Differentiating embryonic stem cells as an in vitro model of early erythropoiesis

Extended studies on mouse embryos during the earliest stages of development are limited owing to both the large number of preimplantation embryos usually required and the difficulties in manipulating embryos after implantation; an in vitro model for embryogenesis would therefore be useful. Recently, blastocyst-derived mouse embryonic stem (ES) cells have been reported to differentiate in vitro to generate embryoid bodies (EBs) which form endoderm, mesoderm and ectoderm, thereby partially mimicking early stages of murine development. After 7 days of differentiation, EBs consisted of 6-10 x 10(3) cells. Confocal laser scanning microscopy of EBs after 13-14 days of differentiation (diameter 650-800 mum) revealed that they were vital, whereas similar-sized human malignant glioma (U 343 MG) cell spheroids showed cell death in the core region. To evaluate the potential of this system as an in vitro model of embryonic erythropoiesis, the EBs were analysed for the presence of erythropoietin (EPO) mRNA by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and we adapted a 2,7-diaminofluorene (DAF)-based staining method to detect haemoglobin in situ. DAF-positive cells appeared at day 5-6 of differentiation, whereas EPO mRNA was found in undifferentiated ES cells and in EBs at all stages of differentiation. Early expression of EPO mRNA prior to the onset of erythropoiesis implies that EPO may have a yet unknown role during early development. Apart from its potential to investigate embryonic erythropoiesis, this in vitro model may be useful for pharmacological and toxicological studies focused on early stages of development.