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

The first ES cell line was derived from mouse embryos [5, 6], while hESCs were derived from human blastocysts [7].. 1.2 Conventional Culture of hESCs Typical culture of hESCs requires t

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

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 post-mitotic 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

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 do 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 5 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

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 5 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 non-human 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

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 6 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

30 minutes and remaining matrices were rinsed 5-6 times with PBS Again, the use of MEF potentially exposes the hES to MEF-derived pathogens, hence the authors proposed sterilizing the MEF-lysed ECM before use Preliminary studies conducted by the authors found that the MEF-lysed ECM could be dehydrated, treated with paraformaldehyde, heat-pasteurized (60°C for over

20 hours) or gamma-irradiated without substantial changes to hESC behaviour However, as the authors acknowledged, the complex array of proteins, growth factors and hormones in the ECM would be denatured and destroyed by the sterilization process

Also, it has been found that Neu5Gc represent 20% of total sialic acid in MEFs [15], and whether Neu5Gc is removed by the detergent lysis process was not yet proven by Klimanskaya et al., hence MEF-lysed ECM might still expose hESCs to this immunogen Moreover, this system does not eliminate the need for continuous harvesting of MEFs from mouse embryos

1.3.4 Commercially Available Matrix Substrates

Besides Matrigel, other matrix substrates commercially available for hESC proliferation include Geltrex (Gibco), Cultrex (R&D Systems), ECM gel (Sigma-Aldrich) and PuraMatrix (BD) Of these, Geltrex, Cultrex and ECM gel are all derived from mouse EHS sarcoma, like Matrigel Hence, like Matrigel, it is to be expected that being of mouse-origin, they will also contain Neu5Gc and possibly animal pathogens On the other hand, PuraMatrix is made of synthetic amphiphilic oligopeptides that self-assemble in physiological salt solutions [37] Since PuraMatrix does not contain any

animal-derived materials, it poses no xenogenic threat However, according to the manufacturer, PuraMatrix forms a relatively soft fibrous matrix, and cannot withstand vacuum aspiration around the hydrogel during medium removal Thus, PuraMatrix is more fragile than Matrigel in terms of handling, and there are currently no data supporting hESC proliferation on this material

1.4 Ideal hESC Culture System

The various culture systems described above have sought to improve the culture of hESCs, but there are still open issues The use of mouse-origin Matrigel, MEF-conditioned medium, expensive purified matrix components, and MEF-lysed matrix need to be replaced by transplant-friendly and cost-effective culture systems In view of the imperfections of existing culture systems, an optimal system for the culture and derivation of hESCs would require feeder-free culture on a human ECM, maintained on chemically defined culture medium containing only human substances and proteins to produce clinical-grade hESCs [16] And such a human ECM can made by human fibroblasts, the professional ECM producer

Advances in ECM deposition in our laboratory have shown that the use of MMC in the culture of human fibroblasts can significantly enhance the production of a collagenous matrix [38, 39] Such a matrix could then be treated with detergents for fibroblast removal, rendering the ECM cell-free As such, this complex ECM, which does not require the use of mouse-origin Matrigel nor dependence on MEFs, would be suitable for hES culture

application Moreover, such a cell-free ECM would serve as a study on the effects of ECM on hESC behaviour

• Variability of

feeders

• Xenogenic threat

Conditioned Medium

Defined Medium

• Defined substrate • Dependence on

MEFs

• Xenogenic threat

• Variability of both

substrate and

conditioned medium

• Oversimplified model of ECM Purified

Matrix

Component

- Fn

Defined Medium

• Defined substrate and medium

• Expensive

• Oversimplified model of ECM

MEF-Lysed

ECM

Defined Medium

• Reduce xenogenic threat

1.5 Hypothesis

A human ECM can be bio-assembled in vitro, decellularized and subsequently applied for the sustained growth and maintenance of pluripotency of hESCs

1.6 Objectives

1.6.1 Enhance in vitro Bio-assembly of Matrix Proteins by Fibroblasts

To manipulate fibroblasts to deposit ECM which is rich in proteins such as collagen, Fn, ligands and growth factors

1.6.2 Complete Removal of Fibroblasts to obtain Cell-Free Matrices

To completely remove fibroblasts while retaining acceptable ECM protein and ECM structure levels

1.6.3 Characterization of Cell-Free Matrices

To characterize cell-free matrices and identify components of the matrix

1.6.4 Propagation of hESCs on Cell-Free Matrices

To cultivate hESCs on cell-free matrices with characterization of the hESCs and to assess the maintenance of their undifferentiated state and pluripotency

Chapter 2: Materials and Methods

2.1 Cell Culture

2.1.1 Matrices

Wi38 fetal lung fibroblasts (ATCC, CCL-75) at passage 20-22 were seeded at 25,000 cells/cm2 in either 24 well or 6 well culture plates (CelStar, Greiner Bio-one, 662160) in high glucose DMEM (Gibco-BRL, 10569-010) supplemented with 10% FBS (Gibco, 10270-106), which was replaced with 0.5% FBS culture medium supplemented with 30µg/ml AcA (Wako, 019-12063) with 100µg/ml DxS (USB corporation, 70796-50G) on the following day After 3 days of incubation at 37°C and 5% carbon dioxide, the fibroblast cell layers were washed with PBS twice and lysed with 0.5% DOC (Prodotti Chimici E Alimentari, S.P.A 2003030085) and 0.5X Complete PrI (Roche Diagnostics Asia Pacific, 11836145001) 3 times 10mins on ice to obtain cell-free DxSDOC matrices while a similar protocol of 6 times of 0.5% DOC treatment was applied to get DxSDOCDOC matrices DxSNP40/DNase matrices were made by incubating fibroblasts with 30µg/ml AcA and 100µg/ml DxS for 3 days, washed with PBS twice and lysed with 1% NP40 (Fluka BioChemika, 74385) and 0.5X PrI 4 times 10mins on ice and treated with DNase I (US Biologicals, D3200) twice for 1 hour each at 37°C DNase was used at dilution 1:50 in a buffer made of 10mM Tris pH 7.5 (J.T.Baker, 4109-02), 2.5mM MgCl2 (Fluka BioChemika, 63068) and 500µM CaCl2 (Merck, 1.02382.1000) FcNP40/DNase was obtained by incubating fibroblasts with 30µg/ml Aca and 37.5mg/ml Fc 70 (GE Healthcare, 17-0310-50) and 25mg/ml Fc 400 (GE Healthcare, 17-0300-50) for 6 days and lysed in

a similar manner as DxSNP40/DNase matrices All matrices were washed with PBS 3 times to remove all traces of the lysis solutions

2.1.2 hESCs

H9 (WiCell, WA09) were passaged using collagenase IV (Gibco, 17104-019)

or dispase (StemCell Technologies, 07913) equally onto Matrigel (BD, qualified, 354277), DxSDOC and DxSDOCDOC matrices and cultured using defined medium mTeSR-1 (StemCell Technologies, 05850) Culture medium was changed daily and subculture was done every 5-7 days

hESC-2.1.3 iPSCs

DL1 cells were generously supplied by Dr Jeremy Crook of Singapore Stem Cell Bank, Singapore Stem Cell Consortium, Institute of Medical Biology The iPSCs were derived by transfecting IMR-90 fetal fibroblasts with lentiviral vector expressing Oct-4, Sox2, Nanog, Lin28 and Large T The iPSCs were cultured on MEFs prior to seeding onto matrices, and subcultured every 5-7 days using dispase

synthesized by fibroblasts in cell culture remains mostly in the soluble form, which accumulates in the culture medium instead of depositing at the cell layer To correctly access the amount of insoluble collagen rather than soluble procollagen in fibroblast cultures, procollagen was first removed by aspiration

of culture medium, followed by 2 washes of PBS In this way, only insoluble collagen found at the cell layer would remain To further isolate collagen from other proteins found also at the cell layer, the samples in 24-wells plate format were digested with 100µl of 0.25mg/ml of pepsin (Roche Diagnostics Gmbh, 10108057) supplemented with 0.5% Triton-X 100 (Bio-Rad, 161-0407), which degrades all proteins into peptides except collagen Pepsin-digested samples were separated under non-reducing conditions using 5% resolving/ 3% stacking polyacrylamide gels as outlined in [38, 39] Protein standards used were the Precision Plus Dual Color (Bio-Rad,161-0374 ) and collagen type I (Koken Co., IAC-13) Protein bands were visualized using a Silver Quest Silver Staining Kit (Invitrogen Life Technologies, LC 6070) according

to manufacturer’s instructions The amount of collagen present in the samples was quantified by densitometry of the α-bands of collagen using a GS-800TM Calibrated Densitometer (BioRad, 170-7980)

2.2.2 Immunofluorescence and Mass Spectrometry

The following antibodies were used to immunolabel DxSDOC, DxSDOCDOC, DxSNP40/DNase, FcNP40/DNase matrices: mouse anti-PDI (Invitrogen, S34200), Alexa-Fluor 594 Phal (Invitrogen, A12381), mouse anti-Col I (Sigma, C2456), rabbit anti-Fn (Dako, A0245), mouse anti-HS

DCN (gift from Dr Larry Fisher, LF-136), rabbit anti-BGN (gift from Dr Larry Fisher, LF-112), rabbit anti-LTBP-1 (gift from Dr Carl Heldin, AB39), Alexa Fluor 594 goat anti-mouse (Invitrogen, A11020), Alexa Fluor 488 chicken anti-rabbit (Invitrogen, A21441) and DAPI (Invitrogen) Mass spectrometry identifications were done by Christopher Stephen Hughes and Prof Gilles Lajoie, using Mascot, OMSSA and X!Tandem after analysis on the mass spectrometer Scaffold was used to validate MS/MS based peptide and protein identifications Protein probabilities were assigned by Protein Prophet algorithm Peptide and protein identifications were accepted if they could be established at greater than 95% and 99% probabilities respectively Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony

2.3 Analysis of hESCs

2.3.1 Preparation of Matrigel Coating

175µl of BD Matrigel hESC-qualified Matrix (BD, 354277) was diluted in 12.5ml of DMEM-F12 medium (Invitrogen, 11330032) overnight at 4°C 1000µl or 200µl of diluted Matrigel was coated onto each well of 6-well culture plates or 24-well culture plates respectively and incubated for 1 hour at 37°C Wells were used immediately for hESC culture

2.3.2 Population Doubling Curve

Representative wells were sacrificed for cell counting at each passage to obtain the number of cells and from there calculate the population doublings The wells were harvested into single cell suspensions using TrypLE Express

(Gibco, 12604-021) and neutralized with 10% FBS culture medium All counting was done using Trypan Blue and a haemacytometer

2.3.3 Flow Cytometry

The hESCs were harvested into single cell suspension using TrypLE and fixed with 4% formaldehyde for 10 minutes at 37°C The following antibodies were used to immunolabel the hESCs: goat anti-Oct-4 (Santa Cruz, sc-8628), mouse anti-TRA-1-60 (Chemicon, MAB 4360), mouse anti-TRA-1-81 (Chemicon, MAB 4381), rat anti-SSEA-3 (Chemicon, MAB 4303), mouse anti-SSEA-4 (Chemicon, MAB 4304) and Alexa Fluor 488 donkey anti-goat (Invitrogen, A11055), Alexa Fluor 488 chicken anti-mouse (Invitrogen, A21200) and Alexa Fluor 488 goat anti-rat (Invitrogen, A11006) Flow cytometry was performed using CyAn

to DMEM/F12 (Invitrogen, 10565-042) supplemented with Non-essential amino acids (Invitrogen, 111140050), N2 (Invitrogen, 17502-048), heparin (Sigma, H3149) and FGF-2 (ProSpec, CYT-557_10µg) and embryoid bodies were seeded onto new standard 6 well plates for 3 days Finally, embryoid bodies were seeded onto wells coated with 20µg/ml laminin (Sigma, L6274) according to manufacturer’s instructions and incubated for 7 days Samples were fixed with formaldehyde and immunolabeled with rabbit anti-β III tubulin (Abcam, ab18207) and Alexa Fluor 488 chicken anti-rabbit (Invitrogen) and DAPI

2.3.7 Karyotype

Matrices were prepared in 25cm2 culture flasks (CelStar, Greiner Bio-one, 690-175) in a similar manner as stated in section 2.1.1 125,000 Wi38 fetal lung fibroblasts were seeded into each culture flask using 10% FBS in DMEM and the culture medium was changed to 0.5% FBS in DMEM supplemented with 30µg/ml AcA and 100µg/ml DxS After 3 days of incubation at 37°C and 5% carbon dioxide, the fibroblast cell layers were washed with PBS twice and

lysed with 0.5% DOC and 0.5X Complete PrI 3 times for 10mins each on ice

to obtain cell-free DxSDOC matrices while a similar protocol of 6 times of 0.5% DOC treatment was applied to get DxSDOCDOC matrices Matrigel was prepared in 25cm2 culture flasks in a similar manner as stated in section 2.3.1 2.5ml of diluted Matrigel was used for each culture flask and incubated for 1 hour at 37°C

1.5 million hESCs were seeded into each culture flask and incubated in 5ml of mTeSR-1 for 3 days with daily changes of culture medium Karyotyping services were obtained from KK Women’s and Children’s Hospital Harvesting was done using colcemid and staining done with Trypsin and Wright’s-Giemsa stain

2.3.8 DNA Methylation

hESCs propagated on Matrigel, DxSDOC and DxSDOCDOC were washed with HBSS twice before the cells were harvested in 500µl HBSS by scraping The cells were pelleted by centrifuging at 1100rpm for 3 minutes and the supernatant was removed The pellet was then transferred into a cryovial and snap frozen in liquid nitrogen The vials were then stored in -80°C before shipping under dry ice conditions to Prof Frank Lyko and Michael Bocker of University of Heidelberg, German Cancer Research Center for DNA methylation studies using the Illumina Methylation Assay – Infinium II Analysis was done using Illumina Beadstudio Software to generate the genome-wide correlation data The methylation states of samples obtained from propagation trial 3.5 and trial 3.6 was inter-chip normalized before