n vitro bioassembled human extracellular matrix and its application in human embryonic stem cell cultivation 3

3.4 Matrices that not maintain hESC pluripotency 3.4.1 Absolute cell numbers hESCs were cultured onto AcADOC, DxSNP40/DNase or FcNP40/DNase matrices by enzymatic passaging with collagenase IV and maintained in defined culture medium, mTeSR-1. Subculture was done every 5-7 days and at each passage, sacrificial replicates of the resultant cultures were harvested by TrypLE treatment and counted. The resultant absolute cell numbers were plotted to observe the proliferation rates of the hESCs (Figure 4A). hESCs on AcADOC matrices proliferated only after the first passage, with an initial lag phase during the first passage. hESCs did not adhere, and hence did not proliferate on either DxSNP40/DNase matrices nor FcNP40/DNase matrices. 3.4.2 Morphology of hESCs Although hESCs proliferated on AcADOC matrices, they did not have morphology typical of pluripotent hESCs (Figure 4B). The majority of hESCs grew in clusters resembling embryoid bodies, which were optically dense, with outgrowths of spontaneously differentiated cells that were large and flattened. Hence, although AcADOC matrices allowed for the adherence of hESCs, the cells that were attached to the matrix were not able to remain pluripotent. Instead these hESCs began to form embryoid bodies that will develop into differentiated structures, and they also spontaneously formed differentiated cells that resemble fibroblasts. Hence, hESCs were not maintained on this matrix for more than passages. 51 hESCs did not adhere to DxSNP40/DNase matrices, and settled on the bottom of the culture well as clusters of cells that were easily moved by mechanical disturbances to the plate (Figure 4B). Likewise, hESCs did not adhere to FcNP40/DNase matrices either, and also settled on the bottom of the culture well in clusters that were easily moved (Figure 4B). Taken together, DxSNP40/DNase and FcNP40/DNase matrices, despite the relatively high amounts of proteins within and their complexity (refer to section 3.3), not allow the adherence of hESCs and also not maintain hESCs in a pluripotent state. This observation is surprising, since these matrices contains more Fn (by immunofluoresence comparison, section 3.3.2), it would be expected that the 52 increased presence of Fn would aid in increasing integrin binding and hence cell binding. This contradiction could possibly be due to the increased presence of DCN and BGN in the DxSNP40/DNase and FcNP40/DNase matrices interfering with TGFB signaling for the hESCs, since DCN and BGN both bind active TGFB, and could lead to a decrease in the amount of active TGFB available for the hESCs. 3.5 Matrices that maintain hESC pluripotency 3.5.1 Population doublings of hESCs maintained on DxSDOC and DxSDOCDOC matrices hESCs were subcultured onto either DxSDOC or DxSDOCDOC matrices using collagenase IV for enzymatic passaging. Control hESCs were subcultured onto Matrigel-coated wells using the same procedure. All hESCs were maintained using commercially available defined culture medium, mTeSR-1, which has been optimized for Matrigel, the standard feeder-free culture system. Subculture was done every 5-7 days depending on the confluency of the hESCs. In this way, hESCs were maintained for up to 20 passages. At each passage, representative cultures of hESCs from each matrix condition were sacrificed for cell counting using a haemacytometer. Population doublings from each passage were calculated and the data plotted for comparison (Figure 5A). 53 54 55 56 57 . immunofluoresence comparison, section 3. 3.2), it would be expected that the 53 increased presence of Fn would aid in increasing integrin binding and hence cell binding. This contradiction could possibly. due to the increased presence of DCN and BGN in the DxSNP40/DNase and FcNP40/DNase matrices interfering with TGFB signaling for the hESCs, since DCN and BGN both bind active TGFB, and could. section 3. 3), do not allow the adherence of hESCs and also do not maintain hESCs in a pluripotent state. This observation is surprising, since these matrices contains more Fn (by immunofluoresence