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Cells Isolated from Regenerating Caudal Fin of Sparus aurata Can Differentiate into Distinct Bone Cell Lineages

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Abstract

Teleosts have the ability to regenerate their caudal fin upon amputation. A highly proliferative mass of undifferentiated cells called blastema forms beneath wound epidermis and differentiates to regenerate all missing parts of the fin. To date, the origin and fate of the blastema is not completely understood. However, current hypotheses suggest that the blastema is comprised of lineage-restricted dedifferentiated cells. To investigate the differentiation capacity of regenerating fin-derived cells, primary cultures were initiated from the explants of 2-days post-amputation (dpa) regenerates of juvenile gilthead seabream (Sparus aurata). These cells were subcultured for over 30 passages and were named as BSa2. After 10 passages they were characterized for their ability to differentiate towards different bone cell lineages and mineralize their extracellular matrix, through immunocytochemistry, histology, and RT-PCR. Exogenous DNA was efficiently delivered into these cells by nucleofection. Assessment of lineage-specific markers revealed that BSa2 cells were capable of osteo/chondroblastic differentiation. BSa2 cells were also found to be capable of osteoclastic differentiation, as demonstrated through TRAP-specific staining and pit resorption assay. Here, we describe the development of the first successful cell line viz., BSa2, from S. aurata 2-dpa regenerating caudal fins, which has the ability of multilineage differentiation and is capable of in vitro mineralization. The availability of such in vitro cell systems has the potential to stimulate research on the mechanisms of cell differentiation during fin regeneration and provide new insights into the mechanisms of bone formation.

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Acknowledgments

The monoclonal antibodies used in this study were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. We thank Dr. Jorge Dias for providing animals for experiment.

Funding

This work was co-funded by the Portuguese Science and Technology Foundation (FCT) through projects PTDC/MAR/105313/2008 (FISHCELL) and UID/Multi/04326/2019 (CCMAR) and by the European Community (EC) through project ASSEMBLE (FP7/227799). PV was supported by a post-doctoral grant (SFRH/BPD/39189/2007) from the FCT.

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  1. João Cardeira

    Present address: Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231, Bad Nauheim, Germany

Authors and Affiliations

  1. Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    Parameswaran Vijayakumar, João Cardeira, Vincent Laizé, Paulo J. Gavaia & M. Leonor Cancela

  2. Department of Biomedical Sciences and Medicine (DCBM) and Algarve Biomedical Center (ABC), University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    M. Leonor Cancela

  3. Centre for Ocean Research, Sathyabama Institute of Science and Technology, Jeppiaar Nagar, Rajiv Gandhi Salai, Chennai, Tamil Nadu, 600 119, India

    Parameswaran Vijayakumar

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  1. Parameswaran Vijayakumar
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Correspondence to Parameswaran Vijayakumar or M. Leonor Cancela.

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Animal handling and experimentation was performed by qualified operators legally accredited by the Portuguese Direção Geral de Alimentação e Veterinária (DGAV) and following the EU and Portuguese legislation for animal experimentation and welfare (Directive 86/609/CEE and 2010/63/EU, Portaria 1005/92, 466/95 and 1131/97). Experimental procedures were performed under authorization (0421/000/000) from the DGAV, complying with the decreto de lei 113/2013 de 7 de Agosto, from the Portuguese legislation.

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Supplementary Fig. 9

Untreated cultures of BSa2 cells. Cells immunolabeled with anti-Osteocalcin 1 antibody (a–d); cells immunolabeled with anti-chondroitin sulphate proteoglycans antibody (e–h); cells immunolabeled with anti-collagen 2 antibody (i-l); Cells labeled with DAPI (blue) (a, e, i); cells labeled with Alexa (red) (b, f, j); DIC image (gray) (c, g, k); merged images of DAPI (blue), Alexa 594 (red) and DIC (gray) (d, h, l). Bar is 10 μm. (PNG 1.51 mb)

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Vijayakumar, P., Cardeira, J., Laizé, V. et al. Cells Isolated from Regenerating Caudal Fin of Sparus aurata Can Differentiate into Distinct Bone Cell Lineages. Mar Biotechnol 22, 333–347 (2020). https://doi.org/10.1007/s10126-019-09937-3

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