COMPORTAMIENTO A FLEXIÓN CONCRETO
12.2 COMPORTAMIENTO A FLEXIÓN
As stated previously, there are two main characteristics of pluripotent stem cells; their ability to self-renew indefinitely, and their pluripotency – the ability to generate cells from the mesoderm, ectoderm and endoderm. During in vivo development, pluripotency is a characteristic of the ICM of the preimplantation blastocyst, existing transiently. In vitro, however, these cells maintain their pluripotency indefinitely (section 1.5.1). Although well studied, the mechanisms governing pluripotency in these cells are still relatively poorly understood.
Thus far, research has identified a group of key transcription factors, playing essential roles in maintenance and control of pluripotency – Oct4, Sox2 and Nanog. Indeed, these factors are used in the reprogramming of somatic cells to a pluripotent state (section 1.5.2). Much of the original work was performed in mESCs, before the derivation of hESCs in 1998, with many of the mechanisms conserved between the two systems.
A member of the POU (Pit-Oct-Unc) transcription factor family and encoded by the Pou5f1 gene, the role of Oct4 was first demonstrated during embryonic development in mice. In vivo, the expression of Oct4 mRNA and protein is restricted to pluripotent cells within the gastrulating embryo, and in germ cells (Rosner et al., 1990), and in vitro to ESCs (Okamoto et al., 1990), and was found to be rapidly downregulation during differentiation and specification of cells to specific germ layers (Okamoto et al., 1990, Pesce et al., 1998). Indeed, introduction of a homozygous Oct4-/- mutation results in peri-implantation
lethality, and cells no longer progress to become mature ICM cells, revealing an essential role for Oct4 in the establishment of ICM pluripotency (Nichols et al., 1998). In hESCs in vitro, knockdown of Oct4 results in rapid changes in morphology, a marked reduction in growth rate and cell surface marker expression, including down regulation of SSEA3, SSEA4, and Tra1-60 (Hay et al., 2004, Matin et al., 2004). Cells deficient in Oct4 also show a clear upregulation of differentiation-associated markers, particularly genes associated with differentiation to trophoectoderm (Niwa et al., 2000, Matin et al., 2004), endoderm (Hay et al., 2004) and mesoderm (Rodriguez et al., 2007). Upregulation of Oct4 was also shown to induce changes in genes associated with mesodermal and endodermal differentiation (Niwa et al., 2000, Rodriguez et al., 2007), thus implying a critical amount of Oct4 is required to efficiently regulate pluripotency. Additionally, RNAi-induced silencing of Oct4 induced a change in >1000 genes, with both positive (e.g. pluripotency-associated TFs) and negative (e.g. mesoderm, endoderm and ectoderm-associated genes) regulation of different gene sets, many of which are implicated in the strict regulation of pluripotency (Babaie et al., 2007).
Oct4 interacts with other TFs, in order to activate and repress the expression of specific genes. One such protein, with which Oct4 can heterodimerise, is Sox2, a member of the SRY-related HMG box (Sox) family, which encodes transcription
factors with a single HMG DNA-binding domain. Similary to Oct4, Sox2 is expressed in the pluripotent cells in the developing embryo and in mESCs and hESCs in vitro. Inhibition of Sox2 expression results in embryonic lethality in vivo, as no ICM develops (Avilion et al., 2003), and in hESC in culture resulted in the loss of pluripotency, indicated by changes in morphology and surface marker expression (Fong et al., 2008). Furthermore, hESCs, in which Sox2 has been knocked down, are most likely to differentiate towards the trophectoderm, with significant upregulation in genes associated with this cell lineage (Adachi et al., 2010). Cells in which Sox2 was transiently overexpressed observed a very similar phenotype, suggesting that tight regulation of Sox2 is needed in order to maintain pluripotency.
The last of the three key transcription factors is Nanog. A homeodomain TF, Nanog was identified in mESCs, using in silico digital differentiation display, and found to be enriched in undifferentiated cells (Mitsui et al., 2003). mESC lacking Nanog acquired morophological changes, and differentiated towards extraendoderm lineages, and Nanog null embryos were epiblast deficient, showing that Nanog is essential for the maintenance of a pluripotent state (Mitsui et al., 2003). Hyslop et al. confirmed this is hESCs, whereby RNAi- mediated silencing of human Nanog induced activation of a number of extraembryonic endoderm associated genes such as GATA4 and GATA6, and trophoectoderm-associated genes (Hyslop et al., 2005). It was also shown that Nanog was expressed in the ICM of blastocyst stage pre-implantation human embryos, but not in some of the earlier-stage embryos, demonstrating a role for Nanog in the maintenance of pluripotency.
Most interestingly, genome-scale location analysis showed that main protein- coding and miRNA genes are targeted by all three of these transcription factors (Boyer et al., 2005). Oct4, Sox2 and Nanog were found to co-occupy the promoter region of 353 different genes, with binding sites occurring in close proximity. The three factors were found to regulate pluripotency by binding and transcriptionally activating genes with roles in pluripotency, including Oct4, Sox2, Nanog and STAT3, and binding and transcriptionally inactivating genes that promote development, such as HOXB1and PAX6. The binding sites for the three TFs are in close proximity in co-occupied promoter regions, and the presence of Nanog was shown to increase the efficiency with which the Oct4-Sox2
heterodimer forms (Boyer et al., 2005, Rodda et al., 2005). Indeed, targeted down regulation of any one of these three factors results in a decrease in the expression of the other two. Thus, a synergy exists between these three factors, forming an autoregulatory loop (Figure 1.4), and working to regulate a large number of differentiation and pluripotency associated genes.
Figure 1.4 –Pluripotency-associated transcription factors form an autoregulatory loop.
Schematic showing the autoregulatory function of the three main pluripotency-associated transcription factors. Blue boxes show genes, red circles show proteins/TFs, dotted arrows represent translation. Adapted from Boyer et al. (2005).