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See detailLong term culture and characterization of chicken primordial germ cells
Tonus, Céline ULg; Cloquette, Karine; Ectors, Fabien ULg et al

Poster (2013, October)

Avian primordial germ cells (PGCs) can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs) can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. Moreover, we recently isolate and cultivate a new PGC line from turkey. All PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS. RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a progressive drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. At day 6, colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. In order to evaluate the germinal differentiation of cultured PGCs during the gonadal development as well as the germline transmission rate, we established a stably expressing GFP line that was successfully injected in emrbyos. Results are in progress. [less ▲]

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See detailThree-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep
Toppets, Vinciane ULg; Piret, Joëlle ULg; Gabriel, Annick ULg et al

in Journal of Neuroimmunology (2013), (262), 79-84

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See detailChapter 13: Adaptation of a Universal Procedure for Cryopreservation of Different Developmental Stages: Is it Conceivable?
Vanderzwalmen, Pierre ULg; Ectors, Fabien ULg; Grobet, Luc ULg et al

in Varghese, Alex C.; Sjöblom, Peter; Jayaprakasan, K (Eds.) A practical guide to setting up an IVF lab, embryo culture systems and running the unit (2013)

A RENEWED INTEREST IN CRYOPRESERVATION: WHY? It is now well recognized that the proportion of births following transfer of cryopreserved gametes or embryos will increase dramatically. Several reasons may ... [more ▼]

A RENEWED INTEREST IN CRYOPRESERVATION: WHY? It is now well recognized that the proportion of births following transfer of cryopreserved gametes or embryos will increase dramatically. Several reasons may explain this rising interest for embryo transfer (ET) after cryopreservation. The improvement of in vitro culture technique enables gaining better quality embryos associated with the policy of single embryo transfer (SET) results in the increasing proportion of supernumerary embryos cryopreserved at different stages of development. Moreover, with the increased efficiency of the cryopreservation technique such as vitrification, there is now a tendency to shift from fresh ET to cryopreserved ET in artificial or natural cycles. This attitude is relevant especially when the progesterone level before oocyte pick-up is above a physiological limiting value, a not optimal thickness of the endometrium or when embryos are originated from an in vitro maturation cycle. Cryopreserved ET is also an opportunity, when fresh ET cycles are cancelled because of hyperstimulation. With the introduction of vitrification, there is also an emergent change in the general attitude towards oocyte cryopreservation offering available solution, for example, in the field of preservation of fertility, ovarian hyperstimulation syndrome, poor responder, absence of sperm at the time of oocyte pick-up, cryo-banking for egg donation program or for social reason and finally to overcome ethical concerns and legal restrictions. [less ▲]

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See detailA method allowing long term culture and expansion of chicken primordial germ cells
Tonus, Céline ULg; Waroux, Olivier ULg; Grobet, Luc ULg

Conference (2013, March 12)

We developed an original method allowing long term culture (up to 20 month) of undifferentiated chicken PGCs, with good proliferation rates. We use a co-culture system: PGCs are cultivated in cell culture ... [more ▼]

We developed an original method allowing long term culture (up to 20 month) of undifferentiated chicken PGCs, with good proliferation rates. We use a co-culture system: PGCs are cultivated in cell culture inserts, in the presence of irradiated Buffalo Rat Liver cells. According to our own experience, physical separation of PGCs and feeder cells limits the differentiation of PGCs. Our PGCs lines keep their original phenotype including their gonadal colonization ability. We also developed a novel cryopreservation method based on vitrification, in complement of a slow rate freezing. [less ▲]

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See detailLower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.
Vanderzwalmen, P.; Connan, Delphine ULg; Grobet, Luc ULg et al

in Human reproduction (Oxford, England) (2013)

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary ... [more ▼]

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). WHAT IS KNOWN ALREADY: To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. STUDY DESIGN, SIZE, DURATION: Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liege. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. MAIN RESULTS AND THE ROLE OF CHANCE: The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was approximately 2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000 degrees C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. LIMITATIONS, REASONS FOR CAUTION: The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. WIDER IMPLICATIONS OF THE FINDINGS: The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. STUDY FUNDING / COMPETING INTEREST(S): The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests. [less ▲]

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See detailLong term culture and characterization of chicken primordial germ cells
Tonus, Céline ULg; Waroux, Olivier ULg; Cloquette, Karine et al

Poster (2012, November)

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species ... [more ▼]

Avian primordial germ cells (PGCs), can keep their germ cells properties and are foreseen as promising tools for developing avian transgenesis and preservation of genetic resources of endangered species. We have developed original methods that allow long term (20 month) expansion of primary cultures of undifferentiated PGCs and their efficient cryopreservation. Blood samples were collected from stage 13-18 embryos, pooled, deposited in cell culture inserts and co-cultivated in the presence of irradiated BRL cells. This physically separated co-culture system along with selective culture medium promoted emergence, selection and proliferation of undifferentiated PGCs lines. Overall, 35% of blood samples gave rise to PGCs cell lines originating from three commercial layer breeds and two Belgian endangered breeds. PGCs lines were first characterised for the expression of the stem cells and PGCs characteristic marker SSEA-1 by FACS (expression rate: 90-99%). RT-PCR confirmed expression of germ-line specific markers (CVH, CDH, DAZL), pluripotency markers (cPouV, cSox2, cNanog), telomerase and CXCR4 receptor. In addition, by means of a quantitative PCR amplification of a chromosome W specific sequence, we demonstrated a drift of all our lines towards the male sex (WL), while they were initially isolated from pooled blood samples with statistically equivalent numbers of male and female embryos (35 females: 29 males). PGCs were subsequently efficiently cryopreserved by slow freezing or by a newly developed vitrification method. Labelled PGCs from 10 lines were injected in recipient embryos. Colonization of the genital ridges confirmed that PGCs retain their gonadal migratory ability, both after long-term culture (min 3, max 20 month) and after cryopreservation. [less ▲]

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See detailOPTIMIZATION OF HUMAN ES CELLS (hESCs) CRYOPRESERVATION
Connan, Delphine ULg; Ectors, Fabien ULg; Grobet, Luc ULg et al

Poster (2012, October 22)

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See detailThe three-dimensional reconstruction of the innervation pattern in the lymphoid compartment of the ovine pharyngeal tonsil highlighted a possible way of neuro-invasion by the scrapie agent.
Toppets, Vinciane ULg; Piret, Joëlle ULg; Grobet, Luc ULg et al

in Proceedings of the 2nd Scientific Meeting of the Faculty of Veterinary Medicine, ULg, Belgium, October 19, 2012 (2012, October 19)

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See detailNeuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion
Toppets, Vinciane ULg; Piret, Joëlle ULg; Kirschvink, Nathalie et al

in Cell & Tissue Research (2012)

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being ... [more ▼]

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being debated. To determine the potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of neurofilaments heavy (200 kDa) (NFH), neurofilaments light (70 kDa) (NFL) and glial fibrillar acidic protein (GFAP) was semi-quantitatively analysed inside the different compartments of the tonsil. The results showed that the most innervated areas were the interfollicular area and the connective tissue located beneath the respiratory epithelium. Even if the germinal centre of the lymphoid follicles was poorly innervated, the existence of rare follicular dendritic cell-nerve synapses inside the germinal centre indicates that this mechanism of neuroinvasion is possible but unlikely to be unique. The host PRNP genotype did not influence the pattern of innervation in these different tonsil compartments, unlike age: an increase of nerve endings in a zone of high trafficking cells beneath the respiratory epithelium occurred with ageing. A minimal age-related increase of innervation inside the lymphoid follicles was also observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells able to transport PrPd, could ensure a more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after amplification of PrPd, or could act as direct site of entry during neuroinvasion. [less ▲]

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See detailFeatures of follicular dendritic cells in ovine pharyngeal tonsil: An in vivo and in vitro study in the context of scrapie pathogenesis
Toppets, Vinciane ULg; Defaweux, Valérie ULg; Piret, Joëlle ULg et al

in Veterinary Immunology and Immunopathology (2011), 141

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal ... [more ▼]

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion “reservoir” during the latency phase, thus playing a key role during the scrapie lymphoinvasion. [less ▲]

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See detailCryopréservation d’ovocytes et d’embryons par congélation ou vitrification dans le cadre de l’assistance médicale à la procréation
Vanderzwalmen, Pierre ULg; Ectors, Fabien ULg; Papras, Y et al

in Poncelet, Christophe; Sifer, Christophe (Eds.) Physiologie, pathologie et thérapie de la reproduction chez l’humain (2011)

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See detailAseptic vitrification of blastocysts from infertile patients, egg donors and after IVM
Vanderzwalmen, Pierre ULg; Ectors, Fabien ULg; Grobet, Luc ULg et al

in Reproductive Biomedicine Online (2009), 19(5), 700-707

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