n vitro bioassembled human extracellular matrix and its application in human embryonic stem cell cultivation 6

88 89 90 91 In the initial passages, hESCs on the three matrices grew at a similar rate, as evident from the similar gradients on the population doublings graph Beyond the 5th passage, hESCs on Matrigel began to slow down their population doubling rates, with a gentler slope hESCs on DxSDOC and DxSDOCDOC continued with the same steady gradient with no apparent loss of doubling rate, finally finishing with 19.9% and 16.8% more than the population doublings achieved by hESCs on Matrigel 92 In agreement with the observations in section 3.5.1 and 3.6.1, DxSDOC and DxSDOCDOC matrices using the enzyme dispase again allowed for higher population doublings of hESCs compared to the control 3.7.2 Morphology of hESCs Phase contrast pictures of hESCs cultured on Matrigel, DxSDOC and DxSDOCDOC matrices up to passage and passage 20 are shown in Figure 7B Arrows indicate areas of spontaneous differentiation At passage 5, hESCs on Matrigel, DxSDOC and DxSDOCDOC grew in tight round colonies The cells within the colonies were small, with high nucleus to cytoplasm ratios and prominent nucleoli The colonies had distinct edges for each sample There were a few areas of differentiation (arrows) for the hESCs on DxSDOCDOC but they were a small minority of the entire population At passage 20, hESCs on Matrigel grew in smaller colonies with diffuse borders and the periphery cells were large and flattened There were widespread areas of differentiation Any pluripotent colonies were almost nondistinguishable, only recognizable by the difference in the sizes of colony cells compared to the differentiated cells hESCs on DxSDOC grew in large round colonies that still had a dense morphology The colonies have distinct borders and the cells within were small with prominent nucleoli and high cytoplasm to nucleus ratios There 93 were however, some areas of spontaneous differentiation at the periphery, albeit less than that seen in the control hESCs on Matrigel The areas of differentiation had large, flattened cells, indicated by arrows In contrast, hESCs on DxSDOCDOC grew in large round colonies that still remained dense The colony cells were also small and tightly packed, with clear distinct colony edges There were areas of differentiation at the colony periphery but these were far less than that seen in the control cells and the hESCs cultured on DxSDOC 3.7.3 Pluripotency markers levels by flow cytometry A panel of five pluripotency markers, Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, were used to characterize the hESCs after passages on either Matrigel, DxSDOC or DxSDOCDOC matrices For each hESC sample, triplicates of 10,000 labeled cells were used for each marker An average of the percentage of positively-labeled cells of each triplicates was plotted, together with the standard deviation (Figure 7C) Oct-4 levels expressed by hESCs on Matrigel were at 58.9% and the levels produced by hESCs on DxSDOC and DxSDOCDOC were decidedly lower at 11.3% and 38.1% SSEA-3 marker levels were similar in hESCs on Matrigel (61.3%) and on DxSDOC (64.4%), but were higher in hESCs on DxSDOCDOC (69.3%) SSEA-4 levels for hESCs on Matrigel and DxSDOC were both similar at 62.9% and 63% respectively, but were higher again for hESCs on DxSDOCDOC at 70.9% TRA-1-60 levels were low for hESCs on 94 Matrigel at 31.2% and comparatively higher for hESCs on DxSDOC (39.6%) and DxSDOCDOC at 45% TRA-1-81 levels were not significantly different in hESCs on Matrigel (38.2%), DxSDOC (43.8%) and DxSDOCDOC (53.7%) Hence, hESCs on DxSDOCDOC scored better in the markers SSEA-3, SSEA4 and TRA-1-60 compared to hESCs on Matrigel, but were lower for Oct-4 The same panel of five pluripotency markers, Oct-4, SSEA-3, SSEA-4, TRA1-60 and TRA-1-81 was used to characterize the hESCs after 20 passages on either Matrigel, DxSDOC or DxSDOCDOC matrices Again, triplicates of 10,000 labeled cells were used for each marker An average of the percentage of positively labeled cells of each triplicate was plotted, together with the standard deviation (Figure 7D) It was noted that hESCs on Matrigel and DxSDOC had relatively higher markers at passage but these were decreased by passage 20 (Figure 7D) For the intracellular pluripotency marker Oct-4, hESCs on DxSDOCDOC showed a higher percentage at 36.5%, than hESCs on Matrigel (29.7%), and DxSDOC (16.8%) It is noted that a population percentage of only 36.5% seems low, but as stated in section 3.5.3, Oct-4 is an intracellular pluripotency marker, which requires extra processing of the cell suspension to permeate the cellular membrane and nuclear membrane, thus resulting in more cells lost in the extra processing steps, which can contribute to the lower percentage seen 95 However, it is also important to note the relatively higher percentage of hESCs cultured on DxSDOCDOC positive for Oct-4 compared to the control hESCs on Matrigel Due to the limitations of using an intracellular pluripotency marker (Oct-4) the other pluripotency markers included were cell surface markers, hence preventing excessive cells lost to processing steps For surface marker SSEA3, hESCs on DxSDOCDOC showed a much higher population percentage at 62.8% Population percentages for hESCs on Matrigel remained low at 13.6%, and remained low also for hESCs on DxSDOC at 28.6% For the surface marker SSEA-4, there were relatively higher population percentages for hESCs on all three matrices hESCs on DxSDOCDOC showed a population percentage at 67.1%, hESCs on Matrigel showed 54.7% and hESCs on DxSDOC showed 74.4% Again, hESCs on DxSDOCDOC surpassed that of hESCs on Matrigel in terms of SSEA-4 expression For the surface marker TRA-1-60, the population percentages for hESCs on Matrigel and on DxSDOC were low at 23.7% and 36.4% respectively In contrast, hESCs on DxSDOCDOC matrices had higher population percentage at 47.5% For the surface marker TRA-1-81, again, population percentages for hESCs on Matrigel and on DxSDOCDOC were low at 36.1% and 21.2% respectively, 96 while hESCs on DxSDOCDOC matrices had relatively higher population percentages at 47.1% Hence, it is clearly evident that hESCs cultured on DxSDOCDOC matrices for 20 passages showed, on average, higher population percentages positive for the five pluripotency markers, than hESCs cultured on Matrigel for 20 passages 3.7.4 Pluripotency markers levels by immunofluorescence At passage 5, hESCs on Matrigel, DxSDOC and DxSDOCDOC were harvested for adherent immunofluorescence analysis It was found that the hESCs under the three conditions grew in tight colonies, with the majority of cells positive for Oct-4 and SSEA-4 (Figure 7E) When the hESCs were labeled for Tra-1-60, it was observed that the hESCs also grew in tight colonies with some areas of differentiation, indicated by arrows (Figure 7F) However, the majority of cells were positive for the pluripotency marker Some of the hESCs on Matrigel were not positive for TRA-1-81 and SSEA-3 (arrow) (Figure 7G), however, the majority of cells under all three conditions were positive for the markers These observations correlate to observations by flow cytometry, that the hESCs on DxSDOCDOC were not any less pluripotent than the control hESCs on Matrigel 97 culture, it would be possible to empirically prove the effects these components have on hESCs To further identify their roles, these components could be supplemented into DxSDOC and DxSDOCDOC matrices, to investigate if that would negate the pluripotency maintaining effect of the DxSDOC and DxSDOCDOC matrices DxSDOCDOC matrices for hESC culture could have commervial potential to be developed into a research tool for hESC culture, with widespread impact on hESC research There is much potential that DxSDOCDOC matrix could replace Matrigel in the market, as the work presented here has proven that the human matrix improves the proliferation of hESCs while maintaining their pluripontency In addition, the DxSDOCDOC matrix not only is from human origin, it does not require reconsitution prior to use, hence improving consumer convenience Currently, DxSDOCDOC matrices are stored in PBS at 4°C for up to three months with no deterioration in their performance, Compared to Matrigel, which has to be stored at -20°C, the storage conditions of DxSDOCDOC matrix has a commercial advantage as it decreases cost incurred from storage conditions Moreover, preliminary data shows that hESCs could be propagated in a pluripotent state on dried DxSDOCDOC matrices, and there is an added possibility of freeze-drying the DxSDOCDOC matrices, allowing for more cost reduction by transporting matrices in dried form The commercial development of DxSDOCDOC matrices would however, require more rigorous testing of the matrices effect on hESCs To provide for 108 a more comprehensive understanding of the hESCs after propagation on matrices, the gene expression of Oct-4 should be analysed and flow cytometry methods for assessing the hESCs could be improved by using fluorophoretagged antibodies as flow cytometry markers Further analysis of the inducibility of hESCs into cardiomycyte, hepatocyte, osteoblastic, chondrogenic and other lineages shoud be proven, to gain market confidence in the DxSDOCDOC matrix 109 Conclusion In summary, this work sought to explore the possibility of developing an alternative embryonic stem cell culture system, with the aim of overcoming current obstacles that hinder the safe clinical implementation of human embryonic stem cell culture technologies Significant refinements were made at each human embryonic stem cell culture step that can contribute to a safer and more efficient human embryonic stem cell culture: For the enhancement of collagenous extracellular matrix, MMC theory was applied successfully and found to increase the amount of insoluble collagen I deposition Both crowding agents, Dextran Sulfate 500kDa and a combination of Ficoll 70kDa and Ficoll 400kDa, resulted in increased collagen I deposition, although the morphologies were different In order to produce a cell-free matrix for more efficient and safer human embryonic stem cell culture, five different matrices were produced by using different permutations of macromolecular crowders and detergent processes Although all five matrices were found to be free from whole cells after detergent lysis, only two matrices, DxSDOC and DxSDOCDOC were found to support human embryonic stem cell proliferation More importantly, this work has also shown that the above two bioassembled matrices actually promoted superior proliferation rates and comparable pluripotency preservation in human embryonic stem cells when compared to the cells grown on the classical feeder-free culture substrate, Matrigel 110 One of the unexpected and interesting findings during the course of this study was that matrices that were richer in proteins, such as those derived using the gentler detergent NP40, appeared to be detrimental to the culturing of embryonic stem cells This is contradictory to the natural expectation that these cells should have been rather more prolific when provided with increased amounts of proteinaceous substrate that may facilitate cell growth by promoting cell signaling and growth factor availability However, subsequent staining and gel electrophoresis also showed that in addition to Fn and collagen, which aids in cell adherence and mechanical stability, other proteins such as DCN and BGN were also left behind on the matrices in increased amounts As these proteins bind active TGFβ and incorporate them into the matrix, this finding may have been contributed to by a paradoxical reduction of free active TGFβ available for preventing cell differentiation An avenue for further study on this paradox may be achieved in future by directly quantifying TGFβ, in both rich and poor culture media, via assay methods Also, mass spectrometry can be utilized to qualify and quantify the protein components of both categories of matrices, which could also provide some insight into this fascinating phenomenon An eventual research aim in this respect would be to fully grasp which proteins in the matrices are beneficial or detrimental, so that each can be added or removed specifically in the design of the perfect cell-free matrix The poor performance of induced pluripotent stem cells on these bioassembled matrices also came as a surprise, since these induced cells are otherwise extremely similar to their embryonic cell counterparts Once again, this opens 111 opportunities for further exploration As stem cell research continues to mature both technically and ethically, the hope of eventually harnessing its full therapeutic potential in a safe and morally commensurate manner will cast future attention on culturing adult induced pluripotent stem cells on matrices which are derived from clinical-grade human fibroblasts, and which are both industrially-efficient and entirely cell-free 112 References Fehrer C and Lepperdinger G Mesenchymal stem cell aging 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Learning from nature: emulating macromolecular crowding to drive extracellular matrix enhancement for the creation of connective tissue in vitro, Tissue Engineering, Croatia: IN- TECH (2010) 44 Minton