Intern
    Institut für Hygiene und Mikrobiologie

    In vitro cultivation of Echinococcus larvae/stem cells and parasite development

    To investigate host-helminth interaction and helminth infection strategies on the molecular level, the availability of suitable in vitro cultivation systems is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out is the larval stage of the fox-tapeworm E. multilocularis. Respective ‘first generation’ cultivation systems, developed by us and others, relied on the co-incubation of larval tissue with host feeder cells (Brehm & Spiliotis, 2008; Brehm, 2010; see figure 1 below). We later introduced the first axenic (host cell-free) system for long-term in vitro maintenance of metacestode vesicles (Spiliotis et al., 2004). In this system, parasite larvae can be kept for several weeks or months in the complete absence of host cells when an anaerobic environment and host-cell conditioned medium are applied. Under these conditions, the larvae proliferate and differentiate towards the protoscolex stage (see figure 2 below). Using the axenic cultivation system, we also solved a classical problem of molecular and cellular helminthology: the establishment of primary cell cultures (Spiliotis et al., 2008; Spiliotis and Brehm, 2009). In this system we use metacestode vesicles as a source of parasite stem cells which, after several weeks of cultivation, completely regenerate new vesicles in a manner that closely resembles the oncosphere-metacestode transition during natural infections (Spiliotis et al., 2008; Olson et al., 2012; see figure 3 below). Very recently, we also demonstrated that parasite development is driven by a population of pluripotent stem cells that are capable of constant self-renewal, that are the only proliferative cell type in Echinococcus, and that can give rise to all differentiated cells of the parasite (Koziol et al., submitted). Furthermore, we characterized the nervous system of the protoscolex stage and demonstrated that the non-motile metacestode stage is equipped with a nervous system (or nerve-net) (Koziol et al., 2013). Our in vitro – cultivation methods constitute important tools for molecular investigations on host-parasite interactions in alveolar echinococcosis and on the roles of totipotent germinative (stem) cells in parasite development and metastases formation. First investigations to establish transgenic techniques and to apply RNA-interference to Echinococcus larvae have also been carried out (Spiliotis et al., 2008; Spiliotis et al., 2010).

     

    In vitro co-cultivation of E. multilocularis metacestode vesicles with host cells (in the collagen sandwich system). Left: starting culture. Right: after 8 weeks of incubation.
    Axenic (host-cell free) cultivation of E. multilocularis metacestode vesicles.
    In vitro cultivation of E. multilocularis germinal cells and complete regeneration of metacestode vesicles. A) In vitro cultivated germinal cells (large nucleus; large nucleolus). B) In vitro vesicle formation after 4 weeks of incubation. Not cell aggregates and forming vesicles (arrow). C) SEM of cell aggregate. D) TEM of cell aggregates. Note forming vesicle with central cavity surrounded by germinal cells.

    References:

    Koziol, U., Rauschendorfer, T., Zanon-Rodriguez, L., Krohne, G. and Brehm, K. The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis.EvoDevo, under review.

    Koziol, U., Krohne, G., Brehm, K. (2013) Anatomy and development of the larval nervous system in Echinococcus multilocularis. Front. Zool. 10: 24.

    Olson, P.D., Zarowiecki, M., Kiss, F., Brehm, K. (2012) Cestode genomics – progress and prospects for advancing basic and applied aspects of flatworm biology. Parasite Immunol., 34: 130-150.

    Spiliotis, M., Mizukami, C., Oku, Y., Kiss, F., Brehm, K., Gottstein, B. (2010) Echinococcus multilocularis primary cells: improved isolation, small-scale cultivation and RNA interference.
    Mol. Biochem. Parasitol. 174: 83-87.

    Brehm, K. (2010) Echinococcus multilocularis as an experimental model in stem cell research and molecular host-parasite interaction.
    Parasitology 137: 537-555.

    Spiliotis, M. & Brehm, K. (2009) Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures.
    Methods Mol. Biol. 470: 245-262.

    Spiliotis, M., Lechner, S., Tappe, D., Scheller, C., Krohne, G. & Brehm, K. (2008)Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles.
    Int. J. Parasitol. 38: 1025-1039.

    Brehm, K. & Spiliotis, M. (2008) Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells.
    Exp. Parasitol. 119: 506-515.

    Spiliotis, M., Tappe, D., Sesterhenn, L. & Brehm, K. (2004) Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions.
    Parasitol. Res. 92: 430-432

    Kontakt

    Institut für Hygiene und Mikrobiologie
    Josef-Schneider-Straße 2
    97080 Würzburg

    Suche Ansprechpartner

    Campus Medizin