• iPSCs
• reprogramming
• ß-catenin
• stem cells
• T-cell factor 3
• Tcf3
• Wnt

During development from zygote to adult, cells become more and more committed to specific functions. This process is known as differentiation and it was thought to be unidirectional and irreversible. Some years ago it has been demonstrated that cell differentiation can be reverted and differentiated cells can be reprogrammed to a pluripotent state, re-acquiring stemness and potency.
The process of direct reprogramming, that leads to the generation of induced pluripotent stem cells (iPSCs) through transduction of somatic cells with a “cocktail” of defined transcription factors, usually takes several weeks to complete and is very inefficient. Somatic cell reprogramming can be achieved even via their fusion with stem cells. Previously published data from our and others laboratories showed that Wnt3a, an activator of the canonical Wnt/β-catenin pathway, strongly enhances both cell-fusion mediated and direct reprogramming.
One of the effects of the activation of the canonical Wnt pathway is the inactivation of the β-catenin destruction complex (APC/Gsk3/Axin) and the stabilization of β-catenin that can translocate into the nucleus. Once in the nucleus, β-catenin exerts its trans-activating transcriptional activity binding transcription factors of the Tcf/Lef family (Lef-1, Tcf1, Tcf3 and Tcf4). In particular T-cell factor 3 (also called Tcf7l1) is the most expressed Tcf/Lef member in embryonic stem cells (ESCs) where it has been showed to co-occupy, along with Oct4, Nanog and Sox2, a consistent subset of common target genes promoters. According to literature Tcf3 has a dual function, depending on the levels of nuclear β-catenin. In absence of β-catenin it works as a repressor by recruiting transcriptional corepressors, chromatin remodeling and histone-modifying complexes whereas, in presence of nuclear β-catenin, it can activate the same or different classes of genes by recruiting different sets of cofactors.
The aim of my thesis project was to investigate the effects of Tcf3 silencing on the iPSCs generation. As somatic cells I used murine adult progenitor neural stem cells (NPCs), carrying a cassette made up of a GFP and a puromycin resistance under the control of the Oct4 promoter. They do not express Oct4, whereas they show quite high levels of Sox2 and c-Myc. It is known that iPSCs can be obtained from NPCs just transducing them with Oct4 (O) and Klf4 (K).
I produced retroviruses carrying a short hairpin against Tcf3 (shTcf3) or Luciferase (shLuc, as negative control). NPCs cells were cultured in NPCs medium for 3, 5, or 7 days after transduction with OKshTcf3 or OKshLuc and then counted and replated in equal number, switched to ESCs medium (containing leukemia inhibitory factor, LIF) and cultured for several weeks. Fifteen days after infection, AP positive clones emerged in plates containing OKshTcf3 infected NPCs in the 7 day time point. Eighteen days post infection, GFP positive clones started to emerge among them. At the same time point, AP positive clones emerged in plates containing control infected NPCs only 22 days after the infection and they started to express GFP at day 28 post infection.
In addition the number of clones was increased about 10-fold in OKshTcf3 infected NPCs cells with respect to the controls, showing that Tcf3 silencing accelerates the reprogramming and increases its efficiency.
Tcf3 silencing also improved timing and efficiency of the reprogramming process in 3 and 5 day time points.
Both OKshTcf3-iPSCs and OKshLuc-iPSCs showed an ES-like morphology, Oct4 driven-GFP fluorescence and reactivation of endogenous Nanog, SSEA1, Gdf3 and Fgf4, together with the loss of the NPCs markers Olig2 and Blp2. Pluripotent nature of iPSCs clones was confirmed by teratoma formation assay in nude mice.
Tcf3 can recruit repressive cofactors such as Groucho, TLE-1 and histone deacetylase to its target genes, inducing heterochromatin formation. The enhancement of reprogramming after Tcf3 silencing could be due to loss of heterochromatin formation. To assess this hypothesis we analyzed histone modifications during iPSCs generation by immunofluorescence and Western-blot against histone 3 Acetylation (AcH3 - an open chromatin marker) and histone 3 lysine-9 trimethylation (H3K9me3 – a chromatin marker associated with transcriptional repression). The cells in which Tcf3 was silenced showed higher levels of AcH3 and lower levels of H3K9me3, indicating the presence of a open-chromatin state in absence of Tcf3. Interestingly, these epigenomic modifications occurred before the transcriptional re-activation of pluripotency marker genes.
I can conclude that Tcf3 silencing is associated with an improvement of cell reprogramming kinetics and efficiency and that this effect is probably achieved through modifications of the epigenetic status of the cells, in order to overcome the epigenetic barrier, represented by heterochromatin, to iPSCs generation