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Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, critically determined by chromatin structure and interactions of chromatin-binding proteins. The dynamics of chromatin structure are lightly regulated by multiple epigenetic mechanisms such as histone modifications and the incorporation of histone variants. In the current work, we induced neural differentiation of a human embryonal carcinoma stem cell line, NTERA2/NT2, by retinoic acid |RA) treatment, primarily according to two different methods of adherent cell culture (rosette formation) and suspension cell culture (EB formation) conditions, and histone modifications and variations were compared through these processes. Western blot analysis of histone extracts showed significant changes in the acetylation and methylation patterns of histone H3, and expression level of the histone variant H2A.Z, after RA treatment in both protocols. Using chromatin immu no precipitation (ChIP) coupled with real-time PCR, it was shown that these epigenetic changes occurred on the regulatory regions of 4 marker genes (Oct4, Nanog, Neslih, and Pax6) in a culture condition dependent manner. This report demonstrates the dynamic interplay of histone modification and variation in regulating the gene expression profile, during stem cell differentiation and under different culture conditions. Key words: epigenetic, H2A.Z, NTERA2, neural differentiation, retinoic acid. La differencials on des cellules souches d'un etat pluripotent a un etat determine implique des changements globaux dans les patrons d'expression du genome, determines de fagon critique par la structure de la chromatine et les interactions des proteines de liaison de la chromatine. La dynamique de la structure de la chromatine est fortement regulee par de multiples mecanismes epigenetiques tels les modifications d'histones et I'incorporation des variants d'histones. Dans ce travail, nous avons induit une differencial ion neurale des cellules de la lignee de carcinome embryonnaire humaine NTERA2/NT2 par un traitement a l'acide retinoique (AR),en suivant deux methodes differentes de culture cellulaire, soit sur des cellules adherentes (formation de rosettes) ou en suspension (formation de CE), et les modifications/variations d'histones ont ete comparees au cours de ces processus. L'analyse d'extraits d'histones en buvardage par Western ont montre des changements significatifs dans les patrons d'acetylation/methy]ation de l'histone H3, et dans les niveaux d'expression du variant d'histone H2A.Z apres un traitement a 1'AR, dans les deux protocoles. A 1'aide d'une immunoprecipitation de la chromatine (ChIP) couplee a une PCR en temps reel, nous montrons que ces changements epigenetiques surviennenl dans les regions regulatriees de quatre genes marqueurs (Oct4, Nanog, Nesiin et Pax6) de (aqon dependanie des conditions de culture. Ce rapport demonlre l'inleraction dynamique de la modification/variation des histones dans la regulation du profil d'expression genique, lors de la differenci ation des cellules souches el sous differentes conditions de culture. Mots-cles : epigenetique. H2A.Z, NTERA2, differenci ation neurale, acide retinoique. [Traduit par la Redaction], Introduction Embryonal carcinoma (EC) cells derived from germ cell tumors are valuable tools for investigating embryogenesis and developmental biology processes. The advantage of reproducibility and rapid in vitro expansion and [...]

Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 µM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (αV, α6, and β1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells. [ABSTRACT FROM AUTHOR]

The recent discovery of genomic reprogramming of human somatic cells to an embryonic stem (ES) cell-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells and represent one of the most promising sources of patient-specific cells for use in disease model, development of pharmacology and toxicology, screening teratogens, and regenerative medicine. Here we describe the detailed methods for the generation of undifferentiated human iPS (hiPS) cells in feeder layer- and serum-free conditions. This system eliminates direct contact of stem cells with MEFs and reduces use of unknown serum factors that may have undesired activities and enables consistency in large-scale and long-term expansion of undifferentiated hiPS cells. Our findings greatly simplify the method for induction of pluripotency and bring it one step closer to clinical applications. Moreover, the established hiPS cells showed chromosomal stability during long-term culture.