The molecular mechanisms that are involved

The molecular mechanisms that are involved in inducing of alternative cell fates by increasing and reducing the amount of Sox2 during reprogramming remain unclear. An interesting finding was that Oct4 changes its partner and targets genes in a dose-dependent manner in human ESCs (Stefanovic et al., 2009). Excessive expression of Oct4 in human ESCs leads to differentiation into a cardiac diltiazem hcl manufacturer through switching of the binding element that is located to Sox17 from the Sox2 promoter. It is possible that switching protein interaction partners and binding affinity to regulatory elements of downstream genes, which changes cell fate, occurs in Sox2 in a dose-dependent manner, similar to that of Oct4. The qPCR analyses that are discussed here were evaluated using RNA that was extracted from heterogeneous populations through reprogramming that was induced using defined factors. Further comparisons of expression profiles between colonies could be helpful for understanding the molecular events that underlie the dose-dependent effect of Sox2. We conclude that the significantly increased generation efficiency of fully reprogrammed iPSCs via OK+LS resulted from efficient reprogramming into fully reprogramming iPSCs following a significant increase in Oct4-GFP-positive iPSCs. Optimizing the dose of reprogramming factors will be a key to realizing practical applications of recently developed integration-free and genetic modification-free reprogramming technologies.
Materials and methods
Acknowledgments
Introduction X chromosome inactivation (XCI) is the mechanism by which dosage compensation of the sex chromosome is achieved in mammals (Leeb and Wutz, 2010). This mechanism is controlled by the noncoding RNA Xist which is expressed from the inactive X chromosome, and coats it in cis. Xist is crucial for initiating silencing, although at a later stage, inactivation becomes independent of Xist and involves a series of chromatin modifications (Lucchesi et al., 2005; Ng et al., 2007). The initiation and maintenance of XCI are extremely important for embryonic processes as well as for adult cell physiology (Ng et al., 2007). In addition, investigating XCI in pluripotent cells may teach us about the epigenetic state of these cells. It has been shown that in mouse embryonic stem cells (mESCs) and mouse-induced pluripotent stem cells (miPSCs) both X chromosomes are active, and inactivation occurs during differentiation in a random fashion (Navarro et al., 2008; Maherali et al., 2007). The analyses of human embryonic stem cells (hESCs) demonstrated a more complex situation (Dhara and Benvenisty, 2004; Hoffman et al., 2005; Adewumi et al., 2007; Shen et al., 2008; Silva et al., 2008; Dvash and Fan, 2009; Dvash et al., 2010), and the status of XCI in different cell lines was determined mainly according to XIST expression levels (Adewumi et al., 2007; Silva et al., 2008; Dvash and Fan, 2009). These studies showed that undifferentiated female hESCs either may possess two active X chromosomes and low levels of XIST or show XCI with low or high levels of XIST expression (Dhara and Benvenisty, 2004; Shen et al., 2008; Silva et al., 2008). Thus, the expression levels of XIST are not sufficient to determine XCI, and a more informative tool is necessary. Little is known about the status of X inactivation in human-induced pluripotent stem cells (hiPSCs) and it is still not clear whether the X chromosome can be activated on reprogramming of somatic cells (Lagarkova et al., 2010; Tchieu et al., 2010). Here, we examined XCI in hESCs and hiPSCs by meta-analysis of the entire set of genes on the X chromosome. Our analysis enabled us to divide the pluripotent stem cell lines based on the level and location of X inactivation, identifying a new category of partial XCI. This broad analysis adds another dimension to the current classification of pluripotent stem cell lines, and enhances our understanding of XCI in hESCs and hiPSCs.