The appendix as a viable source of neural progenitor cells to functionally innervate bioengineered gastrointestinal smooth muscle tissues

Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of ace-tylcholine to both appendix and SI constructs causedarobust contraction that was decreased by pre-treatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pre-treatment with nitric oxide synthase inhibitor (N_ω-nitro-L-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs,entericNPCsderivedfromtheappendix andSIdifferentiateinasimilarmannerandarecapable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations.