Enteric neural differentiation in innervated, physiologically functional, smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells

The enteric nervous system (ENS) controls gastrointestinal (GI) functions, including motility and digestion, which are impaired in ENS disorders. Differentiation of enteric neurons is mediated by factors released by the gut mesenchyme, including smooth muscle cells (SMCs). SMC-derived factors involved in adult enteric neural progenitor cells (NPCs) differentiation remain elusive. Furthermore, physiologically relevant in vitro models to investigate the innervations of various regions of the gut, such as the pylorus and lower oesophageal sphincter (LES), are not available. Here, neural differentiation in bioengineered innervated circular constructs composed of SMCs isolated from the internal anal sphincter (IAS), pylorus, LES and colon of rabbits was investigated. Additionally, SMC-derived factors that induce neural differentiation were identified to optimize bioengineered construct innervations. Sphincteric and non-sphincteric bioengineered constructs aligned circumferentially and SMCs maintained contractile phenotypes. Sphincteric constructs generated spontaneous basal tones. Higher levels of excitatory and inhibitory motor neuron differentiation and secretion of bone morphogenic protein 2 (BMP2) were observed in bioengineered, innervated, sphincteric constructs compared to non-sphincteric constructs. The addition of BMP2 to non-sphincteric colonic SMC constructs increased nitrergic innervations, and inhibition of BMP2 with noggin in sphincteric constructs decreased functional relaxation. These studies provide: (a) the first bioengineered innervated pylorus and LES constructs; (b) physiologically relevant models to investigate SMCs and adult NPCs interactions; and (c) evidence of the region-specific effects of SMCs on neural differentiation mediated by BMP2. Furthermore, this study paves the way for the development of innervated bioengineered GI tissue constructs tailored to specific disorders and locations within the gut.