An in vitro functional assay to predict in vivo muscle stem cell engraftment outcomes

Bella Xu (1, 2), Sadegh Davoudi (1), Majid Ebrahimi (1), Mohsen A. Bakooshli (1), Ziad Al Tanoury (3)

(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto

(2) Department of Chemical Engineering and Applied Chemistry, University of Toronto

(3) Harvard Medical School, Harvard University

Functional validation of promising stem cell endogenous repair candidates constitutes a major translational bottleneck. As a consequence, candidate selection is biased to minimize risk and prioritizes hits most likely to produce dramatic in vivo phenotypes. We have developed an in vitro stem cell mediated skeletal muscle repair platform that predicts in vivo transplantation outcomes and has the potential for parallel stratification of all candidates. We generated thin sheets of mature human skeletal muscle fibers by infiltrating a thin cellulose scaffold with primary patient-derived myoblasts encapsulated in an extracellular matrix hydrogel. Sheets were engrafted with freshly isolated muscle stem cells, injured by exposure to myotoxin, to induce a regenerative microenvironment, and muscle repair was assessed over 10 days of repair. The spatiotemporal dynamics of the in vitro repair process matched those observed in vivo, but only when both stem cells and injury were present. Repair outcomes were modulated using 9 drugs, 2 of which were previously reported and 7 of which were newly identified from a 476-compound kinase inhibitor screen on muscle stem cells and compared to outcomes of standard in vivo transplantation studies. In vivo outcomes, both positive and negative, were predicated in our in vitro muscle stem cell-mediated repair platform. Further, side-by-side comparisons accurately identified a range of repair phenotypes. Finally, we were able to incorporate human iPSC derived Pax7+ cells into the regenerative environment of the microtissue, and assess the effect of various repair conditions on the newly formed human fibers. Our platform represents a powerful opportunity to compress the discovery pipeline by combining drug screening and validation in one step.