Recent efforts in derivation of iPS cells have demonstrated that a relatively small number of genes when introduced into differentiated somatic cells have the ability to reprogram nuclear memory and cause acquisition of a pluripotent phenotype. These observations suggest that there may be only a few prime upstream regulators of the stability of differentiation phenotype, particularly in fibroblasts. We show that by manipulating culture conditions alone, we can achieve changes in fibroblasts that would be beneficial in development of patient-specific cell therapy approaches. First, we demonstrate an increase in the number of population doublings of adult dermal fibroblasts in vitro; second, fibroblasts acquire expression and nuclear localization of several stem cell specific transcription factors, and third, the cells maintain low TERT levels and are not tumorigenic.
Experimental evidence gathered over the past decades for involvement of FGF2 in a number of cellular and developmental processes is extensive. Among others, FGF2 has been shown to regulate human ES cell self-renewal (Levenstein et al., 2006
), is a potent mitogen and morphogen for a variety of cell types (reviewed in Ornitz and Itoh, 2001
), has been shown to initiate regeneration in the eye (Hayashi et al., 2004
), and reprograms primordial germ cells to pluripotency (Durcova-Hills et al., 2006
The increase in in vitro
longevity of fibroblasts cultured in reduced oxygen were consistent with those reported previously (Saito et al., 1995
). An increased life span has been associated with FGF2-facilitated selection and enrichment of multipotent cells (Bianchi et al., 2003
; Quarto and Longaker 2006
); however, FGF2 has not been shown to impact the in vitro
life span of terminally differentiated cell types. The initial FGF2 effect is likely mediated via FGF receptors at the plasma membrane. Nuclear translocation of FGFR1 and FGFR2 upon FGF2 treatment indicates a possibility for involvement of both FGF2 and these receptors at the chromatin level. FGF receptors have been observed in 3T3
cells (Maher, 1996
) and mammary epithelial cells (Bryant and Stow, 2005
), and have been associated with FGF2 mitogenic activity, which may act through a number of FGFR isoforms. Therefore, the increased number of population doublings in our FGF2-treated fibroblasts could be a consequence of FGF2 mitogenic stimulation. The changes we observed in FGFR and FGF2 localization did not appear to coincide with an increase in their mRNA levels. In addition to their mitogenic effects, both FGFR1 and 2 have been associated with FGF2-mediated maintenance of pluripotency in hES cells (Babaie et al., 2007
; Greber et al., 2007a
). Although FGF2 has not been implicated previously in transcriptional activation of OCT4 or SOX2, it has been determined that the maintenance of expression of these genes and cell pluripotency is dependent on FGF2 (Levenstein et al., 2006
). The proposed action of FGF2 involves induction of members of TGF-β pathway; TGF-β ligands maintain expression of OCT4, SOX2, and NANOG, which in turn, activate expression of endogenous FGF2 that completes this regulatory loop (Greber et al., 2007b
). Our data is in agreement with previous hypothesis that this circuit can be initiated and perpetuated by exogenous FGF2, leading to autocrine signaling by endogenous FGF2 (Dvorak et al., 2005
; Dvorak and Hampl, 2005
; Greber et al., 2007a
; Levenstein et al., 2006
; Xu et al., 2005
). BMP signaling has been shown to antagonize FGF2 signaling in maintaining the pluripotent state of human ES cells (Xu et al., 2008
). Inhibition of OCT4 nuclear detection in our culture system in the presence of BMP-2 suggests that a similar pathway may be involved.
Recently, FGF2 has been shown to be involved in remodeling of the chromatin in rat cortical neuronal progenitor cells by methylation of histone H3K4 (K4me3) and repression of methylation of H3K9 (Song and Ghosh, 2004
). Both of these posttranslational histone H3 modifications have been associated with transcriptionally active chromatin (Kimura et al., 2004
It was surprising that detectable levels of stem cell transcription factors were present in control fibroblasts and that at least one of these genes (OCT4) represented the true embryonic form. Despite the presence of transcription factor mRNAs, however, no detectable level of these proteins could be found either by Western blotting or ICC. Although we have not yet determined the functional relationship between FGF2 and stem cell gene expression in dermal fibroblasts, it has become apparent that a combination of FGF2 supplementation, low oxygen culture conditions, and cell culture surface triggered translation of these proteins and their appropriate translocation to cells' nuclei. Our results suggest for the first time that alteration of cell fate may depend not only on induction of new transcription, but on posttranscriptional regulation as well.
The absence of tumor formation after injection into SCID mice indicates that despite stem cell gene expression, subsequent translation and appropriate nuclear localization after 7 days of culture, these cells have not yet acquired a pluripotent phenotype. Events downstream of OCT4, SOX2, and NANOG that may be critically important for acquisition and maintenance of pluripotency may require extended culture of cells under appropriated conditions (Takahashi et al., 2007b
; Yu et al., 2007
). Because only up to 30% of cells demonstrated stem-like nuclear localization of the transcription factors, absence of tumor formation may have been due to insufficient numbers of OCT4/NANOG/SOX2 positive cells injected.
The ability of lowered oxygen together with FGF2 to induce expression of stem cell genes in adult human fibroblasts without hTERT protein expression and with a significantly increased replication potential suggests that sufficient numbers of cells could be produced for therapeutic applications. Our data suggests that mechanisms regulating translation and posttranslational modifications may be critically important in induction of a stem cell phenotype. This suggests that there may be a subpopulation of fibroblasts capable of responding to FGF2 at the translational or signaling level. In addition, the possible synergistic effect of FGF2 and subatmospheric oxygen culture conditions warrants further exploration.
The mechanism of induction of key regulatory genes involved in pluripotency by nontransgenic methods to create truly pluripotent cells will require further investigation. The published studies on transgene induced pluripotency in fibroblasts show that forced expression of exogenous pluripotency genes is required for at least 30 days prior to detection of phenotypic changes in the cells followed by amplification of colonies of cells with truly pluripotent properties (Takahashi et al., 2007b
; Yu et al., 2007
). The long-term stability of this phenotype will likely involve introduction of extra cellular components and specialized media formulations similar to those employed for derivation and in vitro
maintenance of hESCs and IPS cells, and possibly factors yet to be identified. However, this work suggests that it may be possible to dedifferentiate adult human somatic cells by modifying the in vitro
culture conditions. The ability to dedifferentiate somatic cells to a less differentiated (not necessarily pluripotent) state by simply modifying the culture conditions may have value in the utilization of autologous or primary cells for cell therapy and diagnostic applications.