br Microarray based transcriptome analysis Total RNA was iso

Microarray-based transcriptome analysis Total RNA was isolated for each sample. For global gene expression analysis on an Illumina microarray platform quality-checked total RNA was used. 500ng RNA for each sample was used. The cRNA synthesis, BeadChip hybridization and scanning were performed by ATLAS Biolabs GmbH (http://www.atlasbiolabs.com). The dendrogram compares the expression data and the correlation values and was calculated using the Gene Expression Module of the software GenomeStudio (Illumina).
Acknowledgements J.A. acknowledges support from the Max Planck Society, German Federal Ministry of Education and Research (BMBF grant number 01GN1005) and the Medical Faculty of Heinrich-Heine University—Düsseldorf, Germany.
Resource table. Resource details Primary human amniotic fluid Aminoallyl-dCTP - Cy3 (AFCs) were reprogrammed by transduction of retroviruses which express OCT4, SOX2, KLF4 and c-MYC (). The AFiPSCs express the pluripotency associated transcription factors OCT4, NANOG, SOX2 and the cell surface markers SSEA4, TRA-l-60, TRA-1-81 (Fig. 1A). Embryoid body-based differentiation was done to demonstrate in vitro the capability to differentiate to cell types representative of the three germ layers endoderm, mesoderm and ectoderm (Fig. 1B) as well as in vivo it was demonstrated by teratoma formation in immunodeficient mice (Fig. 1C). DNA fingerprinting confirmed the origin of the AFiPSC (Fig. 1D). The transcriptome profile of an AF-derived iPSC line 41 and the human embryonic stem cell lines – H1 and H9 – have a Pearson correlation of 0.953 and 0.941 respectively (Fig. 1E). Genome analysis revealed a normal female karyotype of AFiPSC (Fig. F).
Materials and methods
Acknowledgments J.A. acknowledges support from the Max Planck Society, German Federal Ministry of Education and Research (BMBF grant number 01GN1005) and the medical faculty of Heinrich-Heine University—Düsseldorf, Germany.
Introduction Cultures for human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) are a useful platform in research and regenerative medicine (Yu and Thomson, 2008; Reubinoff et al., 2000; Thomson et al., 1998; Ludwig et al., 2006). However, culturing hESCs/iPSCs is labor-intensive and requires fastidious culture conditions. Media used in hESC/iPSC cultures are expensive because they inevitably contain cytokines and molecules to support hESC/iPSC survival and maintenance of their self-renewing and pluripotent properties. The need to change media every day makes the hESC/iPSC cultures more costly and labor-intensive. We recently reported that doxycycline improves both hESC/iPSC viability and self-renewal (Chang et al., 2014). These effects of doxycycline are not associated with its antibacterial action but are mediated by activation of the intracellular PI3K-Akt pathway, which, in addition to its general cell survival effects, has been reported as the most crucial signal for hESC/iPSC self-renewal (Bendall et al., 2007; Singh et al., 2012). In this study, we examined if the doxycycline effects can be practically utilized to save the cost and labor of hESC/iPSC culturing.
Materials and methods
Results and discussion
Acknowledgements This work was supported by a grant from the Medical Research Center (2008-0062190), funded by the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology (MEST), Republic of Korea, and the research fund of Hanyang University (HY-2014).
Introduction In adult mammals, HSCs give rise to all the blood cells throughout life. During development, HSCs originate in the dorsal aorta, from where they migrate into the FL at E11. The FL is the main site of hematopoiesis until near birth. Around birth, HSCs migrate from the FL to the BM, where they reside during postnatal life. In the FL, HSCs undergo multiple rounds of symmetrical self-renewing cell divisions to give rise to the pool of stem cells required for the lifetime of the organisms. However, in adults, the most primitive HSCs (termed long-term repopulating (LT)-HSCs, that repopulate the hematopoietic system long-term following transplantations, are quiescent, dividing only every 140–180days in mouse; Wilson et al. 2008; Foudi et al. 2009). BM HSC divisions are asymmetrical generating one HSC and one committed progenitor/short-term repopulating (ST)-HSC (Takano et al. 2004). Nevertheless, quiescent LTR-HSCs can respond very rapidly to stress or damage, and quickly exit from quiescence to regenerate the blood system (Cheng et al. 2000). However, this is associated with aging of LT-HSCs, demonstrated by shortening of telomeres and skewing of LT-HSC differentiation towards the myeloid lineage (Vaziri et al. 1994; Sudo et al. 2000).