Figure 3.

Pro-inflammatory IL-1β and TNFα cytokine expression in regenerating bladder tissue.

(A) Detrimental to proper tissue regeneration is the presence of confounding pro-inflammatory cytokines including IL-1β and TNFα. IL-1β (red) and TNFα (red) levels were highly abundant in CD34⁺ HSPC and uPOC grafts at the 4 week time-point. TNFα levels remained elevated 10 weeks post-surgery while IL-1β decreased only moderately in the unseeded samples. IL-1β and TNFα levels in POC/MSC and POC/MSC + CD34⁺ HSPC grafts were approximately half the value found in POC/CD34⁺ HSPC and uPOC grafts at the 4 week time-point. There was a general decrease in pro-inflammatory cytokine expression at the 10 week time-point for all samples; however, TNFα expression was relatively elevated with the POC/CD34⁺ HSPC seeded construct. n = 10 images/animal. Blue = DAPI. Magnification, 400x. Scale bar, 50 µm.

(B) Quantification of pro-inflammatory cytokine expression, shown as percentage of cells staining positive for TNFα (left bars) and IL-1β (right bars) at 4 weeks (left panel) and 10 weeks (right panel). The cytokine expression profile for uPOC graft tissue was predominantly pro-inflammatory, with mean percentages of 41.5 ±2.2% TNFα⁺ and 33.3 ±1.2% IL-1β⁺ at 4 weeks and 34.3 ±3.1% TNFα⁺ and 27.0 ±2.3% IL-1β⁺ at 10 weeks. Significant reduction in both TNFα and IL-1β expression was observed for grafts seeded with MSCs or MSCs/CD34⁺ HSPCs (4W: POC/MSC 20.5 ±0.8% TNFα⁺ and 13.9 ±0.9% IL-1β⁺, POC/MSC + CD34⁺ HSPCs 22.5 ±1.3% TNFα⁺ and 14.2 ±3.7% IL-1β⁺; 10W: POC/MSC 16.0 ±1.6% TNFα⁺ and 7.0 ±1.2% IL-1β⁺, POC/MSC + CD34⁺ HSPCs 14.4 ±2.2% TNFα⁺ and 11.5 ±1.1% IL-1β⁺); grafts seeded only with CD34⁺ HSPCs did not show this effect (4W: 39.2 ±2.6% TNFα⁺ and 30.5 ±1.3% IL-1β⁺; 10W: 26.6 ±1.5% TNFα⁺ and 17.8 ±4.6% IL-1β⁺). Data shown as means ±SE. Significance shown for comparison of cell-seeded groups to uPOC; *P <0.05, **P <0.01, ***P ≤0.001, ****P <0.0001.