A UTF1-based selection system for stable homogeneously pluripotent human embryonic stem cell cultures.
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ABSTRACT:
SUBMITTER: Tan SM
PROVIDER: S-EPMC2094078 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.
| S-EPMC5834688 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) derived from somatic cells (SCs) and embryonic stem cells (ESCs) provide promising resources for regenerative medicine and medical research, leading to a daily identification of new cell lines. However, an efficient system to discriminate the different types of cell lines is lacking. Here, we develop a quantitative system to discriminate the three cell types, iPSCs, ESCs, and SCs. The system consists of DNA-methylation biomarkers and mathematical models, including an artificial neural network and support vector machines. All biomarkers were unbiasedly selected by calculating an eigengene score derived from analysis of genome-wide DNA methylations. With 30 biomarkers, or even with as few as 3 top biomarkers, this system can discriminate SCs from pluripotent cells (PCs, including ESCs and iPSCs) with almost 100% accuracy. With approximately 100 biomarkers, the system can distinguish ESCs from iPSCs with an accuracy of 95%. This robust system performs precisely with raw data without normalization as well as with converted data in which the continuous methylation levels are accounted. Strikingly, this system can even accurately predict new samples generated from different microarray platforms and the next-generation sequencing. The subtypes of cells, such as female and male iPSCs and fetal and adult SCs, can also be discriminated with this method. Thus, this novel quantitative system works as an accurate framework for discriminating the three cell types, iPSCs, ESCs, and SCs. This strategy also supports the notion that DNA-methylation generally varies among the three cell types.
| S-EPMC3572019 | biostudies-literature
Project description:Directed differentiation of human embryonic stem cells (hESCs) has generated much interest in the field of regenerative medicine. While subpopulations of hESCs within pluripotent cultures have been identified based on expression of specific surface antigens, their significance and fates are not well understood. To determine whether such subpopulations indicate specific tissue fates or represent stochastic antigen distributions within proliferating cultures, we isolated CD133(+) or CD135(+) hESCs from proliferating cultures constitutively expressing enhanced green fluorescent protein (GFP), and co-cultured these with unselected GFP(-) hESCs. After passage in culture, GFP(+) hESCs reanalyzed for the persistence of CD133 or CD135 expression, as well as other surface antigens (Tra-1-60, SSEA-4, FGFR-1), demonstrated that these two subpopulations continued to express CD133 or CD135 over serial passage, and that CD133(+) hESCs were enriched for SSEA-4 expression as well. Upon differentiation in vitro, CD133(+)GFP(+) hESCs gave rise solely to ectoderm, as detected by expression of nestin. Tissues representing endoderm (alpha-fetoprotein(+)) and mesoderm (smooth muscle actin(+)) were not seen among GFP(+) tissues. In contrast, selection against CD133 gave rise almost exclusively to mesoderm and endoderm. In contrast, CD135(+)GFP(+) hESCs gave rise to tissues representing all three embryonic germ layers, and were virtually indistinguishable from CD135(-)-derived tissues. Similar results were obtained by in vivo differentiation in teratomas. These data establish that subpopulations of proliferating hESCs whose tissue fate is predetermined exist, and challenge the notion that all cells within proliferating hESC cultures are truly "pluripotent." This co-culture approach also will enable identification of other distinct hESC subpopulations, and selection for these should prove valuable in generating tissue-specific reagents for cell-based therapy.
| S-EPMC2939715 | biostudies-literature
Project description:Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.
| S-EPMC3213153 | biostudies-literature
Project description:BackgroundThe scarcity of pluripotent stem cells poses a major challenge to the clinical application, given ethical and biosafety considerations. While germline stem cells commit to gamete differentiation throughout life, studies demonstrated the spontaneous acquisition of pluripotency by spermatogonial stem cells (SSCs) from neonatal testes at a low frequency (1 in 1.5 × 107). Notably, this process occurs without exogenous oncogenes or chemical supplementation. However, while knockout of the p53 gene accelerates the transformation of SSCs, it also increases risk and hampers their clinical use.ResultsWe report a transformation system that efficiently and stably convert SSCs into pluripotent stem cells around 10 passages with the morphology similar to that of epiblast stem cells, which convert to embryonic stem (ES) cell-like colonies after change with ES medium. Epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and fresh mouse embryonic fibroblast feeder (MEF) are essential for transformation, and addition of 2i (CHIR99021 and PD0325901) further enhanced the pluripotency. Transcriptome analysis revealed that EGF activated the RAS signaling pathway and inhibited p38 to initiate transformation, and synergically cooperated with LIF to promote the transformation.ConclusionThis system established an efficient and safe resource of pluripotent cells from autologous germline, and provide new avenues for regenerative medicine and animal cloning.
| S-EPMC10693117 | biostudies-literature
Project description:BackgroundSomatic cells can be reprogrammed into induced pluripotent stem (iPS) cells. Recently, selection of fully reprogrammed cells was achieved based on colony morphology reminiscent of embryonic stem (ES) cells. The maintenance of pluripotency was analysed.Methodology/principal findingsClonal murine iPS cell line TiB7-4, which was derived from adult fibroblasts, was analysed for maintenance of pluripotency. Colony morphology, expression of pluripotency factors and stage specific embryonic antigen 1 (SSEA1) were analysed by real time PCR and flow cytometry. We found the iPS cell line TiB7-4 and its subclones to be rather diverse and exhibiting a tendency towards spontaneous differentiation and loss of pluripotency independent of their initial colony morphology. In contrast an undifferentiated transcription factor 1 (UTF1) promoter-driven G418 (Neo) resistance significantly improved the quality of these iPS cells. After selection with UTF-Neo for two weeks iPS subclones could be stably maintained for at least 40 passages in culture and differentiate into all three germ layers. As control, a construct expressing G418 resistance under the control of the ubiquitously active SV40 early promoter formed subclones with different colony morphology. Some of these subclones could be cultured for at least 12 passages without loosing their pluripotency, but loss of pluripotency eventually occured in an unpredictable manner and was independent of the subclones' initial morphology and SSEA1 expression. A UTF-Neo-based selection of a whole population of TiB7-4 without further subcloning resulted in the generation of cultures with up to 99% SSEA1 positive cells under stringent selection conditions.ConclusionsOur data indicate that temporary selection using a genetic UTF1-based system can generate homogenous pluripotent iPS cells that can be maintained without permanent selection pressure.
| S-EPMC2833193 | biostudies-literature
Project description:The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.
| S-EPMC2435228 | biostudies-literature
Project description:Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications.
| S-EPMC3273828 | biostudies-literature
Project description:We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.
| S-EPMC2834855 | biostudies-literature
Project description:mouse embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells were compared via metabolomic analysis
2013-01-16 | ST000003 | MetabolomicsWorkbench