Advances in Aquatic Invertebrate Stem Cell Research

The stem cells discipline represents one of the most dynamic areas in biology and biomedicine. The vast majority of research on stem cells is being conducted in vertebrate models. Currently, over 98% of all cell lines are of mammalian origin, which represent only 0.4% of the extant identified metazoan evolution. In particular, aquatic invertebrates as a whole show the largest biodiversity and the widest phylogenetic radiation on Earth but have not yet significantly contributed to cell lines. Yet, with over 500 publications since the 1960s, the current lack of cell lines does not result from a lack of attempts at cultivating these cells but rather from fragmented research efforts in highly taxonomically diverse model species, a paucity in reports of negative results and persistent knowledge gaps in their in vitro metabolic requirements. To promote the establishment of aquatic invertebrate cell lines, there is thus a need for comprehensive knowledge mapping across taxa to identify adequate, possibly cell type-specific, protocols. Here, we review strategies for preparing an optimal inoculum, for optimizing culture conditions and for cell lineage authentication to monitor the quality of cell cultures. Finally, we conclude with our view on promising research perspectives towards establishing aquatic invertebrate cell lines.

Adult stem-cells (ASC) in many aquatic invertebrates not only are very common, but are also morphologically highly diverse, and exhibit a wide range of differentiation states as well as somatic and germline characteristics. One unexpected phenomenon is the presence of intracellular symbionts in the ASCs of some invertebrates. We review the literature on ASCs intracellular symbionts in sponges (phylum Porifera) and others invertebrates. We showed that the intracellular symbionts (either prokaryotic or eukaryotic) of sponges are found only in representative species of only one sponge class Demospongiae. Eukaryotic symbionts are exclusively unicellular photosynthetic algae in sponges, and are found only in pluripotent stem cells, the archaeocytes; they are documented in five orders of Demospongiae. Bacteriocyte-like cells have been reported in four other phyla, indicative of their independent evolutionary origins. Our results would add considerable insight into the establishment and maintenance of intracellular symbioses in ASCs, and provide new insight into the diversity of symbiotic associations seen across the Tree of Life.

Adult stem cells (ASCs) of aquatic invertebrates are involved in important biological processes such as regeneration and asexual reproduction. Unlike vertebrates, they share pluripotency and even totipotency and do not reside in permanent niches. Aquatic invertebrates represent the widest phylogenetic animal radiation on Earth but, up to now, limited research data are available on their ASCs. Although less studied than their vertebrate counterparts, aquatic invertebrate ASCs express orthologues of many vertebrate genes usually associated with stemness. With this review, we aim at providing a data base for current and future studies on ASC properties through a comprehensive literature analysis of intra- and inter-phylum comparisons for gene expressions and their functions in aquatic invertebrate ASCs. We concentrate on major gene families where sufficient data are available; gaps in our results will be filled by future studies on ASCs of aquatic invertebrates.

Oxylipins, oxygenated fatty acid derivatives, are well-established stress mediators acting in auto- and paracrine fashion. The most studied branch of oxylipins, eicosanoids, are produced from twenty carbon polyunsaturated fatty acids (PUFAs). In vertebrates they are synthesized mainly by lipoxygenase (LOX), cyclooxygenase (COX) and cytochrome P450-type monooxygenases. In corals, besides COX and LOX enzymes, the oxidation of arachidonic acid (AA) is catalyzed by natural fusion proteins, comprised of a LOX domain and a catalase related peroxidase domain, allene oxide synthase (AOS) or hydroperoxide lyase (HPL). Although oxylipins are well studied in vertebrate stem cells, their role in stem cells originating from marine invertebrates remains unexplored. Here, we present an overview of major oxylipin pathways in vertebrates and marine invertebrates, and discuss their potential role in invertebrate stem cells.

Multipotent interstitial stem cells in the freshwater polyp Hydra define one of the best-studied pre-bilaterian adult cell lineages. They represent a population of small, fast-cycling cells that give rise to three somatic differentiation products (neurons, nematocytes, and gland cells) under conditions of indefinite asexual growth and reproduction, and they also form the gametes when sexual reproduction is initiated. Interstitial stem cells in Hydra and other marine hydrozoans have been studied intensively using sophisticated cellular and molecular methods over several decades. Here, we discuss the properties of interstitial stem cells in Hydra and the known feedback control mechanisms maintaining tissue homeostasis and spatial distribution of interstitial cells along the polyp’s major body axis. We summarize the current state of knowledge about molecular regulation of self-renewal and somatic differentiation, and put particular emphasis on those molecular factors that have been shown to affect decision-making by using methods of functional interference.

Basic molecular mechanisms that orchestrate stem cell maintenance and fate are widely conserved across kingdoms, allowing for cross-species studies from simple model systems to mammals. In this context, planarians offer extraordinary possibilities containing a reservoir of experimentally accessible adult pluripotent stem cells, “the neoblasts”. Indeed, in vivo reverse genetic manipulation of crucial neoblast regulators allows a fine study of adult stem cell fate in their natural environment. Recent extensive transcriptomics analysis revealed that planarian neoblasts are a widely heterogeneous population including clonogenic and lineage-committed stem cells, constituting a dynamic compartment that talks with differentiated tissue for proper physiological homeostasis and tissue regeneration. In this chapter, we review, in a chronological perspective, the most recent findings in the comprehension of neoblast biology, including their embryonic origin, and compare the most accredited models of pluripotency maintenance and fate determination.

Pigmentation, a natural mechanism, plays an important role in photo-protecting larvae and embryos of sea urchins from harmful impacts of solar radiation, hypoxia, pathogens, metals and toxicants and might be useful as a marker of environmental stresses. The use of sea urchin embryos and gametes in testing developmental and production effects has been successfully developed by a number of laboratories worldwide. The objective of this study was to find the maximal expression level of the genes encoding enzymes expressed in pigment cells throughout the development of Scaphechinus mirabilis and in cell cultures of this sand dollar. Two genes related to different gene families (pks and sult) were selected for analysis in pigmentation, and their expression level was evaluated by quantitative real-time PCR. The naphthoquinoid pigments of echinoderms and related compounds form a new class of highly effective antioxidants of the phenol type, exhibiting high bactericidal, algicidal, hypotonic and psychotropic activity. Studying marine invertebrate stem cells and primarily differentiation processes and growth regulation may open novel biotechnological avenues such as new applications including basic research in translational medicine.

The regeneration process assumes the presence in the body of cells capable of self-renewal and subsequent differentiation into specialized cells. Whether these cells are stem cells or are present in circulating fluids or tissues as a pool of reserve progenitor cells, or whether they appear following dedifferentiation/transdifferentiation of specialized cells of individual tissues, are the main questions that scientists are focusing on. Understanding the origin and pathways of differentiation in coelomic fluid cells and coelomocytes of the starfish Asterias rubens was the aim of this research. The coelomic epithelium is considered as a possible source of coelomocytes. Further effective studies of coelomocyte replenishment are difficult due to the lack of protein markers characterizing various cell morphotypes. Additional difficulties lie in the heterogeneity of analyzed cell populations. In the present study, we separated cells of the coelomic fluid and the coelomic epithelium, and a subpopulation of the coelomic epithelium enriched with poorly differentiated cells, which are proposed precursors of some types of coelomocytes, in a Percoll density gradient. Characterization of the cell morphology of different fractions and their behavior in vitro (functional characteristics) revealed an enrichment of the gradient fractions in two of eight types of coelomocytes and three of eight morphotypes of cells of the coelomic epithelium.

Tunicates, the sister group of vertebrates, are cosmopolitan marine filter-feeding organisms. Various species pertaining to this chordate subphylum have been the subject of several researches, as they show remarkable stem cell mediated regenerative abilities and, in colonial species, a continuous development of new individuals by asexual reproduction. In this chapter, we highlight the current knowledge on the biology of stem cells and their involvement in development and regeneration of both solitary and colonial tunicates, with particular reference to ascidians. In addition, we put a special focus on the methods used to identify stem cells and their niches, their involvement in biological phenomena such as budding, torpor, aging and chimerism, the cellular and molecular basis of regeneration as well as on the possible future directions in the study of stemness in ascidians.

The urochordate Botryllus schlosseri belongs to the Vertebrata’s closest living invertebrate group. This colonial species represents an invertebrate model system that maintain high capacity of adult stem cell activity, where various blood cell types, expressing multipotent or totipotent phenotypes, circulate in vasculature throughout life. While in vitro cultures of isolated blood cells from Botryllus may serve as indispensable tools for studying stem cells biology, up to date, there is no single established cell line available from Botryllus, since drawn cells stop dividing in vitro within 24-72h after isolation and many of the cultures are commonly contaminated by bacteria and protists. Moreover, low yields of blood cells are of significant hindrance to the development of long-term cultures since lower numbers of cells eventually lead to poor results. Tackling these two critical technical obstacles, we present here methodologies for improved aseptic conditions and for higher yields of cells extracted from colonial vasculature. This study was performed on two colonial stocks (Israel, laboratory stocks; Helgoland, Germany- field collected stocks) which resulted with a significant difference in the numbers of cell extractions between the two stocks and significantly different blood cell yields between various blastogenic stages (laboratory stocks), further revealing differences between field/laboratory-maintained colonies

Ascidians are invertebrate chordates and may reveal parallels to vertebrate traits including cellular immunity, tissue rejection, and self-renewal, all functions executed by ascidian blood cells. Understanding their individual properties, functional plasticity, and lineage resemblances among ascidian species is, however, limited by a lack of cytochemical and molecular markers. We performed a lectin-based glycan profiling of haemocytes in three selected ascidian species to compare different blood cell populations and mirror their relatedness. We found differing repertoires of species-specific glycans for blood cells believed to be homologous in their function. Within species, characteristic glycans or glycan combinations mark haemocyte types and support their hematopoietic relatedness or distinguish maturation stages. Strikingly, Ciona and Phallusia haemoblasts have few carbohydrate decorations and drastically differ from differentiated cells, likewise phagocytes from cytotoxic cells, as compared with Botryllus, where a complex role of haemocytes in asexual self-renewal and allorecognition may involve carbohydrates. Cytotoxic cells generally carry most decorations. Within cell types, specific carbohydrates reside on the cell surface including amoeboid extensions, while others are within granules possibly marking molecules important in cytotoxicity and crosslinking. Taken together, these carbohydrate biosensors should further the molecular and functional characterisation of the outstanding properties of the different haemocytes in genetically accessible ascidian species.

This is the second part of the Appendix from Chapter 1.