Generation of Cardiomyocytes from New Human Embryonic Stem Cell Lines Derived from Poor-quality Blastocysts

Raya, Ángel; Rodríguez-Pizà, Ignasi; Arán, Begoña; Consiglio, Antonella; Barri, P. N.; Veiga, Anna; Belmonte, Juan Carlos Izpisúa

doi:10.1101/sqb.2008.73.038

Cited by 45 publications

(38 citation statements)

References 50 publications

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“…6C), comparable with a human embryonic stem cell line (ES6) (35), showing that the obtained iPS cell lines had pluripotency properties similar to human embryonic stem cells.…”

Section: Generation Of Human Ips Cells After Transfection Of the Pcagmentioning

confidence: 65%

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Montserrat

Garreta

González

et al. 2011

Journal of Biological Chemistry

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Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-␤-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-␤-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-␤-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.In 2006, Yamanaka and Takahashi (1) showed for the first time that mouse embryonic fibroblast (MEFs) 3 can acquire a transcriptional profile and phenotype similar to an embryonic stem (ES) cell by ectopically expressing only four transcription factors. These so-called induced pluripotent stem (iPS) cells were generated by infecting MEFs with four retroviruses constitutively expressing Oct4, Sox2, Klf4, and c-myc (OSKM) (1). Since then several groups have confirmed these results and improved the reprogramming technique, generating iPS cells from mouse and human cells (2-7).iPS cells share many characteristic of ES cells. They form colonies with similar proliferation and morphological features, express a similar set of pluripotency markers, show comparable promoter methylation levels and telomerase activities and are able to differentiate both in vitro and in vivo into cell derivatives of the three germ layers. Hence, they represent a novel source of pluripotent stem cells with great potential for basic research, drug discovery, or autologous cell therapy.Retroviruses have been so far the most commonly used vectors to express the OSKM set of transcription factors into fibroblasts (1, 3, 5), neural stem cells, keratinocytes, or cord blood stem cells (8 -11) using sometimes different combinations of factors (7). Other viral gene transfer vectors such as lentiviruses have also allowed successful reprogramming (12, 13).The constitutive retroviral and lentiviral vectors commonly used for reprogramming allow efficient generation of iPS cells from mouse and human somatic cells. However, their permanent integration into the genome limits their use for eventual therapeutic applications due to the risk of both insertional mutagenesis and particul...

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“…6C), comparable with a human embryonic stem cell line (ES6) (35), showing that the obtained iPS cell lines had pluripotency properties similar to human embryonic stem cells.…”

Section: Generation Of Human Ips Cells After Transfection Of the Pcagmentioning

confidence: 65%

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Montserrat

Garreta

González

et al. 2011

Journal of Biological Chemistry

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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%

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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“…These poor-quality embryos (PQEs) are typically discarded following treatment or donated for research after patient consent. Despite impaired implantation and developmental potential, PQEs have been shown to be a viable source for the derivation of new human embryonic stem cell (hESC) lines [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. It is estimated that hundreds of thousands of embryos per year are discarded because of poor quality and the majority of infertility patients are in favor of donating them for stem cell research [7,17,18].…”

Section: Introductionmentioning

confidence: 99%

The Influence of Early Embryo Traits on Human Embryonic Stem Cell Derivation Efficiency

O’Leary

Heindryckx

Lierman

et al. 2011

Stem Cells and Development

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Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

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Generation of Cardiomyocytes from New Human Embryonic Stem Cell Lines Derived from Poor-quality Blastocysts

Cited by 45 publications

References 50 publications

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

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

The Influence of Early Embryo Traits on Human Embryonic Stem Cell Derivation Efficiency

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