2. COMPONENTE URBANO
2.3 ACCIONES URBANISTICAS 1 Suelo de expansión urbana
Having demonstrated that NHNS contain p75 positive cells and putative ENSC, the next stage in the development of a stem cell transplantation technique was to show that these cells could be transplanted into aganglionic distal hindgut and that any new ENS neurons found in the transplant were generated within the neurosphere, rather than being pre-‐existing neurons incorporated into the neurosphere initially.
For transplantation, specimens of distal hindgut were harvested from e11.5 CD-‐1 mouse embryos as shown in Figure 2.7 and cultured on a semi-‐permeable membrane (Millicell, Millipore UK Ltd, Watford, UK). This was done by placing 3ml of warmed tissue culture medium in a 50mm tissue culture dish and placing the culture plate insert within it. The hindgut was dissected free in sterile PBS in the same way and the caecum was removed. The hindgut was then removed using a sterile 100µm pipette and placed on the culture plate insert. A 40µl aliquot of tissue culture medium (containing DMEM-‐high, see Appendix 1) was then placed on the hindgut sample. Tissue culture medium was changed every 3 days and the aliquot of medium on top of the hindgut sample was also changed by carefully removing it with a sterile 40µl pipette and replacing the medium.
Single human neurospheres that had been cultured for a minimum of 28 days were apposed to the proximal end of the isolated hindgut using a 100µl sterile pipette, as shown in Figure 2.7. As controls, gut explants were cultured without neurospheres and explants cultured with the caecum still attached, as described in Figure 2.7A. Specimens were fixed and processed for immunohistochemistry after 8 days in culture.
Tissue culture specimens were fixed by removing the tissue culture medium and gently washing three times in PBS. All specimens were fixed in 4% (w/v) paraformaldehyde in PBS for 1 h at room temperature and then rinsed 3 times with PBS. They were then immersed in 20% (w/v) sucrose for 1 h prior to embedding in 7.5% (w/v) gelatine in 15% (w/v) sucrose. Embedded specimens were frozen at -‐80oC in isopentane and 7μm serial sections cut using a cryostat.
Immunohistochemistry was performed for PGP9.5 and S100 as described previously. To identify human cells in the embryonic mouse gut, mouse anti-‐ human ribonucleoprotein antibody (HRNP, Chemicon) 1:50 was used with Texas Red-‐conjugated goat anti-‐mouse IgG 1:200 (Abcam Ltd, Cambridge, UK). To assess whether cells were generated within the neurosphere, human neurospheres were incubated with 10 mM BrdU for 18 h before transplantation. Incorporated BrdU was detected by a 2 h incubation with mouse anti-‐BrdU (Dako) diluted 1:25 followed by rinsing in PBS and a further 2 h incubation with Texas Red-‐conjugated goat anti-‐mouse IgG (Abcam) diluted 1:200. Dual labelling was performed as before.
2.5.2 Results
2.5.2.1 Growth of NHNS
derived neurospheres, there was no statistically significant difference in size between human and mouse neurospheres when the data for all time points was aggregated (two-‐tailed t=test, p=0.065) or when individual time points were considered separately (Kruskal-‐Wallis 1-‐way ANOVA with Dunns post test, p>0.05 for all time points.
Table 2.3: Yield and size of EMNS and NHNS
EMNS NHNS Day Neurosphere number Neurosphere volume (µm3 x103) Neurosphere number Neurosphere volume (µm3 x103) 0 0 0 0 0 7 20 72 +/- 19 18 39 +/- 9 14 53 702 +/- 146 21 199 +/- 74 21 66 671 +/- 80 48 536 +/- 120 28 70 1190 +/- 364 52 783 +/- 100
Figure 2.8 Growth of human neurospheres
A
B
C
D
E
Figure 2.8 Growth of NHNS
The growth of human neurospheres. Phase contrast micrographs of: (A) Single cell suspension of human cells at D0. (B) Neurospheres at D7 of culture. (C) Neurosphere at D14 of culture. Note neurite outgrowth from the neurosphere (arrowhead). (D) Neurospheres at D21 of culture. Although neuro- sphere size had increased, there also many smaller cell clusters and individual cells present. (E) Neuro- spheres at D28 of culture. Neurosphere size has increased to steady state and there are fewer indi- vidual cells surviving within the culture. (F) Scatter plot (median and range) showing size of NHNS neuro- spheres in comparison with EMNS at the same time- point. Human neurospheres tended to be smaller than their embryonic mouse counterparts, but this
2.5.2.2 Cell marker expression of single cell suspension and NHNS
Immunofluorescence of the single cell suspension derived from digesting the circular and longitudinal muscle layers is shown in Figure 2.9. This demonstrates that within the digested sample there are cell positive for human nuclei, p75, PGP9.5 and S100. The majority of the cells however are positive for SMA, as would be expected. There were few cells positive for p75 and relatively more positive for PGP9.5 and S100B. This raises the possibility that there may be p75 positive ENSC present within the sample.
After 30 days in culture, Immunohistochemistry confirmed the presence of the neural crest cell marker p75 (Figure 2.10A) as well as glial and neuronal cell markers within mature neurospheres (Figure 2.10B and C). BrdU incorporation demonstrated proliferating cells that tended to be uniformly distributed throughout the neurospheres, in contrast to mouse neurospheres (Figure 2.10D). Dual labelling with PGP9.5 and BrdU showed the presence of cells positive for both BrdU and PGP, indicating that new neuronal cells had been generated from actively dividing precursors within the neurosphere (Figure 2.10D inset, arrowhead). Immunofluorescent labelling for neurotransmitters commonly found in the ENS showed the presence of NOS (Figure 2.10E), ChAT (Figure 2.10F), VIP (Figure 2.10G), SP (Figure 2.10H) and CGRP (Figure 2.10I) within the neurospheres. No TH immunoreactivity was seen and the proportion of cells staining for NOS and ChAT was greater than that for the other neurotransmitters.
There appeared to be a positive reaction to c-‐Kit in the primary neurospheres (Figure 2.11A and B) but a similar pattern of staining was seen on the negative control (Figure 2.11C). c-‐kit and PGP9.5 dual labelling of transplanted NHNS (for transplantation and culture method, see section2.5.1.4) demonstrated that defined c-‐kit staining appeared to appear within the neurosphere at the peripheries (Figure 2.11D). It therefore seems likely that there are some c-‐Kit reactive cells within the neurosphere but the results presented here cannot
2.5.2.3 Demonstration of clonality of NHNS
Despite multiple attempts at demonstrating clonality of NHNS in a similar way to that described for mouse neurospheres(Almond et al., 2007), no neurospheres formed under these conditions. However, when NHNS were cultured at clonal density, then new neurospheres began to form (Figure 2.12A and B). These neurospheres were very low in number (typically 1 or 2 per 90mm Petri dish) but displayed the typical morphology of NHNS and produced neurite outgrowth (Figure 2.12C and D).
2.5.2.4 NHNS can be transplanted into embryonic mouse distal hindgut