Initial analysis was performed on unstained frozen sections made from chimeras containing Vybrant-labelled endometrial cells fixed at 2 hours (Day 0), 3 days and 7 days respectively (Figure 21), three of the explants generated from each time point (Day 0, Day 3, Day 7) were sectioned in preparation for analysis with two immunofluorescence runs performed per section. The results presented are representative samples. It can be observed that the distribution of the Vybrant labelling alters as time in culture increases. At Day 0, after only 2 hours of culture, cells displaying bright green fluorescence were located at the periphery of the section, where they could be seen at the base of the tissue pellet, attached to the membrane filter, and also on the pellet surface. By Day 3 the cells appeared to have permeated the tissue such that the full thickness of the section contained green fluorescent cells. At this time point, the fluorescent population comprised cells that displayed bright fluorescence, and cells that displayed weak fluorescence. By Day 7, fluorescence was still present throughout the full thickness of the section, but in comparison to Day 3, it appeared that there were fewer bright green fluorescent cells. Furthermore, it was difficult to tell if the weak fluorescent signal observed throughout the section was from human cells that had depleted their level of fluorescence due to cell division, or whether it was simply background autofluorescence.
96 Figure 21: Distribution of Vybrant Staining Changes With Increased Time in Culture
Fig21. Frozen sections of chimeric explants containing endometrial cells labelled with Vybrant prior to culture were analysed using fluorescence and phase microscopy. Sections demonstrate the distribution of Vybrant fluorescence as at the indicated time points. Arrows mark the membrane on which the chimeras were cultured. Scale bar 100μm.
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4.1.2 Minimal Co-Localisation is Detected Between Vybrant and the Human-Specific Anti- Cytoplasmic Antibody at Day 7 of Culture
One of the observations from analysis of the Vybrant sections of endometrial epithelial- neonatal kidney chimeras was that fewer positive staining areas could be identified on Day 7 than on Day 0. There are a number of possible explanations for this, the most likely being that either the endometrial cells are losing the Vybrant label due to cell division or the Vybrant signal is decreased because the endometrial cells were dying. To distinguish between these two possibilities, frozen sections were immunostained with human specific anti-cytoplasmic antibody at Day 0, Day 3 and Day 7 with the aim of identifying cells co- staining for this antibody and Vybrant (Figure 22). As expected, at Day 0 most cells displayed both green and red fluorescence, indicating that all Vybrant-labelled cells expressed the human-specific cytoplasmic protein. By Day 3, many of the cells expressing the anti-cytoplasmic protein appeared to display weak green fluorescence. By Day 7, there were noticeably fewer cells expressing the anti-cytoplasmic protein and these tended to have little or no green fluorescence. These areas did not often co-localise with red fluorescence, suggesting that was most likely due to background autofluorescence. The observation that many cells expressing the human-specific cytoplasmic protein within the Day 3 chimeras displayed weak green fluorescence suggested that some of the endometrial cells had proliferated between Day 0 and Day 3. The observation that there were noticeably fewer cells expressing the human-specific cytoplasmic protein within the Day 7 chimera suggested that many endometrial cells died between Day 3 and Day 7. However, the fact that the few human cells present at Day 7 displayed lower levels of green florescence than those at Day 3, suggested that surviving human cells were likely to have gone through at least one cell division between the two time points.
98 Figure 22: Anti Cytoplasmic Immunofluorescence Staining of Vybrant Labelled Chimeras
Fig22. Frozen sections of chimeric explants containing endometrial cells labelled with Vybrant fluorescence prior to culture were immunostained with anti-cytoplasmic antibody (A-HCP) (Red) and the nuclear stain DAPI (Blue). Co-localisation can be readily observed after 2 hours of culture (Day 0). At Day 3 and Day 7 co-localising cells are less readily identified and areas of staining for either Vybrant or anti-cytoplasmic antibody that do not co-localise are observed. Arrows identify potentially co-staining cells. Biological and technical repeats were conducted with the images shown being representative. Scale bar 50μm.
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4.1.3 ‘Bright’ Vybrant Staining Present at Day 7 Does Not Represent Viable Cells
Another observation from the sections of chimeras containing Vybrant-labelled human endometrial cells was that there were a number of intense green fluorescent spots present in the day 7 sample that did not co-stain for the human-specific cytoplasmic protein. Further analysis under higher power showed that these spots did not contain a nucleus, suggesting that they were not representing viable cells (Figure 23).
Figure 23: Day 3 and Day 7 Vybrant Labelled Chimera Frozen Sections Stained With Human Anti-Cytoplasmic Antibody
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Fig 23. Frozen section from a Day 7 chimera containing Vybrant labelled endometrial cells (Green) was immunostained with anti-cytoplasmic antibody (Red) and the nuclear stain DAPI (Blue). Areas of non co- staining fluorescence may be identified which are not associated will a cell nucleus. It is possible that these areas represent cell fragments. Scale bar 50μm.
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