Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach

Abstract

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

Regeneration of Starfish Radial Nerve Cord restores animal mobility and unveils a new coelomocyte population

“…The P3 cells differ from P1 cells in terms of DRAQ5 signal intensity and BF size features and differ from P2 cells mostly in BF size features. The two P1 morphotypes previously described by fluorescence microscopy (Andrade et al 2021) were also discriminated by the IA algorithm. Here, the P1S morphotype presents a smaller nucleus-to-cytoplasm ratio and a heavily granulated cytoplasm, surrounded by an uninterrupted bright red membrane.…”

“…These populations are present in similar proportions in all experimental groups, both in the control condition and in the nerve regenerating samples (at all time-points). When analyzed previously (Andrade et al 2021), two morphotypes of the haploid and low mitotic active P1 cell population were detected. One with a less heterogeneous nucleus and a higher nucleus-to-cytoplasm ratio, was hypothesized to be a non-differentiated form of the more mature morphotype.…”

“…These cells are involved in hemostasis, migrating to the site of the lesion and forming a clot that separates the inner from the outer environment to prevent bleeding (Ben Khadra et al 2015). The phagocytes, besides phagocytic activity against pathogens, form a network at the site of the lesion supporting the clot formation by the thrombocytes-like cells (Andrade et al 2021;Khadra et al 2017). Coelomocytes could also be involved in wound repair since they show an increasing level of Hsp70 during this process, and this molecule induces the release of pro-inflammatory cytokines during repair phase in mammals (Ben Khadra et al 2017;Pinsino et al 2007).…”

“…Histological analyses were used to study the cell-tissue pattern during the regeneration of the injured nerve tract. In addition, the role of coelomocytes was assessed by flow cytometry (FC) coupled with imaging flow cytometry (IFC) following the implemented experimental protocol (Andrade et al 2021). The objective being to characterize the dynamics of the coelomocyte populations circulating in the coelomic fluid during the regenerative process.…”

Stem Cells and Innate Immunity in Aquatic Invertebrates: Bridging Two Seemingly Disparate Disciplines for New Discoveries in Biology

“…Echinoderm immune cells are the so-called coelomocytes, a heterogeneous population of cells freely circulating in all coelomic spaces, including the perivisceral coelomic cavities, the open water-vascular system, the perihemal system, but also in the main tissues and organs (95)(96)(97). A general consensus on echinoderm coelomocyte types is lacking, possibly due to the actual diversity of morphotypes among the different classes ( 93) but mostly to the diversity of techniques and protocols used to analyze them (98). For this reason, from two to up to twelve cytotypes are reported in the literature (97,(99)(100)(101)(102)(103)(104)(105).…”