Abstract
BackgroundIt is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal.ResultsWe show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells.ConclusionsIn E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.
Highlights
It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle
By developing methods for analyzing gene expression with cellular resolution in E. multilocularis, we show that differentiated cell types do not proliferate, and that the germinative cells are heterogeneous at the molecular level, showing in addition several differences with the neoblasts from other flatworms
Standard in vitro culture of metacestodes was done in co-culture with rat Reuber hepatoma feeder cells, and primary cell preparations were performed and cultured in cDMEM-A pre-conditioned medium essentially as previously described [34], with the following modifications: 1) cells were detached from the metacestode tissue with a single treatment of 20 minutes with trypsin/ethylenediaminetetraacetic acid (EDTA) and 2) primary cells were cultured in cDMEM-A instead of hydatid fluid
Summary
It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). Classical ultrastructural studies in planarians described the neoblasts as small, round cells with a large nucleus containing little heterochromatin and a large nucleolus, with scant cytoplasm containing mitochondria, abundant free ribosomes and few other organelles [9,10] They possess cytoplasmic electrondense ribonucleoprotein (RNP) granules called chromatoid bodies, which are molecularly and morphologically similar to the well known germ granules present in the germ cells of many animals. Many studies have shown that genes involved in posttranscriptional regulation and chromatin modification are highly upregulated in neoblasts [13,14,15,16,17,18] These include genes that are typically considered markers of germ cells in other model animals, such as the DEAD box RNA helicase vasa and the Argonaute family gene piwi [11]. The development of molecular markers has further shown that the neoblasts are heterogeneous at the molecular level [9,10]
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