Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

SIMPLE SUMMARY: Engineered human skeletal muscle tissue is a platform tool that can help scientists and physicians better understand human physiology, pharmacology, and disease modeling. Over the past few years this area of research has been actively being pursued by many labs worldwide. Significant challenges remain, including accessing an adequate cell source, and achieving proper physiological-like architecture of the engineered tissue. To address cell resourcing we aimed at further optimizing a process called transdifferentiation which involves the direct conversion of fibroblasts into skeletal muscle cells. The opportunity here is that fibroblasts are readily available and can be expanded sufficiently to meet the needs of a tissue engineering approach. Additionally, we aimed to demonstrate the applicability of transdifferentiation in assembling tissue engineered skeletal muscle. We implemented a screening process of protein ligands in an effort to refine transdifferentiation, and identified that most proteins resulted in a deficit in transdifferentiation efficiency, although one resulted in robust expansion of cultured cells. We were also successful in assembling engineered constructs consisting of transdifferentiated cells. Future directives involve demonstrating that the engineered tissues are capable of contractile and functional activity, and pursuit of optimizing factors such as electrical and chemical exposure, towards achieving physiological parameters observed in human muscle. ABSTRACT: The development of robust skeletal muscle models has been challenging due to the partial recapitulation of human physiology and architecture. Reliable and innovative 3D skeletal muscle models recently described offer an alternative that more accurately captures the in vivo environment but require an abundant cell source. Direct reprogramming or transdifferentiation has been considered as an alternative. Recent reports have provided evidence for significant improvements in the efficiency of derivation of human skeletal myotubes from human fibroblasts. Herein we aimed at improving the transdifferentiation process of human fibroblasts (tHFs), in addition to the differentiation of murine skeletal myoblasts (C2C12), and the differentiation of primary human skeletal myoblasts (HSkM). Differentiating or transdifferentiating cells were exposed to single or combinations of biological ligands, including Follistatin, GDF8, FGF2, GDF11, GDF15, hGH, TMSB4X, BMP4, BMP7, IL6, and TNF-α. These were selected for their critical roles in myogenesis and regeneration. C2C12 and tHFs displayed significant differentiation deficits when exposed to FGF2, BMP4, BMP7, and TNF-α, while proliferation was significantly enhanced by FGF2. When exposed to combinations of ligands, we observed consistent deficit differentiation when TNF-α was included. Finally, our direct reprogramming technique allowed for the assembly of elongated, cross-striated, and aligned tHFs within tissue-engineered 3D skeletal muscle constructs. In conclusion, we describe an efficient system to transdifferentiate human fibroblasts into myogenic cells and a platform for the generation of tissue-engineered constructs. Future directions will involve the evaluation of the functional characteristics of these engineered tissues.