Cell cultures for schistosomes – Chances of success or wishful thinking?

International Journal for Parasitology 40 (2010) 991–1002

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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara

Current Opinion

Cell cultures for schistosomes – Chances of success or wishful thinking? T. Quack a, V. Wippersteg b, C.G. Grevelding a,* a b

Institute for Parasitology, Justus-Liebig-University, 35392 Giessen, Germany Biotest AG, 63303 Dreieich, Germany

a r t i c l e

i n f o

Article history: Received 31 March 2010 Received in revised form 27 April 2010 Accepted 29 April 2010

Keywords: Schistosoma Cell culture Invertebrate cell line Oncogene ras Cell ‘immortalisation’

a b s t r a c t Due to their worldwide importance for human and animal health, schistosomes are in the focus of national and international research activities. Their aims are to elucidate the genome, the transcriptome, the proteome and the glycome of schistosomes with the expectation to understand the biology of these blood flukes and to identify new candidate antigens for the development of a vaccine, or target molecules for the design of novel pharmaceutical compounds. All of these efforts have delivered a vast amount of information about the genetic equipment of schistosomes. In the emerging era of post-genomic research, however, methods and tools are necessary to interpret all available data and to characterise molecules of interest in more detail. In addition to transgenesis, it is generally accepted that cell lines for schistosomes are among the requirements to overcome present research limitations. In our commentary the prospect of establishing cell cultures for schistosomes is discussed. To this end we summarise the comprehensive endeavours made in the past regarding the establishment of invertebrate cell lines pointing to critical parameters that should be considered when making new attempts towards schistosome cell culturing. Furthermore, based on preliminary data with pilot-character, we discuss recent advances indicating the possibility of overcoming existing restrictions with respect to the ‘immortalisation’ of cells by oncogenes. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The clade of Platyhelminthes (flatworms) is comprised of the classes Turbellaria (planarians), Cestoda (tapeworms) and Trematoda (flukes), of which the latter is subdivided into ectoparasitic Monogenea and endoparasitic Digenea (Mehlhorn and Piekarski, 2002). Schistosomes belong to the digenean trematodes and exhibit clear sexual dimorphism, which is unique within the class of otherwise exclusively hermaphroditic trematodes. Another unusual phenomenon of schistosomes is the sexual maturation of the female. The differentiation of vitellarium and ovary as a prerequisite for egg production strongly depends on constant pairing-contact with the male (Erasmus, 1973, 1986; Den Hollander and Erasmus, 1985; Popiel, 1986; Kunz, 2001; LoVerde, 2002; Grevelding, 2004; Hoffmann, 2004; LoVerde et al., 2004c; Knobloch et al., 2006). Schistosomes exhibit a complex life-cycle associated with aquatic conditions including asexual and sexual reproduction within invertebrate and vertebrate hosts, respectively. Adult schistosomes live as couples in the mesenteric complex of the intestine. The smaller female is enclosed in the ventral groove (gynaecophoric canal) of the male. If paired the female can produce up to 300 eggs

* Corresponding author. Address: Institute for Parasitology, Justus-Liebig-University, Rudolf-Buchheim-Str. 2, 35392 Giessen, Germany. Tel.: +49 641 9938466; fax: +49 641 9938469. E-mail address: [email protected] (C.G. Grevelding).

per day, some of which penetrate the intestinal wall and enter the gut lumen. The eggs are excreted with the faeces, and upon contact with freshwater and in response to light the miracidia hatch and actively seek and penetrate into their intermediate water-snail host. Here multiplication and differentiation via mother and daughter sporocyst stages to cercariae occur. The cercariae are released into the water, actively seeking a mammalian host to penetrate the skin and transform into schistosomula, which then enter the capillaries. Following the circulation, they pass through the heart and lungs, developing to adult worms via juvenile stages. Finally, male and female schistosomes reach the portal veins of the liver where pairing occurs. As couples, they actively migrate to the mesenteric veins to complete the natural lifecycle. Approximately 30–50% of the released eggs are not excreted with the faeces, but are swept into the circulation and deposited mainly in the spleen and liver, but also in the lungs (Moore and Sandground, 1956). Secreted egg antigens evoke host-immune responses in these tissues, leading to inflammatory, granulomatous reactions, which are characteristic for schistosomiasis or bilharzia, the disease caused by schistosomes (Ross et al., 2002). This neglected parasitic disease affects approximately 700 million people in 74 countries, mainly in tropical and sub-tropical regions of Africa, America and Asia. Over 207 million people are infected (Chitsulo et al., 2000, 2004; Mayer and Fried, 2002; Zhou et al., 2002; Savioli et al., 2004; LoVerde et al., 2004b; WHO fact sheet fs115 (www.who.int/mediacentre/factsheets/fs115)) and the annual

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number of deaths is estimated to be at least 200,000 (Engels et al., 2002). Besides their medical importance for humans, schistosomes also afflict wild and domestic animals, thus they have developed a zoonotic potential and give rise to economic losses (De Bont and Vercruysse, 1997, 1998; Ross et al., 2001; Engels et al., 2002; Chitsulo et al., 2004; Sutherst, 2004; Huang and Manderson, 2005; Quack et al., 2006). Praziquantel is the only drug capable of combatting all Schistosoma spp. in humans and animals, but is only effective in killing adult worms (Cioli and Pica-Mattoccia, 2003). As praziquantel does not prevent re-infection, it has to be administered at frequent intervals in endemic regions with high re-infection rates. An emerging problem is the appearance of praziquantel-resistant Schistosoma strains (Bennett et al., 1997; Fallon, 1998; Liang et al., 2000, 2001; Doenhoff et al., 2002, 2008; Fenwick and Webster, 2006; Melman et al., 2009). Oxamniquine as an alternative applicable drug is exclusively effective against Schistosoma mansoni (Pitchford and Lewis, 1978; Pica-Mattoccia et al., 1997), however, resistance has also been reported (Cioli et al., 1989, 1992; PicaMattoccia et al., 1993; Coelho et al., 1997). Although considerable efforts have been made in the past and continue in vaccine development and the discovery of new target molecules (Waine et al., 1993, 1994; Suri et al., 1997; McManus et al., 2001; Verity et al., 2001; Fenwick et al., 2003; Pearce, 2003; Carvalho-Queiroz et al., 2004; LoVerde et al., 2004a; Gan et al., 2006; Tran et al., 2006; Loukas et al., 2007; Hotez et al., 2008; McManus and Loukas, 2008; Driguez et al., 2010) a vaccine against schistosomes is currently not available, as the present model vaccines are perceived to not provide sufficient protection (Bergquist et al., 2002; Bergquist, 2004; Lebens et al., 2004). With respect to the fear of emerging drug resistance and because a potent vaccine is not expected in the near future, there is an urgent need to undertake more basic research on schistosomes. In the pre-genomic sequencing era, many genes that might be of importance for the development of schistosomes or for host–parasite interactions have been identified (LoVerde et al., 1989, 2004c; LoVerde and Chen, 1991; Ramachandran et al., 1996; Skelly and Shoemaker, 1996; Davies et al., 1998; Mastroberardino et al., 1998; de Mendonca et al., 2000; Inal and Sim, 2000; Rao and Ramaswamy, 2000; Fantappie et al., 2001; Osman et al., 2001; Caffrey et al., 2002; Hoffmann et al., 2002; Knobloch et al., 2002; Schramm et al., 2003; Vicogne et al., 2003; Kapp et al., 2004). To date, such efforts have been dependent on empirical approaches, mainly constrained by the lack of substantial genomic and biological information. To overcome these limitations different programmes have been initiated by governmental and educational institutions, industry and the World Health Organization (WHO) (Foster and Johnston, 2002; Hoffmann and Dunne, 2003; Hu et al., 2003, 2004; Verjovski-Almeida et al., 2003, 2004, 2007; McManus et al., 2004; Berriman et al., 2009). Their specific aims are to elucidate the genome, the transcriptome, the proteome, the glycome and the lipidome of schistosomes to identify potential candidates for the development of vaccines, or for the design of novel pharmaceutical compounds (Davies and Pearce, 1995; Davies et al., 1998; van Hellemond et al., 2006; Hokke et al., 2007; Dissous et al., 2007; Han et al., 2009; LoVerde et al., 2009; Quack et al., 2009; Swierczewski and Davies, 2009, 2010, Beckmann and Grevelding, 2010; Beckmann et al., 2010). Genomic data are now available, marking the beginning of the post-genomic era of schistosome research. This requires the development and application of techniques to exploit these data and, among other things, to study the function of genes of interest. Several methods to isolate and culture developmental stages of S. mansoni have been established. Eggs, sporocysts, schistosomula and adults can be maintained in vitro for short or long time periods, and are amenable to genetic and molecular manipulation

(Mann et al., 2009). Utilising biolistics, square-wave electroporation and soaking procedures, schistosomes have been transiently transformed with mRNA, plasmid DNA, reporter-gene constructs or virions, enabling among other topics, both gene expression analyses and promoter studies (Heyers et al., 2003; Correnti and Pearce, 2004; Yuan et al., 2005; reviewed in Grevelding, 2006; Correnti et al., 2007; Mann et al., 2009; Dvorak et al., 2010; Kines et al., 2010). The RNA interference (RNAi) principle of gene silencing, originally developed for Caenorhabditis elegans (Fire et al., 1998), has been successfully adapted to schistosomes (Correnti et al., 2005; Krautz-Peterson et al., 2007; Ndegwa et al., 2007; Yoshino et al., 2009; Beckmann et al., 2010) in providing new perspectives for reverse genetic approaches. Furthermore, progress has been made to stably transform schistosomes (Grevelding, 2006; Beckmann et al., 2007; Brindley and Pearce, 2007; Mann et al., 2008). Besides evidence for germ-line transmission of transgenes, their genomic integration has been demonstrated by different approaches (Kines et al., 2006, 2008; Morales et al., 2007). As metazoan parasites, however, schistosomes are not ideal organisms for laboratory manipulation. The complete life-cycle of this blood fluke has to be maintained in intermediate and definitive hosts to gain access to all stages (schistosomula, adults, miracidia, sporocysts and cercariae), which is labour-intensive. Furthermore, the numbers of parasites that can be generated are limited and any scale-up of production is costly. Different in vitro culture systems for schistosomal life-cycle stages, such as schistosomula (Eveland et al., 1979; Lin and Zhou, 1986; Clemens and Basch, 1989), adults (Clegg, 1965; Fu et al., 1976; Basch and Humbert, 1981; Basch, 1981a,b, 1984; Basch and Basch, 1982; Irie et al., 1983; Barth et al., 1996), eggs (Newport and Weller, 1982a,b; Jurberg et al., 2009), sporocysts (DiConza and Hansen, 1972, 1973; DiConza and Basch, 1974a,b; Cohen and Eveland, 1984; Coustau and Yoshino, 2000; Bixler et al., 2001; Bender et al., 2002) and cercariae (Basch and DiConza, 1977) have been developed; a universal cultivation method that allows the maintenance of the complete life-cycle in vitro has not yet been established. Research studies also aimed to develop methods for the in vitro generation of cercariae from sporocysts (Ivanchenko et al., 1999) and for the transformation of miracidia to sporocysts (Coustau et al., 1997) and cercariae (Muftic, 1969), respectively. Furthermore, protocols for cloning sporocysts and transplantation techniques for sporocysts (Jourdane and Theron, 1980; Jourdane, 1990; Bayne and Grevelding, 2003; Kapp et al., 2003) have been developed by different research groups, demonstrating that an intermediate hostindependent in vitro propagation is, at least, basically feasible. Some earlier studies successfully demonstrated the transformation of cercariae to schistosomula (Gilbert et al., 1972; Eveland and Morse, 1975; Wang et al., 2006) and even to egg-producing adult schistosomes (Wang and Zhou, 1986). Current research projects are reinforcing the attempt to achieve in vitro propagation from schistosomula to fertile adult schistosomes independent of a definitive vertebrate host (Haas, personal communication). By refining protocols it may become possible in the future to complete the schistosome life-cycle in vitro. A further aspect which hampers basic research on schistosomes is the lack of established cell lines, although initial approaches to isolate and maintain cells isolated from adult and juvenile schistosomes have been reported (Weller and Wheeldon, 1982; Coles and Fitzgerald, 1986; Hobbs et al., 1993; Bayne et al., 1994; Bayne and Barnes, 1997; Dong et al., 2002). Therefore, the establishment of cell lines is an important long-term aim, and may provide the basis for stable resources for genomic DNA (gDNA), mRNA and proteins for genomic, transcriptomic and proteomic analyses under strictly controlled conditions. More importantly, cell lines will be pivotal to functional characterisation of genes of interest in the homologous system, as well as to facilitate high-throughput screenings

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of pharmacological compounds to discover novel candidate drug targets and to determine their mechanisms of action. Furthermore, schistosomal cell lines will be beneficial for homologous protein expression in supporting functional studies and vaccine development, as genus-specific differences related to codon usage as well as post-translational modifications, such as glycosylation and lipidation, are crucial for the appropriate function and immunogenicity of proteins. This article will summarise previous attempts and current progress concerning the isolation of schistosomal cells and the establishment of indispensable, required cell lines. Furthermore, it is intended to outline ideas for future strategies to overcome the current limitations to accomplish this ambitious aim.

2. Status quo Historically, attempts to isolate and maintain cells from schistosomes were initiated in the early 1970s by the basic work of Capron and Dupas (1972), who reported the limited migration of cells from the cut ends of trypsinized worms cultured in mammalian cell culture media. The results were successfully reproduced by Weller and Wheeldon (1982) some years later, demonstrating that migrating cells survived only as long as physical contact was maintained with a viable parental fragment. The isolation of cells from adult schistosomes was performed by mincing worms in a trypsin-containing solution employing long-handled, sharppointed scissors followed by further trypsinization of the resulting fragments (Weller and Wheeldon, 1982). This procedure yielded single cells of different types and discrete cell plaques, but mainly fragments which appeared semi-transparent and refractile, revealing muscular activity for weeks. Fragments with an intact tegumental covering exhibited a repair of cut surfaces by cellular and tegumental outgrowth and a tendency to re-association. Some cells showed active pseudopodal and phagocytic activity. However, degenerative changes occurred in some plaques within 7 days, most of which had disintegrated by day 28. The plaques consisted of different types of cells, which according to their morphology were characterised as ‘‘deltaic fan cells” exhibiting polar, fan-like cytoplasmic extrusions of delicate, transparent and membranous material probably representing tegumental cells elaborating the glycocalyx. ‘‘Round granular cells” appeared as large round or irregular oval cells of fine granular cytoplasm and delicate nuclei. Such cells have been characterised as indicators of medium toxicity, as large cytoplasmic vacuoles rapidly appeared under unfavourable conditions. Vitelline cells were isolated as well, containing yellow–brown cytoplasmic granules. Initially these cells were round-shaped developing bipolar lanceolate cytoplasmic processes on attachment to the substrate. A further type of cell exhibited flagellar activity and was depicted as ‘‘flagellated cells”. These occurred separately or in sparsely distributed, loosely associated groups of 2 to 8 or more cells, which were most frequently pear- or lancet-shaped with a prominent refractile, cytoplasmic body at their basal ends. In cell culture ‘‘flagellated cells” were identified by their flagellar activity generated by a single anterior flagellum or two or more flagella. Some other cell types, such as multinucleate syncytial cytoplasmic aggregations of diverse morphology, grape-like masses of developing ovarian cells and rounded or cuboidal oocytes, were also observed. The latter remained free-floating as separate cells, in small groups or occasionally in short chains. Whether the maturation processes of oocytes continued in vitro could not be determined by the authors. Moreover, while amitotic cell division was commonly observed for developing plaques, mitotic activity itself was extremely rare. Following the trailblazing work of Weller and Wheeldon (1982) few research groups made further attempts at establishing cell lines

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from S. mansoni or Schistosoma japonicum in the ensuing decades. Their efforts differed in many aspects, such as the initial disruption method, the media employed for in vitro cultivation of the isolated cells and the supplementation with a variety of additives. Moreover, the studies varied in respect to the sources from which the cells or tissue fragments were isolated. Concerning cell isolation procedures, scissors (Weller and Wheeldon, 1982; Dong et al., 2002; Ming et al., 2006a), scalpel or razor blades and glass homogenisers (Coles and Fitzgerald, 1986; Blair et al., 1991), as well as electric tissue disrupters (Hobbs et al., 1993; Bayne et al., 1994) were employed. Subsequent homogenisation of initial cells or cell plaques isolates was occasionally facilitated by utilising Pasteur pipettes (Blair et al., 1991; Dong et al., 2002; Ming et al., 2006a). The disruption protocols usually included a supportive treatment with different proteases whereby trypsin was mainly applied (Weller and Wheeldon, 1982; Coles and Fitzgerald, 1986; Dong et al., 2002; Ming et al., 2006a). Various combinations of different proteases including collagenase have been tested, demonstrating a preference for the employment of the cysteine-protease papain, which resulted in a higher number of vital cells (Blair et al., 1991). Other protocols made use of a special crab collagenase, which was shown to be better suited in digesting the interstitial collagen matrix of schistosomes than collagenases derived from mammalian or bacterial sources. Furthermore, crab collagenase also exhibited chymotrypsin-, trypsin- and elastase-like activities, which was speculated to be supportive (Hobbs et al., 1993; Bayne et al., 1994). At the beginning of schistosomal cell culture research, scientists employed media used for the in vitro cultivation of mammalian cells as a logical starting point because media, which previously had been shown to be beneficial for the growth of intact schistosomes in vitro, were not supportive or even toxic for isolated schistosomal cells. Initial experiments utilising modified mammalian cell culture media were not successful and encouraged Weller and Wheeldon (1982) to systematically develop media optimised for the cultivation of schistosomal cells. In their heroic work, the authors tested over 1900 different media formulations, varying the composition of diverse inorganic and biological additives. Furthermore, they analysed the influence of osmolarity and the effect of different CO2-concentrations and buffers on the control of pH in the culture system as physicochemical parameters. Finally, even the influence of the glass- and plastic-ware employed as cell culture material and different culture substrates used for surface coating to promote cell attachment or growth were investigated. The exploration of media providing near physiological conditions for cells derived from mature schistosomes was predominantly guided by emerging knowledge on schistosome physiology, metabolism and on biochemistry. Compared with mammalian cell cultures, schistosome cells were shown to be more susceptible to trace toxicants, which required great care in selecting chemicals, nutrients and other compounds, as well as the employed cell culture material. Investigations related to the addition of classical growth-promoting supplements, such as sera and different extracts derived from various tissues or embryos, showed remarkable results. None of the tested extracts showed supportive effects; moreover, some proved to be overtly toxic. Sera from different sources were tested, finally, routinely employing foetal or newborn calf serum. Unexpectedly, individual lots of sera varied considerably in their supportive capacity and toxicity. As a consequence, proactive validation of sera is strongly recommended. Interestingly, the authors discovered that a serum concentration of 10%, commonly used for in vitro culture systems, was inhibitory. Therefore, the amount of serum in the media was decreased to 2%. Weller and Wheeldon (1982) omitted antibiotics from the culture system, as the presence of penicillin and streptomycin had been shown to impair development of schistosomules in vitro (Eveland et al., 1979).

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In contrast, detrimental effects of antibiotic application were not observed by others (Blair et al., 1991; Hobbs et al., 1993). The empirically developed ‘‘schisto” salt solution differs significantly from salt solutions commonly used for human cells, especially with respect to the requirement of lower phosphate and higher Mg2+, Ca2+ and K+ concentrations. The drastic increase of glucose concentration to 12,000 mg/ml was shown to be beneficial. The addition of glycerol and trehalose, which have been found in schistosomes, seemed to have positive effects, whereas other carbohydrates were not supportive. Enrichment of the ‘‘arbitrarily derived nucleotide mixture” with orotic acid showed promising results. Studies on lipid metabolism revealed that schistosomes are host-dependent for sterols and long-chain fatty acids, and that cholesterol and linoleic acid were taken up by adult worms. Therefore, a lipid mixture was applied to the medium containing cholesterol, sodium linoleate, sodium oleate and four different vitamins prepared in the presence of different carrier substances to facilitate their solubility in aqueous media. Many media judged as good included yeast extract representing a classical source of essential growth factors, which additionally may provide a basic vitamin supply. Addition of meat extract also resulted in beneficial effects, presumably for similar reasons. Elevated levels of choline chloride and DL-carnitine HCl at low concentrations obviously led to better growth. The supplementation of media with different hormones, such as insulin, triiodo-L-thyronine, serotonin, as well as with certain metabolic intermediates, were also shown to be helpful. The cells were routinely maintained in a 5% CO2–air atmosphere in a horizontal position at 35 °C and the culture medium was renewed twice or even three times per week, depending on the resultant pH. In summary, the work of Weller and Wheeldon (1982) revealed that, at the cellular level, schistosomal metabolic requirements differ from those of their vertebrate host. Even if largely optimised, the culture system described lacked essential elements for continued cell division in vitro. Cell cultivation protocols of other researchers differed in respect to the media or the supplements employed. Furthermore, the results regarding the supportive or non-supportive effects of different additives applied remained controversial. Coles and Fitzgerald (1986) initially conducted the culturing of schistosomal tissues or cells in insect-tissue culture medium containing 20% FBS, whereas subsequent work was performed with the medium developed by Weller and Wheeldon (1982). In addition, Coles and Fitzgerald (1986) analysed a range of growth factors with respect to a putative stimulatory effect on cell division. No improvement was observed except for supplementation of the medium with an extract derived from Hymenolepis diminuta (rat tapeworm), which promoted cell adhesion. These results coincide in principle with the findings of Weller and Wheeldon (1982), who observed some growth-promoting activity, only if haemolymph derived from the intermediate snail host Biomphalaria glabrata was added to the medium. Interestingly, in other studies the addition of haemolymph yielded no improvement (Hobbs et al., 1993). Various media were used by different research groups, such as HEPES-supplemented RPMI-1640 (Blair et al., 1991; Ming et al., 2006a), a medium based on serum- and phosphate-free DMEM, which has been extensively modified according to informations from Weller, Wheeldon and Newport (Hobbs et al., 1993), as well as a special 1:1-mixture of S. mansoni (SM)-medium and medium F (Bayne et al., 1994). Furthermore, for cells derived from adults and schistosomula DMEM/F-12 and TC-199 were used, respectively, both supplemented with 10% calf serum (Dong et al., 2002). Other media, such as DMEM, F-12:DMEM, Schneider’s medium, Lebowitz’s L-15, Ham’s F-12, medium F (Stibbs et al., 1979), Swimm’s S-77, medium 199 and RPMI were less successful in maintaining viability (Hobbs et al., 1993). In addition to Weller and Wheeldon, other researchers performed extensive studies

regarding the putative supportive effects of several biological compounds. No improvement was observed by modifying medium SM with lipids, trace elements, hydrolysates or supplementing with serum, tissue extracts from vertebrates or invertebrates, purified attachment factors, hormones and growth factors (Hobbs et al., 1993). Moreover, conditioned medium from cultures of whole worms, schistosomula, sporocysts and a variety of cell lines derived from vertebrates and invertebrates did not improve viability. Evidence was obtained that the adherence and growth of S. japonicum cells can be improved by co-culturing with mouse embryonic fibroblast layers and collagen of rat tails (Li et al., 2006), by spermidine (Chen et al., 2006), or using extracellular matrix material from rabbit lungs or liver (Chen et al., 2002; Qin et al., 2005). Studies of Bayne et al. (1994) with S. mansoni cells demonstrated that vitronectin, fibronectin, collagen types I and IV and laminin had no adherence-promotion capacity. Furthermore, factors with putative growth-promoting effects, such as pituitary extracts, insulin, ecdysone, farnesol, vertebrate cytokines and platelet-derived growth factor (PDGF), as well as lipid-rich extracts of blood and egg yolk, neither improved the viability of the cultures nor enhanced cell growth (Bayne et al., 1994). Exposure of sporocyst cells to epidermal growth factor (EGF), with or without insulin also had no beneficial effect. Interestingly, it was demonstrated that cellular viability could be extended from 1 week to up to 6 months by co-culturing juvenile worm cells on a feeder layer of irradiated buffalo rat liver (BRL) cells, which had been plated 12–24 h earlier (Hobbs et al., 1993). Feeder layers of bovine endothelial or mouse embryo (3T3) cells were less effective. It is reasonable to assume that BRL-cells might have provided some liver-specific factors, which were necessary for the survival of cells derived from juvenile worms, as they had been isolated from mouse liver. Such growth-promoting factors were probably not provided by other cell types. Importantly, the above feeder layer could not be replaced by conditioned medium from BRL-cells, even if concentrated. This could probably be explained by instability of the hypothesized factors or by the presence of the feeder cells as a kind of detoxifying agent to remove components from the medium, which were detrimental to the worm cells. Another possibility was that direct contact between the schistosomal and feeder cells was crucial as they provided an insoluble matrix not released into the medium. With respect to the cultivation of cells from in vitroderived sporocysts, co-cultivation (synxenical cultivation) of schistosomal cells with either ganglia cells isolated from snails (B. glabrata) or B. glabrata embryonic (Bge) cells was shown to be beneficial to the vitality of parasite cells, compared with axenic controls (Bayne et al., 1994). This effect was not observed if the direct contact between schistosomal and snail cells was prevented or Bge cell-conditioned medium was employed. This supports the argument that (germ) cells of sporocysts may require growth factors produced in snail neurones. Obviously, direct contact of the cells was crucial, possibly because this factor was not released into the medium. Interestingly, medium SM conditioned by Sf9 insect cells also had beneficial effects (Bayne et al., 1994). Many cells isolated from adult worms 21 days p.i. appeared either small and adherent or larger and non-adherent (Coles and Fitzgerald, 1986), resembling cell types described by Weller and Wheeldon (1982). Lateral spreading of the isolated cells did not occur, but apparent cell multiplication continued for a few days that developed into an attached layer several cells thick. Provided that the initial cell concentration was high enough, relatively large numbers of cells accumulated within 1 month, but only a few of those seemed to be alive. However, continued growth did not occur for the adherent or non-adherent cells and the authors failed to generate any long-term cell cultures/lines. Besides a variety of unidentifiable cell types, Blair et al. (1991) isolated numerous spindle-shaped muscle fibres and flame cells from 45 to 60 days p.i.

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adult schistosomes, which appeared to survive for at least 24 h. Employing 18 days p.i. juvenile worms as the source for cell isolation prepared from liver, Hobbs et al. (1993) generated adherent and non-adherent single cells together with clusters of cells. The cultures showed a predominance of small, round cells adherent to the plastic-cell culture surface. Some tightly adherent cells had the shape of ‘‘fried eggs”, whereas others resembled the deltaic fan cells described by Weller and Wheeldon (1982), appearing poorly attached and suspended like round or pear-shaped balloons. Flame cells were occasionally observed, which maintained their activity throughout the life of the culture. Finally, larger cells with rapidly moving flagella, as well as arrhythmically contracting individual bi- or multi-polar cells were also detected. Remarkably, the survival rate for cells derived from schistosomula was significantly lower than that for cells originating from juveniles, whilst cell cultures from adult worms rapidly degenerated (Hobbs et al., 1993). Adaptation of their isolation protocol for in vitro-derived primary sporocysts, all the cell types described for intact sporocysts (Pan, 1980) were successfully isolated, including germ cells, flame cells, interstitial cells, muscle cells, nerve cells, multinucleate subtegumental cells and cells of the penetration glands. Flame cells were viable for at least 30 days as regards their rhythmical beating. Cell doublets resembling interstitial or germ cells appeared within 2 weeks of culture, possibly indicating a single round of replication. Although indefinite propagation had not been achieved, cultures remained viable for several months (Bayne et al., 1994). Cell preparations from 42 to 43 days p.i. and 20 to 21 days p.i. schistosomes resulted in the isolation of 10 different cell types in total, which could be extensively characterised by their ultrastructure (Dong et al., 2002). Vitelline cells, flame cells, multinucleate subtegumental cells, nerve cells and germ cells were isolated from adults, whereas experiments employing schistosomula yielded sustentacular cells, flame cells, nerve cells, mast cells, muscle cells, multinucleate subtegumental cells, interstitial cells, penetration gland cells and germ cells. In contrast to the cells isolated from adult schistosomes, at least a few cells derived from schistosomula were observed to be dividing and appeared to be in the prophase of the cell-cycle. This indicated that schistosomula cells might exhibit a greater potential to proliferate in vitro than cells derived from adult worms. Although the authors demonstrated survival of healthy cells for at least 18 days, degenerative processes occurred progressively during culture-time prolongation. After 36 days of culture, evidence for more heterochromatin than euchromatin was detected, which indicated a decrease in transcriptional activity and consequent impairment of cell proliferation. Since variation in the methods of Weller and Wheeldon (1982) failed to improve the culture of schistosomal cells, Coles and Fitzgerald (1986) followed the strategy of producing tumour-like schistosome cells by employing the mutagen ethyl methanesulphonate (EMS). Exposure of trypsinized cells to EMS did not lead to a dividing sub-population, whereas treatment of intact worms resulted in four different morphological phenotypes (Coles and Fitzgerald, 1986). The most obvious effect observed was a phenomenon described as ‘‘ballooning”, which was detected after 1 week of treatment. Morphologically, it resembled the ‘‘blubbing” of male worms following treatment with oxamniquine in vitro (Kohn et al., 1982). Some worms showed increased numbers of ‘‘large round cells”, occasionally detectable as moving freely within the body space, which had developed following treatment. A few treated worms developed bulges on their surface, which were distinct from ‘‘ballooning” in that they were filled with tissue. Finally, some individuals exhibited a disruption of the tegument to reveal a ‘‘mass of worm tissue” attached to the living worm. This outgrowth of tissue was interpreted as the growth of undifferentiated cells. However, no type of cell appeared to grow after the death of the worm, so that the original aim of obtaining continuously dividing

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cells of schistosomes was not achieved. Towards this aim further attempts were performed by Bayne and colleagues, who tested the effects of two different combinations of tumour-promoting substances. However, neither the combined treatment with cadmium and phorbol myristate acetate (PMA) nor with a calcium ionophore and PMA induced proliferation in the cultures (Bayne et al., 1994), although it had been reported by others that the induction of neoplasia in flatworms is possible (Hall et al., 1986). Similar attempts by treating cells of S. japonicum with phytohaemagglutinin (PHA), a mitogen capable of inducing mitosis, did not result in continuously dividing cells (Dong et al., 1998). To promote mitogenic activity Dong and colleagues employed N-methyl-N-nitro-N-nitrosoguanidine (MNNG; Gichner and Veleminsky, 1982); a monofunctional, alkylating reagent acting directly on nucleic acids to cause chromosomal DNA damage. As a strong mutagen, MNNG was expected to induce cell transformation leading to ‘immortalised’ cell lines (Ming et al., 2006a). Although an extensive experimental design was followed using varying MNNG concentrations, exposure times, and application time-points, the results of these experiments, finally, revealed only beneficial effects with respect to cell growth, prolonged cell survival and cell division frequency, when cells were treated 4 days after isolation with 3 lg/ml MNNG for 48 h with RPMI-1640 as basic medium. Under these conditions cells survived for at least 246 days or more. A combination of MNNG treatment and liver-matrix addition improved carbohydrate metabolism of cultured cells (Ming et al., 2006b). However, despite all this progress, Ming et al. (2006a,b) failed to passage dividing cells, possibly because the number of mutated cells induced by MNNG was not high enough to dominate in the mixedcell culture. In summary, the results described remain contradictory, which could be explained by the different parasite material used as a source for cell isolation in the diverse studies. Besides in vitro-derived sporocysts, juveniles and mature adult schistosomes were used, which had been perfused between 18 and 60 days p.i. Obviously, cells derived from different developmental stages or timepoints could differ significantly with respect to their individual physiological requirements. This view is supported by the finding that culturing juvenile cells (prepared from host liver) on a feeder layer of rat liver cells was beneficial to cell viability, whereas snailderived ganglia cells or Bge cells exhibited positive effects on cells isolated from sporocysts. The controversially discussed supplementation of media with different amounts of, for example, serum ranging from 0% to 20%, also possibly reflects such individual requirements.

3. What can be learned from other invertebrate cell lines? Cell lines established in the past have been predominantly derived from Dipteran (Schneider, 1969, 1972; Hsu et al., 1970, 1972; Mosna and Dolfini, 1972) and Lepidopteran insects (Hink and Ignoffo, 1970; Hink, 1970; Hink and Ellis, 1971; Vaughn et al., 1977; Lynn, 2007a), as well as the Coleoptera (Lynn, 1995; Fernon et al., 1996; Iwabuchi, 1999; Hoshino et al., 2009), whereas cell lines from other invertebrates and arachnids have rarely been generated (Hink, 1979; Bayne, 1998). Other than some cell lines developed from shrimps (Kasornchandra et al., 1999; Chen and Wang, 1999a), oysters (Wen et al., 1993; Chen and Wang, 1999b) and at least one nematode (Manousis and Ellar, 1990), there are some 40 cell lines from ticks (Bell-Sakyi et al., 2007, 2009). None or just a few cell lines exist from parasite vectors, intermediate hosts of parasites and from parasites themselves (Hansen, 1976; Kurtti and Brooks, 1977; Furuya, 1991; Yamashita et al., 1997; Liu et al., 1998). However, to date only 11 cell lines are commercially available, including the popular Sf9- and Schneider-Drosoph-

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ila cell lines mainly utilised for heterologous protein expression (www.atcc.org). Bge cells derived from the S. mansoni intermediate snail host B. glabrata represent the only cell line derived from nonarthropods. Attempts at establishing cell cultures and cell lines from invertebrates revealed some interesting features and probably underestimated aspects. A variety of successfully established cell lines have been derived from embryonic tissue or embryonated eggs (Schneider, 1972; Bhat and Yunker, 1977; Kurtti and Brooks, 1977; Holman and Ronald, 1980; Munderloh and Kurtti, 1989; Bell-Sakyi, 1991; Munderloh et al., 1994; Fernon et al., 1996; Simser et al., 2004; Esteves et al., 2008; Bell-Sakyi et al., 2009), larval stages (Iwabuchi, 1999; Eguchi and Iwabuchi, 2006; Lynn, 2007a, 2007b; Hoshino et al., 2009) and germinal cells (Manousis and Ellar, 1990; Yamashita et al., 1997; Liu et al., 1998; Kasornchandra et al., 1999). This has indicated that tissues and organs from early developmental stages are more promising sources for the establishment of cell lines. This conclusion has been supported by the findings from schistosomal cell isolation approaches that indicated cells from juvenile worms and in vitro-derived sporocysts were superior to cells of adults (Hobbs et al., 1993; Bayne et al., 1994). A further important aspect has been the choice of an appropriate medium, as the different specific formulations represent critical parameters influencing cell growth and viability. Requirements varied not only between cell lines derived from different organisms, but even between cell lines from the same species as was demonstrated for the cultivation of insect cells (Hink et al., 1973). The same held true for supplementation with growth factors, hormones and other additives. Regarding attempts at the cultivation of schistosomal cells, this could be confirmed by the different growth requirements of cells prepared from juvenile worms and in vitro-derived sporocysts, for which haemolymph and liver feeder cells have proved to be supportive, respectively. Comparable effects have been observed regarding the cultivation of primary cells from Echinococcus multilocularis. These cells were maintained in vitro for several months and proliferated only in the presence of vesicle fluid derived from Echinococcus cysts (Spiliotis et al., 2008). A further parameter often discussed in different studies was the osmolarity of the medium, which represents the total concentration of different mono- and bi-valent metal ions, sugars, as well as proteins. As an important parameter in media formulations for invertebrate cell lines, osmotic pressure has already been discussed by Weller and Wheeldon (1982), who described an osmolarity in the range of 301–330 mOsm as optimal for maintaining schistosomal cells in vitro (Weller and Wheeldon, 1982). Studies with adult-derived cells from moths, for example, have indicated a reduction in cell growth if the osmotic pressure of 316 mOsm varied more than ±40 mOsm (Kurtti et al., 1974, 1975; Hink, 1979). In contrast, media for maintaining embryonic cells derived from the snail B. glabrata had been designed to parallel molluscan haemolymph osmolarity, which is significantly lower by varying between 108 and 266 mOsm (Lee and Cheng, 1972; Hansen, 1976). These findings should be considered for any future media formulations towards the establishment of cell lines from schistosomal sporocysts. Body fluid could be analysed as a basis for design of media for invertebrate cell culture as it was speculated for Ascaris suum (Coles and Fitzgerald, 1986). But with the exception of the fluid from cestode cysts, body fluid is not found in the Platyhelminthes. The occurrence of ‘‘ballooning” in cultured S. mansoni adult males, which had been treated with EMS, might offer the possibility of collecting sufficient liquid to analyse ionic composition and osmolarity of the extracellular fluid from schistosomes, which could provide valuable data for novel media compositions (Coles and Fitzgerald, 1986). Another important physicochemical parameter was the pH of the media, ranging between 6.2 and 7.0 for most invertebrate media and as high as 7.4

for some cockroach cell lines (Kurtti and Brooks, 1977). The optimal pH for moth cells (Heliothis zea) was found to be between 6.5 and 7.0 (Kurtti and Brooks, 1972), whereas for two leafhopper (Aceratagallia sanguinolenta and Agallia constricta) cell lines it was 6.3–6.4 (Martinez-Lopez and Black, 1977). Regarding suspension cultures of cabbage looper (Trichoplusia ni) cells the determined pH-optimum ranged from 6.0 to 6.5, whereas pH 6.7 was clearly detrimental (Hink, 1970). Adjustment and stability of the appropriate pH can be achieved by different buffer systems (e.g., bicarbonate and phosphate) and is closely linked to the atmosphere used for cell cultivation. Many vertebrate cell lines are grown in a gas phase of 5% CO2 in air to reduce the loss of CO2 from the bicarbonate buffer. Since invertebrate cell culture media have been mainly buffered with alternative buffers (e.g., HEPES), the cells were incubated under normal (normoxic) atmosphere (Hansen, 1976; Hink, 1979). However, the total elimination of bicarbonate was not generally advisable, as it comprises an essential metabolic precursor. Concerning phosphate-based buffers, high phosphate-ion concentrations may be damaging at supraphysiological levels of PO43 in such buffers by unbalancing normal physiology that would lead to unhealthy cells. Hyperoxic cultivation conditions are possibly deleterious, for example, due to the oxidation of lipids that renders them toxic. Echinococcus multilocularis larvae have been shown to be highly sensitive to reactive oxygen species that were formed during conventional cell cultivation. Therefore, the cultivation of metacestode vesicles under reducing and oxygenfree conditions for the establishment of an axenic in vitro cultivation system marked a crucial break-through and was achieved by cultivation of vesicles in a nitrogen atmosphere, and employed a culture medium supplemented with reducing substances, such as b-mercaptoethanol and L-cysteine (Spiliotis et al., 2004; Spiliotis and Brehm, 2009). Similarly, hypoxic culture conditions with lower O2-levels of up to 6% were shown to be beneficial for axenic cultivation of S. mansoni sporocysts (Bixler et al., 2001), which paralleled the O2-level of 9% as determined for B. glabrata haemolymph (Lee and Cheng, 1971). Prevention of oxidative stress was achieved by the addition of reducing compounds, such as DTT, L-cysteine and reduced glutathione (Buecher et al., 1974). Notably, for the cultivation of cells derived from adult schistosomes it was shown that a gas mixture consisting of CO2 (0.71%), O2 (9.2%) and N2 (>90%) was not beneficial. Moreover, a pure N2-atmosphere even proved lethal (Weller and Wheeldon, 1982). In summary, with respect to the described physicochemical parameters, cultivation conditions for future attempts on establishing cell lines from schistosomes should reflect the physiological environment from which the cells are isolated. For example, cells derived from sporocysts may prefer conditions similar to snail haemolymph, whereas the conditions found in venous blood could be more favourable for cells prepared from adult schistosomes. Another critical point controversially discussed is the supplementation of culture media with serum, whose effect strongly depends on age and species of the donor, as well as on the quality of the appropriate lot. Therefore, the design of new media should probably start from serum-free media. Alternatively, media could be employed including a ‘‘synthetic serum-equivalent” of defined composition (e.g., GIT-medium, Wako Chemicals, Japan). This would guarantee a charge-independent quality of the medium and, simultaneously, the absence of viruses, cytokines, toxicants or other contaminants supposed detrimental for the in vitro cultivation of cells. A crucial aspect in cell line development, especially for cells derived from parasites isolated from their hosts, is their origin. This importance was reflected in findings from Echinococcus-derived cells (Fiori et al., 1988), which were finally proven to be of bovine origin by karyotype analyses (Howell and Matthaei, 1988). Therefore, the identity of cultivated cells has to be scrutinised by species-specific PCR approaches, karyotyping, or other appropriate

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methods as was successfully accomplished for the B. glabrata-derived Bge cell line (Hansen, 1976). Although cells from different schistosome life stages have been successfully isolated and maintained in vitro, continuously dividing cell populations have not been obtained as yet even though mutagenic and mitogenic substances were applied. Accordingly, further ‘immortalisation’ approaches will have to be envisaged, for example starting with cells isolated from life stages exhibiting a high developmental potential, such as juvenile stages or sporocysts. Utilisation of isolated germinal tissues enriched with stem cells may also be favourable for such purposes. However, innovative strategies for ‘immortalising’ schistosome cells also have to be considered, for example, by transformation with DNA-constructs stimulating mitotic activity (Tapay et al., 1995).

4. ‘Immortalisation’ by transfection with oncogenes; a practicable approach? The limitation of the proliferative potential of cultured cells is a consequence of pathways that exist in a cell actively controlling its mitogenic activity. Methods designed to ‘immortalise’ vertebrate cells have aimed to subvert the mechanisms controlling cell-cycle checkpoints or the inactivation of tumour-suppressor genes. Such manipulations allow an expansion of the cell’s life-span. Researchers have fooled such cell-cycle regulation and differentiation by the ectopic expression of viral oncogenes, which were introduced by transfection (Houweling et al., 1980; Sdek et al., 2006), viral infection (Chang et al., 1982; Scholte et al., 1989) and via retroviral vectors (Cone et al., 1988; Emami et al., 1989). Mucosal human

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papillomaviruses (HPVs) infect human genital and oral epithelial cells causing malignant lesions. Expression of HPV oncogenes E6 and E7 was sufficient for primary human keratinocyte ‘immortalisation’ and for the initiation and all subsequent stages of carcinogenic progression (Wise-Draper and Wells, 2008). In addition to HPV E6 and E7, the simian virus 40 large-T antigen (SV40 LT) exerted oncogenic potential and was capable of ‘immortalising’ primary cells (Wang et al., 1989; Truckenmiller et al., 1998). Epithelial cell lines were generated by the use of SV40 sequences, either by infecting cells with complete viruses (Hronis et al., 1984) or through the expression of the SV40 LT antigen alone (Agarwal and Eckert, 1990; Schiller et al., 1992). Although ‘immortalisation’ was achieved by the ectopic expression of oncogenes, results were quite variable, depending on the cell type (Kuppuswamy and Chinnadurai, 1988), whilst in most cases the occurrence of differentiated phenotypes was altered or even absent. Human corneal epithelial cells (Araki-Sasaki et al., 1995) were also ‘immortalised’ by the ectopic expression of SV40 viral sequences, but although the obtained cell lines shared biochemical and morphological features typical for corneal epithelial cells, their differentiation pattern was disturbed, exhibiting low K3/K12 keratin levels and two to three layered epithelia, in contrast to high K3/ K12 keratin levels and five to six layers found in human corneal epithelia (Hogan et al., 1971). The loss of status of differentiation following SV40 transfection approaches can be overcome, however, by co-transfection with plasmids coding for signal transduction molecules such as Ras. This proto-oncogene controls cell proliferation and differentiation processes, and its mutant form (G12V and/or Q61L) is a dreaded oncogene involved in the ontogenesis of different types of tumours (Der et al., 1986; Young

Fig. 1. Microscopic analyses of posterior regions from adult male schistosomes transiently transformed by particle bombardment (see Wippersteg et al., 2002b for technical details) with a fusion construct of GFP and a Ras proto-oncogene variant ((A and B) Q61L; (C and D) G12V/Q61L). Ras was cloned downstream of GFP to ensure farnesylation at the C-terminal end, which is a prerequisite for the membrane association of Ras and its subsequent activity (Young et al., 2009). Areas of morphological aberrations and GFP activity overlap ((A and C) Fluorescence microscopy; (B and D) Bright-field microscopy; t, dorsal tegument; g, gut area).

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et al., 2009). Co-transfection experiments with mutant forms of ras and SV40 demonstrated the possibility of ‘immortalising’ granulosa cells from the rat while simultaneously maintaining the differentiation status of these manipulated cells (Amsterdam et al., 1988). Furthermore, permanent adipocyte cell lines maintaining a status of differentiation were also established using a combination of SV40 LT and ras (Benito et al., 1993). According to the genomic information available at present, a number of well-known proto-oncogenes occur in the schistosome genome (Berriman et al., 2009; Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium, 2009). Many years ago, we and others had already cloned and characterised the Ras protein of S. mansoni, providing evidence that only one ras-gene (SmRas) was present, which was transcribed throughout development (Kampkötter et al., 1999; Osman et al., 1999). In a pilot experiment, we examined whether mutated forms of SmRas may also have oncogenic potential in worms. To this end, mutations were introduced by site-directed mutagenesis into SmRas at positions G12 and Q61, known from the literature to negatively influence the GTPase activity of Ras in changing its proto-oncogenic form into the oncogenic form. For plasmid vector construction, SmRas variants carrying either one or two mutations (G12V and/or Q61L) were placed under the control of a promoter of the ER60 gene, known to be expressed in excretory/secretory tissues (Finken-Eigen and Kunz, 1997; Wippersteg et al., 2002a). Following cloning, the vectors were used for transformation experiments by particle bombardment of adult worms. After approximately 10 days, in the gut area of transformed worms morphological changes were detected which resembled neoplastic aberrations as indicated by tissue enlargement. To monitor whether these morphological changes were really due to the activity of the SmRas variants, GFP reporter-gene constructs were cloned containing the Ras variants in translational fusions with GFP. After particle bombardment and an incubation period of about 10 days, neoplastic aberrations were again detected in the gut of transformed worms. Furthermore, within the morphologically aberrant areas GFP fluorescence was also observed confirming the activity of the Ras variants in this area (Fig. 1). Reverse transcription (RT)-PCR analyses using primers directed against the chimeric ras/GFP confirmed transcription of the fusion construct in transgenic worms (Fig. 2). These results confirmed that mutated proto-oncogenes might have the capacity to exert oncogenic potential in schistosomes. In addition, this experiment may serve as encouraging proof of principle for the idea to ‘immortalise’ isolated schistosome cells by transfection with oncogenes. However, one prerequisite for cell ‘immortalisation’ experiments by oncogenes is the availablity of transfection methods for primary cells of schistosomes. Recently, it has been shown that primary cells from vertebrates can be transfected (Goldbard, 2006), demonstrating that the ‘immortalised’ status of a cell line is not a prerequisite for transfection success. In fact, the possibility of transfecting primary cells points a way to ‘immortalise’ schistosome cells. It even seems likely that such an enterprise can be accomplished since Schneider cells of Drosophila have been successfully transfected (Suske, 2000; March and Bentley, 2007). Beyond that, there is initial evidence for the successful transfection of primary cells from S. japonicum by electroporation using a cytomegalovirus (CMV) promoter-GFP construct as vector (Yuan et al., 2005). Further promising results for the ‘‘transfectability” of worm cells have been obtained with primary cells from metacestode vesicles of the cestode E. multilocularis. It was not only shown that these cells can be maintained under reducing conditions, but also that they proliferate in culture in the presence of vesicle fluid from larval cysts. Moreover, these cells started differentiation processes induced by co-cultivation with host hepatocytes in a trans-well system (Spiliotis et al., 2008; Spiliotis and

Fig. 2. Diagram of the reporter-gene construct used for transformation and transcriptional analysis of transiently transformed worms. (A) GFP-Ras protooncogene fusion construct consisting of the regulatory promoter (50 ) and terminator (30 ) elements of the cysteine-protease gene ER60 (Wippersteg et al., 2002b), GFP and Ras. Binding sites of the primers, which were used for reverse transcription (RT)-PCR analysis are indicated, as well as the size of the expected product of 480 bp. (B) RT-PCR analysis with total RNA extracted from adult worms shown in Fig. 1 (A and B) and wildtype control worms. Amplification using the ras and GFPspecific primers indicated in (A) resulted in a 480 bp product as predicted (lane 1). No product was obtained using RNA from wildtype worms as control (lane 2). M, Molecular weight marker; size indicated in bp.

Brehm, 2009). The same group showed that Echinococcus cells can be transfected by the bacterium Listeria monocytogenes, which opens up an alternative way to shuttle transgenes into parasite cells (Spiliotis et al., 2008). 5. Outlook The substantial work performed to isolate and maintain schistosomal cells in the past has revealed that a variety of parameters influence the success of establishing cell cultures from this parasite. A lot can be learned from previous experiences regarding the composition of media, the benefit of growth-supporting supplements or co-cultivation with feeder cells or bio-matrixes as well as the influence of basal physicochemical parameters that have been demonstrated to be critical (e.g., pH and osmolarity of the medium, gas phase composition, etc.). Furthermore, for ‘immortalisation’ approaches the source for cell isolation has to be carefully chosen. Besides treatment of isolated cells with mutagenic substances, transfection of such cells with DNA-constructs exhibiting oncogenic potential, as exemplarily shown, presumably represents one of several feasible approaches to overcome existing limitations towards permanently dividing cells. Combining classical knowledge with new tools available today provides excellent prerequisites to successfully establish cell lines from schistosomes in the near future. Acknowledgements The authors gratefully acknowledge critical reading of the manuscript by Dr. Roger D. Dennis. Financial support was obtained from the Deutsche Forschungsgemeinschaft (GR 1549/1-4). References Agarwal, C., Eckert, R.L., 1990. Immortalization of human keratinocytes by simian virus 40 large T-antigen alters keratin gene response to retinoids. Cancer Res. 50, 5947–5953. Amsterdam, A., Zauberman, A., Meir, G., Pinhasi-Kimhi, O., Suh, B.S., Oren, M., 1988. Cotransfection of granulosa cells with simian virus 40 and Ha-RAS oncogene

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