Entrada del paramilitarismo al Caquetá

Embryogenesis is often viewed as a progressive loss of developmental capacity from a ‘totipotent’ zygote. However, in reality, the mammalian egg is a highly specialized and restricted cell, and its developmental potential is unlocked through the formation of epiblast cells in the inner cell mass (ICM) of the blastocyst (Selwood and Johnson, 2006).

differentiate into most cell types within an organism. Thus, ES cells hold great promise for regenerative medicine and serve as an excellent in vitro model system with which to study early development in mammals. Because ES cells can give rise to functional gametes, they have been used extensively to create genetically engineered lines of mice for developmental, genetic, and biomedical research (Capecchi, 2005). ES cells are pluripotent stem cells derived from the ICM of a mammalian blastocyst, an early-stage embryo approximately 3.5 days post coitum (Nichols and Smith, 2012). ES cells were first derived in 1981 by explanting blastocysts or ICMs from mice, and their pluripotency was demonstrated by their ability to contribute to the development of all tissues in mice after the injection of isolated ES cells into host blastocysts (Evans and Kaufman, 1981, Martin, 1981). Human ES cells, first isolated in 1998 by Thomson and colleagues, have the potential to form derivatives of all three embryonic germ layers (Thomson, 1998). In 2004, a new standard protocol was first established to generate human ES cells efficiently by supplementing FGF/activin into the cell culture system (Cowan et al., 2004).

Studies in experimental mammalian embryology have mainly focused on mouse embryos. Structures in the mouse embryo, including the placenta, extra-embryonic membranes, and the egg cylinder, are very similar to the corresponding structures in the human embryo. Nonetheless, human ES cells will be particularly valuable for investigating human embryonic development.

For ES cell studies, the initial challenge scientists need to overcome is maintaining the undifferentiated status of ES cells and their two major properties, self-renewal and pluripotency, during in vitro culture and maintenance. As mentioned earlier, mouse ES cell lines were initially obtained from the blastocysts of the Sv129 strain as round colonies on a layer of chemically arrested mouse embryonic fibroblasts (MEFs), known as a

‘feeder layer’ (Evans and Kaufman, 1981), indicating that MEFs might provide essential factors for maintaining undifferentiated ES cells. ES cell lines have also been established from single cells isolated following epiblast microdissection at embryonic day 4.5 (E4.5) (Brook and Gardner, 1997). Leukaemia inhibitory factor (LIF) derived from feeder layer cells is the critical factor in inhibiting ES differentiation and supporting the proliferation of undifferentiated stem cells (Williams et al., 1988). LIF is a cytokine produced by the endometrium that promotes blastocyst implantation (Stewart et al., 1992). GP130, the receptor for LIF, is expressed in ES cells and mediates ES cell self-renewal and pluripotency by phosphorylating the transcription factor STAT3 (Williams et al., 1988). Using a constitutively activated form of STAT3, Yokota’s group has shown that activation of this transcription factor alone is sufficient to support ES cell self-renewal at a high density in serum-supplemented medium (Matsuda et al., 1999). Although STAT3 signalling is sufficient to support mouse ES cell self-renewal, this pathway is not responsible for the self-renewal of human ES cells (Smith, 2001, Thomson, 1998, Sato et al., 2004). Instead, Sato et al. found that activation of the Wnt pathway, mediated by 6- bromoindirubin-3′-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), can support the undifferentiated phenotype of both human and mouse ES cells and sustain the expression of some ES cell-specific transcription factors, such as Oct-3/4, Rex-1, and Nanog (Sato et al., 2004).

Evidence suggests that not only GP130, but also a class I cytokine receptor, the low- affinity LIF receptor (LIF-R), can mediate the actions of LIF via heterodimerization (Gearing et al., 1991, Gearing and Bruce, 1992, Davis et al., 1993). Utilising self-renewal and pluripotency as surrogate markers to evaluate whether ES cells remain in the undifferentiated state, studies have shown that oncostatin M (OSM), cardiotrophin (CT-1),

and ciliary neurotrophic factor (CNTF), which are LIF-related cytokines, can act through the same receptor complex (in the case of CNTF, additionally including the CNTF-Rα subunit) and similarly sustain ES cell self-renewal (Conover et al., 1993, Rose et al., 1994, Wolf et al., 1994, Yoshida et al., 1994, Pennica et al., 1995). Furthermore, ES cells can also be derived and maintained in vitro using a combination of interleukin-6 and soluble interleukin-6 receptor (IL-6/sIL-6R) (Nichols et al., 1994, Yoshida et al., 1994). Hitoshi et al. found that granulocyte colony-stimulating factor receptor (G-CSF-R) could signal ES cell self-renewal (Niwa et al., 1998). Surprisingly, G-CSF-R is a cytokine receptor that is evolutionarily related to GP130 and LIF-R, but is not expressed in ES cells (Gearing et al., 1991, Chambers et al., 1997).

Because pluripotency is considered one of the most important ES cell properties, it is critical to retain pluripotency by preventing differentiation. Studies over the past few years have revealed the role of some transcription factor networks and epigenetic processes in the maintenance of ES cell pluripotency; these include OCT3/4, Nanog, SOX2, Esrrb, and Klf4 (Pesce and Scholer, 2001, Pesce et al., 1998, Ambrosetti et al., 1997, Niwa et al., 2000, Mitsui et al., 2003, Boyer et al., 2005, Boyer et al., 2006, Kaji et al., 2009, Kalmar et al., 2009). In addition, a paper published in Nature Genetics suggested that DNA modification, histone or chromatin structure changing also can more or less affect the activities of the genes targeted by these transcription factors (Jaenisch and Bird, 2003).

Oct3/4, a member of the POU transcription factor family, is encoded by Pou5f1 and is a central player in ES cell self-renewal and differentiation into specific lineages (Pardo et al., 2010, Nichols et al., 1998). It has been reported that Oct3/4 can directly inhibit ES cell differentiation towards the trophectoderm by interacting with Cdx2, an inducer of trophectoderm differentiation, to form a repressor complex (Niwa et al., 2005). Sox2 is

widely believed to cooperate with Oct3/4 in activating Oct3/4 target genes (Yuan et al., 1995). Nevertheless, OCT3/4 can associate with SOX2 to form a complex that suppresses the expression of Cdx2 (Niwa et al., 2005). Interestingly, Oct3/4 and Sox2 are transcriptionally regulated by an enhancer containing a composite element recognized by Oct4 and Sox2 (Chew et al., 2005, Okumura-Nakanishi et al., 2005). Nanog, the variant homeodomain-containing protein, which was isolated in a functional cDNA screen in ES cells, cooperates with STAT3 to drive ES cell self-renewal (Chambers et al., 2003). As mentioned earlier, three essential transcription factors, OCT3/4, SOX2, and Nanog, have essential roles in early development and are required for the propagation of undifferentiated ES cells in culture. In this respect, some studies have provided new evidence to support that OCT3/4, Nanog, and SOX2 co-regulate stem cell pluripotency by co-occupying a substantial portion of their target genes, including STAT3, HESX1, FGF-2, and TCF (Pan and Thomson, 2007, Boyer et al., 2005, Chew et al., 2005, Pan et al., 2006). Estrogen-related-receptor beta (Esrrb) has also been found to coordinate with two master regulators of the ES cell genetic program, Oct4 and Nanog, by binding to the proximal 5′- untranslated region of the Oct4 gene (van den Berg et al., 2008, Zhang et al., 2008). Kruppel-like factor 4 (KLF4) has been identified as a direct target of STAT3. Importantly, regulation of KLF4 by either LIF or Stat3 supports LIF-independent ES cell self-renewal (Hall et al., 2009, Niwa et al., 2009). KLF4 can also bind to the promoters of Oct3/4 target genes, such as left-right determination factor 1 (Lefty1) which is a related members of the TGF-β family of growth factors (Meno et al., 1996), and regulate ES cell pluripotency in co-operation with Oct3/4 and Sox2 (Nakatake et al., 2006).