In Vitro Cellular Dynamics of Skin-Derived Fibroblasts of an MSTN-/-Gene-Edited Buffalo.

Abstract

Fibroblasts are widely used cell type in cellular reprogramming studies, including genetic engineering, due to their easy accessibility and cellular plasticity. The present work aimed to study the in vitro cellular dynamics of fibroblasts derived from an MSTN^-/- (myostatin knockout) gene-edited buffalo. Established MSTN^-/- fibroblasts exhibited higher cell and nuclear size, reduced doubling time, and shortened telomere length compared with wild-type cells. Assessment of mitochondrial function using JC-1 and MitoSOX dyes revealed preserved mitochondrial membrane potential; however, MSTN^-/- fibroblasts showed a higher proportion of MitoSOX-positive cells, suggesting elevated mitochondrial reactive oxygen species production, potentially associated with altered cellular morphology such as vacuolation. Epigenetic profiling demonstrated significantly reduced levels of active histone acetylation marks H3K18ac and H4K5ac in MSTN^-/- fibroblasts, while the repressive mark H3K9me2 remained unchanged. To evaluate their translational potential, MSTN^-/- fibroblasts were used as donor cells for somatic cell nuclear transfer. In vitro developmental competence, including cleavage rates, blastocyst production rates, and total blastocyst cell number, was comparable to cloned embryos derived from wild-type cells. Overall, MSTN^-/- fibroblasts display distinct cellular and epigenetic features with normal SCNT embryo production, supporting their utility for cellular reprograming studies.