Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche

Abstract

The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst progenitor cells) which directs the typical behavior of the male regenerative center. Previously we demonstrated the necessity of the apical cell for the survival of the germline stem cell. From these studies we are now able to culture viable isolated germline stem cells and all cells of its niche complex, although DNA synthesis stops after Day 1 in culture. This enables us to examine the effects of supplements to our standard medium on the interaction of the germline stem cell with its niche, the apical cell. The supplements we evaluated included conditioned medium, tissues, organs, and hemolymph of male locusts, insect hormones, mammalian growth factors, Ca(2+) ion, and a Ca(2+) ionophore. Although biological effects on the germline stem cell and apical cell could be detected with the additives, none of these supplements restored the in vivo behavior of the incubated cell types. We conclude that the strong adhesion between germline stem cells and apical cells in vivo is actively maintained by peripheral factors that reach the apical complex via hemolymph...

Recent Progress of In Vitro 3D Culture of Male Germ Stem Cells

“…Therefore, artificial technology to simulate such an in vitro microenvironment is the primary condition for the 3D culture of SSCs. On this basis, it will be of great value to develop a new in vitro 3D cell culture system to induce the differentiation of SSCs into functional male germ cells [6,7].…”

Dynamic cytoplasmic projections connect mammalian spermatogonia in vivo

“…Recent reports reveal that spermatogonia also have cellular projections in lower organisms. In locusts, male germline stem cells (GSCs) use filopodia to interact with somatic apical cells (Dorn and Dorn, 2011). In Drosophila testes, short microtubule-based nanotubes extend from the GSCs into the hub to facilitate ligandreceptor interactions between GSCs and terminally differentiated somatic supportive hub cells and cyst stem cells (Lin, 2002).…”

Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies

“…The most notable examples are cell lines derived from embryos of migratory grasshopper Melanoplus sanguinipes (Tsang et al 1981;Munderloh et al 1994) and cricket Gampsocleis gratiosa (Zhang et al 2011), and from midgut of painted grasshopper Poekilocerus pictus (Kharat et al 2010). Most reported studies which utilized the primary cell cultures of locust embryonic tissues, muscle, gonads, brain, and neuronal cells targeted rather at elaborating a vertebrate mirrored physiology than at exploring the locust itself (Duce and Usherwood 1986;Rossler and Bickmeyer 1993;Dorn and Dorn 2011;Ostrowski et al 2011). This is without undermining the intensive past efforts made to establish locust stable cell lines with an intention of studying locust-pathogen interactions, screening locust pathogens, and developing viral bio-pesticides (Khurad et al 1991;Hernandez-Crespo et al 2000).…”