Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules

Abstract

The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

“…This is especially crucial because adult fibroblasts do not express Sox2. This study, together with others is an important step forward in defining the critical determinants for the generation of iPS cells from differentiated fibroblasts [25][26][27]. Future studies will determine if we can combine our direct reprogramming procedure with small molecule compounds to activate endogenous expression of Oct4, knock down Oct4-specific suppressor(s), or achieve reprogramming with Oct4 recombinant protein alone.…”

Concise Review: Culture Mediated Changes in Fate and/or Potency of Stem Cells

“…As Ko et al [28] found that conversion of testis-derived cells (0.01%) was only possible when cells were plated at specific densities, one might speculate that the culture conditions themselves play a role in this dedifferentiation process. It is worth noting that GSC are the only postnatal cells that continue to express high levels of Oct4, the only TF that cannot yet be replaced by other TFs or small molecules for the generation of iPSC [13]. Moreover, clinical teratomas, which contain cells from the three germ layers, occur almost exclusively in the gonads, and during development, EG cells, derived from the genital ridge that ultimately generate GSC, have a differentiation potential similar to that of ESC [33].…”

“…Although it is clear that the reprogramming factors must enable cells to push backward in the so-called valleys described by Waddington [2], thereby removing the epigenetic hurdles that stabilize cells in their differentiated status, we still have a long way to go before we comprehend the epigenetic changes required for full reprogramming. Nevertheless, several small molecules, including epigenetic modifiers and signaling pathway inhibitors, have been used successfully to enhance reprogramming or replace TFs to the point where all original TFs, except Oct4, can be replaced by a cocktail of small molecules [12,13].…”

Of mice and men – differential mechanisms of maintaining the undifferentiated state in mESC and hESC

“…GSK3β is the beta isoform of the glycogen synthase kinase 3, a serine/threonin kinase phosphorylating target proteins such as p53, Axin, Notch, and SMAD3 (see below). Inhibition of GSK3β in mESC results in growth acceleration, producing tumour-like structures [42]. Inhibition of GSK3 together with inhibition of ERK in very early murine embryos supports derivation of 'naïve' mESC [43,44].…”