Neonatal beta cell but not acinar cell neogenesis evidenced by lineage tracing

Background and aims: Stimulation of beta cell formation and beta cell transplantation offer hope as curative diabetes therapies. Yet, these are hindered by insufficient knowledge in the mechanisms of pancreas cell formation after birth. In adult mice, self-duplication is the main mechanism of beta cell formation. Though, the origin of different pancreas cells formed during the major pancreas expansion in the first month of life remains unclear and controversial. We wanted to uncover the source and quantity of the newly formed beta cells and to unravel cellular contributions during physiological neonatal pancreas growth. Materials and methods: To specifically trace the fate of beta, duct, acinar and alpha cells in the first month of life, we used transgenic RIPCreER Rosa26LoxSTOPLoxYFP, Hnf1bCreER R26LacZ, ElastaseCreER R26YFP and Glucagon-rtTA TetO-Cre R26YFP mice, respectively. Pups received tamoxifen on the day of birth or continuous doxycycline from day 1 on and were followed during the next four weeks. Results: In tamoxifen-treated RIP YFP mice, beta cells are labelled with a high efficiency and specificity. 82.2 ± 1.7% beta cells express YFP at postnatal day 7 (P7). Without tamoxifen, there is only limited ‘leaky’ expression. From week 1 to week 4, there is a 3- to 4-fold increase in beta cell mass. The islet size distribution shows a decrease in the fraction of small islets (1-5 beta cells) and an increase in the fraction of larger islets (11-50 beta cells and >50 beta cells). To quantify beta cell neogenesis, we assessed the beta cell labelling index. The percentage of labelled beta cells remains unchanged between week 1 and week 2, but starts to decrease from week 3 on and falls significantly to 70.8 ± 2.6% at week 4. The YFP+ fraction of beta cells are beta cells which were already present at birth or originate from self-duplication of beta cells. This decrease indicates that between 2 and 4 weeks of age new beta cells (about 16-17%) arise from non-beta cells (progenitor cells or stem cells). No further reduction in the beta cell labelling is observed from week 4 to week 6 (70.4 ± 0.9% YFP+ beta cells at P42) indicating that the period during which beta cell neogenesis occurs under physiological growth is restricted to the neonatal period between 2- and 4-weeks of age. Further, our lineage tracing experiments reveal that nor duct cells nor acinar cells nor alpha cells contribute to the neonatal beta cell neogenesis. And we show that physiological neonatal growth of exocrine acinar cells occurs primarily by self-duplication (and hypertrophy), as the labelling index of acinar cells in Ela YFP mice did not decrease during the first 4 weeks of life (35.2 ± 5.4% YFP+ acinar cells at P7). In line with this, duct cell-tracing in Hnf1b LacZ mice did not show significant increase in acinar labelling. Interestingly, we obtained indirect evidence that somatostatin-producing delta cells may be the origin of the newly formed neonatal beta cells as the percentage somatostatin/insulin double positive cells increases specifically at P21 and P28, coinciding with the timing of neonatal beta cell neogenesis, and declines again at P42. Conclusion: We provide direct evidence (via genetic lineage tracing) which shows that new formation of beta cells from non-beta cells occurs in young mice and that self-duplication is the main mechanism for the increase in acinar cell number. Grant Acknowledgement: FWO: project and research fellowship, VUB: price Ignace Vanderschueren 2012