Signalling, cell cycle and pluripotency in embryonic stem cells

International audienc

Directed Pancreatic Acinar Differentiation of Mouse Embryonic Stem Cells via Embryonic Signalling Molecules and Exocrine Transcription Factors

Pluripotent embryonic stem cells (ESC) are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh) and bone morphogenetic protein (BMP) pathways, fibroblast growth factors (FGF) and retinoic acid (RA) in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC. © 2013 Delaspre et al.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII)-FEDER (PI052738 and PI080511 to A.S.; PI100094 and Tercel RD06/0010/ 0025 to B.S.). A.S. was supported by ISCIII and the Health Department of the Generalitat de Catalunya; F.D. was the recipient of a Graduate Fellowship from ISCIII and was also supported by Tercel; M.M. was the recipient of a Graduate Fellowship from the Generalitat de Catalunya.Peer Reviewe

Temperature Imaging using Quadriwave Shearing Interferometry. Applications in Thermoplasmonics

International audienceThe use of illuminated gold nanoparticles as ideal nanosources of heat is the basis of numerous research activities and applications in physics, chemistry, biology and medicine. This field defines the area recently named Thermoplasmonics [1]. In most of the activities related to Thermoplasmonics, probing the temperature at the vicinity of the metal nanoparticles is not an easy task. In this context, we recently developed a novel optical microscopy technique, named TIQSI, aimed at mapping the temperature around plasmonic nanoparticles [2]. The approach is based on the measure of the thermal-induced variation of the refractive index surrounding the sources of heat. The TIQSI technique cumulates all the advantages a thermal microscopy technique may require: i) high resolution (diffraction limited), ii) high readout rate (less than one image per second), iii) high temperature sensitivity (<1°C), iv) large accessible temperature range, v) temperature can be measured without fluorescence labelling or any other kind of thermal probe, v) no need to use sophisticated devices such as heterodyne detection, acousto-optic modulator, spectrometer, etc, like previous thermal imaging techniques. In this presentation, we will first introduce the TIQSI technique, its principle and capabilities. We will then present several recent applications made it possible by this new thermal imaging technique. In particular, we shall explain how this technique have been already used to quantitatively measure the absorption cross section of gold nanoparticles [3] and graphene sheets, how it can be used to map the temperature in real time in living cells [4], how it can help to design temperature distributions at will at the microscale using gold nanoparticles [5,7], and how it can be used to investigate thermal-induced phenomena in hydro- dynamics and phase transitions [6]

Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

AbstractThe imprinted genes of primate embryonic stem cells (ESCs) often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs), SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates

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

Cell cycle features of primate embryonic stem cells.

International audienceUsing flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after gamma-irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent

Krüppel-like transcription factors and control of pluripotency

Recent papers have demonstrated a role for Krüppel-like transcription factors 2, 4 and 5 in the control of mouse embryonic stem cell pluripotency. However, it is not clear whether each factor has a unique role or whether they are functionally redundant. A paper by Parisi and colleagues in BMC Biology now sheds light on the mechanism by which Klf5 regulates pluripotency

Synchronisation et calibrage entre un Lidar 3D et une centrale inertielle pour la localisation précise d'un véhicule autonome

International audienceLaser remote sensing (Lidar) is a technology increasingly used especially in the perception layers of autonomous vehicles. As the vehicle moves during measurement, Lidar data must be referenced in a fixed frame which is usually done thanks to an inertial measurement unit (IMU). However, these sensors are not designed to work together natively thus it is necessary to synchronize and calibrate them carefully. This article presents a method for characterizing timing offsets between a 3D Lidar and an inertial measurement unit. It also explains how to implement the usual methods for pose estimation between an IMU and a Lidar when using such sensors in real conditions.La télédétection par laser (Lidar) est une technologie de plus en plus utilisée en particulier dans les fonctions de perception et localisation nécessaires à la conduite autonome. L'acquisition des données Lidar doit être couplée à la mesure du mouvement du véhicule par une centrale inertielle. Ces capteurs n'étant pas conçus pour fonctionner ensemble nativement, il est nécessaire de maitriser leur synchronisation et leur calibrage géométrique. Cet article présente une méthode pour caractériser les décalages temporels entre un Lidar 3D et une centrale inertielle. Il explique aussi comment mettre en œuvre les méthodes de la littérature pour le calcul de la pose entre centrale inertielle et Lidar sur un véhicule utilisé en conditions réelles

Apoptosis, G1 Phase Stall, and Premature Differentiation Account for Low Chimeric Competence of Human and Rhesus Monkey Naive Pluripotent Stem Cells

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization