Induction of Stem Cell Gene Expression in Adult Human Fibroblasts without Transgenes

CellThera, Inc., Worcester, Massachusetts, USA.
Cloning and Stem Cells (Impact Factor: 2.66). 08/2009; 11(3):417-26. DOI: 10.1089/clo.2009.0015
Source: PubMed


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.

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    • "It is interesting that similar changes occur in reverse during acquisition of regeneration competence in adult human fibroblasts. After a short term culture in reduced oxygen and supplementation with FGF2 [35], [38] human fibroblast cease expressing OCT4 pseudogenes in favor of OCT4A. We have reported previously that these cells do not acquire the pluripotent phenotype [35], but rather undergo a phenotype switch that demonstrates regeneration competence in vivo [38]. "
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    ABSTRACT: 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.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "We have observed that adult human fibroblasts demonstrated FGF2- and surface-mediated induction of some endogenous stem cell genes and a capacity to acquire a more developmentally plastic phenotype. This low level of activation of stem cell genes was not sufficient for induction of a phenotypic conversion into a pluripotent cell phenotype [18]. However, when transplanted into skeletal muscle injury, adult human fibroblasts grown in low oxygen and with supplementation of FGF2 had the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation [19]. "
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    ABSTRACT: Adult 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. We 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. Transcriptome 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.
    Full-text · Article · Sep 2013 · BMC Genomics
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    • "This family of transcription factors also affects neuronal differentiation (Wang et al., 2013). Furthermore, human fibroblasts grown under 5% O 2 demonstrate an increased cell life span and begin to express low levels of Sox2, Oct4 and Nanog (Page et al., 2009), potentially becoming more easy to reprogram. This would partially explain the increase in iPSC reprogramming observed by Yoshida et al. (2009) under low O 2 conditions. "
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    ABSTRACT: We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in the O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fire action potentials in low O2 relative to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and is not synergistic with blockade of GSK3 beta and Smad-mediated pathways. Our results show that lowering the O2 tension at initiation of reprogramming is a simple and efficient manner to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.
    Full-text · Article · Apr 2013 · Journal of Neuroscience Methods
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