Engineering the Robust Transdifferentiation of Human Dermal Fibroblasts into Skeletal Muscle

Abstract

Skeletal muscle has a remarkable capacity for self-repair, however, it is unable of restoring volumetric tissue loss due to traumatic injury, congenital defects or tumor ablation [1]. Skeletal muscle tissue engineering aims to create in vitro constructs that mimic native skeletal muscle tissue in terms of both structure and function. Such tissue engineered constructs can be either therapeutically relevant or used as in vitro microphysiological models for disease modeling, and toxicological or pharmacological studies. Current in vitro models of human skeletal muscle lack the physiology and functional output of native muscle due to the cell source being difficult to derive to sufficient numbers and a maturity level. Here, I utilize the ability of the MYOD1 transcription factor to induce trans differentiation of human dermal fibroblasts (HDFs) into skeletal muscle. This process requires significant improvement in efficiency to derive sufficient cells and to form 3D biosynthetic skeletal muscle tissues. Since, in vivo myogenesis is regulated by specific signaling pathways (WNT, TGFβ, RTK) and extracellular matrix components, I hypothesized that manipulation of the soluble and cell-adhesion based microenvironment would significantly enhance the transdifferentiation process. Trans differentiation was evaluated based on the number of cells that fused and formed multinucleated skeletal muscle cells, the length and diameter of the derived myotubes, sarcomeric organization, and functional characteristics of trans differentiated cells. HDFs were treated with small molecule inhibitors and ligands activators of WNT, TGFβ, and RTK signaling. Transdifferentiation efficiency was also evaluated in HDFs seeded on Fibronectin, Laminin, Collagen type 1, Poly-D-Lysine, and Poly-L-Ornithine. I determined that manipulating specific molecular signaling pathways and seeding transduced HDFs on certain substrates significantly enhanced the trans differentiation efficiency to a level where I derived an almost pure functional population of human skeletal muscle cells.

Date of Award	Dec 2015
Original language	American English
Supervisor	Nicolas Christoforou (Supervisor)