The interplay of metabolism and signaling in the differentiation pattern of 3D embryo-like models

Three-dimensional in-vitro models from mouse embryonic stem cells have improved vastly in recent years. But variability in, and between, experiments still poses a great challenge in this field. To use these models as reliable tools to study embryonic development and other questions regarding cell differentiation, it is important to understand which variables affect organoid morphologies and gene expression patterns. Here we aim to better understand the connection between metabolic changes in cells, their pluripotency state and how those changes influence the differentiation potential of cells. Moreover, these metabolic changes may affect cell viability and it’s potential to keep differentiating without further intervention. Here, we employ a skeletal muscle differentiation protocol that is based on the `neglect` of the organoids for long periods of time. This protocol can yield a substantial amount of skeletal muscle fibers with minimal usage of growth factors and handling. Interestingly, we observe an interchangeable effect of Bmp4 addition and basal medium components on organoid viability and muscle generation efficiency. Using this protocol, we intend to establish a connection between metabolic changes and external signaling and how the two may be interchangeable.