Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona

Aran, Begoña; Rodríguez-Pizà, Ignasi; Raya, Ángel; Consiglio, Antonella; Muñoz, Yolanda; Barri, Pere N.; Izpisúa, Juan Carlos; Veiga, Anna

doi:10.1007/s11626-010-9288-0

Cited by 7 publications

(6 citation statements)

References 5 publications

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“…The three human (h) ESC lines used, ES[2], ES [3] and ES [4], were derived as published (Aran et al, 2010;Raya et al, 2008) at the CMR [B]. Lowpassage cells of each line were thawed and cultured for one passage over a feeder layer of human foreskin fibroblasts (HFFs; CCD1112Sk ATCC, Manassas, VA, USA) in hES medium at 37°C and 5% CO 2 .…”

Section: Human Esc Culturementioning

confidence: 99%

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Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

et al. 2011

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SUMMARYThe events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

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Section: Human Esc Culturementioning

confidence: 99%

“…The three ESC lines used in the study were derived in the same laboratory using the same method (Aran et al, 2010), had the same karyotype (46XY) and were fully characterized. Moreover, ESCs were derived from embryos that were handled in the same way as those used in the study.…”

Section: Transcriptome Analysis Of Human Embryos Reveals Substantial mentioning

confidence: 99%

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

et al. 2011

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“…MCF-7/Rep colonies (or regions of colonies) were cultured in suspension, so that they formed large aggregates as described elsewhere. [97][98][99] Quantitative real-time polymerase chain reaction (qRT-PCR)…”

Section: Immunofluorescence Staining and High-content Confocal Imagingmentioning

confidence: 99%

Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

et al. 2013

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Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg's theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka's stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR--the master switch of cellular catabolism and anabolism--in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1-60, and TRA-1-81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44(+) and ALDEFLUOR-stained ALDH(bright) cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.

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“…From 47 blastocysts and partially surviving embryos that were seeded only one cell line was derived. It is not possible from our data to establish a correlation between derivation efficiency and ICM quality, but as shown by our own results in previous reports [1,24], low quality embryos can be successfully used in hESC derivation, with derivation efficiency rates similar to the ones obtained with good quality embryos. Although many authors report the number of embryos and derivation methodology used, few reports provide information about the quality of the embryos used for derivation.…”

Section: Discussionmentioning

confidence: 50%

“…All whole embryos and ICMs were cultured on irradiated human foreskin fibroblasts monolayers (HFF-1, CCD1112Sk ATCC) in derivation medium. This medium is composed of 50 % hES medium and 50 % hES conditioned medium (hES medium exposed to growing culture of hESC for one day with 2.5 % of hES cell tested FBS, Hyclone) supplemented with 2.5 % of ES cell tested FBS, following the methodology previously described [1,6]. The derivation medium was gradually substituted for hES medium between first and third passages.…”

Section: Derivation Of Hesc Linesmentioning

confidence: 99%

Vitrified blastocysts from Preimplantation Genetic Diagnosis (PGD) as a source for human Embryonic Stem Cell (hESC) derivation

Arán

Solé

Rodríguez-Pizà

et al. 2012

J Assist Reprod Genet

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Embryos diagnosed as abnormal in Preimplantation Genetic Diagnosis (PGD) cycles are useful for the establishment of human Embryonic Stem Cells (hESC) lines with genetic disorders. These lines can be helpful for drug screening and for the development of new treatments. Vitrification has proved to be an efficient method to preserve human blastocysts. One hundred and three abnormal or undiagnosed vitrified blastocysts from the PGD programme at Institut Universitari Dexeus were donated for human embryonic stem cell derivation. The overall survival rate after warming was 70.6 %. Our results showed better survival rates when blastocysts have not started the hatching process (initial/expanded 87.8 %, hatching 68.3 % and hatched 27.3 %). Thirty-five blastocysts and 12 partially surviving embryos were seeded. One hESC line with the multiple exostoses type 2 paternal mutation was obtained.

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Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona

Cited by 7 publications

References 5 publications

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

Vitrified blastocysts from Preimplantation Genetic Diagnosis (PGD) as a source for human Embryonic Stem Cell (hESC) derivation

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