Sponges (phylum porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases. Sponges (Phylum Porifera) are key components of many benthic marine ecosystems. There are more than 9,000 described species that occur worldwide, from the intertidal to the deep sea 1. Among the oldest metazoans, sponges have evolved a variety of strategies to adapt to different environments. Because they are sessile as adults, they have evolved sophisticated chemical systems for communication, defense from predators, antifoulants to prevent other organisms from growing over them, and to prevent infection from microbes filtered out of the water 2,3. These chemicals interact with molecules that have been conserved throughout evolutionary history and are involved in human disease processes, for example, cell cycling 4 , immune and inflammatory responses 5 , and calcium and sodium regulation 6,7. Vertebrate, insect, and plant cell lines are important tools for research in many disciplines, including human health, evolutionary and developmental biology, agriculture, and toxicology. Although cell lines have been established for freshwater and terrestrial invertebrates (e.g., Hydra, Caenorhabditis), and long-term (>1 month) primary cultures have been reported for cells derived from tissues of the cnidarian Anemonia viridis and the shrimp Penaeus 8,9 , attempts to establish cell lines from marine invertebrates have been unsuccessful 9-11. Marine sponges, including some of the species in this study, are the source of thousands of novel chemicals with pharmaceutically relevant properties 12-14. Supply of these chemicals is a bottleneck to development of sponge-derived drug leads: wild harvest is not ecologically sustainable, and chemical synthesis is challenging due to the complexity of many of the bioactive chemical compounds. In vitro production has been proposed as an option...

The insulin/IGF-1 signaling pathway is evolutionarily conserved and its function is mediated largely by FOXO transcription factors. Reduced insulin/IGF-1 signaling leads to translocation of FOXO proteins from the cytoplasm to the nucleus where they activate a set of genes that mediate oxidative stress, heat shock, and xenobiotic responses, innate immunity, metabolism, and autophagy. Disruptions in the insulin/IGF-1 signaling pathway affect lifespan in many species. Over the past two decades, the function of these FOXO proteins in age-related diseases has been extensively studied, in the model organism Caenorhabditis elegans as well as in humans. In this review we investigate the mechanisms by which FOXO proteins influence the development of age-related disease pathways in both species.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Sponges and their associated microorganisms are the most prolific source of marine natural products, and many attempts have been made at creating a marine sponge cell line to produce these products efficiently. However, limited knowledge on the nutrients sponge cells require to grow and poor genetic accessibility have hampered progress toward this goal. Recently, a new sponge-specific nutrient medium M1 has been shown to stimulate sponge cells in vitro to divide rapidly. In this study, we demonstrate for the first time that sponge cells growing in M1 can be genetically modified using a CRISPR/Cas12a gene editing system. A short sequence of scrambled DNA was inserted using a single-stranded oligodeoxynucleotide donor template to disrupt the 2′,5′-oligoadenylate synthetase gene of cells from the boreal deep-sea sponge Geodia barretti. A blue fluorescent marker gene appeared to be inserted in an intron of the same gene and expressed by a small number of G. barretti cells. Our results represent an important step toward developing an optimized continuous sponge cell line to produce bioactive compounds.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 minutes, more than 2-fold higher than the already rapid growth rate of 0.74 population doublings in 30 minutes in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.