FIG. 2.

Biochemical characterization of the bioengineered human pylorus constructs. Bioengineered innervated human pylorus SMC constructs form (A) rings of tissue around (B) 0.7-cm-diameter Sylgard posts (40× magnification). Levels of neural differentiation markers over time were measured in RNA isolated from lysates of bioengineered autologous human pylorus constructs using quantitative polymerase chain reaction (qPCR). (C) Significant increases in transcript levels of βIII-tubulin, neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT) within bioengineered constructs were observed by day 10, and no further increase was observed at day 12, indicating that peak neural gene expression occurred at 10 days of coculture. Protein lysates collected from bioengineered constructs were analyzed for contractile SMC markers and neural differentiation markers by Western blot analysis. **p < 0.01, ***p < 0.001. (D) SMC marker smoothelin and pan-neuronal marker βIII-tubulin were expressed in bioengineered innervated human pylorus constructs demonstrating the presence of contractile SMCs and differentiated neurons. Neural subset markers, nNOS and ChAT, were observed after 10 days of in vitro coculture of SMCs and NPCs within bioengineered constructs. Smoothelin expression was detected in SMCs only cultures, however, no expression of neural differentiation markers was observed in SMC monocultures. NPCs cultured alone did not express smoothelin, nNOS, or ChAT, but did express low levels of βIII-tubulin (Fig. 2D). n.s., non significant.