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Presenter: W.J, Hawthorne, Westmead, Australia
Authors: E. Jimenez-Vera, P. Phillips, S. Davies, P. J. O’Connell, W.J. Hawthorne
Molecular profiling demonstrates better outcomes for reversal of diabetes in long-term culture of neonatal islet cell clusters
E. Jimenez-Vera, P. Phillips, S. Davies, P. J. O’Connell, W.J. Hawthorne
Westmead Hospital, Millennium Institute, CTRR, Westmead, Australia
Objective. Porcine neonatal islet-like cell clusters (NICC) may provide a limitless source of beta-cells for transplantation into patients to cure type 1 diabetes. However,significant delays in the time to function are encountered because of their mature state at the time of isolation. The aim of this study was to determine the optimal culture time for NICC in order to achieve optimal functional outcomes.
Methods: NICC isolated from 1-3 day old pig pancreases were cultured for up to 27 days. On days 6, 12, 19 and 27 the following parameters were examined: number (IEQ), beta cells viability index by FACs, stimulation index, Insulin: DNA ratio, ATP activity, viability, and RT PCR of insulin, glucagon, Caspase 3, and tissue factor (TF). At the various time points cultured NICC were also transplanted under the renal capsule of streptozotocin induced diabetic SCID mice.
Results: NICC cultured for 12, 19, and 27 days achieved normal blood glucose levels within a mean of 38, 26 and 22 days respectively. NICC cultured for 6 days achieved normoglycaemia in only 50% of animals and took a mean of 57 days. Longer culture time produced a significant loss of IEQ over time. Preliminary RT PCR data indicates that Insulin expression levels were higher at days 12, 19, 27compared to day 6. Glucagon gene expression was highest at day 12. Tissue factor (TF) gene expression was highest on day 6.
Conclusion: Culture of NICC for 12days provided the best in-vivo functional outcome for transplantation with a shorter time to in vivo function and higher levels of insulin and glucagon gene expression and lower levels of TF expression. Furthermore, all mice transplanted with NICC cultured for 12 days normalized within a mean of 38 days.
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