Reprogramming of differentiated somatic cells into induced pluripotent stem (iPS) cells has potential for derivation of patient-specific cells for therapy as well as for development of models with which to study disease progression. Derivation of iPS cells from human somatic cells has been achieved by viral transduction of human fibroblasts with early developmental genes. Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. Here we show that primary adult human fibroblasts have basal expression of mRNA for OCT4, SOX2, and NANOG. However, translation of these messages into detectable proteins and their subcellular localization depends on cell culture conditions. Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency. Further investigation of the factors involved in inducing this response could lead to discovery of defined culture conditions capable of altering cell fate in vitro. This would alleviate the need for forced expression by transgenesis, thus eliminating the risk of mutagenic effects due to genetic manipulation.

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We

The transcription factor NANOG is essential for maintaining pluripotency in embryonic stem cells. We have previously reported the expression of NANOG in adult human fibroblasts; here we present a more thorough investigation into the expression of NANOG in a panel of both differentiated and undifferentiated human cells. We utilize RT-PCR, qRT-PCR, cloning and sequencing, sequence alignment, restriction digestion, immunocytochemistry, Western blotting, and EMSA to investigate expression of NANOG in a variety of somatic, transformed and stem cell phenotypes. RT-PCR and qRT-PCR analysis revealed the presence of NANOG transcripts in all the cell types examined, albeit at magnitudes lower than human embryonic stem cells. Further investigation by single nucleotide polymorphism analysis of expressed transcripts in several cell types detected a NANOG pseudogene, NANOGP8, one of only two NANOG pseudogenes with the potential of encoding a similar size protein to embryonic NANOG (eNANOG). Our analysis demonstrates that although the NANOG protein is detected in nearly all cells examined, expression of the eNANOG and/or NANOGP8 transcript as well as the sub-cellular localization of the protein is cell type-specific. Additionally, smooth muscle cells, which express exclusively NANOGP8, display nuclear localization of NANOG protein, indicating that NANOGP8 is a protein coding gene possibly functioning as a transcription factor. Lastly, all cell types expressing eNANOG and/or NANOGP8 were found to be capable of binding a NANOG consensus sequence in vitro. We conclude that eNANOG is not exclusively expressed in undifferentiated cells and that both eNANOG and NANOGP8 may function as transcription factors in a cell type-specific manner.

BackgroundAdult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence.ResultsWe identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment.ConclusionsTranscriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential.

The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.

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Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in X. laevis. We characterize 115 miRNAs that have been described either in X. tropicalis (85), X. laevis (9) or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. Additionally, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identifies target miRNAs for future functional studies.

The ability of cells to sense and respond to the mechanical stiffness of the surrounding matrix is important to support normal cell function, wound healing, and development. Central to the process of durosensing is the cytoskeleton composed of three classes of filaments: F‐actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that contributes significantly to cell mechanics and cell traction force, which is required to probe extracellular matrix. The role of vimentin in how cells sense and respond to the mechanical rigidity of extracellular matrix is largely unclear. To investigate the role of vimentin in durosensing, we knocked down the vimentin expression level in 3T3 fibroblasts using shRNA transfection and measured cellular responses as functions of substrate stiffness. We quantified durosensitivity by the rates at which cell area and traction force change with substrate stiffness. Our results show that that vimentin plays a role in durosensing by modulating traction force and knocking out vimentin did not significantly affect durosensitivity. These results indicate that vimentin may be a redundant component of the machinery that cells use to sense substrate stiffness.