Provision of Optimal Conditions for In vitro Differentiation into Peristaltic Smooth Muscle from a Murine Induced Pluripotent Stem Cell Line

Background/Purpose: The aim of this study was to search for the optimum conditions for differentiating murine induced pluripotent stem cells (iPSCs) into contracting smooth muscle, as a precursor to utilizing in vitro tissue engineering techniques to generate functioning intestinal tract wall for patients with short bowel syndrome. Method: iPSCs were cultured for 6 days in low adherent 96 well-plates (5005,000 cells per well) in a differentiation medium comprising KnockoutTM DMEM supplemented with 10% serum replacement, activin A, L-glutamine, non-essential amino acids, 2-mercaptoethanol, penicillin and streptomycin. After 6 days, the embryoid body that had formed in each well was transferred into a 12 well plate, each well containing 8 embryoid bodies. The influence of various additives on the patterns of differentiation was studied. Results: Outgrowth culture produced differentiated cell clusters, including cardiac-like cells, sheets of smooth muscle with peristaltic-like activity, and mucosal cells, according to the culture conditions. Conclusion: Activin A and serum replacement increase the reproducibility for producing sheets of peristaltic smooth muscle from 10.8% to 53.3%. Further refinement in this technique has the potential to allow patients to “grow” their own small bowel wall in vitro for subsequent transplantation.