RESUMEN
One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Vectores Genéticos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Lentivirus/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Southern Blotting , Western Blotting , Proliferación Celular , Células Cultivadas , Electroporación , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Citometría de Flujo , Células HEK293 , Corazón/embriología , Corazón/fisiología , Humanos , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinación Genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , TransgenesRESUMEN
Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte based screening platform is a useful tool for discovery of cytoprotective molecules.
Asunto(s)
Células Madre Embrionarias/citología , Isquemia/tratamiento farmacológico , Miocitos Cardíacos/citología , Óxido Nítrico/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacología , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
Intergeneric nucleus transfer (ig-NT) is a promising technique to produce offspring of endangered species. The objectives of this study were to (1) investigate the in vitro development of marbled cat (MC; Pardofelis marmorata) and flat-headed cat (FC; Prionailurus planiceps) ig-NT embryos reconstructed from domestic cat (DC; Felis catus) oocytes (Experiment 1), (2) evaluate the effect of individual FC donor cell lines on NT success (Experiment 2), and (3) assess the developmental ability of FC-cloned and DC-IVF embryos in vitro and in vivo after oviductal transfer (Experiment 3). In Experiment 1, the morula rate of FC-reconstructed embryos was significantly higher than those of MC and DC embryos but lower than that of parthenogenic DC embryos. However, blastocyst rate was not different. In Experiment 2, FC-ig-NT embryos reconstructed from female muscular tissue had significantly higher morula rate in comparison with those derived from other donor cell lines. However, there was no difference in blastocyst rate among cell lines. In Experiment 3, in vitro development of FC-ig-NT embryos was lower than that of DC-IVF embryos. The competency of in vivo development of FC-ig-NT and/or DC-IVF embryos was investigated by assessing pregnancy rate after their transfer into DC recipients. Domestic cat recipients receiving only FC-ig-NT embryos, FC-ig-NT embryos in one side of the oviduct and DC-IVF embryos contralaterally (co-transfer), and only DC-IVF embryos were observed. No pregnancy was detected in all recipients receiving FC-ig-NT embryos. One recipient receiving co-transferred embryos became pregnant, then delivered DC-IVF dead fetuses (n=2) and live kittens (n=6). All recipients receiving DC-IVF embryos became pregnant, and three of six recipients delivered five DC-IVF kittens. These results illustrate the developmental capacity of MC- and FC-ig-NT embryos up to the blastocyst stage. Individual donor cell line affects the developmental success up to the morula stage of FC-ig-NT embryos. Improving the developmental competence and quality of FC-ig-NT embryos may be required for implantation and development to term of FC-ig-NT offspring.