Section 1.1 of measuring gene activity was represented in a simplified manner assuming that there are linear steps from gene to mRNA to protein levels. In a real biological system, these steps are not linear as there are many levels that control the amount protein present in a cell. With our current understandings and
technological abilities, the final level of protein activities has to be approximated using data from mRNA experiments.
In the context of this thesis we concentrate on subsets of genes that are capable of regulating the expression of other genes or mediate the signal in stem cells and during differentiation.
1.2.1 Embryonic stem cells
Embryonic stem cells (ESCs) are pluripotent cells that are capable of differen-tiating to any cell type in the organism. The first human embryonic stem cells (hESCs) were derived from the inner cell mass of a blastocyst in 1998 by Thom-sonet al.(1998). Human embryonic stem cells are the basis of three primary germ layers: ectoderm, endoderm and mesoderm. These layers develop further to more than 200 different cell types in the human body (Figure 2).
Blastocyst
Embryonic stem cells
Inner cell mass
Ectoderm Endoderm Mesoderm
Figure 2: Pluripotent embryonic stem cells are derived from the inner cell mass of a blastocyst. These cells are capable of differentiation first to three primary germ layers (ectoderm, endoderm, mesoderm) and finally to over 200 distinct cell types in the human body.
The pluripotent stem cells at each cell division give birth to another pluripotent stem cell and to one cell that starts to differentiate towards one of the three main germ layers. This self-renewal property keeps stem cells in an undifferentiated state.
Human embryonic stem cells are kept in the pluripotent state by expressing three transcription factors – OCT4, SOX2 and NANOG (Boyer et al., 2005).
These three transcription factors regulate each other and a variety of target genes
(Boyeret al., 2005; Macarthur, Ma’ayan, & Lemischka, 2009). In order to start differentiation, the activity of these three genes needs to be decreased and the activity of the differentiation driver genes has to increase (Yeo & Ng, 2013).
Exit from pluripotency state can be achieved by knocking down the core regu-lators of OCT4, SOX2 and NANOG (Greber, Lehrach, & Adjaye, 2007; Matinet al., 2004). Alternatively, overexpressing cell type specific genes individually or in combinations leads also towards differentiation, e.g. overexpression of HEX for hepatoblasts (Inamuraet al., 2011); APP for neural differentiation (Freudeet al., 2011); GATA4 and TBX5 for cardiac specification (Dixonet al., 2011).
The commonly used term pluripotency covers at least two sub-states - naïve and primed. The naïve or ground state describes embryonic stem cells that have no differentiation bias. These cells are capable of forming blastocyst chimeras when additional cells are added to preimplantation embryos; have two activeX chromo-somes (if female karyotype); express Rex1, NrOb1 and FGF4 markers. Primed pluripotent embryonic stem cells, however, cannot form blastocyst chimeras any more, they have an inactivated chromosomeXand also have a differentiation bias.
(Nichols & Smith, 2009)
According to these properties, mouse embryonic stem cells are considered to be in the naïve state, while the majority of human embryonic stem cells repre-sent the latter, primed, state. However, it is possible for hESCs to adopt the naïve phenotype, at least for limited number of passages (Hannaet al., 2010). Also, primed mouse embryonic stem cells resembling hESCs can be produced when isolated from the post-implantation epiblast (Tesaret al., 2007). The differences between mESCs and hESCs, attributed for a long time to species differences, may be due to differences of cell states, instead. It has been shown recently that when human embryonic stem cells are treated with 5i/L/A (MEK inhibitor, GSK3 in-hibitor, ROCK inin-hibitor, BRAF inin-hibitor, SRC inin-hibitor, Activin, hLIF in N2B27 medium) then primed hESCs are converted to the naïve state (Theunissenet al., 2014).
Although the mechanisms of cell state transition from naïve to primed are not clear yet, it can be expected to take place through gradual changes of the activity levels of key factors. For example, it has been shown that an alternative OCT4 enhancer is used only in naïve ESCs and it is inactivated in primed state (Theunis-senet al., 2014). Also, gradual changes can be encoded in proteins changing their dimerisation partners and, thus, the regulated genes. For example, OCT4 changes its known dimerisation partner SOX2 to SOX17 during the first differentiation steps (Kallaset al., 2014).
Alternative cellular systems used for studying gene regulation in pluripotent stem cells are embryonic carcinoma cells. These cells are derived from germ cell tumours, like the testicular tumour. Embryonic carcinoma cells share cell characteristics with hESCs, and core regulatory proteins are the same as in hESCs (Clark, 2007). Embryonic carcinoma cell lines are easier to culture and were available as a model system for studying pluripotency before the first hESC lines were established (Andrews et al., 2005). Embryonic carcinoma cell lines like
NCCIT and NTERA2 have been used for studying self-renewal and pluripotency regulatory networks (Andrews et al., 2005; Greber, Lehrach, & Adjaye, 2007;
Josephsonet al., 2007).
1.2.2 Adipocytes
Adipocytes are cells of mesenchymal origin and their main function is storing energy as fat. Mesenchymal stem cells originate from the mesoderm and differ-entiate into bone, muscle, connective tissue and adipocytes. There are three main types of adipose tissue – brown, brite (beige) and white (Giralt & Villarroya, 2013;
Harms & Seale, 2013).
White adipocyte
Brite adipocyte
Brown
adipocyte Myocyte White
pre-adipocyte
Mesenchymal stem cell differentiation towards adipocytes
Myogenic precursors
Figure 3:Differentiation of adipocyte lineages from mesenchymal stem cells. White and brite adipocytes are differentiated from white pre-adipocytes while brown adipocytes, like monocytes, are derived from myogenic precursors.
During adipocyte differentiation from the mesenchymal stem cells, first pre-cursory cells are formed. These cells cannot differentiate any more towards alter-native lineages such as muscle or bone. In the final step, terminal differentiation into mature and functional adipocytes takes place (Figure 3).
Mouse embryonic stem cells can be differentiated directly into adipocytes when treated with retinoic acid (Daniet al., 1997). Also PPARγ is a key reg-ulator of adipocyte development (Rosenet al., 1999) and is capable of converting non-adipose cells to adipocyte-like cells together with C/EBPα(Wuet al., 1999).
Although some regulators that drive differentiation of adipocytes are known, the detail regulatory steps and participating genes are still poorly described.