digestive tract, since it accounts for a significant percentage of an animal microbiota, and at the same time provides an outstanding system to study microbiota effects on regeneration. Lastly, while this review serves to highlight echinoderms, primarily holothuroids, as models for regeneration studies, it also provides multiple examples of microbiota-related interactions in other processes in different organisms.

Spawning induction, development and culturing of the solitary ascidian Polycarpa mytiligera, an emerging model for regeneration studies

BackgroundAscidians (phylum Chordata, class Ascidiacea) represent the closest living invertebrate relatives of the vertebrates and constitute an important model for studying the evolution of chordate development. The solitary ascidian Polycarpa mytiligera exhibits a robust regeneration ability, unique among solitary chordates, thus offering a promising new model for regeneration studies. Understanding its reproductive development and establishing land-based culturing methods is pivotal for utilizing this species for experimental studies. Its reproduction cycle, spawning behavior, and developmental processes were therefore studied in both the field and the lab, and methods were developed for its culture in both open and closed water systems.ResultsField surveys revealed that P. mytiligera’s natural recruitment period starts in summer (June) and ends in winter (December) when seawater temperature decreases. Laboratory experiments revealed that low temperature (21 °C) has a negative effect on its fertilization and development. Although spontaneous spawning events occur only between June and December, we were able to induce spawning under controlled conditions year-round by means of gradual changes in the environmental conditions. Spawning events, followed by larval development and metamorphosis, took place in ascidians maintained in either artificial or natural seawater facilities. P. mytiligera’s fast developmental process indicated its resemblance to other oviparous species, with the larvae initiating settlement and metamorphosis at about 12 h post-hatching, and reaching the juvenile stage 3 days later.ConclusionsPolycarpa mytiligera can be induced to spawn in captivity year-round, independent of the natural reproduction season. The significant advantages of P. mytiligera as a model system for regenerative studies, combined with the detailed developmental data and culturing methods presented here, will contribute to future research addressing developmental and evolutionary questions, and promote the use of this species as an applicable model system for experimental studies.

Ocular tentacle regeneration and associated malformations in Stramonita haemastoma (Gastropoda: Muricidae)

The present study aimed to elucidate the causal factor of tentacular malformations observed in Stramonita haemastoma. For this purpose, an annual, spatial and interspecific survey of the tentacular malformations was conducted in addition to the experimental semi-amputation of the tentacles of S. haemastoma. This study reports, for the first time, the occurrence of tentacular malformations in S. haemastoma collected for one year from the Bizerta Channel. Tentacle malformations were detected in 12 out of 17 Mediterranean and Atlantic coastal sites. Besides S. haemastoma, two sympatric muricid species (Hexaplex trunculus and Bolinus brandaris) also showed the occurrence of tentacular malformations. The experimental semi-amputation of the tentacles of S. haemastoma confirmed the capacity of regeneration in this gastropod; however, the process was often deficient and the neo-formed anterior segment was usually shorter, thinner and provided with a small ocellus. Sometimes, individuals were unable to regenerate a number of structures like the posterior segment and the ocellus, which may explain the tentacular malformations observed previously in S. haemastoma. This study provided new data about the incidence of tentacular malformations in marine gastropods. Our results further demonstrate the capacity of regeneration in S. haemastoma, which can be used as a new model in regeneration studies.

Whole-mount in situ hybridization using DIG-labeled probes in planarian.

In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts.

Space Research Program on Planarian Schmidtea Mediterranea’s Establishment of the Anterior-Posterior Axis in Altered Gravity Conditions

Planarians of the species Schmidtea mediterranea are a well-established model for regeneration studies. In this paper, we first recall the morphological characters and the molecular mechanisms involved in the regeneration process, especially focussing on the Wnt pathway and the establishment of the antero-posterior axial polarity. Then, after an assessment of a space-experiment (run in 2006 on the Russian Segment of the International Space Station) on planarians of the species Girardia tigrina, we present our experimental program to ascertain the effects that altered-gravity conditions may have on regeneration processes in S. mediterrnea at the molecular and genetic level.

Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis

BackgroundThe contribution of cell proliferation to regeneration varies greatly between different metazoan models. Planarians rely on pluripotent neoblasts and amphibian limb regeneration depends upon formation of a proliferative blastema, while regeneration in Hydra can occur in the absence of cell proliferation. Recently, the cnidarian Nematostella vectensis has shown potential as a model for studies of regeneration because of the ability to conduct comparative studies of patterning during embryonic development, asexual reproduction, and regeneration. The present study investigates the pattern of cell proliferation during the regeneration of oral structures and the role of cell proliferation in this process.ResultsIn intact polyps, cell proliferation is observed in both ectodermal and endodermal tissues throughout the entire oral-aboral axis, including in the tentacles and physa. Following bisection, there is initially little change in proliferation at the wound site of the aboral fragment, however, beginning 18 to 24 hours after amputation there is a dramatic increase in cell proliferation at the wound site in the aboral fragment. This elevated level of proliferation is maintained throughout the course or regeneration of oral structures, including the tentacles, the mouth, and the pharynx. Treatments with the cell proliferation inhibitors hydroxyurea and nocodazole demonstrate that cell proliferation is indispensable for the regeneration of oral structures. Although inhibition of regeneration by nocodazole was generally irreversible, secondary amputation reinitiates cell proliferation and regeneration.ConclusionsThe study has found that high levels of cell proliferation characterize the regeneration of oral structures in Nematostella, and that this cell proliferation is necessary for the proper progression of regeneration. Thus, while cell proliferation contributes to regeneration of oral structures in both Nematostella and Hydra, Nematostella lacks the ability to undergo the compensatory morphallactic mode of regeneration that characterizes Hydra. Our results are consistent with amputation activating a quiescent population of mitotically competent stem cells in spatial proximity to the wound site, which form the regenerated structures.

Hydrobia ulvae (Gastropoda: Prosobranchia): a new model for regeneration studies.

Within 2 weeks of decapitation, Hydrobia ulvae was able to regenerate new head structures including buccal ganglia. It was also capable of regenerating propodial ganglia after anterior foot amputation. The functional regeneration of the buccal ganglia was demonstrated by behavioural observations and by electrophysiological experiments. The presence of the oesophagus was shown to be important for regeneration of the buccal complex. H. ulvae provides a new model for regeneration studies, so details of the topographic anatomy and biology of this species are described. To standardize experimental animals in future studies, the effects of age, sex and trematode infestation on the regeneration capacity of H. ulvae have been evaluated. The high capacity for regeneration together with the possibility of using electrophysiological techniques makes H. ulvae a favourable model in which to study neurogenesis in adult animals.

Characterization of a new in vitro model for studies of reepithelialization in human partial thickness wounds.

Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5-7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this human in vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement to in vivo studies on epidermal healing.

Autologous peripheral nerve grafting into murine brain as a model for studies of regeneration in the central nervous system

Autologous sciatic nerve was grafted into rat brain by (i) passing an 8-mm segment of nerve tied to a straight surgical needle through two craniotomy holes (“through-and-through” model); (ii) inserting a small tube of polyethylene containing the 8-mm nerve piece (“nerve-within-tube” model). Longitudinally oriented neurofilament-positive fibers were consistently observed within the graft. Compared with the through-and-through model, axonal sprouting in the nerve-within-tube model followed a slow-motion pattern so that a growing front of regenerating axons could be easily identified and more easily related to the cellular events occurring in Wallerian degeneration. In the through-and-through model, regenerated axons at the brain-nerve interface followed a disorganized, tortuous course so that direct continuity between brain and graft was difficult to demonstrate. The reverse was true in the nerve-within-tube model, i.e., axons penetrated directly into the graft. The difference in orientation of axonal growth at the brain-graft interface appeared to be related to the glial reaction. In the through-and-through model, reactive astrocytes formed a mesh of randomly oriented fibers in the damaged brain tissue facing the graft (anisomorphic gliosis). Conversely, longitudinally oriented fibers extended directly from the brain to the graft in the nerve-within-tube model, where brain damage was substantially reduced (isomorphic gliosis). A different type of glial fibrillary acidic (GFA) protein-positive fibrous structures was identified in the graft. Compared with reactive astrocytes, these structures were more elongated, more uniform in diameter, and less brightly immunofluorescent. Moreover, they were present throughout the graft, whereas astrocytes were confined to the distal end of the transplant, i.e., the part of nerve close to the brain-graft interface. Based on previous reports in the literature we interpret these GFA protein-positive structures as reacting Schwann cells.