n vitro bioassembled human extracellular matrix and its application in human embryonic stem cell cultivation 1

Chapter 1: Introduction 1.1 Human Embryonic Stem Cells Stem cells are cells that are at an early stage of development, which can proliferate by self-renewal and retain the potential to differentiate into two or more cell types [1, 2, 3, 4] There are two types of stem cells - adult stem cells and ES cells Adult stem cells are found within adult organs whereas ES cells are derived from the inner cell mass of pre-implantation embryos and have a high nucleus to cytoplasm ratio, prominent nucleoli and distinct colony morphology The first ES cell line was derived from mouse embryos [5, 6], while hESCs were derived from human blastocysts [7] hESCs characteristically have high telomerase activity and express known surface markers - SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 [7, 8] The POU transcription factor, Oct-4 [9, 10] and Nanog [11, 12] are also highly expressed in hESCs In contrast to adult stem cells, these cells are able to differentiate into cells of all three germ layers - the endoderm, mesoderm and ectoderm; when implanted into SCID mice, they form teratomas and when allowed to grow to over-confluence, hESCs differentiate to endodermal and trophoblastic lineages [7] When cultured as suspended small clumps in differentiation medium and subsequently plated onto gelatin-coated plates, they form embryoid bodies with heterogenous morphology in the outgrowths which are positive for differentiation markers β-tubulin III, cardiac troponin I and α-fetoprotein [4] Given the hESCs’ ability to proliferate extensively and their capacity to differentiate into lineages that are otherwise unavailable, hESCs are considered an attractive therapeutic tool for tissue engineering, cellular transplant therapy and as drug discovery tools [2, 3, 4, 7, 13, 14] In tissue engineering, stem cells can possibly be used to generate healthy tissue to replace those damaged by trauma or disease It is believed that diseases that involve specialized cells known for limited regeneration, such as Parkinson’s disease, Alzheimer’s disease, heart diseases, stroke, arthritis, diabetes and spinal cord damage, can potentially be treated by cellular transplant therapy To test a large library of drugs in the process of drug discoveries, large numbers of cells of the known target disease have to be available In the case of specialized cells with limited proliferation potential, such large numbers are difficult to attain hESCs, with their ability to proliferate and subsequently differentiate into the required cell type, promise to alleviate this problem in drug discovery Despite the promise of hESCs in tissue engineering and cellular transplant therapy, the gap between scientific research and clinical applications for safe and effective stem cell based therapies is still wide Generally, for hESCs to be applied for human clinical trials, various bottlenecks have to be cleared before the process is considered safe hESCs have to undergo proliferation to generate a large number of pluripotent cells, which are then induced to undergo differentiation into the desired cell type Issues that need to be addressed include limiting the transfer of infectious agents from the donor cells to the host, reducing host immune responses by limiting immunogen exposure and retention in donor cells, reducing the risk of tumour generation upon implantation and attaining the desired efficacy of the differentiated cell type All these issues are dependent on the proliferation and differentiation process that the hESCs will undergo before implantation 1.2 Conventional Culture of hESCs Typical culture of hESCs requires the use of mitotically inactivated MEF feeder layers and fetal bovine serum to maintain their undifferentiated state [3, 4, 7] The feeder layers are presumed to provide soluble factors and an anchoring substrate for hESCs, hence providing a substitute growth-conducive microenvironment that maintains hESCs’ pluripotency However, the use of MEF and fetal bovine serum has proven disadvantageous for the clinical applications of hESCs Although MEFs are arrested in a postmitotic state with mitomycin C treatment prior to hESC co-culture, there is no way to remove them completely when the cells are brought into suspension Typically, MEF constitute 9% to 38% of the confluent co-culture As a result, MEFs are unavoidably implanted together with the hESCs into the host, even though this may only be a small admixture The presence of MEFs in the cellular graft could lead to host immune rejection and also the transfer of infectious agents MEFs are of animal origin, which exposes animal pathogens to hESCs It was found that hESCs exposed to animal-derived products expressed Neu5Gc, an immunogenic non-human sialic acid, thus rendering them unsuitable for clinical transplantation into humans [15] MEFs were also found to contain Neu5Gc [15] Although other feeder layers using human cell types have been developed, they are fibroblasts derived from fetal skin and muscle of aborted embryos, or from adult tissues such as Fallopian tubes and foreskin [16] However, at present, no GMP-grade xeno-free human feeder cells are available [16] Additionally, the use of feeder layers can confound downstream analysis of hESCs For example, during flow cytometry analysis of pluripotency marker expression by hESCs grown on MEFs, the MEFs have to either be separated from the hESCs prior to analysis, or labeled with a marker specific for MEFs and subtracted during the analysis process In reverse transcriptase-polymerase chain reaction analysis and other characterization analyses, MEFs can contribute 9% to 38% of the entire co-culture population, therefore adding significantly to background noise Even the use of human cells as feeders poses challenges for hESC culture, as the dependence on a co-culture system makes it technically challenging for large-scale expansion, such as in bioreactor settings [4, 16] To generate clinically-relevant numbers of differentiated cells from hESCs, a large number of undifferentiated hESCs are first required, as only a fraction of the undifferentiated hESCs will differentiate into the desired lineage To generate these undifferentiated hESCs, a large number of MEFs or human feeders are hence required As MEFs have to be harvested from developing mouse embryos and have a limited culture lifespan (6-8 passages), a large number of mouse embryos must be sacrificed making this a technically challenging and resource-wasting process Human feeders such as foreskin fibroblasts also have a limited lifespan, fibroblasts from fallopian tubes are limited in number, and the acquiring of human feeders from aborted embryos faces the same problems as the acquisition of MEFs Finally, feeder layers have batch-to-batch variability and the undefined quality of feeders makes reproducibility difficult One of the major criteria for developing cellular therapies using hESCs is to provide cells with defined quality characteristics that are safe for the patient In order to so, GMP need to be employed Other than preventing microbial contamination in the product, GMP requires the development of validated standard operating procedures to ensure that the cells are produced in a reproducible manner [16] As such, the batch-to-batch variability of feeder layers would present difficulties in achieving GMP standards 1.3 Feeder-Free Culture of hESCs To tackle the limitations of feeder layer culture, feeder-free culture systems have been developed Substitutes for soluble growth factors and anchoring substrates are the underlying principles of these systems, which try to mimic the microenvironments of the hESCs Indeed, keeping in mind the origin of hESCs, the neighbouring cells and their ECM serves as environmental cues for the developing hESCs Laminin, a basement membrane protein, and collagen I and III are produced as early as the 8-cell stage mouse embryo, while other ECM proteins, such as Fn, HSPG and collagen type IV appear later [17] In day old mouse blastocysts, laminin, entactin, collagen IV, HSPGs and Fn were found in the basement membrane between the ectoderm and the primitive endoderm which later becomes the inner cell mass [17] As the embryo develops, new germ layers will emerge, generating new epithelium and basement membrane, providing anchorage for the hESCs [18] The presence of ECM proteins in developing embryos indicates that these proteins play a role in maintaining ES cell As such, feeder-free culture systems typically consist of various ECM substrates supplemented with medium containing various growth factors 1.3.1 Matrigel One commonly used feeder-free culture substrate is Matrigel™, a solubilized basement membrane preparation extracted from EHS mouse sarcoma [19] Its major component is laminin, followed by collagen IV, HSPGs, and entactin [19, 20] The membrane is harvested using 2M urea and 0.05M Tris-HCL, pH 7.4 [19] and reconstituted as a gel at temperatures above 10ºC, with an optimum temperature of 35°C [20] The culture of hESCs on Matrigel requires the use of conditioned medium from MEF feeder layers or chemically defined medium Matrigel with MEF Conditioned Medium hESCs can be propagated for long periods of time on Matrigel with conditioned medium (up to 130 population doublings) [4] Conditioned medium is obtained by harvesting culture medium from MEFs, which contains growth factors and cytokines secreted by the MEFs The need for conditioned medium still represents a major limitation Firstly, culture of hESCs still requires the culture of MEF, albeit in a separate system, for conditioned medium As stated earlier, one of the problems with large scale expansion using feeder systems that require MEFs is the limited culture lifespan of MEF and hence the continued need for harvesting MEFs from mouse embryos As such, the large-scale expansion of hESCs using Matrigel with conditioned medium still faces the same problem faced by the standard feeder culture system Secondly, the use of conditioned medium from non-human MEFs can still introduce animal pathogens to hESCs, which can cause the expression of immunogens in these hESCs, rendering them unsuitable for clinical application Thirdly, conditioned medium is still undefined, and hence subject to batch-to-batch variability Matrigel with Defined Medium hESCs express receptors for various growth factors such as FGF-2, stem cell factor and fetal liver tyrosine kinase-3 ligand [21] Xu et al found that hESCs could maintain undifferentiated proliferation on Matrigel in serum-substituted medium supplemented with 500ng/ml noggin and 40ng/ml FGF-2 [22] Ludwig et al also found that factors, FGF-2, TGFβ, LiCl, γ-aminobutyric acid and pipecolic acid I medium with human serum albumin were critical for optimal undifferentiated proliferation of hESCs on Matrigel [23] These studies have shown that hESCs can be cultivated on Matrigel in culture medium supplemented with defined concentrations of recombinant factors, hence removing the dependence on MEFs for conditioned medium Culturing of hESCs on Matrigel removes the dependence on MEFs, thereby eliminating the risk of accidental MEF transplantation, and reducing downstream analysis complexity However, Matrigel is of mouse origin, which still poses a xenogenic threat As described earlier, Neu5Gc is a nonhuman sialic acid that can elicit an immune response in humans, and Matrigel has been found to contain Neu5Gc [24] In addition, as a product of a tumour cell line, Matrigel’s components, and their relative proportions, might be atypical of the native microenvironment for hESCs Since Matrigel is of mouse origin, the proteins that it presents to the hESCs are xenogenic Moreover, the process of harvesting Matrigel destroys lysine-derived cross-links and disulphide bonds required for the stability of collagen and other protein assemblies The components of reconstituted Matrigel appear to be linked by noncovalent bonds only, and although the proportions of components are constant, there were no parallel multilamellar structures typical of native basement membranes [20], indicating that the exact original morphology of the ECM cannot be regained 1.3.2 Purified Matrix Components as coating material Various groups have cultured hESCs on single matrix components, such as laminin and Fn Xu et al found that hESCs can be cultured on commercially obtained laminin 111 using MEF-conditioned medium for more than months [4] However, it was later reported by other groups that there was significant variability in results, depending on the source and batch of laminin [22] Amit et al showed that hESCs could be propagated for more than 15 passages on recombinant Fn in serum-substituted medium supplemented with TGFβ, LIF and FGF-2, although these hESCs exhibited lower cloning efficiency as compared to those grown on MEFs [25, 26] In addition to the variability of substrate and lower cloning efficiency, such purified matrix components are expensive for large-scale expansion, and are oversimplified mimics of the complex ECM that hESCs are exposed to in their natural microenvironments Native in vivo ECM is made up of numerous different components, namely glycoproteins (such as collagens, laminins, nectins, elastin and microfibrillar components), proteoglycans (such as decorin, biglycan, syndecan etc) and hyaluronic acid Not only does the ECM serve as a scaffold for cell attachment and provide strength for tissue integration, it also serves as a reservoir of growth factors and cytokines Cell attachment to ECM via cell surface receptors such as integrins results in cell-matrix interactions, which regulate cell survival, proliferation, motility and differentiation [27] The ECM stores growth factors, such as TGFβ and FGF 2, which have been demonstrated to have profound effects on ES cell culture Fbn and Fn, components of the ECM, binds to LTBP-1 [28, 29], which in turn, associates with LAP [28, 30] LAP binds to TGFβ in its latent form, and the latter becomes activated when released from LAP [31, 32] TGFβ prevents ES cell differentiation by maintaining Smad2/3 phosphorylation [33] FGF-2 interacts with heparin sulfate proteoglycans in the ECM [34] and maintains undifferentiated proliferation of hESCs [22] The biological action of FGF-2 is prolonged when it is bound to the matrix [35] Furthermore, the binding of FGF-2 to ECM protects it from proteolytic degradation [34] A pure single-component substrate such as laminin or Fn, would lack be lacking in the complex array of ECM proteins and other components, whereas in vivo ECM is bio-assembled as a complex mixture of components Therefore, such single-component substrates can hardly be termed a mimic of the complex microenvironment for hESCs, leading recent research to focus on the use of animals cells-derived ECM, which contain the complex array of proteins in their in situ architecture 1.3.3 MEF-Lysed ECM Klimanskaya et al derived and cultured hESCs on MEF-lysed ECM with medium supplemented with 20ng/mL LIF and 16ng/ml FGF-2 [36] The MEFs were lysed with 0.5% DOC in 10mmol/L Tris-HCL, pH 8.0 on ice for 10 granular and reticular under Fc70/400 crowding (Figure 1C and F) Hence, it has been postulated that the presence of negatively charged DxS causes an aggregation of the otherwise reticular Fn, which in turn, causes the granular deposition of Col I It has been reported that Fn assembly starts with the extension of the compacted Fn dimer, which can be elicited via an interaction with a negatively charged molecule [55], such as DxS It has been reported that a negatively charged macromolecule, PSS, was able to initiate Fn assembly, resulting in Fn adsorption onto PSS-coated wafers [56] Conversely, due to the lack of negatively charged motifs in Fc70 and Fc 400, the Fn assembly is not affected and thus the deposition pattern remains reticular, similar to the uncrowded controls 3.2 Detergent lysis removes intracellular structures 3.2.1 Removal of fibroblasts allows for the study of ECM effects without co-culture influences To fully study the role of the ECM on hESC proliferation and pluripotency maintenance, it was necessary to separate the fibroblasts from their ECM In any co-culture system, there are several undefined factors, which can affect the hESCs behaviour Feeder cells allows for cell-cell interactions with hESCs while providing soluble growth factors and cytokines The removal of fibroblasts from this system eliminates these two factors, fully defining the system such that only the in situ ECM can play a role in affecting hESCs Furthermore, in light of the future clinical applications of hESCs, the removal of fibroblasts would also prevent accidental transplantation of fibroblasts together with hESCs 31 Therefore, following the successful enhancement of ECM laid down by fibroblasts in the previous section, it was necessary to remove the fibroblasts in order to obtain a cell-free matrix Unlike the harvesting of Matrigel, which uses urea to solubilize proteins by denaturing them, prior to subsequent reconstitution, in situ detergent lysis was employed as the method of choice as it can remove the fibroblasts without complete solubilization of the ECM Two detergents, DOC and NP40, were used to lyse the fibroblasts 3.2.2 Five types of bioassembled cell-free matrices An illustration of how different types of bioassembled matrices can be obtained was presented in Figure 2A 32 In the previous sections 3.1.1 and 3.1.2, two morphologically different ECM were obtained by using either DxS or a cocktail of Fc 70 kDa and 400 kDa In addition, ECM lacking in Col I can also be produced under control conditions using fibroblasts cultured with only AcA without any crowders After three days of incubation under the above three conditions, the fibroblasts can be removed either by detergents DOC or NP40 Three protocols for lysis have been developed Cell layers were lysed by either 0.5% DOC three times on ice for ten minutes each (DOC), or lysed by 0.5% DOC six times on ice for ten minutes each (DOCDOC) or lysed by 1% NP40 four times on ice for ten 33 minutes each and subsequently incubated with deoxyribonuclease I (DNase) for two times one-hour treatments (NP40/DNase) DOC is an ionic detergent, hence it is a harsher treatment than non-ionic detergents and can modify protein structure to a greater extent NP40 is a nonionic detergent, which is non-denaturing, although it is less effective for membrane disruption Hence, it was paired with DNase to remove the remaining nuclei Matrices that were obtained from cell layers incubated with only AcA and subsequently lysed with DOC were termed “AcADOC” The other four types of matrices obtained from cell layers incubated with the respective crowder and subsequently lysed with the respective detergent were termed in a similar manner (Figure 2A) To confirm that these matrices had their fibroblasts completely removed, three intracellular components, i.e nuclei, ER and cytoskeleton F-actin, were visualized by immunofluoresence using DAPI, PDI antibody and phal respectively (Figure 2B) DAPI binds to double-stranded DNA found in the nucleus PDI is an enzyme associated with the ER in eukaryotes, which catalyzes the formation and breakage of disulfide bonds during protein folding Thus, the immunostaining of the ER is commonly performed using antibodies against PDI Phal is a bicyclic peptide that is isolated from the Amanita phalloides mushroom and it binds at the interface between F-actin 34 subunits Control cell layers (CL) that were not lyzed by detergents were also similarly stained for the intracellular components Nuclei, ER and actin were not detectable in all five types of matrices, indicating that both detergent treatments were sufficient for complete removal of the intracellular components and hence viable fibroblasts (Figure 2B) Positive controls of cell layers showed positive staining for nuclei, ER and Factin in a typical morphology 3.3 Bioassembled cell-free matrices are complex mixtures of proteins 3.3.1 Col I Retention in cell-free matrices Cell layer samples and detergent-treated matrices were pepsin-treated to isolate collagens, separated by SDS-PAGE, and the protein bands were quantified by silver-stain and densitometry By normalizing the densities of collagen α-bands of the various detergenttreated matrices to the respective cell layer samples, the percentage of collagen retained after detergent lysis can be obtained In comparison to the cell layer samples of DxS-crowded fibroblast cultures, DxSDOC matrices retained about 15.7% of Col I (Figure 3A and B) Such a low percentage was expected since the ionic nature of DOC treatment is expected to cause more protein loss (such as Col I) DOCDOC treatment exposes fibroblast cultures to double the treatments of DOC, and as expected, 35 more Col I was lost in this treatment process, with only a 2.2% retention of Col I compared to the cell layer control samples (Figure 3A and B) The non-denaturing nature of NP40 helped to preserve more of the Col I content in DxSNP40/DNase matrices; NP40/DNase treatment of DxScrowded matrix retained 91.8% of Col I (Figure 3A and B) Evidently, NP40 treatment was a much gentler cell removal process, which is able to retain more of Col I in these matrices 36 Fc-crowded fibroblast cultures were lysed using the gentler detergent NP40 and paired with DNase treatment As expected, FcNP40/DNase matrix retained about 70.9% of Col I (Figure 3C and D) In summary, the harsher ionic detergent DOC and double lysis resulted in a more substantial loss of Col I content in the matrices, compared to using nonionic NP40/DNase treatment However, despite the harsh detergent lysis, Col I 37 levels in DxSDOC and DxSDOCDOC matrices were detectable by SDSPAGE and silver staining 3.3.2 Immunofluorescence analysis and mass spectrometry reveals the presence of several proteins in matrices One of the advantages of bio-assembled matrices compared to singlecomponent matrices such as Fn or laminin, or multi-component matrices, is that, fibroblasts would also lay down several other proteins in addition to Col I Moreover, these proteins will be allowed to assemble as they would in nature, and in similar proportions to that of in vivo ECM is formed from many different structural macromolecules and plays a crucial role in maintaining tissue structure and modulating cell behavior The proper functioning of the ECM depends not only on a single molecule but also rather on its multiple constituents and the scaffolds they form There have been developments in the field of culture surfaces for hESC culture towards simple single- or oligo-component surfaces Villa-Diaz et al found that a synthetic polymer coating was sufficient for hESC culture [57] Two hESC lines, BG01 and H9 were maintained on this coating for 15 passages using conditioned medium However, results were less fruitful when the medium used was defined culture medium Using StemPro, H9 could only be maintained for 10 passages, but BG01 could not exceed passage 3; both cell lines could not be maintained using mTeSR-1 In the second paper, Melkoumian et al maintained H7 hESCs on peptide acrylate surface [58], and this was later developed into a product called SynthemaxTM-T Surface, by 38 Corning The peptides used were vitronectin and bone sialoprotein, containing RGD sequences Other RGD sequence peptides from laminin and Fn were attempted but found to be unsuitable for hESC culture, indicating that RGD sequences alone are not sufficient It was suggested that non-RGD regions of the peptides might play a role in recruiting other components into the substrate [58], so as to substantiate the substrate sufficiently for maintaining hESCs By extrapolation, the other components’ source would have to be either the hESC line itself or the culture medium [59], hence, only certain combinations of cell lines and culture medium would work in tandem with simple component culture substrates Taken together, the findings by Villa-Diaz et al., appear to support this hypothesis, suggesting that simple component culture substrates are less robust than complex multiple component substrates, as their efficacy would be easily affected by culture medium or cell line characteristics The use of simple substrates for hESC culture would then create a selection for subpopulations of hESCs, inflicting an evolutionary bottleneck Considering that hESCs are to be applied clinically, it would be far wiser to preserve a large variety of hESCs rather than a subpopulation Although simple component substrates might be temptingly convenient, complex culture substrates could prove, over long term, to be more stable, as it acts as a buffer against several variable environmental cues coming from culture medium or cell lines themselves The macromolecules of the ECM can be subdivided into collagen, noncollagenous glycoproteins and proteoglycans [60] There are at least two ways in which the ECM modulates cell behavior, through direct ECM-cell contact 39 or through the binding of growth factors or growth factor binding proteins The ECM serves as a storage for growth factors, concentrates them, enhances their effect, and even regulates the activity of these growth factors [61] To test for the presence of some of these proteins, a battery of antibodies were used to immunolabel them in the different types of matrices that were already made in earlier sections The proteins tested for include Col I, Fn, LTBP-1, Fbn, DCN, BGN and HS (Figure 3B) The HS antibody detects all HSPGs Col I was faintly present in DxSDOC matrices and was found in aggregates In contrast, Col I was not detected by immunolabeling in DxSDOCDOC matrices although, as mentioned in section 3.3.1, it was detected by SDSPAGE and silver staining It is possible that the amount of Col I was so low in DxSDOCDOC matrices that it was not visible compared to the background fluorescence and hence not discernible Col I was present in abundance in DxSNP40/DNase matrices, and was found to be in aggregates similar to that seen in DxS-crowded cell layer samples (refer to section 3.1.1) Col I was also immunolabeled in FcNP40/DNase matrices, and could be seen as reticular networks of thin fibrils, resembling that seen in section 3.1.2 Collagens are the most abundant of the macromolecules found in the body [61] and are formed by the varying combinations of three α-chains The amino acid sequence of these α-chains have variable numbers of the classical “Glycine-XY” repetitive motifs that form the collagenous, triple-helical portions of the protein, and these α-chains also contain non-collagenous portions of different amino acid sequences of different lengths and of different locations Collagens 40 can be grouped into two classes - the fibril-forming and the non-fibrillar collagens Three representatives of fibril-forming collagens are Col I, Col III and collagen V, which associate to fibrils Collagen V constitutes the core and Col I and III polymerize around it [60] Hence, Col I and III contribute to the fibril’s strength, and these fibrils build one of the main scaffolds of the interstitial ECM and allow other macromolecules to attach [61] Fn was found, by immunofluorescence, to be present, in the matrices DxSDOC, DxSDOCDOC, DxSNP40/DNase and FcNP40/DNase Fn staining in DxSDOC matrices showed uneven aggregates of Fn, with small elliptical areas that were devoid of Fn These elliptical areas were similar in dimensions to nuclei, but this cannot be confirmed by DAPI staining as all nuclei were already removed by the detergent treatments Fn staining in DxSDOCDOC matrices showed fewer aggregates of Fn, but an uneven layer of Fn still remained, with similar elliptical areas devoid of Fn Fn was present in DxSNP40/DNase matrices in relatively higher amounts than in the previous two matrices, with bright aggregates of Fn Fn was also present in FcNP40/DNase matrices, laid down in a reticular network similar to that seen in Fc-crowded cell layers (refer to section 3.1.2) Fn is a glycoprotein, which are proteins with oligosaccharides covalently attached to it The dimer Fn forms the second main scaffold in the interstitial ECM Both scaffolds of collagen and Fn interact with each other, thus stabilizing the architecture of the ECM Fn, with its many binding sites for integrins, also serves for cell attachment and the control of cellular behavior, such as cell migration and differentiation [62, 61] 41 LTBP-1 was found to be present in each of the matrices The morphology of LTBP-1 in DxSDOC and DxSDOCDOC matrices were similar, with an uneven staining of comparatively large aggregates of irregular shapes Conversely, LTBP-1 staining in DxSNP40/DNase matrices consists of relatively smaller aggregates that speckled the surface of the matrix LTBP-1 was found to be in a reticular network in FcNP40/DNase matrices, not unlike the stainings of Fn and Col I LTBP is a glycoprotein, which can be associated with collagens, Fn and Fbn [28, 29] It is the binding partner of LAP [28, 30], which binds TGFβ in a biologically inactive state [31, 32] The activation or release of TGFβ is mediated through proteolysis The activated TGFβ can also be bound to other ECM molecules such as DCN and BGN [28, 63] Fbn was found to be present in DxSDOC matrices by immunofluorescence but was undetectable in DxSDOCDOC matrices In DxSDOC matrices, Fbn consisted of large aggregates of irregular shapes spaced unevenly Fbns, together with elastin, form the elastic fibers of the connective tissue Fbns are able to associate with each other to form microfibrils, and then bind to elastin to form the third main scaffold in interstitial ECM [60] The LAP-TGFβ complex can be associated with Fbn through the mediating partner LTBP [28] DCN was found to be present in the matrices In DxSDOC matrices, DCN was laid down in small speckles spread out fairly evenly In DxSDOCDOC matrices, similarly sized speckles were also seen, albeit fewer and further between compared to DxSDOC matrices In contrast, DxSNP40/DNase 42 matrices had DCN laid down in larger aggregates of irregular shapes, and situated more frequently than in DxSDOC and DxSDOCDOC matrices DCN in FcNP40/DNase matrices were similar to DxSDOC and DxSDOCDOC matrices, the protein was found in small speckles, but were in far less quantities than those of DxSDOC and even DxSDOCDOC matrices BGN was faintly detected in DxSDOC, DxSDOCDOC and FcNP40/DNase matrices In these three matrices, BGN was laid down in small speckles that were of varying sizes and shapes The amounts appear to be similar in all three matrices, but were far less than that seen in DxSNP40/DNase matrices In the DxSNP40/DNase matrix, BGN was found in irregularly sized aggregates of varying shapes The amount of BGN was much more in comparison to the former three matrices Both DCN and BGN are members of the proteoglycan family, with a small leucine-rich protein core and a glycosaminoglycanbinding N-terminus DCN and BGN are able to bind to collagen fibrils and also to other glycoproteins such as Fn, forming a connection between the two main scaffolds of the ECM [60, 63] Moreover, the two molecules play a role in moderating the activity of TGFβ by binding active TGFβ [28, 63] HS was found to be strongly present in all four matrices, showing the existence of HSPGs In DxSDOC matrices, HSPGs were laid down as fine aggregates evenly spaced out throughout the matrix, with strong positive immunofluorescence staining HSPGs were found to be comparatively less in DxSDOCDOC matrices, but still moderately detectable by immunofluorescence, in comparison to the DxSDOC matrix The HSPGs in 43 DxSDOCDOC matrices were in larger and more diffuse aggregates On the other hand, HSPGs in DxSNP40/DNase matrices were found to be in defined aggregates, with smaller speckles of HSPGs in between the relatively larger aggregates HSPGs in FcNP40/DNase matrices were found to be in a reticular network similar to that of Col I, Fn and LTBP-1, which were also found in the same matrix HSPGs are proteoglycans with glycosaminoglycan chains of HS Glycosaminoglycans are linear polysaccharides formed by repeating units of disaccharides Proteoglycans modulate the hydration of the ECM, and are able to bind growth factors FGF-2 have been shown to bind to HSPGs of the ECM [34], and HSPGs prolong the biological action of FGF-2 when it is bound [35], and binding also protects FGF-2 from proteolytic degradation [34] Further characterization of DxSDOC and DxSDOCDOC matrices by mass spectrometry revealed the presence of several proteins (Table 2) There were extracellular matrix proteins such as collagens, EMILIN-1, Fn, Fbn I, tenascin, secreted proteins transglutaminase 2, remnants of detergent lysis such as cytoplasmic heat-shock protein 70kDa, cytoskeletal proteins actins and tubulins and nucleas-associated proteins such as histones In a similar mass spectrometric analysis of Matrigel, the components of the murine ECM was studied [64] and by comparing the differences between DxSDOCDOC matrix versus Matrigel, it was found that collagen XII, transglutaminase and TGFβinduced protein ig-h3 were found in DxSDOCDOC matrix but not in Matrigel Collagen XII belongs to a subgroup of non-fibrillar-collagens termed fibrilassociated collagens with interrupted triple-helices and may be involved in 44 basement membrane regulation providing specific molecular bridges between fibrils and other matrix components [65] Transglutaminase catalyzes protein crosslinking, apoptosis, differentiation and cell adhesion [66-68] ig-h3 binds to collagen VI [69], biglycan and decorin [70] and mediates cell adhesion The roles that these three components may play to maintain hESCs’ pluripotency is as yet unclear and can be further investigated In addition, there are components in murine Matrigel that are a different version from human proteins († marked, Table 2), such as collagens, EMILIN1, Fbn 1, Fn, tenascin, thrombospondin-1 and vitronectin, all of which play significant roles in regulating several cellular functions [62, 61] The use of mouse homologous proteins rather than human proteins might negatively impact on the regulation of hESC cellular functions 45 ... changed to DMEM/F12 (Invitrogen, 10 565-042) supplemented with Non-essential amino acids (Invitrogen, 11 114 0050), N2 (Invitrogen, 17 502-048), heparin (Sigma, H 314 9) and FGF-2 (ProSpec, CYT-557 _10 µg)... harvested using collagenase and seeded onto CoStar non-adherent plates (Corning, 34 71) to form embryoid bodies using 20% Knockout Serum (Invitrogen, A1099202) in Knockout DMEM (Invitrogen, 10 829- 018 )... immune responses by limiting immunogen exposure and retention in donor cells, reducing the risk of tumour generation upon implantation and attaining the desired efficacy of the differentiated cell