Sponge Cell Culture

Sponge cell culture Klaske J. Schippers Sponge cell culture Klaske J. Schippers Thesis committee Promotors Submitted in fulfillment of the requirements for the degree of doctor Prof. dr. ir. R.H. Wijffels at Wageningen University Professor of Bioprocess Engineering by the authority of the Rector Magnificus Wageningen University Prof. dr. M.J. Kropff, in the presence of the Co-promotors Thesis Committee appointed by the Academic Board to be defended in public Prof. dr. S.A. Pomponi on Friday 15 February 2013 Professor of Marine Biotechnology, Wageningen University / Professor Harbor at 4 p.m. in the Aula. Branch Oceanographic Institute-Florida Atlantic University, USA Dr.ir. D.E. Martens Assistant professor, Bioprocess Engineering Wageningen University Other members Prof. dr. J. van der Oost, Wageningen University Dr. N.J. de Voogd, Museum Naturalis, Leiden Prof. dr. M.J. Uriz, Centro de Estudios Avanzados de Blanes, Spain Dr. ir. G.P. Pijlman, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advances studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Table of contents Chapter 1 Introduction and thesis outline 7 Chapter 2 Cultivation of sponges, sponge cells and symbionts: 21 achievements and future prospects. Advances in Marine Biology 2012. 62: 273-337. Chapter 3 Cell cycle analysis of primary sponge cell cultures. 87 In Vitro Cellular & Developmental Biology – Animal 2011. 47:302-311. Chapter 4 Toward development of a sponge cell line: comparison 105 of cell proliferation in sponge cell and tissue cultures. Submitted for publication. Chapter 5 Methods for insertion and expression of heterologous 127 genes in sponge cells. Submitted for publication. Chapter 6 General discussion: Genomic-based approach for 149 sponge cell culture. Submitted for publication. Klaske J. Schippers Summary 173 Sponge cell culture Samenvatting 179 Dankwoord 185 PhD thesis, Wageningen University, Wageningen, NL, 2013 With summaries in English and Dutch Curriculum Vitae 188 List of Publications 189 ISBN 978-94-6173-441-9 Overview of completed training activities 190 Chapter 1 Introduction and thesis outline 1 GENERAL INTRODUCTION 1.1 Biotechnological potential of sponges Sponges are simple multicellular animals and historically had commercial value as bath sponges, because of their absorptive properties. Due to the invention of the less expensive synthetic bath sponge, farming of the natural bath sponge decreased 1 (Hogg et al., 2010). Current biotechnological applications of sponges are more directed towards the production of sponge-derived bioactive compounds (Pomponi, 2001). As sponges are benthic and sessile as adults, they have evolved a sophisticated chemical communication and defense system based on secondary metabolites (Müller et al., 2004; Hogg et al., 2010). These secondary metabolites can protect the sponge against fouling (Proksch, 1994), predation (Becerro et al., 1997), or microbial infection (Sipkema et al., 2005a) and accordingly have pharmacological activities, such as antibiotic, antiviral, anti-inflammatory and antitumor activity. Figure 1 shows examples of sponge-derived bioactive compounds with pharmaceutical potential. Consequently, marine sponges are the most prolific source of newly discovered bioactive compounds, with more than 7,000 sponge-derived, novel molecules Introduction and thesis outline thesis and Introduction discovered in 40 years (Blunt et al., 2009). A. B. C. discodermolide avarol halichondrin B antitumor antipsoriasis antitumor Figure 1 Examples of sponge-derived bioactive compounds with pharmaceutical potential. A: Discodermolide is found in the sponge Discodermia dissoluta and is a potent inhibitor of tumor cell growth (Pomponi, 2001); B: Avarol is found in the sponge Dysidea avara and is found to be effective in treating psoriasis (Müller, 1991); C: Halichondrin B has been obtained from Lissodendoryx spp. and has a strong anticancer effect (Pomponi, 2001). 1.2 What are sponges? Sponges (phylum Porifera) are sessile benthic animals and are the most simple (they lack true tissue) and ancient multicellular animals on earth (Bergquist, 1978). They inhabit a wide range of ecosystems, varying from marine to freshwater, polar to tropical and shallow to deep waters. Around 8,000 sponge species are described, with an estimated species number of more than 15,000 (Hooper and Soest, 2002). 9 To support life, sponges pump huge amounts of water through their bodies and filter the sponge, are called pinacocytes. There are many more sponge cell types, such it to capture particulate organic carbon (POC), such as bacteria, micro algae and as granulocytes, thesocytes, oocytes or spermatocytes, which are described by dead organic particles (Koopmans et al., 2010). In addition they are also able to Simpson (1984). absorb dissolved organic carbon (DOC) from the water (de Goeij et al., 2008). The organization of the body of the sponge is designed for efficient filtration (see Fig 2A). Sponges reproduce either sexually or asexually (Bergquist, 1978). When they Water is pumped through small pores (ostia), which are on the outside of the sponge. reproduce sexually the sponge will develop larvae, which consist of a package of 1 The water current inside the sponge is generated by flagellated cells, called stem cells (archaeocytes), and are released via the oscula. The larvae are free- choanocytes, which are clustered in so-called choanocyte chambers inside the swimming before they attach on a substratum and develop into a juvenile sponge. sponge. The water current leaves the sponge via a large outflowing opening Asexual reproduction occurs either via fragmentation, budding or gemmule formation. (osculum). Food particles that are trapped by the microvilli of the choanocytes are Fragmentation can occur for example after a storm, and the dispersed fragments can partly digested by the choanocytes and then transferred to the archaeocytes, which reattach and form new individuals. Budding is similar to fragmentation, but then further digest them. The archaeocytes are the most important cellular component of organized by the sponge itself. Some sponges form gemmules to survive. Gemmules the mesohyl, as they distribute the nutrients over the rest of the sponge and are able are small spheres with a coating, which are located at the base of the sponge. This to differentiate into any other cell type (totipotent). The mesohyl is the tissue layer coating can resist harsh environmental circumstances, such as low temperatures or between the channels, choanocyte chambers and outer layer of the sponge (colored desiccation. Just like larvae, gemmules contain a package of stem cells (thesocytes), yellow in Fig 2B) and contains besides cellular components, also non-cellular and after hatching on a substratum they can develop into juvenile sponges. components, such as spicules, needle like structures made of silica or calcite, Introduction and thesis outline thesis and Introduction sponging or collagen fibers (see Fig 2B). These non-cellular components form the Many sponges live in symbiosis with microorganisms, such as bacteria, algae, framework of the sponge and are made by sclerocytes, spongocytes and collencytes, cyanobacteria or fungi (Taylor et al., 2007). These symbionts mostly reside in the respectively. The flat skin cells, which cover the channel walls and the exterior of mesohyl and can contribute up to 40% of the mesohyl volume. They have the ability to recycle nutrients or can fix carbon and nitrogen due to their photosynthetic activity. Another role of the symbiont is the production of bioactive compounds. For many spi sponge-derived bioactive compounds, it is not clear whether they are produced by the sponge or the symbiont. Metabolites known to be produced by the sponge are c c for example, avarol, found in Dysidea avara (Uriz et al., 1996) and stevensine, found a in Axinella corrugata (Andrade et al., 1999). On the other hand, the cyanobacterial symbiont Oscillatoria spongelia, is known to produce antimicrobial polybrominated biphenyl ethers (Unson et al., 1994). spo 1.3 How to produce sponge-derived bioactive compound? Despite the enormous biotechnological potential of sponge-derived bioactive p p compounds, only a few compounds have been successfully developed into products. A B A major obstacle is the lack of sufficient supply of biological material for preclinical and clinical development (Osinga et al., 1999; Munro et al., 1999). Although controlled Figure 2 Schematic representation of morphology and feeding physiology of sponges chemical synthesis of the bioactive molecules (or their analogues) is the preferred (Osinga et al., 1999). A: Outer appearance of a simple, vase-shaped sponge with several small production method for pharmaceutical application, most bioactive natural compounds inflowing pores (ostia) and one large outflowing opening (osculum). A cross-section shows the are chemically complex structures that cannot be synthesized easily. Also, harvesting aquiferous system (channels and choanocyte chambers). Arrows indicate the water flow through the sponge body. B: Detailed overview of canals and choanocyte chamber. A number of sponges with bioactive compounds from nature is neither environmentally nor of different cell types and skeletal elements are shown: choanocytes (c), archaeocytes (a), economically feasible (Pomponi, 1999). Cultivation of the sponge that produces the pinacocytes (p), spicules (spi), spongin fibers (spo). bioactive

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