Early embryoid body differentiation

Embryonic stem (ES) cells, when grown in suspension culture without feeders, spontaneously form round structures known as embryoid bodies. Given the appropriate conditions, these cells can differentiate over time into precursors of all three germ layers. Embryoid bodies, in a disorganized way, mimic early embryonic development to a certain extent and can be used as a synchronously differentiating large scale source of tissue for the study of biological determinants of early differentiation. Embryoid bodies have been used as a source for most early protocols that derive specific differentiated cell types from undifferentiated ES cells, although some protocols, notably those that derive neurons from ES cells, have moved on from EBs as a result of varying replicability and yield. We have decided to look at the transcriptomic profiles of embryoid bodies during the initial stages of embryoid body formation and differentiation, in order to pinpoint novel determinants of key developmental stages. Keywords: time course We have compared embryoid bodies that have been grown in the presence and absence of LIF, on days 1-4 of differentiation. LIF does not inhibit the development of an outer layer of embryonic endoderm and the maintenance of a core of undifferentiated stem cells in embryoid bodies is continued. The absence of LIF is permissive for differentiation of precursors of all 3 germ layers formed in the embryo proper. We have also included a comparison of differentiating EBs with undifferentiated IMT11 ES cells from which the EBs were derived (Day 0). Arrays were hybridised with experimental sample from Day0-4, and a pooled control derived from equal quantities of Day 1-4 EBs. Fluor switches were carried out. Analysis of the results included comparisons of experimental samples with the pooled control, and also comparisons with each other. The conditions for the growth of the EBs were tightly controlled, we formed EBs of a predetermined size via aggregation of 1000 cells in individual hanging drops, in order to minimize variation. The size was chosen after study of key marker genes of differentiation, which revealed that smaller EBs tended to differentiate too fast and not to express markers of all germ layers, while larger EBs tended to show some signs of cell death and also to maintain increasing numbers of undifferentiating ES cells in the inner core. We chose to use our standard 20% medium serum concentration after studies varying the serum showed less strong growth at early stages, making it difficult to obtain enough sample for the arrays. Concurrent studies with which this one is to be compared (GSE8625, and another to be submitted) also use this serum concentration. Furthermore, preliminary results from a concurrent study of undifferentiated ES cells versus microdissected early embryos has shown that our standard medium recipe gives a good correlation between the transcription profiles of early embryonic compartments and the ES cells themselves. This correlation is not maintained in ES lines grown with feeders, knockout medium and serum replacement (SMH, unpublished data). Since we wish to faithfully mimic early embryonic development as far as possible in these studies, we have therefore proceeded with the ES medium described in the GSM submissions accompanying this entry. We have identified approximately 130 ESTs that showed significant changes as a result of analysis of this timecourse. Following rejection of duplicate genes (the NIA set contains many different clones that represent the same gene in some instances), and ESTs where sequence quality was too poor for primer design, we were left with 101 genes that we tested across 6 biological cDNA sample replicates, 3 from 1000 cell EBs and 3 from 750 cell EBs. Of these, 25 were confirmed as differentially regulated from the -LIF sample set and 12 from the + LIF dataset (9 of these were present in both datasets). 3 genes were expressed only in - LIF samples.