Owing to their extremely complex anatomical structures

Owing to their extremely complex anatomical structures in mammals and humans, identification of stem cell niches has proven to be difficult (Li and Xie, 2005). Consequently, in vivo studies to elucidate signals that regulate stem cell activity suffered from this circumstance. Medical application of stem tie2 for tissue replacement and gene therapy requires a detailed understanding of how the choice between stem cell self-renewal and the onset of differentiation is determined. This knowledge may facilitate the expansion of adult stem cell populations in vitro while maintaining essential stem cell characteristics (Jones, 2004). The simplicity of insect GSC/niche arrangement in both sexes and the advanced knowledge of molecular signals from the niche to GSCs make it very tempting to probe this system in vitro to potentially gain basic information that can support the understanding of human systems. Recently, Niki et al. (2006) established a stable cell line of female Drosophila GSCs. They used a bam (bag of marbles) mutant, in which the ovaries show an expansion of GSCs and GSC-like cells (Kai et al., 2005). Since Bam initiates the differentiation of GSCs and cystoblasts, the lack of Bam results in an accumulation of GSC-like cells. GSC maintenance, self-renewal, and division require the growth factor Dpp (decapentaplegic) which is provided by the cap cells (Xie et al., 2005). Cap cells/terminal filament represent the niche of the female GSCs. In the absence of the niche, Dpp had to be substituted in the culture of GSCs/GSC-like cells (Niki et al., 2006). Thus far to our knowledge, there is no report on the culture of male GSCs of Drosophila or any other insect. The testes of the L. migratoria, with its components of the testicular regenerative center, were selected for primary in vitro culture as a continuation of our previous studies on specific laser ablation of the niche cell in L. migratoria testes (Zahn et al., 2007). The GSCs, the apical cell, and the CPCs, including spermatogonia, CCs, and the cells of the follicle wall, have distinct structural characteristics which are maintained in primary culture and allowed us to identify the different cell types in vitro. The coincubation of the different components of the regenerative center and studies on partially and completely dissociated apical complexes resulted in unexpected behavior between the different components and may be indicative of the complexity of the regulation of early spermatogenesis.
Results
Discussion Insects probably offer the simplest structure of a cellular niche known. In L. migratoria the GSC niche consists of a single large cell, the AC, surrounded by about 60 GSCs in the adult. The simplicity, size, and distinct structural characteristics of the cell types of the regenerative center prompted us to study their behavior in primary cultures. Since enzymatic dissociation of the apical complex was not successful, we dissociated the tissue mechanically. Although many cells were injured by this procedure, it had the advantages of preparing ACs (i.e., niches) with varied numbers of remaining GSCs, ranging from the full set to completely naked ACs. This allowed us to address the question of what happens when a niche has space available.
Conclusion Intriguingly the in vitro culture conditions described in this study induced a dissociation of the GSCs from their niche, presumably initiated by the inactivation of the adhesive components between AC and GSCs. Consequently the observed formation of long and motile filopodia by the GSCs may reflect a search of the stem cell to reenter their niche, which most likely has lost its markers. Therefore we postulate that in the intact male, the integrity and proper functioning of the apical complex is supported by factors reaching the complex from the surrounding milieu such as the hemolymph. Since our culture system ensures the survival of the GSC, the system represents an ideal tool for the examination of peripheral factors that are necessary for the maintenance and proper function of the stem cell–niche entity. The mandatory peripheral factors for the circuitry of feedback systems that guarantee tissue homeostasis can be easily examined in this culture system as demonstrated on an exploratory set of additives.