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

Abstract

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 Lamina Propria of Adult Human Oral Mucosa Harbors a Novel Stem Cell Population

“…hOMSC produce and secrete FGF-2, EGF, VEGF, and NGF. These growth factors, particularly FGF-2 [51][52][53], are known to support cell proliferation. Thus, their activity may explain the high-renewal capacity of hOMSC.…”

Generation of pluripotent stem cells without the use of genetic material

“…Some examples of the gene expression levels of Oct4, Sox2, and Nanog in human fibroblasts, keratinocytes, CD133 þ cells from cord blood, and NSCs are described in Figure 5. 13,85,86 Several researchers tried to use endogenously expressing Sox2, Klf4, and/or c-Myc and to generate iPSCs with fewer numbers of pluripotent TFs. Kim et al succeeded in preparing miPSCs from mouse NSCs using two exogenous TFs (Oct4 and Klf4 or Oct4 and c-Myc).…”

“…Figure 5 Relative gene expression levels of Oct4, Sox2, and Nanog in human fibroblasts, keratinocytes, CD133 þ cells from cord blood, and NSCs. The relative gene expression levels in fibroblasts, keratinocytes, and CD133 þ cells compared with hESCs were calculated from the data reported by Giorgetti et al 85 and Page et al 86 The relative gene expression levels in human NSCs compared with hESCs were used from the data reported by Kim et al 13 NSCs endogenously express Sox2, Klf4, and/or c-Myc to some extent, as was discussed in the previous section ( Figure 5). Therefore, Kim et al reprogrammed human fetal NSCs into hiPSCs by the transduction of only Oct4.…”

Advancements in reprogramming strategies for the generation of induced pluripotent stem cells

“…Interestingly, Page et al [77] has reported successful reprogramming of primary adult human fibroblasts by manipulating cell culture conditions alone. In the study, the stem cell genes OCT4, SOX2 and NANOG were shown not to be completely dormant in untreated skins cells, as was initially presumed.…”