Cellules souches pluripotentes induites de lapin : caractérisation moléculaire et fonctionnelle des états naïf et amorcé

Pluripotent stem cells (PSCs) can self-renew at two distinct states, the naive and primed states. Many differences exist between these two states, the most striking is the unique ability of PSCs naïve to colonize the preimplantation embryo and form chimeras. The purpose of my doctoral project was to study pluripotency in rabbits. In this context, I initially manufactured and characterized induced pluripotent stem cells (RbiPSCs) and then evaluated their ability to colonize the embryo and form chimeras. Three RbiPSCs lines were obtained by rabbit fibroblasts reprogramming. Their molecular characterization revealed mixed characteristics, naïve and primed. However, functionally, they are unable to colonize the rabbit embryo, a feature of primed pluripotency. The second part of my doctoral project was to reprogram RbiPSCs to the naïve state. To this end, I have overexpressed Klf2 and Klf4, two genes belonging to the naïve pluripotency network and the mouse PSCs culture conditions. These new cell lines have a gene expression profile closer to that of the rabbit ICM, particularly due to the reactivation of specific markers of naïve pluripotency. Finally, the reverted cells have an increased capacity of colonization of the preimplantation embryo rabbit. My work represents the first example of pluripotent stem cells reprogramming toward the naive state in rabbits. The cells thus produced pave the way for the production of somatic and germline chimerasDeux états d'autorenouvellement des cellules souches pluripotentes (PSCs) ont été définis, à savoir les états naïf et amorcé. De nombreuses différences existent entre ces deux états dont la plus marquante est la capacité unique des PSCs à l'état naïf, de coloniser l'embryon préimplantatoire et former des chimères. L'objectif de mon projet doctoral a été d'étudier la pluripotence chez le lapin. Dans ce cadre, j'ai d'abord entrepris de fabriquer et de caractériser des cellules souches pluripotentes induites (RbiPSCs), puis d'évaluer leur capacité à coloniser l'embryon et à former des chimères. Trois lignées de RbiPSCs dépendantes du FGF2 ont été obtenues par reprogrammation de fibroblastes de lapin. Leur caractérisation moléculaire et fonctionnelle a révélé des caractéristiques mixtes, naïves et amorcées. En revanche, sur le plan fonctionnel, elles sont incapables de coloniser l'embryon de lapin, une caractéristique de la pluripotence amorcée. La seconde partie de mon projet doctoral a consisté à reprogrammer des RbiPSCs vers l'état naïf. Dans ce but, j'ai surexprimé KLF2 et KLF4, deux gènes appartenant au réseau de pluripotence naïf, et utilisé les conditions de culture des PSCs de souris. Les cellules ainsi obtenues présentent un profil d'expression génique plus proche de celui de l'ICM de lapin, dû notamment à la réactivation de marqueurs spécifiques de la pluripotence naïve. Enfin, les cellules ainsi reprogrammées présentent une capacité accrue pour la colonisation de l'embryon préimplantatoire de lapin. Mes travaux constituent le premier exemple de reprogrammation de cellules souches pluripotentes vers l'état naïf chez le lapin. Les cellules ainsi produites ouvrent la voie à la fabrication de chimères somatiques et germinale

Millennial Recurrence of Large Earthquakes on the Haiyuan Fault near Songshan, Gansu Province, China

International audienceThe Haiyuan fault is a major active left-lateral fault along the northeast edge of the Tibet-Qinghai Plateau. Studying this fault is important in understanding current deformation of the plateau and the mechanics of continental deformation in general. Previous studies have mostly focused on the slip rate of the fault. Paleo-seismic investigations on the fault are sparse, and have been targeted mostly at the stretch of the fault that ruptured in the 1920 M ϳ8.6 earthquake in Ningxia Province. To investigate the millennial seismic history of the western Haiyuan fault, we opened two trenches in a small pull-apart basin near Songshan, in Gansu Province. The excavation exposes sedimentary layers of alternating colors: dark brown silty to clayey deposit and light yellowish brown layers of coarser-grained sandy deposit. The main fault zone is readily recognizable by the disruption and tilting of the layers. Six paleoseismic events are identified and named SS1 through SS6, from youngest to oldest. Charcoal is abundant, yet generally tiny in the shallowest parts of the trench exposures. Thirteen samples were dated to constrain the ages of paleoseismic events. All six events have occurred during the past 3500–3900 years. The horizontal offsets associated with these events are poorly known. However, events SS3 to SS6 appear to be large ones, judging from comparison of vertical separations and widths of fault zones. The youngest event SS1 instead seems to be a minor one, probably the 1990 M w 5.8 earthquake. Thus, four large events in 3500–3900 years would imply a recurrence interval of about 1000 years. Three events SS2 to SS4 prior to 1990 occurred sometime during 1440–1640 A.D., shortly after 890–1000 A.D. and 0–410 A.D., respectively. We tentatively associate them with the 1514 A.D., 1092 A.D., and 143 or 374 A.D. historical earthquakes. Taking 10 ‫ע‬ 2 m of slip for large events (SS3 and SS4), comparable to the 1920 M Ͼ8 Haiyuan earthquake, their occurrence times would be consistent with the long-term 12 ‫ע‬ 4 mm/yr estimate of Lasserre et al. (1999). However, a more realistic evaluation of slip rate and its possible change with time requires a more rigorous determination of coseismic slip amounts of past earthquakes

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency

AbstractA short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling

Rabbit induced pluripotent stem cells : molecular and functional characterisation of the naive and primed states

Deux états d'autorenouvellement des cellules souches pluripotentes (PSCs) ont été définis, à savoir les états naïf et amorcé. De nombreuses différences existent entre ces deux états dont la plus marquante est la capacité unique des PSCs à l'état naïf, de coloniser l'embryon préimplantatoire et former des chimères. L'objectif de mon projet doctoral a été d'étudier la pluripotence chez le lapin. Dans ce cadre, j'ai d'abord entrepris de fabriquer et de caractériser des cellules souches pluripotentes induites (RbiPSCs), puis d'évaluer leur capacité à coloniser l'embryon et à former des chimères. Trois lignées de RbiPSCs dépendantes du FGF2 ont été obtenues par reprogrammation de fibroblastes de lapin. Leur caractérisation moléculaire et fonctionnelle a révélé des caractéristiques mixtes, naïves et amorcées. En revanche, sur le plan fonctionnel, elles sont incapables de coloniser l'embryon de lapin, une caractéristique de la pluripotence amorcée. La seconde partie de mon projet doctoral a consisté à reprogrammer des RbiPSCs vers l'état naïf. Dans ce but, j'ai surexprimé KLF2 et KLF4, deux gènes appartenant au réseau de pluripotence naïf, et utilisé les conditions de culture des PSCs de souris. Les cellules ainsi obtenues présentent un profil d'expression génique plus proche de celui de l'ICM de lapin, dû notamment à la réactivation de marqueurs spécifiques de la pluripotence naïve. Enfin, les cellules ainsi reprogrammées présentent une capacité accrue pour la colonisation de l'embryon préimplantatoire de lapin. Mes travaux constituent le premier exemple de reprogrammation de cellules souches pluripotentes vers l'état naïf chez le lapin. Les cellules ainsi produites ouvrent la voie à la fabrication de chimères somatiques et germinalesPluripotent stem cells (PSCs) can self-renew at two distinct states, the naive and primed states. Many differences exist between these two states, the most striking is the unique ability of PSCs naïve to colonize the preimplantation embryo and form chimeras. The purpose of my doctoral project was to study pluripotency in rabbits. In this context, I initially manufactured and characterized induced pluripotent stem cells (RbiPSCs) and then evaluated their ability to colonize the embryo and form chimeras. Three RbiPSCs lines were obtained by rabbit fibroblasts reprogramming. Their molecular characterization revealed mixed characteristics, naïve and primed. However, functionally, they are unable to colonize the rabbit embryo, a feature of primed pluripotency. The second part of my doctoral project was to reprogram RbiPSCs to the naïve state. To this end, I have overexpressed Klf2 and Klf4, two genes belonging to the naïve pluripotency network and the mouse PSCs culture conditions. These new cell lines have a gene expression profile closer to that of the rabbit ICM, particularly due to the reactivation of specific markers of naïve pluripotency. Finally, the reverted cells have an increased capacity of colonization of the preimplantation embryo rabbit. My work represents the first example of pluripotent stem cells reprogramming toward the naive state in rabbits. The cells thus produced pave the way for the production of somatic and germline chimera

Paléosismologie spatiale : segmentation et scénarios de ruptures sismiques (applications à deux failles actives décrochantes asiatiques : la faille de Fuyun et la faille du kunlun, Chine)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Generation of induced pluripotent stem cells in rabbits

International audienceWe describe a procedure for generating induced pluripotent stem cell lines in rabbits, using retroviral vectors expressing Oct4, Sox2, Klf4, and c-Myc of human origin to reprogram rabbit fibroblasts prepared from an ear skin biopsy. We also provide detailed procedures for characterizing the resulting iPSC lines, including the analysis of pluripotency marker expression by RT-qPCR, immunolabeling, and fluorescent-associated cell sorting, the evaluation of pluripotency by teratoma production and genetic stability by karyotyping

Rupture de surface du séisme de Bihar-Népal 1934 (implications pour l'aléa sismique en Himalaya du Népal)

Although the Main Himalayan Frontal Thrust (MHT/MFT), largest and fastest slipping continental megathrust, poses a major threat to the northern Indian sub continent, seismic hazard along it remains to be quantified. Based on historical descriptions of the two main 20th century earthquakes (1905, 1934), a consensus has emerged that neither produced surface ruptures, a view recently reinforced by paleo seismological investigations in which only faulting much older than 1900 was found. This leaves us with fundamental, unanswered questions (recurrence times, rupture lengths, geomorphic signature of large events), and the ominous perspective of even greater quakes with displacements in excess of 15m, potentially on par with M 9 oceanic subduction events. Knowing the precise geometry and history of earthquake ruptures along this very active fault is thus more than ever critical to assess seismic hazard in the area. To address such questions we have engaged high resolution geomorphic and paleo seismic studies of Main Frontal Thrust (MFT) in eastern Nepal. Long wavelength warping of river terraces show that late Pleistocene/Holocene deformation is well expressed across frontal folds above the thrust, which have been successfully used to determine a shortening rate on order of 2 cm/yr, but the surface trace of the MFT, where sharpest, remains the best location to document whether large earthquakes break the ground and to determine their sizes and recurrence times. Our survey of the area between the Mahara Khola and Arun/Sun Khosi valleys shows many clear outcrops with young, pristine scarps, challenging the consensus that no surface rupture took place in the 19th century. This area is also located at the centre of the region comprised entirely within the 1934 isoseismal VIII. In the valley of the Sir Khola, which crosses the northern branch of the MFT, we logged in detail a refreshed river cut face across the 26 m high cumulative thrust scarp. Newly dated charcoal samples collected in the gravel layers of an uplifted strath terrace offset by thrusts reaching the surface confirm the young age of the last event. Six distinct 14C calibrated dates indicate that the terrace was emplaced less than 250 years ago, in the 18th or early 19thcentury, and was subsequently offset by F1, with a vertical throw of 1.5 m ( 3 m of slip). This same terrace is offset again by another fault at the base of the main scarp. Since no other large earthquake than the Bihar Nepal event was recorded locally in the 19th and 20th century, it must be concluded that the Sir Khola rivercut exposes the first unambiguous surface trace ever found of the 1934 earthquake. Similarly, a trench excavated 30 m east of the Natural river cut shows two events approximately 700 years apart. We thus interpret the great 1934 earthquake to be a repeat of the 1255 AD event that destroyed Kathmandu. Other charcoal ages in lower fluvial units of the footwall rapidly jump to 3000 years BP, and to older ages just below (up to 7000 yrs), nearly at the same level as that of the present river. This implies that stratigraphic section is missing, but also that there has been no significant longterm incision by the river just south of the thrust. In the Charnath Khola area, the dating of different uplifted terraces on the MFT hanging wall, and of one on the footwall, supports our findings at the Sirkhola and Ratu Nadi. Here, the 14 16 m high Tintale terrace, whose age is only 1 1.25 kyr, appears to have been uplifted by at least two events (possibly three) with 4 5m of vertical coseismic throw in each event since its abandonment. 14C dating of the lowest hanging wall terrace ( 4 m high) in Tintale creek, which was abandoned less than three centuries ago supports the existence of the 1934 rupture. Geophysical surveys including three shallow seismic profiles and Electrical Resistivity Tomographic (ERT) sections, 1.5 km long each, in the Charnath, Sirkhola and Ratu Valleys, and one Ground Penetrating Radar (GPR) profile at Thapatol near Bardibas, add invaluable information at various depths, complementing our morpho tectonic interpretation of the area. In particular, the seismic profiles shot across the MFT image well the shallow part of the thrust plane down to 400mepth. To our knowledge, at least in Nepal, this is the first effort of this kind to study the MFT at such detailed scale in combination with paleo seismological trenching.PARIS-BIUSJ-Sci.Terre recherche (751052114) / SudocSudocFranceF

Obtention de cellules souches pluripotentes induites chez le lapin

National audienc

Krüppel like factors reprogram rabbit induced pluripotent stem cells to naïve like pluripotency

International audienc

Génération de cellules souches pluripotentes induites (iPS) chez le lapin : Mise au point des conditions d'utilisation de cellules souches pluripotentes induites (cellules iPS) pour la création de lapins transgéniques, modèles animaux de maladies humaines

National audienc