Bioengineered Human Pyloric Sphincters Using Autologous Smooth Muscle and Neural Progenitor Cells

Abstract

Gastroparesis leads to inadequate emptying of the stomach resulting in severe negative health impacts. Appropriate long-term treatments for these diseases may require pyloric sphincter tissue replacements that possess functional smooth muscle cell (SMC) and neural components. This study aims to bioengineer, for the first time, innervated human pylorus constructs utilizing autologous human pyloric sphincter SMCs and human neural progenitor cells (NPCs). Autologous SMCs and NPCs were cocultured in dual-layered hydrogels and formed concentrically aligned pylorus constructs. Innervated autologous human pylorus constructs were characterized through biochemical and physiologic assays to assess the phenotype and functionality of SMCs and neurons. SMCs within bioengineered human pylorus constructs displayed a tonic contractile phenotype and maintained circumferential alignment. Neural differentiation within bioengineered constructs was verified by positive expression of bIII-tubulin, neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT). Autologous bioengineered innervated human pylorus constructs generated a robust spontaneous basal tone and contracted in response to potassium chloride (KCl). Contraction in response to exogenous neurotransmitter acetylcholine (ACh), relaxation in response to vasoactive intestinal peptide (VIP), and electrical field stimulation (EFS) were also observed. Neural network integrity was demonstrated by inhibition of EFS-induced relaxation in the presence of a neurotoxin or nNOS inhibitors. Partial inhibition of ACh-induced contraction and VIP-induced relaxation following neurotoxin treatment was observed. These studies provide a proof of concept for bioengineering functional innervated autologous human pyloric sphincter constructs that generate a robust basal tone and contain circumferentially aligned SMCs, which display a tonic contractile phenotype and functional differentiated neurons. These autologous constructs have the potential to be used as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders.

Biomechanical properties of an implanted engineered tubular gut-sphincter complex

“…In the present study, 10 cm duodenal tissues were used to isolate smooth muscle cells and neural progenitor cells. Our previous proliferation assays have demonstrated the potential of isolating these cells from human tissues and growing them to a sufficient number in order to engineer the constructs (Rego et al, ). Currently, the gut is being used as source of cells.…”

“…The present study provided a bioengineered complex that has both a gut segment and a sphincteric tissue with appropriate neuromuscular function. The implanted engineered sphincteric tissues maintained their ability to generate spontaneous basal tone when compared with engineered sphincters pre‐implantation (Rego et al, ). The contractile phenotype of the sphincteric smooth muscle was maintained.…”

“…Human innervated pyloric smooth muscle sphincters were engineered using a previously described technique (Rego et al, ). A suspension of 200 000 enteric neural progenitor cells was suspended in a gel mix similar to the one described above.…”

“…The tissues were kept in a warm tissue bath throughout the experiments. Establishment of basal tone by the sphincters, electromechanical coupling integrity of the smooth muscle of both the sphincters and the gut tissues, and the functionality of the differentiated neurons were evaluated as described previously (Rego et al, ; Zakhem et al, ).…”

Cigarette smoke enhances initiation and progression of lung cancer by mutating Notch1/2 and dysregulating downstream signaling molecules

“…Total RNA was isolated from cultured cells or tissues using the TRIzol® Reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. qPCR using SYBR green I was carried out to compare the relative expression of specific mRNAs, as previously described [ 23 ]. GAPDH was used as an endogenous control.…”