UC Davis

In vitro muscle differentiation has had a renewed interest in the context of cultivatedmeat. One potential cell source for this product is embryonic stem cells (ESCs). Though myogenic differentiation has been studied in mouse and human ESCs, research on livestock species, namely bovine, is still limited. In this study we sought to gain a wider understanding of bovine embryonic myogenesis through genetic analysis of early embryos from gestation day 40 to 65 as well as adult muscle stem cells. With said understanding we gained important context of differentiation in vitro. In that effort, we adapted previous differentiation protocols in human and mice based on WNT and BMP signaling modulation to bovine ESC (bESC) and obtained cells in early paraxial mesodermal state under a serum free myogenic differentiation protocol. This protocol was then improved upon by implementing a more defined cell substrate and a pre-differentiation step. Lastly bESCs were differentiated under spheroid formation identifying that spheroid culture could be used in differentiation but at a restricted capacity. Our findings were limited to expression of the early paraxial mesoderm genes TBX6 and PAX3 while other myogenic transcripts remained undetectable. This study provides novel information about the specific myogenic transcripts of bovine myogenic differentiation during the critical period of somitogenesis and beyond. Our in vitro studies highlight the many challenges that still need to be overcome for efficient differentiation of bESCs into muscle in vitro. These findings should aid in future approaches to develop methods for the myogenic differentiation of bovine ESCs.