Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation (original) (raw)

Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live twophoton imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a 'developmental clock' using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. The coordinated progression of different cell lineages is essential for the formation of functional tissues and organs. Recent efforts have been focused on developing and optimizing in vitro platforms to study the mechanisms underlying stem cell differentiation as well as multi-lineage progression, using two-and three-dimensional cultures 1-4. Better definition of the constraints on multi-lineage differentiation processes in these systems would enhance their use in the study of development and developmental defects. Embryonic stem cells can be aggregated into embryoid bodies (EBs), which have the potential to differentiate to a diverse population of adult specialized cells 2,5-8. Even though EBs differentiate in a less strictly defined fashion than embryos, they display embryogenesis-like processes such as germ layer formation, ECM secretion, and primitive streak formation 2,6-8. The timing and pattern of these processes are influenced by a profusion of variables such as medium composition, growth surfaces and physical constraints 2. For example, initial EB size affects the extent of mesodermal (and subsequently cardiac) vs. ectodermal differentiation 9. The differentiation of EBs into cells of the three germ layers, even in the absence of externally added directive signals, indicates that the required signals for these processes can autonomously build up within each EB. BMP, Wnt, FGF and activin signaling pathways have all been shown to play important roles in inducing these transitions, both in vivo and in vitro 10-12. FGF signaling was shown to be essential for the onset of neural ectoderm markers 13,14. Wnt and BMP signaling components are essential for the establishment of primitive streak and mesendoderm formation and, in EBs, influence mesendoderm differentiation and axis formation 15-17. How these signals interact to coordinate the growth and relative composition of multiple lineages is not fully characterized. EBs can serve as a valuable tool for studying cell to cell signaling mediated development, and are particularly compatible with high-throughput analysis of post-implantation differentiation processes.