Abstract
BackgroundUnderstanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBsResultsAn initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.ConclusionES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.
Highlights
Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration
Undifferentiated Embryonic stem (ES) cell arrays We studied expression differences between 3 ES cell lines, IMT11, HM1 and SMHBl6 (GEO, GSE8625). 21 genes, including Hprt, were identified from the arrays as possibly differentially regulated, four were confirmed by RT-PCR; Hprt, Mki67, Pter and Scmh1
Analysis of embryoid body (EB) size measured at day 4 shows little variation in diameter between EBs initiated with a cell number of 500 or greater; size constraints may begin to apply once a certain diameter is reached (Figures 4a + b)
Summary
Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Many in vitro ES differentiation protocols rely on embryoid bodies (EBs); floating aggregates of ES cells which, when grown without LIF, mimic to some extent the early stage embryo, giving rise to precursors of a large number of tissues[7,8]. Growth of EBs with LIF favours stem cell renewal and the differentiation of embryonic endoderm [9], while removal of LIF allows the generation of precursors representative of all three germ layers [10]. Protocols for deriving neural, haematopoietic, muscle, bone, pancreatic, hepatic and many other precursor and mature cell types from ES cells in culture [11,12,13] have been developed, many of which still use EBs as a starting point. Subsequent growth, followed by disassociation and plating on adherent surfaces, permits the derivation of terminally differentiated cell types
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