ECM Proteins and Corresponding Integrin Interactions Mediating hESC Activity Using Matrigel TM and Feeder Cells

1.3.6.1 Laminin

During embryonic development, Laminin is the first basement membrane (BM) component of the ECM to be laid down [Mostafavi-Pour et al., 2012]. BM’s function as structural barriers and act as substrates for cellular behaviours including; cell polarity, proliferation, differentiation, migration and chemotaxis [Poschl et al., 2004]. Therefore, BMs allow the isolation of cells whilst at the same time connect them to their interstitial matrix. Examples of BM’s include: perlecan, collagen IV and nidogen [Kruegel and Miosge, 2010].

Laminin is a heterotrimeric glycoprotein with three chains consisting of α, β and γ which can able to give rise to 16 different isoform types of laminin, which all help regulate tissue structure and the behaviour of cells [Kruegel and Miosge, 2010]. During embryonic development, the key laminin isoforms that are involved include laminin-111 and laminin- 511, which are also expressed by hESC lines; although on Matrigel™ substrate, laminin- 111 is the isoform that is present [Rodin et al., 2012]. However, in vitro investigations have also shown the ability of recombinant laminin-511 to promote hESC self-renewal and to be more effective in mediating hESC adhesion, expressing standard hESC markers, maintaining a reasonable proliferation rate, and retention of pluripotency (up to 20 passages) relative to other laminin isoforms such as laminin-111 and laminin-332. Furthermore, the extensive use of laminin-511 has included its ability to support derivation of new hESC lines simply by plating ICM’s onto laminin-511 which permitted ICM attachment and the outgrowth of hESCs [Azarin and Palecek, 2010].

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Recent investigations have confirmed the ability of laminin to support hESC growth similar to Matrigel™ during in vitro culture [Young and Carpentar, 2002]. Thus, laminin is considered to be an essential protein in promoting cell adhesion, migration, spreading and stimulating the proliferation of hESCs whilst retaining their undifferentiated state [Mostafavi-Pour et al., 2012]. Many interacting integrin receptors for laminin have been identified and include: α1β1, α2β1, α2β2, α3β1, α6β1, α6β4, α7β1, α9β1, α10β1 and αVβ1; these are able to give rise to heterodimers specific for adhesion to other ECM proteins including fibronectin, vitronectin and collagen IV [Prowse et al., 2011].

However, for laminin, α6β1 is considered to play a crucial role in hESC adhesion and is a highly expressed surface receptor [Humphries et al., 2006; Kruegel and Miosge, 2010; Young and Carpentar, 2002]. Specifically, there are two isoforms of the α6 sub-unit, α6A and α6B with the differences visible in the cytoplasmic domains but in undifferentiated hESCs the α6B isoform is expressed.

26 1.3.6.2 Fibronectin

Fibronectin is an omnipresent, structural glycoprotein with a high molecular weight, consisting of peptide sub-units which comprise three types of repeats including type I, II and III. Fibronectin contains binding sites for various ECM proteins including: collagen, fibrin, fibronectin itself and heparin. Functions of fibronectin include; organising the ECM, therefore it plays a vital role in matrix assembly, cell adhesion, spreading, migration, morphology and organisation of the cytoskeleton. Within the cell, fibronectin is initially synthesised as a monomer but is instantly dimerised inside the endoplasmic reticulum and secreted into the ECM as a disulphide-bonded dimer in an inactive form; upon binding to its interacting integrin (α5β1) [Mostafavi-Pour et al., 2012], this extends the fibronectin dimer via the RGD (Arg-Gly-Asp) peptide binding sequence [Coppolino and Dedhar, 2000] resulting in its activation and subsequent fibrillar network formation [Labat-Robert, 2012]. Furthermore, out of the 24 integrin heterodimers, 8 of these integrins have been identified to be able to bind to the RGD peptide sequence [Labat-Robert, 2012].

During hESC culture, fibronectin is secreted by feeder cells (MEFs) into ES conditioned media but is also present as a component of MatrigelTM; hESCs cultured on Matrigel™ in combination with ESC conditioned media express corresponding interacting integrins for fibronectin, specifically α5β1 .

1.3.6.3 Vitronectin

Vitronectin (molecular weight of ~ 75kDa) is a multi-functional glycoprotein and can be found in blood, plasma (concentration of 200µg/ml) and ECM; it has the ability to bind and anchor onto proteins such as glycosaminoglycans (GAGs), collagen, plasminogen as

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well as the urokinase receptor. Major biological functions of vitronectin include: fibrinolysis, hemostasis, immune defence and more relevant functions with respect to cell behaviour. These include; cell adhesion, spreading, ECM anchoring (to collagen and GAGs), proliferation, proteolytic degradation of matrix and cell migration when coupled with interacting cell integrins and growth factors, which together have a synergistic effect on cell growth [Schvartz et al., 1999].

Cell adhesion, spreading and migration are primarily mediated via the RGD peptide within vitronectin; in vitro the RGD peptide has demonstrated its ability to bind to and interact with several integrins such as; αVβ3, αVβ5, αVβ1, αVβ6, and αVβ8 resulting in subsequent protein phosphorylation triggering the activation of the MAPK pathway [Braam et al., 2008; Schvartz et al., 1999]. However, recent studies have reported the αVβ5 vitronectin receptor to be important in mediating hESC adhesion and pluripotent expansion when cultured using MatrigelTM or Vitronectin substrates[Braam et al., 2008]

28 1.3.6.4 Collagen IV

Collagen IV is another type of BM protein that is present in all BMs including Matrigel™ [Poschl et al., 2004]. It is comprised of 6 different alpha chains that are able to assemble into 3 different heterotrimers. Collagen IV is unique compared to other types of collagen. As a basement membrane, collagen IV has an important role in mediating cell adhesion, migration and differentiation by acting as a scaffold which provides mechanical stability, structural integrity and central cohesiveness to BMs in situations where greater mechanical stability is required and there is an increase in mechanical forces [Poschl et al., 2004]. The network of collagen IV fibres functions as a foundation scaffold with the ability to form a complex arrangement by the incorporation of other components such as Laminin, Nidogen- 1 and 2 (mediate the formation of ternary complexes between Laminin and collagen IV in

vitro) and Perlecan. Together, this combination of proteins produces a highly

supramolecular architecture that form sheet-like BM complex structures [Poschl et al., 2004].

Collagen IV contains binding sites for numerous cell types including: platelets, hepatocytes, keratinocytes, endothelial/pancreatic cells and hESCs [Kruegel and Miosge, 2010]. There are many interacting integrins that can bind to collagen IV including; α1β1, α2β1, α3β1, α6β1, α10β1, α11β1 and αVβ5 [Kruegel and Miosge, 2010]. However, the collagen receptor identified in playing a major role in adhesion of hESCs to MatrigelTM specifically includes α2β1 [Mostafavi-Pour et al., 2012] and α9β1, which is a receptor not only for collagen IV but also laminin and VCAM-1 [Lee et al., 2010].

29 1.3.6.5 Heparin Sulphate Proteoglycans

Heparin sulphate protegolycans (HSPGs) are cell surface and ECM proteins; containing a core protein they are surrounded by covalently linked glycosaminoglycan (GAG) chains. Based on the core protein of HSPGs, they can be classified into 3 types; perlecan, glypican (disulfide-stabilised globular core protein linked to the plasma membrane) and syndecan (transmembrane protein which can also bind chondroitin sulphate), the latter two having a greater importance in cell surface HSPGs [Lin, 2004].HSPGs are able to incorporate into a network of other BM proteins including Laminin and Collagen IV. Other critical functions of HSPGs in relation to BMs include; retaining BM integrity, BM filtration functions and the ability of BMs to lock storage of growth factors [Poschl et al., 2004]. HSPGs are able to manage GFs, but also function as GFs themselves and more importantly, they are able to facilitate the interaction of GFs with the extracellular matrix [Symes et al., 2010]. HSPGs play an important role in regulating the formation of signalling pathways such as Wnt, Hedgehog, TGF-β and specifically bFGF. bFGF receptor activation is known to mediate hESC pluripotency and is also strongly linked with integrin signalling pathways [Braam et

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Table 1.4 Summary of the critical integrins and interactive ECM ligands which promote the adhesion and undifferentiated expansion of hESCs [Meng et al., 2010; Mostafavi-Pour

et al., 2012 Rowland et al., 2010]

Integrin Integrin Function Corresponding ECM Protein (Ligand)

Reference

α6β1 Maintain hESC stemness Laminin Meng et al., 2010,

Mostafavi-Pour et

al., 2012.

α5β1 Maintain hESC stemness Fibronectin Rowland et al., 2010, Mostafavi- Pour et al., 2012. αVβ5 Mediates hESC adhesion and

maintenance of pluripotency

Vitronectin Rowland et al., 2010

α2β1 Major role in hESC adhesion to Matrigel™

Collagen IV Meng et al., 2010

In summary, numerous ECM proteins are present in MatrigelTM and are also secreted by hESCs. These proteins play a crucial role in mediating hESC attachment and proliferation when cultured in vitro (Figure 1.6). There are various integrin receptors located on the membrane of hESCs which mediate the connection to these proteins and support the bi- directional talk between the cytoplasmic region of a cell and its extracellular matrix. The critical integrins that have been identified in mediating hESC attachment and proliferation whilst retaining pluripotency are summarised in Table 1.4. Furthermore, the impact of ECM protein availability, concentration and corresponding hESC surface integrin expression on hESC differentiation is not fully understood and yet to be fully elucidated. This would help to determine the exact integrin-protein interactions which induce hESC differentiation. However, the key interactions involved in hESC adhesion have partially been identified.

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Figure 1.6 Schematic representing the connection between ECM proteins and intracellular components proteins through integrins. Adapted from Humphries et al., 2006.