The North West Embryonic Stem Cell Centre

Pluripotency and self renewal

Embryonic stem cells have the characteristics of dividing to give further stem cells and, under the appropriate conditions, of giving rise to precursors of different differentiated cell types from all the three major groups of cells (also known as germ lineages) within the body.

Several transcription factors are involved in maintaining the undifferentiated dividing stem cell population. These include Oct-4, Nanog and Sox-2. Our researchers assess and monitor our new hES cell lines by immunofluorescence and FACs analysis for these markers together with a variety of cell surface markers. An hES cell line engineered in the Centre with green fluorescent protein under the Oct-4 promoter, is a useful tool to assess the hES cell population by FACs analysis in experiments. This helps to determine the role of particular signalling pathways involved in maintenance of self renewing stem cells.

We are also investigating the role of extracellular matrix (see section on stem cell niche), analysing components which are optimal for maintaining the cells as undifferentiated pluripotent cells by proteomic analysis. For instance, candidates like fibronectin have been confirmed to have specific stem cell maintenance properties through such experiments.

Induced Pluripotent Stem (iPS) Cells

A rapidly emerging strategy for the generation of pluripotent cells is induced Pluripotent Stem (iPS) cells. The seminal studies of Yamanaka and Thomson showed that it is possible to genetically reprogram mammalian somatic cells to a pluripotent state strongly resembling that of ES cells by expressing a cocktail of only four transgenes; OCT4, SOX2, C-MYC and KLF4 or LIN28 from retroviral vectors. The attraction of this strategy is that patient-specific autologous iPS cells can be generated from easily obtainable somatic cells such as skin fibroblasts thereby avoiding issues of immune rejection. iPS technology is currently precluded from the clinic due to the nature of genetic modification and the lack of a comprehensive understanding of the mechanism. C-MYC and KLF4 are potent oncogenes raising serious pre-clinical safety concerns. Furthermore, the low efficiency of iPS generation indicates that there is a significant as yet undefined block to reprogramming when using transgenic over-expression. However, these cells currently provide a valuable model system for the study of human development, disease and pharmacological toxicity. At NWESCC we have generated iPS cell lines using the Yamanaka protocol from human Dermal Fibroblasts. We are currently investigating safer and more efficient methods of generating iPS cells avoiding the use of oncogenes. Our research is directed toward developing a clinically efficacious protocol for iPS reprogramming of patient’s somatic cells.

Principal investigators

Collaborator

Postdoctoral research associate

Research technician

Pluripotency and self-renewal

We are investigating different factors that regulate pluripotency and self-renewal of human embryonic stem cells.