Pancreatic islet cell transplantation is proven to restore functional beta cell mass to achieve normoglycemia in diabetics, but is limited by low availability of human cadaveric donor tissue. A strategy to build capacity is to use stem cells (SCs), and induce their expansion and differentiation into functional islets in vitro. Islet cell neogenesis has been linked to transient and sequential expression of specific transcription factors (TFs) and the actions of endogenous growth factors. We used a model of streptozotocin-induced beta cell depletion in the neonatal rat resulting in immediate hyperglycemia. However, over three weeks blood glucose levels decrease accompanied by an increased cellular immunoreactivity for nestin, Nkx2.2, Nkx6.1, and finally insulin as islet cell neogenesis is enhanced. Similarly, beta cell generation can be achieved in vitro starting with immature, endocrine hormone-negative pancreatic epithelial precursor cells. Temporal changes in TF expression suggest initiation of a cascade of signals inducing islet cell formation. We hypothesised that over-expression of candidate TFs would drive islet production with beneficial functional outcome. To test this we have developed inducible, targeted Ngn3 and Nkx2.2 constructs. Targeted expression is achieved using the Pdx1 promoter to drive an inducible form of Cre recombinase. Upon addition of tamoxifen, Cre will catalyse loxP mediated excision of the second construct allowing LacZ gene removal and promoter alignment to Ngn3 or Nkx2.2 coding region linked with EGFP. These inducible constructs have been electroporated into embryonic SCs, from which Nkx2.2 chimeric animals have been generated for studies of islet cell regeneration. In vitro studies using these ES cells have confirmed inducible TF expression, and characterised their individual roles in targeted islet cell formation.
22 - 24 Mar 2004
British Endocrine Societies