The aim of this work was to obtain greater insight into the differentiation capabilities of the embryonic stem (ES) cell line currently being employed in a broader study of haematopoiesis within the UQ Bioengineering Laboratory.
A block in the developmental progression of these cells toward the haematopoietic lineages had been observed prior to the commencement of this work. A delay in onset of mesoderm formation, indicated by a delay in the transcription of the brachyury gene was observed and it was desired that transcription profiles for other genes indicating progression toward haematopoiesis be established. By assaying for transcription of Flk-1 and CD34 using real-time quantitative PCR, data was obtained that allowed transcription of a greater range of pre-haematopoietic markers to be quantified. Flk-1 and marks for an intermediate subset of mesoderm and CD34 is expressed by immature haematopoietic cells.
Transcription profiles were obtained with respect to brachyury, Flk-1 and CD34 for ES cells differentiated under two different culture regimes: suspension and methylcellulose culture. The resulting transcription profiles for the ES cells in suspension culture indicated delays in the onset of brachyury up-regulation, and little up-regulation of the Flk-1 and CD34 transcripts. Methylcellulose culture appeared capable of hastening the onset of haematopoietic development: primitive haematopoietic colonies were observed in culture on day 7 and while the transcription profile still indicates a delay in development compared to data obtained in other laboratories, an improvement was seen in comparison with the suspension culture results.
While this work has not provided data that allows the cause of the developmental delay to be pinpointed, useful profiling data has been added to that already obtained relating to this particular cell line. Steps toward optimising the culture for haematopoietic development have been made with the introduction of the methylcellulose culture technique employed.