LA NECESIDAD DE DEFINIR LOS PARA QUÉ, (HORIZONTES)

Adult stem cells (ASCs), which also includes fetal and somatic stem cells, are found in tissues such as bone marrow, blood, liver, skin and muscle amongst other tissues and are present as undifferentiated cells amongst other differentiated cells (Fujimaki et al., 2013). Adult stem cells within tissues have the ability to support repair and differentiate into other representative specialized cell types (Kondo, 2010). The typical role of an adult stem cell is to maintain and replenish their tissue of origin throughout an individual’s life (Fujimaki et al., 2013).

In general, adult stem cells can be classified depending on (1) The ability to differentiate into different cell types where adult stem cells can have two kinds of cell potency including

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multipotent stem cells such as, human mesenchymal stem cells (hMSCs) and unipotent stem cells such as muscle stem cells (Meirelles, 2009). (2) The germ layer they come from, for example, endoderm layer makes up pancreas, lung, intestine and liver while ectoderm layer gives rise the skin, nervous system, hair and mammary glands. On the other hand, muscle, mesenchymal stem cells and circulatory system generated from mesoderm layer (Haasters et al., 2009).

1.2.2.1 Mesenchymal stem cells

Bone marrow (BM) is the most common source for isolation of mesenchymal stem cells, but other tissues such as adipose tissue, compact bone, liver, intestinal tract, kidney, and umbilical cord blood have also been utilised as a source for isolation of mesenchymal stem cells (Gao et al., 2014). Mesenchymal stem cells (MSCs) are a specific rare population of adherent cells that constitute about 1 in 10,000 of bone marrow mononucleated cells. These cells are characterized by the ability to produce single cell colonies, and their capacity to adhere to tissue culture plastic. These colonies are termed the CFU-F (Colony Forming Unit– fibroblast) (Wang and Wagers, 2011). The first descriptions of human mesenchymal stem cells (hMSCs) were discovered by Friedenstein and colleagues in the early 1970s as a non- hematopoietic stem cell. It was established that hMSCs were able to differentiate into a range of lineages including osteoblasts (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells) (Friedenstein et al., 1968; Karystinou et al., 2009). Furthermore, several recent studies have observed that hMSCs can differentiate into other lineages such as myocytes and neurons (non-mesenchymal lineages) (Bossolasco et al., 2005). In addition to multipotency, one of the main properties of MSCs is their inability to self-renew and that the maintenance of the non-differentiated state is lost over several cell divisions (Sethe et al., 2006). In vitro, BM-derived MSCs can be cultured easily and can be expanded to relatively high numbers for use in different applications (Bernardo et al., 2007). In general, hMSCs have three

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primary functions linked to their potential therapeutic use including tissue repair via molecule secretion, tissue replacement through multipotent properties, and their immunomodulatory functions (Devine et al., 2003; Ryan et al., 2005). By contrast, information about hMSCs in vivo is very poor for two reasons, rarity in different tissues as well as the difficulty of isolating a large quantity and high purity of hMSCs (Boguest et al., 2005). However, MSCs usually exist in a very low concentration within a given tissue. In order to distinguish them from other cell populations, it is possible to exploit the cell surface markers which are unique for each cell type. These are proteins molecules, also known as “receptors” that coat the surface of all cells, and are able to bind to other cells, surfaces or proteins. CD (cluster of differentiation) markers are commonly used to identify stem cell types in bone marrow, but additionally a number of antibody-binding receptors, antigens, can also be used (Chippendale et al., 2011). However, the International Society for Cellular Therapy has defined minimal guidelines for MSCs characterisation including; plastic adherence, multi-lineage differentiation into fat, bone, and cartilage, together with a surface expression of stem cell markers (CD73, CD90, and CD105), with lacking expression of haematopoietic markers (CD34, CD45, CD11a, CD19 or CD79a, CD14 or CD11b and histocompatibility locus antigen (HLA)-DR) (Dominici et al., 2006). It must also be considered that many different cell types may have a number of markers in common, so in order to isolate a specific stem cell population contained within the highly heterogeneous bone marrow, or to further separate the sub-populations of the adherent fraction, a combination of different markers must be used (Chippendale et al., 2011).

1.2.3 The role of DNA methylation in stem cell regulation

DNA methylation plays a major role in the regulation of stem cell differentiation and is essential for maintenance of stem cell characteristics (Huang et al., 2013). hESCs have been shown to have unique DNA methylation marks when compared to DNA methylation status

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of both differentiated cells and cancer cells (Bibikova et al., 2006; Altun et al., 2010). However, high expression levels of the de novo methyltransferase enzymes (DNMT3A and DNMT3B) occur during development especially in undifferentiated cells which reduce during cellular differentiation (Li et al., 2007; Totonchi et al., 2017). In addition, the pluripotency-associated genes such as OCT-4 and NANOG become methylated (silenced) to allow stem cell to differentiate into specific lineages (Gopalakrishnan et al., 2008; Pelosi et al., 2011). Moreover, as differentiation continue, the global DNA methylation decreases but the fundamental methylation profile is maintained by DNMT1 (Ludwig et al., 2014). The significance of methylation on the genes that associate with pluripotency is dependent on the stages of embryogenesis and on specific cell types. For example, promoter regions of DNA can be hypomethylated in mouse ES cells but are hypermethylated in trophoblast ES cells (Gopalakrishnan et al., 2008). Furthermore, Ho et al found that some genes were unmethylated in ESCs with no transcriptional, but that these genes could be supressed by DNA methylation during differentiation (He et al., 2018). Changes in DNA methylation are important for stem cell differentiation as methylated DNA alters the interaction between transcription factors and their binding sites (TFBSs) on DNA (Chen et al., 2011) or by formation of complex with other molecules such as histone modification enzymes that alter chromatin activity (Hutnick et al., 2009).

However, knockout of DNMT leads to loss ESCs the ability to differentiate while retaining the ability to replenish themselves (self-renewal). These results suggested that DNMTs play a prominent epigenetic role in pluripotency (Okano et el., 1999; Hu and Rosenfeld, 2012). In addition, another DNA methylation mechanism is involved in regulation of hESCs pluripotency; TETs enzymes (Moore et al., 2012). It has reported that global 5hmC levels significantly changed during lineage commitment of pluripotency stem cells into neural cells (Kim et al., 2014). On the other hand, knockout of TET1 in mESCs results in reduced levels

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of 5hmC and an associated slight increase in global 5mC level (Dawlaty et al., 2011). Recently, Samadian et al found that the expression of TET1 was significantly upregulated during embryonic stem cells (ESC) derivation. Moreover, they also demonstrated that maintaining DNA methylation at low levels was essential to establish the ground-state of pluripotency during the early days of ESC derivation (Samadian et al., 2018).