Kylie Van Deinsen Hesp

Sponges are ancient filter‐feeding animals that seem to have barely changed since they evolved over 600 million years ago. Water enters through pores connecting to a myriad of channels, pumped through chambers where choanocytes take up particles or dissolved nutrients, and then expelled through oscula. However, this seemingly simple system conceals a complex conglomerate of chemical compounds, many of which are biologically active. These chemicals allow sponges to interact with their surroundings and protect themselves. For example, antibacterial and antiviral compounds ward off potential pathogens, while cytostatic compounds prevent other competing organisms from growing. When such compounds were first discovered, sponges were quickly recognized as a treasure trove of potential new drugs. However, a bottleneck emerged that would stymie sponge cell culture for decades: obtaining enough biomass to produce compounds at a large enough scale for clinical trials. Many studies focused on developing sponge cell lines, which would provide a scalable platform to produce compounds in a controlled environment. Despite all efforts and promising results, no sponge cell lines were established.

In Chapter 2 we reported a long‐awaited breakthrough: cells of 9 sponge species divided rapidly in amino acid‐optimized nutrient medium M1, based on mammalian cell culture medium 199 (MN99). The fastest dividing cells doubled in under an hour. Cells of species that responded to M1 medium could also divide in the more basal marine‐adjusted MN99, albeit usually slower and to a lower density. Among these species were 3 members of the genus Geodia, G. neptuni, Geodia sp., and G. barretti, which showed most consistent results between different individuals. The Geodia spp. were subcultured 3 ‐ 5 times over a period of 21 ‐ 35 days and reached an average total of 6 population doublings. All 3 species were able to proliferate at both 4 °C, the temperature used for G. barretti, and 22 °C, at which G. neptuni and Geodia sp. were incubated. The finite cell lines we developed represented the first real leads to develop stable or continuous marine sponge cell lines.

We followed up on this lead for G. barretti in Chapter 3 and established the first continuous marine sponge cell line. Cells of 3 individuals G. barretti were cultured in OpM1 medium, the successor of M1 with added growth factors, lipids, vitamins, and other nutrients. In OpM1, G. barretti cells proliferated even more rapidly and to a higher density than in M1 medium. We analyzed the impact of the individual components in OpM1 and determined that phytohemagglutinin (PHA) was a critical component. Besides this finding, all‐but‐one of the added nutrient mixtures in OpM1 contributed to the differences in cell division between OpM1 and M1. In subculturing experiments, G. barretti cells of 3 individuals could double nearly 100 times, compared to 5 doublings cells of the same individuals reached in M1. The maximum number of doublings of the G. barretti cell line has yet to be determined. Subcultured cells could be cryopreserved and thawed to inoculate new cultures. These results brought us one step closer to sponge cells producing biopharmaceuticals at industrial scale.

Although we now had proliferating sponge cells, we still did not know much about what stimulates them to divide, how they respond to nutrients, or deal with stress, among others. In Chapter 4 we started to bridge this knowledge gap by comparing genes expressed by G. barretti cells in fragments of an intact sponge, in cells after being dissociated and cryopreserved, and in cells cultured in OpM1 medium. Cells in all 3 states expressed genes that drive the cell cycle and prevent apoptosis at equal levels. Telomerase reverse transcriptase, the gene that determines the number of times the genome can be replicated, was also expressed in all samples, providing further evidence that sponge cells may be naturally immortal. Few differences were observed between sponge fragments and dissociated cells, suggesting dissociating cells does not strongly affect them. Genes expressed in cultured cells and sponge fragments were most different, showing the extensive changes needed for sponge cells to adjust to in vitro culture, either because cells actively adapt or because a specific cell type is selected by culture conditions. Cultured cells reorganized their cytoskeleton, downregulated genes needed to adhere to the extracellular matrix, synthesized more proteins and lipids, and broke down fewer lipids and glycogen reserves for energy, likely favoring soluble energy sources in the medium. Phagocytosis was also downregulated, further indicating that cells use soluble energy sources that can diffuse or be transported across the plasma membrane, either because cells adapt or because the culture conditions favor cell types with these traits. These results gave us unprecedented insight into the inner workings of sponge cells.

In Chapter 5 we demonstrated that cultured G. barretti cells can be genetically modified with the CRISPR/Cas12a system. We used CRISPR/Cas12a to introduce a double‐stranded break at a target site in the G. barretti 2',5'‐oligoadenylate synthetase gene, and inserted a scrambled DNA sequence through homology‐directed repair by providing a single‐stranded DNA donor with short homology arms. A longer double‐stranded DNA donor was used to insert and express blue fluorescent marker gene TagBFP, although efficiencies were low. Introducing longer constructs as single‐stranded donors may allow more efficient gene editing. Our results represent an important step towards developing a molecular tool box for sponge cells.

Chapter 6 reflects on what we have learned in the previous chapters, which questions have arisen from this new knowledge, and what next steps should be taken. Our results seem to implicate bad luck in choosing model organisms as the main reason why sponge cells were not cultured successfully before. Sponge cells can divide incredibly rapidly, and we hypothesize that the cells may be dividing without replicating their genome, either because tetraploid cells are present to immediately start regenerating the sponge after damage, or because the sudden change in the presence of nutrients may trigger the cells to undergo meiosis, a trait found in a phylogenetically‐related group, the unicellular choanoflagellates. Active telomerase found in sponge cells and the age sponges are reported to reach in the wild suggest sponges may be naturally immortal. Studying telomere length in the G. barretti cell line may help shed light on this matter, as well as elucidating how sponges repair DNA damage to maintain genomic integrity during their long lifespan. If sponge cells are indeed immortal, we will not need CRISPR‐Cas12a to immortalize sponge cells. However, this versatile tool has many other uses in developing production strains from sponge cells, for example by upregulating metabolic pathways to increase product yields or knocking out other pathways to direct resources to the product.

Finally, we discuss the future of sponge biotechnology. Now that we can culture sponge cells consistently and possibly continuously, many new lines of research have opened up. Methods used to develop the G. barretti cell line can be applied to other species. However, high levels of inter‐ and intraspecies variation in sponges means model organisms and source material will need to be assessed carefully using preliminary experiments, and establishing cell lines will require species‐specific optimizing of media and culture conditions. Sponge cell lines are an ideal model system for all kinds of experiments, for example to predict the impact of warmer and more acidic oceans due to climate change, study animal‐microbe (endo)symbiosis, or determine how bioactive compounds are produced. Understanding these pathways will help improve yields and produce sufficient supply of bioactive compounds for clinical trials and treatment. Before sponge cell lines can produce bioactive compounds on an industrial scale, different bioreactors will need to be tested at a small scale to determine the most efficient design. If sponge cell lines can be axenic and how this would affect desired compounds still needs to be determined. Many new challenges await, but we now have the required tools and knowledge to overcome these challenges. It is the dawn of a new era in sponge biotechnology.