Human intestinal smooth muscle progenitors generate orientated muscle layers for intestinal regeneration

Abstract

Background/Objectives: Several severe gastrointestinal disorders involve functional and/or structural disruption of the neuromuscular compartment. Therefore, cell therapy could be beneficial for the treatment of such diseases. The aim of this project was to identify a pool of intestinal smooth muscle progenitor cells (SMPCs) and determine their ability to generate functional smooth muscle for regenerative medicine purposes. Methods: SMPCs were isolated from foetal and paediatric intestine and characterized by immunostaining, flow cytometry, SNP arrays and RNA-seq analyses. In vitro, their ability to produce smooth muscle cells was examined via activation/inhibition of the TGFß pathway, by immunostaining, WB, qPCR, RNA-seq and calcium imaging. Finally, SMPCs were used to regenerate the muscular compartment of decellularised foetal human intestine in ex vivo culture, and analysed by immunohistochemistry. Results: Cultured SMPCs demonstrated high proliferative capacity (66.4% ± 9.1 Ki67+), maintenance of genomic stability, a mesenchymal origin (98.6% ± 1.3 CD90+) and features in common with pericytes (88.6% ± 10.6 NG2+, 98.0% ± 1.7 CD146+ and 51.6% ± 39.8 PDGFRß+). Immunostaining, RNA-seq, qPCR and WB data targeting the expression of SM22, Calponin and MYHC confirmed smooth muscle differentiation of SMPCs that varied depending on the levels of TGFß activation. Upon TGFß inhibition, cells displayed some level of differentiation due to an activation of the IGF-pathway as evidenced by RNA-seq analyses. Differentiated SMPCs also exhibited calcium transients in response to carbachol. SMPCs, when seeded onto decellularised scaffolds, distributed in two orthogonal layers of SM22+/Calponin+ cells, matching the anatomical structure of healthy intestinal muscle layers. Conclusions: Our results confirm that SMPCs generate mature smooth muscle cells in vitro, mostly via activation of the TGFß pathway. Ex vivo, these cells have the capacity to self-organize in two orthogonally aligned smooth muscle layers. Future experiments are investigating the functionality of these layers via organ bath contractility, plus the involvement of the IGF-pathway. Thus, SMPCs show good potential for the treatment for enteric smooth muscle disorders.L6320787052020-07-29 <br />