Atrapamiento celular

MSCs, CCs and CC-RA cells were evaluated via RT-qPCR for the recommended ISCT CD markers for the characterisation of Wharton’s Jelly-derived MSCs. These included CD73, CD90, CD105, CD34 and CD45. The samples used to perform this characterisation were cells cultured and differentiated from Cords 5, 6 and 13. The average Cq of each CD marker is shown in Table 3.2 and the raw data is available in Appendix III. CD90 and CD105 were detected during the early cycles of the RT-qPCR for all the three cell types. In MSCs, CD73 was detected during the final cycles of the RT-qPCR and both CD 34 and CD45 were beyond the 40th cycle and so not detected by the system, while in CCs and CC-RAs, expression of CD73, CD34 and CD45 registered values very close to the 40th cycle.

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Cell Type Sample CD73 CD90 CD105 CD34 CD45

MSC Cord 5 31.65 24.43 26.11 39.85 40.00 Cord 6 33.80 27.62 28.63 40.00 40.00 Cord 13 32.82 26.97 31.36 40.00 40.00 CC Cord 5 38.95 22.07 24.00 31.82 40.00 Cord 6 32.98 20.27 23.67 32.87 34.62 Cord 13 40.00 24.60 25.64 33.76 40.00 CC-RA Cord 5 35.69 27.01 26.19 38.59 40.00 Cord 6 40.00 29.43 29.69 40.00 40.00 Cord 13 40.00 27.43 26.20 35.83 40.00

Table3.2: Characterisation by CD Markers

The RNA extracted from the cultured MSCs, CC and CC-RA cells was analysed by RT-qPCR for the expression of a panel of CD markers. The table shows the average cycling quantification (Cq) values detected during the analysis. The Cq is detected when the sample reaction surpasses that of pre-assigned threshold. The maximum Cq value reading is 40.00. When a sample Cq reaches this value, the expression which is being sought is said to be absent (value is inversely proportion to the expression). The average Cq for the expression of CD73 is seen to decrease in both CC and CC-RA cells. CD34 and CD45 expression are relatively close to the 40.00 limit for all the three cell types. On the other hand, CD 90 and CD105 were detected during much early cycles of the RT-qPCR run.

CD – Cluster of differentiation, MSC – Mesenchymal Stem Cells, CC – Conditioned Cells, CC-RA – Retinoic Acid treated CC, Cq – cycle quantification, RT-qPCR – Real Time quantification polymerase chain reaction

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Figures 3.7 and 3.8 show the average fold change of these CD markers expressed by CC and CC-RA cells when these are compared to MSCs and CCs respectively. Graphs are plotted with the fold change on the y-axis and the CD of interest on the x-axis. The baseline (1.00) for comparing MSCs and CCs were the MSCs. When comparing CC and CC- RAs the baseline (starting point) was the CD expression in CCs. This means that in the case of Figure 3.7, the bars are showing whether the fold changed in CCs increased or decreased in respect to CD expression of the MSCs. In Figure 3.7A, CD73 in CCs was not detected and therefore the fold change decreased from 1.00 to 0.00. CD90 was also decreased by a fold change of 0.20, while as per Figure 3.7B, CD105 had a fold change increase of 1964.57. It was not possible to calculate the fold change of CD34 and CD45 because these where not expressed.

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Figure 3.7: Fold change in expression from Mesencyma Conditioned Cells

The figure illustrates the average between the CD markers detecte during RT-qPCR analysis. The s calculated fold change was the M considered to be the baseline (1.00 difference of less than 1.00 repr expression, while a fold change gre an increase in marker expression. markers expressed by MSCs and C for CD73 (A) was 0.00, CD90 (A) difference and CD105 (B) had a fo CD34 and CD45 were not expresse represents the number of technica represent the RQmin and RQmax

significance in the fold difference b (P<0.05).

Legend: MSCs – Mesenchymal Conditioned Cells, CD - Cluster o qPCR – Real Time quantificatio reaction

in CD marker RNA ymal Stem Cells to

ge fold change difference tected in MSCs and CCs e starting point of this e MSCs, and which was 1.00). Thus a fold-change represents a decrease in greater than 1.00 shows ion. When comparing CD d CCs, the fold difference (A) resulted in a 0.20 fold a fold difference of 5.76. ssed by MSCs and CCs. N nical replicates. Error bars

max. The * denotes a

ce between the cell types al Stem Cells, CCs - er of differentiation, RT- cation polymerase chain

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Similarly, in Figure 3.8, graphs are representing the Fold change in expression in CC-RA with respect to CCs. In Figure 3.9, both CD90 and CD105 have a fold change decrease of 4.2 and 0.79 respectively. It was not possible to calculate the fold change of CD34 and CD45 in the three cell types because these where not expressed. The same hold for CD73 in CC and CC-RA cells.

Figure 3.8: Fold change in Neural Marker RNA expression from Conditioned Cells to Retinoic Acid-treated Conditioned Cells.

The figure illustrates the average fold change difference between the CD markers detected in CCs and CC-RA cells during RT-qPCR analysis. The starting point of this calculated fold change was the CCs, which was considered to be the baseline (1.00). Thus, a fold-change difference of less than 1.00 represents a decrease in expression, concurrently a fold change greater than 1.00 shows an increase in marker expression. When comparing CD markers expressed by the CCs and CC-RA cells, the fold difference for CD90 was 0.42 while CD105 had a fold difference of 0.79. CD73, CD34 and CD45 were not expressed by CCs and CC-RA. N represents the number of technical replicates. Error bars represent the RQmin and RQmax. The * denotes a significance in the fold difference

between the cell types (P<0.05).

Legend: CC - Conditioned Cells, CC-RA – Retinoic Acid treated CC, CD- Cluster of differentiation, RT-qPCR – Real Time quantification polymerase chain reaction

-3.00 -2.50 -2.00 -1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 CD90 CD105 F o ld C h an g e ( R Q ) CD Markers N = 9

*

105