Induced Pluripotent Stem Cell (iPSC) derived myogenic progenitors cultured in equine skeletal muscle decellularized matrix

Abstract

Equine represents an attractive animal model for diseases that affect the musculoskeletal tissue, exhibiting much similarity to the injuries that occur in humans. Cell therapy and Tissue Bioengineering, especially with the use of natural biomaterials derived from decellularized extracellular matrix (dECM), have been widely used as a therapeutic alternative by Regenerative Medicine in musculoskeletal diseases.Thus, the aim of this study will be to produce an acellular biomaterial of equine skeletal muscle and to evaluate the effectiveness in supporting the in vitro culture of equine induced pluripotent stem cells (iPSC). Biceps femoris (1.5 x 1.5 cm) samples will be frozen at -20°C for 4 days and incubated in 1% SDS (7 days), 5 mM EDTA + 50 mM Tris (2 days) and 1% Triton X-100 (2 days); the effectiveness of the decellularization will be evaluated by the absence of remnant cells (histological analysis), preservation of matrix proteins (immunofluorescence), organization of ECM ultrastructure (scanning electron microscopy) and quantification of remnant DNA (Picogreen Assay). The biomaterials will be recellularized with iPSCs at the concentration of 50,000 cells/cm2 using a rotatory cell culture system in a miogenic differentiation medium. As a control, iPSC will cultured on gelatin and matrigel layer. The presence of the cells in the biomaterial will be evaluated by quantification of nuclei (DAPI) and DNA. To confirm the differentiation in muscle lineage of iPSCs, analysis of the gene expression of myogenic progenitors (PAX 3, Myo-D, myogenin, desmin, myosin) will be performed in three different periods of in vitro differentiation. We hypothesize dECM biomaterial produced from the equine skeletal muscle could preserve potential for myogenic differentiation, representing a promising biomaterial for Regenerative Medicine in the therapy of musculoskeletal diseases. (AU)