n vitro bioassembled human extracellular matrix and its application in human embryonic stem cell cultivation 5

74 75 76 77 3.6.3 Morphology of hESCs The morphology of the hESCs were checked after 13 passages and the phase contrast images shown (Figure 6B) hESCs cultured on DxSDOCDOC matrix still retained a pluripotent morphology with round, tight colonies with clearly defined margins These colonies consist of cells with high nuclear to cytoplasm ratio In contrast, hESC cultured on Matrigel grew in larger colonies, albeit still with defined colony edges These control cells still retained their high nuclear to cytoplasm ratio, but there were more pockets and areas consisting of differentiated cells with flatter and larger morphologies (arrows) 3.6.4 Pluripotency markers levels by flow cytometry At the 5th passage, hESCs cultured on DxSDOCDOC and on Matrigel were harvested and analyzed by flow cytometry in a similar fashion to that of section 3.5.4 The same pluripotency markers were used to characterize the hESC samples (Figure 6C) It was found that 60.5% of the hESCs cultured on Matrigel were positive for the marker Oct-4, while 76.4% of hESCs on DxSDOCDOC were positive for Oct-4, which showed a significant increase compared to the control Conversely, SSEA-3 levels were lower in hESCs on DxSDOCDOC at 44% compared to 57.2% in hESCs on Matrigel SSEA-4 levels were similarly strong in both samples of hESCs on Matrigel and DxSDOCDOC, at 93.6% TRA-1-60 levels were both high in both samples on Matrigel and 78 DxSDOCDOC, at 73.3% and 77.1% respectively 84.8% of hESCs on DxSDOCDOC were positive for the marker TRA-1-81, which is higher than that of hESCs on Matrigel at 75.2% Hence, at passage 5, hESCs on DxSDOCDOC expressed comparable pluripotency marker levels compared to the control and markers Oct-4 and TRA-1-81 were markedly increased, whereas SSEA-3 expression was decreased At the 13th passage, again, the hESCs cultured on either Matrigel or DxSDOCDOC were harvested for immunolabeling using the same pluritpotent markers and analyzed by flow cytometry Oct-4 levels were very much reduced at 39.9% for hESCs on Matrigel and 39.3% for hESCs on DxSDOCDOC, but the two samples were comparable to each other SSEA-3 levels were also not significantly different between the two samples at 41.8% and 38.3% for hESCs on Matrigel and DxSDOCDOC respectively SSEA-4 levels were much higher in hESCs on DxSDOCDOC at 82.7% compared to 73.7% in hESCs on Matrigel TRA-1-60 levels were similar in both hESCs on Matrigel and DxSDOCDOC at 65% and 68.8% respectively TRA-1-81 levels were also comparable in hESCs on Matrigel at 71.2% versus 74.7% in hESCs on DxSDOCDOC 79 Hence, at passage 13, hESCs on DxSDOCDOC produced comparable pluripotency marker levels compared to the control, except for SSEA-4, where significantly more hESCs on DxSDOCDOC had the expression of SSEA-4 3.6.5 Pluripotency markers levels by immunofluorescence The colonies of hESCs were harvested after 13 passages for adherent immunofluorescence and stained for same panel of pluripotency markers as the previous propagation trial - Oct-4, SSEA-4, TRA-1-81, SSEA-3, and TRA-1-60 hESCs cultured on DxSDOCDOC grew mostly in tight colonies that had cells positive for the pluripotency markers (Figure 6E-G) Control hESCs also mostly grew in colonies positive for the pluripotency markers, but there were some cells that were not positive for the markers (arrows) The above observations confirmed the pluripotent morphology seen under phase contrast microscopy 3.6.6 In vitro induced differentiation hESCs cultured on DxSDOCDOC for 13 passages were induced into neural differentiation using a protocol modified from WiCell Research Institute, National Stem Cell Bank In this protocol, the hESCs formed embryoid bodies using non-adherent cell culture plates and were transferred onto laminincoated plates and incubated in neural induction medium Under these conditions, hESCs were able to form extensions between embryoid bodies that were stained positive for β III tubulin, a marker for neural differentiation These results indicate that after 13 passages on DxSDOCDOC, the hESCs 80 were still receptive to neural induction and differentiation, showing their retention of differentiation capabilities 3.6.7 Karyotype Both hESCs cultured on Matrigel and on DxSDOCDOC were found to have normal female karyotype All 20 tested metaphases of hESCs on Matrigel had normal karyotypes, while 19 out of 20 tested metaphases of hESCs on Matrigel had normal karyotypes; metaphase showed non-clonal random loss Again, as mentioned in section 3.5.6, these cells can be considered as karyotypically normal due to only metaphase out of 20 having non-clonal random loss, which could have been a result of technical artifacts or random mitotic error 3.6.8 DNA Methylation Epigenetic programming has been shown to play an important role in regulating cellular differentiation, and because little is known about the epigenetic factors that define the differentiation state or potency of stem cells in comparison to differentiated cells, DNA methylation studies were done The goal was to identify methylation changes between hES cells grown on the human matrices versus Matrigel Samples from this propagation trial were collected and analyzed by Illumina Methylation Assay – Infinium II (see section 3.5.7.) Genes that were found to be overexpressed in iPSCs were selected to act as a pluripotency signature and the methylation status of the respective CpGs were presented in a heatmap (Figure 6J) Samples from the previous propagation trial were combined into the same heatmap and labeled 81 with a suffix (3.5.7), while samples from this propagation trial were labeled with a suffix (3.6.8) In addition, neurally-differentiated hESCs that were previously cultured on DxSDOCDOC were also included in the analysis and labeled with a suffix (3.6.6) From the heatmap, it can be seen that there were no major changes in the signature, which is in contradiction to observations seen for the differences in cellular behaviour Again, the epigenetic analysis of the samples was performed in the same way as in section 3.5.7, which only covers CpGs out of all the CpGs found to be present in each gene of the signature, as such, it only serves as an incomplete representation of the epigenetic changes that can be found Interestingly, in the neurally differentiated hESCs, one of the two CpGs representing the gene coding for Oct-4 (POU5F1) was found to be highly methylated at 0.91 (arrow) compared to the other pluripotent samples ranging from 0.45 to 0.57 It is possible that the neural differentiation of the hESCs has led to DNA methylation changes in that particular CpG, which could lead to changes in transcription levels It is also interesting to note that the assay used was able to pick epigenetic differences only in the situation when there are major changes to the hESCs such as the changes brought about by neural differentation Conversely, when cellular changes are comparatively minor, such as poor pluripotency maintenance on Matrigel, the epigenetic assay was unable to detect any major epigenetic changes This inadequacy of the assay could be due to it only representing a small fraction of the CpGs found within the genome, and unless major advances in technology allows for a far larger representation of the genome, the assay may not be sufficient to detect 82 epigenetic changes that truly reflects observations seen of cellular morphology changes Deep (454) bisulfite sequencing was used to analyze the methylation status of 14 CpG sites found within the Oct4 promoter region The statuses of hESCs on Matrigel or human matrices are presented in a heatmap (Figure 6K) In all conditions, the DNA methylation of 14 CpG sites remained low, hence the Oct-4 promoter were unmethylated, indicating that the Oct-4 promoter region was not yet silenced in all three conditions Oct-4 is an important transcription factor shown to be highly expressed in undifferentiated cells, and its expression leads to the expression of other genes that maintain hES cells in an undifferentiated state DNA methylation status of the 27,578 CpG sites tested for using the Illuminium Infinium Chip revealed a high overall similarity between Matrigel versus DxSDOC hESC cultures, and Matrigel versus DxSDOCDOC hESC cultures (Figure 6L) However, there still remains methylation differences between Matrigel-propagated hESCs and the DxSDOC-propagated hESCs, as 426 markers were found to be differentially methylated (Figure 6M) 194 markers were found to be differentially methylated between Matrigelpropagated hESCs versus DxSDOCDOC-propagated hESCs Between the two groups of differential markers, there is an overlap of 49 markers there were differentially methylated (Figure 6M) These 49 markers represent a diverse group of genes with many different functions While the functional significance of this observation will have to be further analyzed, it is possible 83 that the differential methylation of these 49 genes could have contributed to the superior proliferation of pluripotent hESCs on the DxSDOC and DxSDOCDOC matrices 3.6.9 DxSDOCDOC matrices are able to maintain pluripotent hESCs In this repeat propagation trial based on population doubling observations, morphology, pluripotency marker expression, in vivo differentiation studies and karyotype results, it was concluded once again that hESCs cultured on DxSDOCDOC showed superior population doubling rates, while maintaining pluripotent-like morphology compared to control hESCs on Matrigel Pluripotency marker expressions were comparable to the control hESCs by flow cytometry and by adherent immunofluorescence studies, hESCs on DxSDOCDOC had fewer areas of differentiation compared to the control In vitro differentiation studies showed that hESCs cultured on DxSDOCDOC matrices retained their neural differentiation capacity after 13 passages and yet maintained their karyotype despite a prolonged culture period of 18 passages 3.7 Matrices that maintain hESC pluripotency using dispase 3.7.1 Population doublings In section 3.4, 3.5 and 3.6, all hESCs were passaged by enzymatic passaging using collagenase IV There is another enzyme, dispase, which is commonly used for enzymatic passaging of hESCs In the propagation trial described in this section, dispase is used for subculturing of the hESCs to illustrate that irregardless of whether dispase or collagenase IV is used, DxSDOCDOC will still prove superior at increasing the population doublings and also in 84 maintaining the pluripotent characteristics of the hESCs during their propagation Once again, hESCs were cultured onto Matrigel, DxSDOC and DxSDOCDOC matrices for up to 20 passages hESCs were maintained in defined culture medium, mTeSR-1, and subcultured every 5-7 days depending on the confluency of the cultures At each passage, replicates of the cultures were sacrificed for cell counting and the population doublings were calculated and plotted (Figure 7A) 85 86 87 ... and were transferred onto laminincoated plates and incubated in neural induction medium Under these conditions, hESCs were able to form extensions between embryoid bodies that were stained positive... that maintain hESC pluripotency using dispase 3.7.1 Population doublings In section 3.4, 3 .5 and 3.6, all hESCs were passaged by enzymatic passaging using collagenase IV There is another enzyme,... Methylation Epigenetic programming has been shown to play an important role in regulating cellular differentiation, and because little is known about the epigenetic factors that define the differentiation