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1.
Comput Methods Programs Biomed ; 221: 106857, 2022 Jun.
Article de Anglais | MEDLINE | ID: mdl-35597201

RÉSUMÉ

BACKGROUND AND OBJECTIVE: Independent living and transportation are crucial aspects for people living with a disability. After an injury, it is important to assess driving ability, in terms of physical and psychological conditions, and to test the effects of prescribed drugs. Within this framework, driving simulators are suitable tools for training driving skills; however, available tools are expensive or lack appropriate sets of behavioral measures to fully characterize the drivers' ability. METHODS: This work presents the first step toward the development of ADRIS, a new open-source, accessible, realistic virtual reality simulator for training and testing driving skills of people with sensory-motor disability. This includes a prototype based on an open-source simulator for autonomous driving research (CARLA), with the addition of customized features such as adaptable driving controllers, a virtual reality headset, and the possibility to collect behavioral and physiological data. Also, the new system allows to set different environmental conditions, to include and control the timing of potentially dangerous situations, and to set scenarios with various difficulty levels. RESULTS: Tests on 17 healthy participants demonstrated that the simulator is well tolerated in terms of discomfort, physical fatigue, and mental effort. Also, the system is easy to use and is capable of providing a realistic driving experience, allowing the extraction of reliable behavioral parameters. CONCLUSIONS: ADRIS combines a high-fidelity virtual world, with customizable features specifically designed for the training and testing of people living with a disability, thus making it usable in many contexts such as home training, rehabilitation, education, and research.


Sujet(s)
Conduite automobile , Personnes handicapées , Troubles moteurs , Réalité de synthèse , Conduite automobile/enseignement et éducation , Conduite automobile/psychologie , Simulation numérique , Humains
3.
J Assist Reprod Genet ; 37(5): 1029-1036, 2020 May.
Article de Anglais | MEDLINE | ID: mdl-32198717

RÉSUMÉ

Cell pluripotency, spatial restriction, and development are spatially and temporally controlled by epigenetic regulatory mechanisms that occur without any permanent loss or alteration of genetic material, but rather through modifications "on top of it." These changes modulate the accessibility to transcription factors, either allowing or repressing their activity, thus shaping cell phenotype. Several studies have demonstrated the possibility to interact with these processes, reactivating silenced genes and inducing a high plasticity state, via an active demethylating effect, driven by ten-eleven translocation (TET) enzymes and an overall decrease of global methylation. In agreement with this, TET activities have been shown to be indispensable for mesenchymal to epithelial transition of somatic cells into iPSCs and for small molecule-driven epigenetic erasure. Beside the epigenetic mechanisms, growing evidences highlight the importance of mechanical forces in supporting cell pluripotency, which is strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, through the activation of specific mechanosensing-related pathways. In this review, we discuss and provide an overview of small molecule ability to modulate cell plasticity and define cell fate through the activation of direct demethylating effects. In addition, we describe the contribution of the Hippo signaling mechanotransduction pathway as one of the mechanisms involved in the maintenance of pluripotency during embryo development and its induction in somatic cells.


Sujet(s)
Différenciation cellulaire/génétique , Reprogrammation cellulaire/génétique , Développement embryonnaire/génétique , Cellules souches pluripotentes/cytologie , Méthylation de l'ADN/génétique , Épigenèse génétique/génétique , Fibroblastes , Humains , Mécanotransduction cellulaire/génétique , Transduction du signal/génétique
4.
Theriogenology ; 145: 77-85, 2020 Mar 15.
Article de Anglais | MEDLINE | ID: mdl-32004821

RÉSUMÉ

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency.


Sujet(s)
Bovins , Liquide folliculaire/composition chimique , microARN/métabolisme , Ovocytes/métabolisme , Animaux , Femelle , Régulation de l'expression des gènes au cours du développement , microARN/génétique
5.
Animal ; 13(12): 2830-2839, 2019 Dec.
Article de Anglais | MEDLINE | ID: mdl-31199215

RÉSUMÉ

Pig intestinal epithelium undergoes a complete renewal every 2 to 3 days that is driven by intestinal stem cells (ISCs) located at the crypt base in their niche. Intestinal stem cells generate a pool of highly proliferative transit-amplifying cells, which either migrate up the villus and differentiate into enterocytes and secretory cells or migrate towards the base of the crypt where they differentiate into Paneth cells that secrete antimicrobial peptides. The balance between ISCs' self-renewal and differentiation controls intestinal epithelial homeostasis; therefore, ISCs are essential for ensuring intestinal epithelial integrity. Detailed knowledge of these mechanisms in pig and other domestic species is very limited. Therefore, the aim of this work was to characterize ISC from birth to weaning. We analysed the duodenum, jejunum and colon of six piglets at birth, 6-day-old nursing piglets and 28-day-old weanlings, one week after weaning. We immunolocalized homeobox only protein+ (HOPX) and sex-determining region Y-box 9+ (SOX9) cells that identify quiescent and active ISC, respectively. The volume of ISCs was quantified with stereological methods and was compared to that of mitotic cells expressing proliferating cell nuclear antigen and apoptotic cells identified by the presence of cleaved caspase-3. Furthermore, we compared all these values with crypts and villi measurements and their ratio. Our results indicated that both quiescent and active ISCs are present in pig intestine from birth to weaning and are localized in the crypts of the small and large intestine. However, both markers were also observed along the villi and on the colon luminal epithelium, suggesting that at these stages, pig mucosa is still immature. Weaning induced a dramatic reduction of both HOPX+ and SOX9+ cells, but SOX9+ cells underwent a significantly greater reduction in the small intestine than in the colon. This suggests that the two ISC types are differentially regulated along the intestinal tracts. Overall, the pig ISC complex has many similarities with its murine counterpart, but also has some differences. These include active ISC not showing the typical columnar base morphology as well as the absence of bona fide Paneth cells. This is the first description of ISC dynamics during pig's early life and provides useful reference data for future studies, aimed at targeting ISC for the development of efficient alternatives to in-feed antibiotics for preserving intestinal integrity.


Sujet(s)
Suidae/physiologie , Animaux , Différenciation cellulaire , Prolifération cellulaire , Entérocytes/physiologie , Femelle , Muqueuse intestinale/cytologie , Muqueuse intestinale/physiologie , Intestins/cytologie , Intestins/physiologie , Mâle , Parturition , Grossesse , Cellules souches/cytologie , Cellules souches/physiologie , Suidae/génétique , Sevrage
6.
Reprod Fertil Dev ; 29(8): 1545-1555, 2017 Aug.
Article de Anglais | MEDLINE | ID: mdl-27623773

RÉSUMÉ

MicroRNAs (miRNAs) are known to control several reproductive functions, including oocyte maturation, implantation and early embryonic development. Recent advances in deep sequencing have allowed the analysis of all miRNAs of a sample. However, when working with embryos, due to the low RNA content, miRNA profiling is challenging because of the relatively large amount of total RNA required for library preparation protocols. In the present study we compared three different procedures for RNA extraction and prepared libraries using pools of 30 bovine blastocysts. In total, 14 of the 15 most abundantly expressed miRNAs were common to all three procedures. Furthermore, using miRDeep discovery and annotation software (Max Delbrück Center), we identified 1363 miRNA sequences, of which bta-miR-10b and bta-miR-378 were the most abundant. Most of the 179 genes identified as experimentally validated (86.6%) or predicted targets (13.4%) were associated with cancer canonical pathways. We conclude that reliable analysis of bovine blastocyst miRNAs can be achieved using the procedures described herein. The repeatability of the results across different procedures and independent replicates, as well as their consistency with results obtained in other species, support the biological relevance of these miRNAs and of the gene pathways they modulate in early embryogenesis.


Sujet(s)
Blastocyste/métabolisme , Analyse de profil d'expression de gènes/méthodes , Régulation de l'expression des gènes au cours du développement , Séquençage nucléotidique à haut débit , microARN/métabolisme , Animaux , Bovins , Épigenèse génétique , Femelle , microARN/génétique , Grossesse
7.
Clin Epigenetics ; 8: 119, 2016.
Article de Anglais | MEDLINE | ID: mdl-27891192

RÉSUMÉ

In the presence of different environmental cues that are able to trigger specific responses, a given genotype has the ability to originate a variety of different phenotypes. This property is defined as plasticity and allows cell fate definition and tissue specialization. Fundamental epigenetic mechanisms drive these modifications in gene expression and include DNA methylation, histone modifications, chromatin remodeling, and microRNAs. Understanding these mechanisms can provide powerful tools to switch cell phenotype and implement cell therapy. Environmentally influenced epigenetic changes have also been associated to many diseases such as cancer and neurodegenerative disorders, with patients that do not respond, or only poorly respond, to conventional therapy. It is clear that disorders based on an individual's personal genomic/epigenomic profile can rarely be successfully treated with standard therapies due to genetic heterogeneity and epigenetic alterations and a personalized medicine approach is far more appropriate to manage these patients. We here discuss the recent advances in small molecule approaches for personalized medicine, drug targeting, and generation of new cells for medical application. We also provide prospective views of the possibility to directly convert one cell type into another, in a safe and robust way, for cell-based clinical trials and regenerative medicine.


Sujet(s)
Thérapie cellulaire et tissulaire/méthodes , Épigenèse génétique , Médecine de précision/méthodes , Bibliothèques de petites molécules/pharmacologie , Animaux , Différenciation cellulaire , Systèmes de délivrance de médicaments , Hétérogénéité génétique , Humains , Bibliothèques de petites molécules/usage thérapeutique , Cellules souches/cytologie , Cellules souches/effets des médicaments et des substances chimiques
8.
Vet J ; 211: 52-6, 2016 May.
Article de Anglais | MEDLINE | ID: mdl-27033591

RÉSUMÉ

Diabetes is among the most frequently diagnosed endocrine disorder in dogs and its prevalence continues to increase. Medical management of this pathology is lifelong and challenging because of the numerous serious complications. A therapy based on the use of autologous viable insulin-producing cells to replace the lost ß cell mass would be very advantageous. A protocol to enable the epigenetic conversion of canine dermal fibroblasts, obtained from a skin biopsy, into insulin-producing cells (EpiCC) is described in the present manuscript. Cells were briefly exposed to the DNA methyltransferase inhibitor 5-azacytidine (5-aza-CR) in order to increase their plasticity. This was followed by a three-step differentiation protocol that directed the cells towards the pancreatic lineage. After 36 days, 38 ± 6.1% of the treated fibroblasts were converted into EpiCC that expressed insulin mRNA and protein. Furthermore, EpiCC were able to release insulin into the medium in response to an increased glucose concentration. This is the first evidence that generating a renewable autologous, functional source of insulin-secreting cells is possible in the dog. This procedure represents a novel and promising potential therapy for diabetes in dogs.


Sujet(s)
Techniques de reprogrammation cellulaire/méthodes , Reprogrammation cellulaire , Épigenèse génétique , Fibroblastes/cytologie , Cellules à insuline/cytologie , Animaux , Différenciation cellulaire , Lignage cellulaire , Chiens , Culture de cellules primaires , Peau/cytologie , Peau/croissance et développement
9.
J Chromatogr A ; 1406: 59-67, 2015 Aug 07.
Article de Anglais | MEDLINE | ID: mdl-26129985

RÉSUMÉ

The water framework directives (WFD 2000/60/EC and 2013/39/EU) force European countries to monitor the quality of their aquatic environment. Among the priority hazardous substances targeted by the WFD, short chain chlorinated paraffins C10-C13 (SCCPs), still represent an analytical challenge, because few laboratories are nowadays able to analyze them. Moreover, an annual average quality standards as low as 0.4µgL(-1) was set for SCCPs in surface water. Therefore, to test for compliance, the implementation of sensitive and reliable analysis method of SCCPs in water are required. The aim of this work was to address this issue by evaluating automated solid phase micro-extraction (SPME) combined on line with gas chromatography-electron capture negative ionization mass spectrometry (GC/ECNI-MS). Fiber polymer, extraction mode, ionic strength, extraction temperature and time were the most significant thermodynamic and kinetic parameters studied. To determine the suitable factors working ranges, the study of the extraction conditions was first carried out by using a classical one factor-at-a-time approach. Then a mixed level factorial 3×2(3) design was performed, in order to give rise to the most influent parameters and to estimate potential interactions effects between them. The most influent factors, i.e. extraction temperature and duration, were optimized by using a second experimental design, in order to maximize the chromatographic response. At the close of the study, a method involving headspace SPME (HS-SPME) coupled to GC/ECNI-MS is proposed. The optimum extraction conditions were sample temperature 90°C, extraction time 80min, with the PDMS 100µm fiber and desorption at 250°C during 2min. Linear response from 0.2ngmL(-1) to 10ngmL(-1) with r(2)=0.99 and limits of detection and quantification, respectively of 4pgmL(-1) and 120pgmL(-1) in MilliQ water, were achieved. The method proved to be applicable in different types of waters and show key advantages, such as simplicity, automation and sensitivity, required for the monitoring programs linked to the WFD.


Sujet(s)
Surveillance de l'environnement/méthodes , Paraffine/analyse , Extraction en phase solide , Polluants chimiques de l'eau/analyse , Eau/composition chimique , Europe , Chromatographie gazeuse-spectrométrie de masse , Limite de détection , Reproductibilité des résultats , Plan de recherche , Température
10.
Reprod Fertil Dev ; 27(5): 776-83, 2015 Jun.
Article de Anglais | MEDLINE | ID: mdl-25739562

RÉSUMÉ

Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a 'highly permissive' state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed 'epigenetic conversion'. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.


Sujet(s)
Lignage cellulaire/physiologie , Plasticité cellulaire/physiologie , Reprogrammation cellulaire/physiologie , Épigenèse génétique , Phénotype , Cellules souches pluripotentes/cytologie , Animaux , Transdifférenciation cellulaire/physiologie
11.
J Assist Reprod Genet ; 32(4): 645-52, 2015 Apr.
Article de Anglais | MEDLINE | ID: mdl-25620022

RÉSUMÉ

PURPOSE: In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated. METHODS: We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy. RESULTS: STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle. CONCLUSIONS: The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.


Sujet(s)
Calréticuline/métabolisme , Cytoplasme/métabolisme , Protéines du cytosquelette/métabolisme , Ovocytes/métabolisme , Ovogenèse/génétique , Protéines de liaison à l'ARN/métabolisme , Calréticuline/génétique , Cryoconservation , Protéines du cytosquelette/génétique , Femelle , Humains , Méiose/génétique , ARN messager/génétique , ARN messager/métabolisme , Protéines de liaison à l'ARN/génétique , Appareil du fuseau/génétique , Appareil du fuseau/métabolisme
12.
Stem Cell Rev Rep ; 10(1): 31-43, 2014 Feb.
Article de Anglais | MEDLINE | ID: mdl-24072393

RÉSUMÉ

Large animal models provide useful data for pre-clinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.


Sujet(s)
Azacitidine/pharmacologie , Reprogrammation cellulaire/effets des médicaments et des substances chimiques , Fibroblastes/cytologie , Fibroblastes/effets des médicaments et des substances chimiques , Cellules à insuline/cytologie , Cellules à insuline/effets des médicaments et des substances chimiques , Peau/cytologie , Animaux , Apoptose/effets des médicaments et des substances chimiques , Glycémie/analyse , Prolifération cellulaire/effets des médicaments et des substances chimiques , Transplantation cellulaire , Diabète expérimental/induit chimiquement , Diabète expérimental/thérapie , Fibroblastes/métabolisme , Cellules à insuline/métabolisme , Mâle , Souris , Souris SCID , Streptozocine/administration et posologie , Relation structure-activité , Suidae , Facteurs temps
13.
Hum Reprod ; 29(1): 114-24, 2014 Jan.
Article de Anglais | MEDLINE | ID: mdl-24135077

RÉSUMÉ

STUDY QUESTION: Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER: Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY: Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION: We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION: The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS: Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S): Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.


Sujet(s)
Cryoconservation/méthodes , Ovaire/cytologie , Animaux , Prolifération cellulaire , Cryoconservation/médecine vétérinaire , Altération de l'ADN , Réparation de l'ADN , Femelle , Congélation , Protéines du choc thermique/biosynthèse , Histone/biosynthèse , Techniques de culture d'organes , Follicule ovarique/physiologie , Ovaire/métabolisme , Rad51 Recombinase/biosynthèse , Ovis , Ovis aries
14.
Reprod Domest Anim ; 47 Suppl 5: 11-7, 2012 Aug.
Article de Anglais | MEDLINE | ID: mdl-22913556

RÉSUMÉ

Pluripotent stem cells are the focus of an extremely active field of investigation that is bringing new light on our understanding of the mechanisms that control pluripotency and differentiation. Rodent and primates are the only species where true, or bona fide, pluripotent stem cells have been derived. The attempts to derive pluripotent stem cells from domestic ungulates have been going on for more than 20 years with little progress. Cell lines from these species present a series of limitations that have precluded their use for both basic and clinically oriented studies. However, in the last 3 years, some substantial progress have been made making the currently available ungulate pluripotent stem cells closest than ever before to their human and mouse counterpart. This result has been achieved through both conceptual and technical progress that will be illustrated and discussed in this review.


Sujet(s)
Cellules souches pluripotentes , Ruminants , Sus scrofa , Animaux , Lignée cellulaire , Cellules souches embryonnaires/cytologie , Feuillets embryonnaires/cytologie , Humains , Souris , Cellules souches pluripotentes/cytologie , Primates , Rodentia , Ovis aries
15.
Reprod Domest Anim ; 47 Suppl 4: 86-91, 2012 Aug.
Article de Anglais | MEDLINE | ID: mdl-22827355

RÉSUMÉ

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals.


Sujet(s)
Cellules souches pluripotentes/physiologie , Suidae/embryologie , Animaux , Marqueurs biologiques , Lignée cellulaire , Embryon de mammifère/cytologie , Embryon de mammifère/métabolisme , Régulation de l'expression des gènes au cours du développement
16.
J Dairy Sci ; 95(4): 1885-93, 2012 Apr.
Article de Anglais | MEDLINE | ID: mdl-22459835

RÉSUMÉ

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.


Sujet(s)
Facteur-9 de croissance et de différenciation/analyse , Mammite bovine/physiopathologie , Follicule ovarique/physiopathologie , Animaux , Bovins , Maladies des bovins/étiologie , Maladie chronique , Industrie laitière , Femelle , Infertilité féminine/étiologie , Infertilité féminine/médecine vétérinaire , Mammite bovine/complications , Mammite bovine/anatomopathologie , Ovocytes/composition chimique , Follicule ovarique/anatomopathologie
18.
Theriogenology ; 77(4): 766-72, 2012 Mar 01.
Article de Anglais | MEDLINE | ID: mdl-22217572

RÉSUMÉ

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.


Sujet(s)
Différenciation cellulaire , Développement embryonnaire , Parthénogenèse , Animaux , Lignée cellulaire , Centrosome/physiologie , Stade de la segmentation de l'oeuf , Cellules souches embryonnaires , Femelle , Humains , Mâle , Ovocytes/physiologie , Cellules souches pluripotentes
19.
Theriogenology ; 75(8): 1416-25, 2011 May.
Article de Anglais | MEDLINE | ID: mdl-21463721

RÉSUMÉ

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.


Sujet(s)
Cardiopathies/chirurgie , Myocarde/cytologie , Transplantation de cellules souches/méthodes , Cellules souches/cytologie , Cellules souches/physiologie , Animaux
20.
Theriogenology ; 74(4): 544-50, 2010 Sep 01.
Article de Anglais | MEDLINE | ID: mdl-20570327

RÉSUMÉ

The establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines. These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC. Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells.


Sujet(s)
Lignée cellulaire , Cellules souches embryonnaires/cytologie , Cellules souches pluripotentes/cytologie , Sus scrofa/embryologie , Animaux , Marqueurs biologiques/métabolisme , Cellules de la masse interne du blastocyste/cytologie , Différenciation cellulaire , Techniques de culture d'embryons , Développement embryonnaire , Cellules souches embryonnaires/métabolisme , Cellules souches pluripotentes/métabolisme , Spécificité d'espèce , Facteurs temps
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