n vitro bioassembled human extracellular matrix and its application in human embryonic stem cell cultivation 4

58 59 60 In the initial passage, hESCs cultured onto DxSDOCDOC matrices showed a decrease in their population doublings, which could be due to the initial adaptive state of the hESCs However, with subsequent passages, hESCs began to proliferate and increase population doublings at a similar rate to the control hESCs on Matrigel, as evident from the gradients of the two series hESCs cultured on DxSDOC matrices also showed a decrease in the population doublings in the first two passages, but quickly regained population doublings similar to the control hESCs By the 11th passage, population doublings of hESCs on DxSDOC matrices dipped slightly compared to the control hESCs, but again regained the same proliferation rate 61 Control hESCs that were cultured on Matrigel proliferated at approximately the same rate for the first 13 passages, before losing the fast proliferation rate at the 14th passage From passage 14 to 15, population doublings remained stagnant, and only began to increase at the 16th passage By this passage, hESCs on DxSDOC and DxSDOCDOC matrices began to surpass the control hESCs in terms of population doublings (16.8% versus 13.2%)), and up till the 23rd passage, the population doubling rate of control hESCs continued to remain lower than the hESCs on the two bioassembled matrices, such that the latter two far exceeded the control hESCs (24.8% versus 19.8%) From these observations, it was concluded that in long-term passaging, using collagenase IV and defined culture medium, mTeSR-1, hESCs on either DxSDOC matrices or DxSDOCDOC matrices were able to proliferate more than control hESCs on Matrigel hESCs have long been reported to be able to proliferate for up to 100 passages, albeit under feeder co-culture systems [3, 4, 7] hESCs have also been reported to proliferate for up to 20 passages on Matrigel-coated wells, using enzymatic passaging techniques and MEFconditioned medium [4] According to mTeSR-1’s manufacturers, StemCell Technologies, hESCs can be cultured on Matrigel in the defined culture medium for up to 18 passages [23] When proliferation rate of hESCs begins to slow down, it is a sign of the aging of the cells hESCs on feeder-free culture systems tend to acquire senescence at earlier passages compared to those on feeder co-culture systems, and senescence is usually accompanied by morphological changes Hence, phase contrast images of the hESCs were taken to observe these morphological differences 62 3.5.2 Morphology of hESCs Phase contrast images of hESCs on both bio-assembled matrices and also the control hESCs were taken at an earlier passage, passage +5, and compared to a later passage at passage +20 (Figure 5B) At the earlier passage, hESCs on all three matrices grew in tight, round colonies, with clearly-defined edges, and each cell showed a high nuclei to cytoplasm ratio The colony sizes for hESCs cultured on DxSDOC and DxSDOCDOC matrices appeared smaller compared to the controls At the later passages, the majority of hESCs on all three matrices still grew in tight colonies, but for hESCs that were cultured on either Matrigel or DxSDOC, there were significant fractions that grew on the periphery of colonies, with flattened morphology that did not have high nuclei to cytoplasm ratios, resembling spontaneously differentiated cells Although hESCs on DxSDOCDOC did have some spontaneously differentiated cells in the population, these cells were not common Colonies of hESCs on DxSDOCDOC were larger in the later passage compared to the earlier passage, but still remained round and tight with defined colony margins As reported earlier, the population doublings of the hESCs on bioassembled matrices DxSDOC and DxSDOCDOC exceeded the control hESCs, and yet, despite the extensive passaging, the hESCs on DxSDOCDOC matrices still retained morphology more similar to earlier passage hESCs 63 3.5.3 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 was 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 5C) Comparing the population percentage of hESCs positive for the Oct-4 marker, 62.4% of hESCs on Matrigel expressed Oct-4, while 62.6% of hESCs on DxSDOC matrix expressed the marker and only 42.2% of hESCs on DxSDOCDOC matrix expressed the marker Oct-4 is a POU transcription factor responsible for activating several genes important for maintaining the pluripotency of hESCs Oct-4 has been found to be highly expressed in pluripotent hESCs [9] The pattern of Oct-4 expression in human embryos is similar to that of mouse embryos [10], where Oct-4 is expressed in totipotent embryonic cells, then becomes restricted to the ECM of the blastocysts Oct-4 becomes down-regulated in trophoectoderm and primitive endoderm of the mouse embryo Later, it is maintained in the embryonic ectoderm at the cylinder stage, and down-regulated at gastrulation in an anterior-posterior pattern Germ cells continue to maintain the expression of Oct-4 until the initiation of sexual differentiation [10] The under-expression of Oct-4 in ES cells leads to their differentiation into trophoectoderm lineages [10], indicating the crucial role that Oct-4 plays in maintaining the pluripotent state of ES cells As Oct-4 is a transcription factor, naturally it is found within the nuclei 64 of hESCs, and so, in order to obtain successful Oct-4 immunostaining, the cells have to be first permeabilized using 0.2% Triton-X 100 - this extra processing step results in markedly increased sample loss, which possibly accounts for the low population percentage seen in the flow cytometry readings On the other hand, a much higher percentage for the surface markers SSEA-3 and SSEA-4 was seen 86.9% of hESCs on Matrigel expressed SSEA-3, with 88.9% and 88.7% for hESCs on DxSDOC and DxSDOCDOC respectively Similarly, 85.7% of hESCs on Matrigel expressed SSEA-4, with 84.2% and 88.1% for DxSDOC and DxSDOCDOC hESCs respectively Only 34.6% of hESCs cultured on Matrigel, 36.8% of hESCs cultured on DxSDOC and 37.2% of hESCs cultured on DxSDOCDOC were positive for TRA-1-60 Conversely, 77.7% and 75.1% of hESCs cultured on Matrigel and DxSDOC respectively were positive for TRA-1-81 50.6% of hESCs cultured on DxSDOCDOC were positive for TRA-1-81 Overall, other than for the markers Oct-4 and TRA-1-81, hESCs on DxSDOC and DxSDOCDOC scored comparatively similar to hESCs on Matrigel in the expression of pluripotency markers hESCs that were cultured on the three various matrices were harvested into single cell suspensions at passage +20 and immunolabeled with the same antibodies as those used in the earlier passage samples Across the panel of the 65 five pluripotency markers, flow cytometry of the hESCs under the three conditions showed comparable expression, except for Oct-4 (Figure 5D) Oct-4 positive cells were found to be present in 47.2% of hESCs cultured on Matrigel, compared to only 23.4% of hESCs on DxSDOC and 31.5% of hESCs on DxSDOCDOC However, for all the other four the surface markers, the three populations had comparable expression 77.5% of hESCs on Matrigel were positive for SSEA3, with 72.2% for hESCs on DxSDOC and 70.6% for hESCs on DxSDOCDOC 78.9% of hESCs on Matrigel were positive for SSEA-4, and 85% for hESCs on DxSDOC and 75.1% of hESCs on DxSDOCDOC TRA-160 markers expression was present in 52.4% of hESCs on Matrigel, 40.9% for hESCs on DxSDOC and 49.4% of hESCs on DxSDOCDOC 36.9% of hESCs on Matrigel showed positive staining for TRA-1-81, 31.1% in hESCs on DxSDOC and 41.7% in hESCs on DxSDOCDOC Taking into account that the surface marker levels were similar in the three population samples, hESCs on DxSDOC and DxSDOCDOC did not differ much from hESCs on Matrigel in terms of pluripotency marker expression However, if the population doubling results mentioned in section 3.5.1 were considered, there was a marked increase in the number of population doublings in hESCs cultured on DxSDOC and DxSDOCDOC matrices, in comparison to the control hESCs, and yet, there was comparable expression of pluripotency markers even at a late passage of 20, this observation showed that the bio-assembled matrices 66 DxSDOC and DxSDOCDOC were able to increase the population doublings and yet maintain the pluripotency of hESCs, in comparison to the controls 3.5.4 Pluripotency markers levels by immunofluorescence To confirm the morphological observations in section 3.5.2 and to confirm the flow cytometry results in section 3.5.3, adherent colonies of hESCs cultured on the three matrices were labeled using antibodies against the pluripotency markers of hESCs, Oct-4, SSEA-4, SSEA-3, TRA-1-81 and TRA-1-60 As observed earlier, hESCs cultured on Matrigel, DxSDOC and DxSDOCDOC for passages grew in colonies that exhibited pluripotent morphological characteristics, and these colonies were composed of cells that were positive for all five pluripotency markers (Figure 5E-G) At later passages (passage +20) there were fractions of hESCs on Matrigel and DxSDOC that were not positive for one or more of the markers (arrows) (Figure 5H-J) There were also fractions of hESCs on DxSDOCDOC that were not positive for the pluripotency marker TRA-1-60 (Figure 5I), but as mentioned earlier, these cells were not common Hence, the immunofluorescence studies of the adherent cultures corroborate with the earlier observations 67 3.5.5 In vivo induced differentiation hESCs that were cultured for 18 passages on DxSDOC, DxSDOCDOC and control hESCs on Matrigel were harvested by collagenase IV and implanted into the right hind limb of a SCID mouse The left hind limb was untouched and designated as the control limb The SCID mice were incubated for weeks, after which teratomas from hESCs on DxSDOC and DxSDOCDOC were easily palpable The teratomas formed from hESCs cultured on DxSDOC and DxSDOCDOC matrices were harvested for formaldehyde fixation, microsectioning and H&E staining Differentiated structures were observed in both teratomas, with identifiable neural rosettes (NR), mucous secreting epithelium (MSE), cartilogenous structures (C) and osseous structures (B) (Figure 5K) These observations indicate that after 18 passages on DxSDOC or DxSDOCDOC, these hESCs were still able to differentiate into complex differentiated structures, demonstrating their retention of differentiation capability after prolonged culture hESCs that were cultured on Matrigel did not form any obvious teratomas, and despite an extension of incubation time by another two weeks, no teratomas were palpable A repeat implantation was made using frozen hESCs that were cultured on Matrigel These hESCs were allowed to recover from thawing for passages before the implantation was made, but remained unable to form teratomas It is likely that the control hESCs had lost their differentiation capacity by the 18th passage Teratoma assays were previously proven to be 68 positive for H9 hESCs cultured in this particular way for only passages, as stated by mTeSR-1’s manufacturers, and have not yet been proven positive for more extensive periods However, hESCs cultured on Matrigel using TeSR-1, the original animal-free culture medium that led to the development of mTeSR-1, were able to form teratomas after 20 passages [23] Thus it is possible that the use of animal components such as BSA to replace human serum has led to the failure of this culture medium to maintain differentiation capacity of hESCs when cultured on Matrigel In contrast, hESCs cultured on DxSDOC and DxSDOCDOC continued to retain their teratoma formation capacity, indicating that the bio-assembled human matrices are providing hESCs with the necessary environmental cues, which are lacking in the mTeSR-1 and Matrigel culture systems 3.5.6 Karyotype hESC cultured on the three matrices were tested karyotypically after 18 passages Of the three samples, hESCs cultured on DxSDOCDOC matrix showed normal metaphases on all 20 metaphases tested (Figure 5L) 18 of the 20 metaphases of hESCs cultured on DxSDOC matrix showed normal karyotypes, but metaphases showed non-clonal abnormalities 19 out of 20 metaphases of hESCs cultured on Matrigel for 18 passages showed normal karyotype, but metaphase showed trisomy in chromosome Because only or out of 20 metaphase showed the chromosomal abnormality, the cells were considered as normal, and the abnormality may have been due to technical artifacts or random mitotic errors [71] 69 3.5.7 DNA Methylation hESCs cultured on Matrigel, DxSDOC and DxSDOCDOC were analyzed using the Illumina Methylation Assay – Infinium II platform to profile DNA methylation states of the genes on a genome-wide basis The assay measured the methylation levels of 27,578 CpG dinucleotides of 14,495 genes DNA methylation plays an important part in epigenetic regulation In DNA methylation, methyl groups are added to DNA, typically at CpG sites, and highly methylated areas of the genome tend to be less transcriptionally active It has been shown that changes to the methylation pattern and levels can contribute to cancer and other various developmental diseases [72] The methylation states of hESCs on Matrigel versus the respective methylation states of the same CpG of hESCs on DxSDOC was plotted (Figure 5M) The same was repeated for hESCs on Matrigel versus hESCs on DxSDOCDOC and for hESCs on DxSDOC versus hESCs on DxSDOCDOC The coefficient of determinant R2, which gives an indication of the goodness of fit between the two samples, was calculated (Figure 5M) An R2 value of 1.0 indicates a perfect fit, while an R2 value of more than 0.95 indicates similarity In all three comparisons, the R2 values were found to be more than 0.95, therefore hESCs under all the three matrix conditions were similar to each other, with only slight differences Interestingly, the R2 value for hESCs on Matrigel versus hESCs on DxSDOC (0.9707) and the R2 value for hESC on DxSDOC versus hESC on DxSDOCDOC (0.9682) was lower than the R2 value of hESCs on Matrigel versus hESCs on DxSDOCDOC (0.9808), 70 indicating that hESCs on DxSDOC was the more dissimilar to the hESCs on the other two matrix conditions, and hESCs on DxSDOCDOC was more like hESCs on Matrigel This epigenetic analysis shows that on the epigenetic level, there are no major differences between hESCs on Matrigel, DxSDOC and DxSDOCDOC This observation is in contrast to the differences in cellular behaviour, such as morphology, expression of pluripotency markers, and retention of differentiation capacity This contradiction could be due to the fact that the assay does not completely encompass all the 17,052 genes identified to date Moreover, with only about CpGs to represent each gene, and with several CpGs found in each gene, the assay does not cover all the CpGs in the entire genome Hence, the epigenetic analysis at this point is still limited and not complete, and as a result, it might not have captured the true epigenetic differences between the samples 3.5.8 DxSDOCDOC matrices were able to maintain pluripotent hESCs Based on population doubling observations, morphology, pluripotency marker expression, in vivo differentiation studies and karyotype results, it was concluded 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, and the hESCs on DxSDOCDOC had fewer areas of differentiation compared to the control In vivo differentiation studies showed that hESCs 71 cultured on DxSDOCDOC matrices retained their differentiation capacity and yet maintained their karyotypic integrity despite prolonged culture periods 3.6 Matrices that maintain hESC pluripotency (Repeat) 3.6.1 Repeat propagation trial A repeat of the above-mentioned propagation trial was conducted, with hESCs cultured on DxSDOCDOC matrices, and control hESCs cultured on Matrigel, for up to 13 passages Collagenase IV was used for enzymatic passaging, and defined culture medium, mTeSR-1 was used to maintain the hESCs The purpose of this trial was to confirm the earlier observations that DxSDOCDOC matrices were able to maintain hESCs in a pluripotent state compared to Matrigel with increased population doublings 3.6.2 Population doublings Similar to the earlier propagation trial, representative wells were sacrificed for cell counting at each passage, to obtain the population doublings, which was plotted (Figure 6A) In the first eight passages, population doublings were similar in both hESCs cultured on Matrigel and DxSDOCDOC matrices The dip in population doublings in the 2nd passage could be due to the adaptation of the hESCs to the matrices From the 9th passage onwards, the population doubling rate of hESCs cultured on DxSDOCDOC began to surpass the control hESCs By the 13th passage, hESCs cultured on DxSDOCDOC matrices showed a 42.5% increase in population doublings in comparison to the control Hence, it was confirmed that DxSDOCDOC matrix enables the higher proliferation rate of hESCs compared to control hESCs on Matrigel 72 73 ... gastrulation in an anterior-posterior pattern Germ cells continue to maintain the expression of Oct -4 until the initiation of sexual differentiation [10] The under-expression of Oct -4 in ES cells... their differentiation into trophoectoderm lineages [10], indicating the crucial role that Oct -4 plays in maintaining the pluripotent state of ES cells As Oct -4 is a transcription factor, naturally... is found within the nuclei 64 of hESCs, and so, in order to obtain successful Oct -4 immunostaining, the cells have to be first permeabilized using 0.2% Triton-X 100 - this extra processing step