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Lu Z, Quack T, Hahnel S, Gelmedin V, Pouokam E, Diener M, Hardt M, Michel G, Baal N, Hackstein H, Grevelding CG. Isolation, enrichment and primary characterisation of vitelline cells from Schistosoma mansoni obtained by the organ isolation method. Int J Parasitol 2015; 45:663-72. [PMID: 25937359 DOI: 10.1016/j.ijpara.2015.04.002] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2015] [Revised: 04/01/2015] [Accepted: 04/06/2015] [Indexed: 10/23/2022]
Abstract
In the emerging era of post-genomic research on schistosomes, new methods are required to functionally analyse genes of interest in more detail. Among other tools, schistosome cell lines are needed to overcome present research constraints. Based on a recently established organ isolation protocol for adult Schistosoma mansoni, we report here on the successful enrichment of vitellarium tissue and isolation of vitelline cells. Morphological analyses performed by bright field, fluorescence, scanning and transmission electron microscopy showed typical features of S1 to S4 stage vitelline cells. In addition, molecular analyses using reverse transcription-PCR confirmed the identity of vitelline cells. Cytological and physiological studies included staining experiments with viability dyes and a neutral lipid stain, as well as calcium (Ca2+) imaging. Together they demonstrated cell viability, the possibility to define the differentiation stage of individual vitelline cells, and the suitability to investigate Ca(2+)-associated processes herein. Finally, fluorescence-activated cell sorting was shown to be a convenient way to separate and enrich S1 to S4 stage vitelline cells. In summary, these results demonstrate the expedience of the organ isolation protocol to obtain vitellarium tissue. Importantly, the protocol allows vitelline cells representing defined differentiation stages to be purified, which can be cultured in vitro and used to investigate diverse aspects of schistosome reproductive biology in the post-genomic era.
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Affiliation(s)
- Zhigang Lu
- BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany
| | - Thomas Quack
- BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany
| | - Steffen Hahnel
- BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany
| | - Verena Gelmedin
- BFS, Institute of Parasitology, Justus-Liebig-University, Giessen, Germany
| | - Ervice Pouokam
- Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany
| | - Martin Diener
- Institute for Veterinary Physiology and Biochemistry, Justus-Liebig-University, Giessen, Germany
| | - Martin Hardt
- BFS, Imaging Unit, Justus-Liebig-University, Giessen, Germany
| | - Gabriela Michel
- Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany
| | - Nelli Baal
- Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany
| | - Holger Hackstein
- Institute for Clinical Immunology and Transfusion Medicine, BFS FACS Unit, Justus-Liebig-University, Giessen, Germany
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Ye Q, Dong HF, Grevelding CG, Hu M. In vitro cultivation of Schistosoma japonicum-parasites and cells. Biotechnol Adv 2013; 31:1722-37. [DOI: 10.1016/j.biotechadv.2013.09.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2013] [Revised: 09/06/2013] [Accepted: 09/08/2013] [Indexed: 11/27/2022]
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Hahnel S, Lu Z, Wilson RA, Grevelding CG, Quack T. Whole-organ isolation approach as a basis for tissue-specific analyses in Schistosoma mansoni. PLoS Negl Trop Dis 2013; 7:e2336. [PMID: 23936567 PMCID: PMC3723596 DOI: 10.1371/journal.pntd.0002336] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2013] [Accepted: 06/14/2013] [Indexed: 12/11/2022] Open
Abstract
Background Schistosomiasis is one of the most important parasitic diseases worldwide, second only to malaria. Schistosomes exhibit an exceptional reproductive biology since the sexual maturation of the female, which includes the differentiation of the reproductive organs, is controlled by pairing. Pathogenicity originates from eggs, which cause severe inflammation in their hosts. Elucidation of processes contributing to female maturation is not only of interest to basic science but also considering novel concepts combating schistosomiasis. Methodology/Principal Findings To get direct access to the reproductive organs, we established a novel protocol using a combined detergent/protease-treatment removing the tegument and the musculature of adult Schistosoma mansoni. All steps were monitored by scanning electron microscopy (SEM) and bright-field microscopy (BF). We focused on the gonads of adult schistosomes and demonstrated that isolated and purified testes and ovaries can be used for morphological and structural studies as well as sources for RNA and protein of sufficient amounts for subsequent analyses such as RT-PCR and immunoblotting. To this end, first exemplary evidence was obtained for tissue-specific transcription within the gonads (axonemal dynein intermediate chain gene SmAxDynIC; aquaporin gene SmAQP) as well as for post-transcriptional regulation (SmAQP). Conclusions/Significance The presented method provides a new way of getting access to tissue-specific material of S. mansoni. With regard to many still unanswered questions of schistosome biology, such as elucidating the molecular processes involved in schistosome reproduction, this protocol provides opportunities for, e.g., sub-transcriptomics and sub-proteomics at the organ level. This will promote the characterisation of gene-expression profiles, or more specifically to complete knowledge of signalling pathways contributing to differentiation processes, so discovering involved molecules that may represent potential targets for novel intervention strategies. Furthermore, gonads and other tissues are a basis for cell isolation, opening new perspectives for establishing cell lines, one of the tools desperately needed in the post-genomic era. As a neglected disease, schistosomiasis is still an enormous problem in the tropics and subtropics. Since the 1980s, Praziquantel (PZQ) has been the drug of choice but can be anticipated to lose efficacy in the future due to emerging resistance. Alternative drugs or efficient vaccines are still lacking, strengthening the need for the discovery of novel strategies and targets for combating schistosomiasis. One avenue is to understand the unique reproductive biology of this trematode in more detail. Sexual maturation of the adult female depends on a constant pairing with the male. This is a crucial prerequisite for the differentiation of the female reproductive organs such as the vitellarium and ovary, and consequently for the production of mature eggs. These are needed for life-cycle maintenance, but they also cause pathogenesis. With respect to adult males, the production of mature sperm is essential for fertilisation and life-cycle progression. In our study we present a convenient and inexpensive method to isolate reproductive tissues from adult schistosomes in high amounts and purity, representing a source for gonad-specific RNA and protein, which will serve for future sub-transcriptome and -proteome studies helping to characterise genes, or to unravel differentiation programs in schistosome gonads. Beyond that, isolated organs may be useful for approaches to establish cell cultures, desperately needed in the post-genomic era.
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Affiliation(s)
- Steffen Hahnel
- Institute of Parasitology, Justus-Liebig-University Giessen, Giessen, Germany
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Abstract
Schistosomiasis and food-borne trematodiases are chronic parasitic diseases affecting millions of people mostly in the developing world. Additional drugs should be developed as only few drugs are available for treatment and drug resistance might emerge. In vitro and in vivo whole parasite screens represent essential components of the trematodicidal drug discovery cascade. This review describes the current state-of-the-art of in vitro and in vivo screening systems of the blood fluke Schistosoma mansoni, the liver fluke Fasciola hepatica and the intestinal fluke Echinostoma caproni. Examples of in vitro and in vivo evaluation of compounds for activity are presented. To boost the discovery pipeline for these diseases there is a need to develop validated, robust high-throughput in vitro systems with simple readouts.
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Brindley PJ, Pearce EJ. Genetic manipulation of schistosomes. Int J Parasitol 2007; 37:465-73. [PMID: 17280677 DOI: 10.1016/j.ijpara.2006.12.012] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2006] [Revised: 12/10/2006] [Accepted: 12/11/2006] [Indexed: 10/23/2022]
Abstract
In contrast to the situations with model organisms and parasitic protozoa, progress with gene manipulation with schistosomes has been delayed by impediments that include our inability to maintain the life cycle in vitro, absence of immortalized cell lines, large genome sizes, unavailability of drug resistance markers and other difficulties. However, in the past few years, tangible progress has been reported towards development of tools for gene manipulation and transgenesis of schistosomes, and there is reason to believe that the field is on the verge of transformation into an era where genetic manipulation is routine. Recent reports dealing with approaches and tools to manipulate the genome and gene expression in schistosomes are reviewed here.
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Affiliation(s)
- Paul J Brindley
- Department of Tropical Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
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Ming Z, Dong H, Zhong Q, Grevelding CG, Jiang M. The effect of a mutagen (N-methyl-N-nitro-N-nitrosoguanidine) on cultured cells from adult Schistosoma japonicum. Parasitol Res 2005; 98:430-7. [PMID: 16385406 DOI: 10.1007/s00436-005-0083-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2005] [Accepted: 11/07/2005] [Indexed: 11/26/2022]
Abstract
The transforming effect of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on cultured cells from Schistosoma japonicum (S. japonicum) was studied using mono-factor and orthogonal tests. Under the influence of MNNG, cultured cells grew well, and cell survival time was more than 246 days in low-serum medium. When treated with 3 mug/ml MNNG for 48 h, the number of dividing cells increased significantly as determined by bright-field and scanning electron microscopy (SEM). Under these conditions, abundant microvilli, ruffles, microridges, papillae and blebs were observed on the surface of the induced cells. Treatment with MNNG may overcome existing limitations to get continually proliferating schistosome cells and open the possibility to immortalize isolated cells.
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Affiliation(s)
- Zhenping Ming
- Department of Medical Parasitology and Research Laboratory of Schistosomiasis, Wuhan University School of Medicine, Wuhan, Hubei Province, 430071, People's Republic of China
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Dong HF, Chen XB, Ming ZP, Zhong QP, Jiang MS. Ultrastructure of cultured cells from Schistosoma japonicum. Acta Trop 2002; 82:225-34. [PMID: 12020896 DOI: 10.1016/s0001-706x(02)00014-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Ultrastructures and their dynamic changes of the cultured cells from Schistosoma japonicum were observed in the present experiments. Several types-including polygonal, round granular, deltaic fan-shaped and flagellated cells-were found in the cultures. The polygonal cells took a major ratio in the cultures from adult S. japonicum, while the majority from schistosomula was round granular cells. The ultrastuctures on the cell surface were different between the cells from adults and schistosomula. Some papilla-like tubercula, microvilli and pinocytotic vesicles were observed on the surface of adult cells, but none were found on schistosomula cells. However, more or less mitochondria, endoplasmic reticula, ribosomes and glycogen were observed in the cytoplasm of the cultured cells from both adults and schistosomula. Golgi complexes were rarely found. The nucleus was round, with round nucleolus inside and clear pores on the unit membrane. There was much lumpish heterochromatin located near to the nuclear membrane. Cells from different worm tissues had their own organelles. The germ cells, vitelline cells, flame cells, multinucleate subtegumental cells and nerve cells could be observed in the cultures from adults. The vitelline cells were the greatest in number and nerve cells were the least in number among them. Similarly, there were germ cells, sustentacular cells, flame cells, nerve cells, mast cells, muscle cells, multinucleate subtegumental cells, interstitial cells and penetration gland cells in the cultures from the schistomomula. In addition, a few division cells were also found. It indicated that the schistosomula cells had greater potential ability to proliferate than the adult cells in in vitro culture. Along with the prolongation of the culture time, degeneration of schistosomal cell occurred more and more. Generally, the electron density of cultures gradually got lower, the cristae of mitochondria blurred and disappeared and the mitochondria themselves swelled and finally vacuoled completely. Vitelline cells were most sensitive to the changes of the in vitro condition in all cultures. Their degeneration showed the following characteristics: (1) vitelline globules fused each other, the space between vitelline globules and the membrane surrounding them broadened gradually and vitelline globules were released and uncovered; (2) rough-surfaced endoplasmic reticula enlarged, vacuolated and the ribosomes dropped; and (3) the number and volume of lipid increased. The ultrastructural changes of most of the cultures from schistosomula had the following trends: (1) heterochromatin increased and euchromatin decreased gradually; and (2) endoplasmic reticula changed into short tubes and vacuoles and disappeared finally. The degenerative process of the cultures from S. japonicum consisted of necrosis according to the ultrastructural changes of the mitochondria, vitelline globules, chromatin and endoplasmic reticula within the cells. The changes of the above structures could be used to estimate whether the culture conditions were appropriate.
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Affiliation(s)
- Hui-Fen Dong
- Department of Medical Parasitology and Research Laboratory of Schistosomiasis, Medical School, Wuhan University, Wuhan, Hubei Province 430071, People's Republic of China
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Ivanchenko MG, Lerner JP, McCormick RS, Toumadje A, Allen B, Fischer K, Hedstrom O, Helmrich A, Barnes DW, Bayne CJ. Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni. Proc Natl Acad Sci U S A 1999; 96:4965-70. [PMID: 10220402 PMCID: PMC21800 DOI: 10.1073/pnas.96.9.4965] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.
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Affiliation(s)
- M G Ivanchenko
- Department of Zoology, Oregon State University, Corvallis, OR 97331, USA
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Abstract
Establishment of cell lines from insect and arachnid invertebrates has become routine, whereas other invertebrate taxa have been frustratingly unproductive of cell lines. None is available for any marine invertebrate, despite a strong and well-recognized need for cell lines from species that are important in aquaculture, from parasite vectors and intermediate hosts of parasites, from parasites themselves, from certain biomedical models, and from other species that are pests. Drawing on experiences gained attempting to establish cell lines from molluscs and trematodes and on published and ongoing research with diverse invertebrates, this chapter attempts to anticipate the problems that are likely to be encountered in such endeavors and discusses possible solutions. Criteria to be considered in the selection of basic culture media, temperature, pH, and media additives; approaches that have been developed to yield sterile primary cultures; and factors to consider in decisions about feeding schedules, retention of tissue fragments and nonadherent cells, use of heterologous feeder layers, and other variables are described. Suggestions are made concerning means to objectively score the success of tested variables and means to induce cell replication. The chapter ends with notes on conventional means to characterize cell lines and an account of contemporary efforts to immortalize cells by means of genome manipulation. Enduring success with a single molluscan cell line, transient successes with crustacean and helminth cell lines, and promising developments in transgenesis with invertebrates all lead to the hopeful conclusion that the invisible barrier to cell propagation in historically refractory species will soon be a thing of the past.
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Affiliation(s)
- C J Bayne
- Department of Zoology, Oregon State University, Corvallis 97331, USA
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