Characterisation of nascent mesoderm derived from mouse embryonic stem cells grown in 3-D and 2-D culture systems

Mouse embryonic stem cells (mESCs) are derived from the inner cell mass of the blastocyst. When cultured in 3-D suspension in the absence of feeder cells, they form aggregates called embryoid bodies (EBs) that spontaneously differentiate to generate an outer layer of extra-embryonic endoderm with underlying basement membrane, an inner layer of primitive ectoderm, as well as a central proamnioticlike cavity. Furthermore, EBs can undergo a process that resembles gastrulation and can generate derivatives of the three embryonic germ layers. EBs are therefore very useful as model systems for investigating the early stages of mammalian development, and also for generating specific lineages such as ectoderm, mesoderm and endoderm, which can be used to investigate the mechanisms regulating the differentiation of specific lineages, as well as for regenerative medicine research. However, when used as a source for generating specific cell types of interest, EBs can be problematic, because only a proportion of cells within each EB will differentiate to become the required cell type. For this reason, there has been much interest in developing more efficient 2-D culture systems for directing the differentiation of mESCs to specific cell types. However, it is not clear whether cell types differentiating in EBs are equivalent to the corresponding cell types generated in 2-D differentiation cultures and have the same differentiation potential. To address this question, this study has compared the properties of nascent mesoderm arising in EBs with nascent mesoderm arising in 2-D differentiation culture. In order to do this, a mESC reporter line was created where a gene encoding the far-red fluorescent protein E2-Crimson (E2C) was knocked into the Rosa26 locus of an E14-Bra-GFP mESC line. This line enabled GFP nascent mesoderm cells to be isolated using fluorescence activated cell sorting so that the expression of key genes could be analysed, and then the fate of the cells could be tracked in ii living mice in vivo or following incorporation into developing organs ex vivo due to the fact that they constitutively express E2C. After confirming that the novel reporter E14-Bra-GFP/Rosa26-E2C mESC line displayed typical mESC properties and behaved similarly to unmodified mESC lines, the effectiveness of the E2C reporter for tracking cells in vivo and in vitro was assessed. Although E2C expression was stable, the fluorescence signal was quite weak, which meant that while it was possible to detect E2C in cells in vitro and on histological sections, tracking them in living mice was not feasible. For this reason, the study focused on comparing the gene expression profile of mesoderm isolated from EBs and 2-D cultures using quantitative reverse transcription PCR (RT-qPCR), and then their differentiation potential was assessed by incorporating the mesodermal cells into mouse kidney rudiments ex vivo. The most striking result from the RT-PCR analysis was that the mesodermal cells isolated from the EBs expressed >20-fold higher levels of the lateral plate mesoderm gene, Foxf1, compared to the mesoderm cells derived from the 2-D culture system. Surprisingly, neither the mesodermal cells isolated from the EBs, nor those isolated from the 2-D system integrated into developing nephrons within kidney rudiments cultured ex vivo. This result was unexpected because the kidney is derived from the mesodermal lineage, specifically the intermediate mesoderm, and so it was anticipated that nascent mesoderm cells would be able to contribute to the developing kidneys. However, further analysis showed that the EB-derived mesoderm cells differentiated into PECAM endothelial-like cells within the rudiments. It is known that the lateral plate mesoderm gives rise to the vasculature in the developing embryo, so the fate of the EB-derived nascent mesodermal cells accompanied with their high levels of Foxf1 suggested that they were likely to have been already specified as lateral plate mesoderm. In contrast, the mesodermal cells derived from the 2-D culture system showed a limited capacity to generate PECAM-1 cells, and instead, appeared to integrate into the renal stroma. It can therefore be concluded that Bra mesodermal cells generated using iii different in vitro culture systems have different properties, and might already be specified to more differentiated mesodermal lineages, such as paraxial, intermediate or lateral plate mesoderm. To facilitate future progress, it would be useful to generate dual reporter mESC lines that enabled the expression of Bra and a marker of either paraxial, intermediate or lateral plate mesoderm to be monitored simultaneously in real-time during in vitro differentiation culture.