During the fetal stage, differentiation is the major mechanism for forming new β-cells, but replication or self-duplication is enhanced during the perinatal period. During the neonatal period, β-cell replication continues and significant neogenesis occurs under these normal physiological conditions, as has been demonstrated using various methods, including Cre-lox lineage tracing experiments.
Bouwens et al. (21
) studying neonatal rat pancreas in the first 2 weeks after birth suggested that cytokeratin 20+ (a marker of rat duct epithelium) cells at the periphery of islets served as islet progenitors. Between 2 and 5 days of age, the β-cell mass more than doubled, but the cross-sectional area of the β-cells was unchanged, indicating that new cells rather than larger cells accounted for the increased mass. The BrdU incorporation in β-cells of 2.4% at day 2 could account for only 12% of the observed growth by day 5. It was concluded that most of the new β-cells originated from neogenesis. Their observation that CK20+ cells had higher BrdU incorporation than hormone-positive cells is consistent with the finding that duct cell replication precedes neogenesis (22
Our mathematical modeling study (5
) predicted two waves of neogenesis: one immediately after birth and the other 2–3 weeks after birth. This model used existing data on β-cell mass and its determinants (cell volume, replication, and apoptosis frequency) to estimate turnover of β-cells. In our subsequent longitudinal study of β-cell mass and its determinants over the first month after birth (6
), we documented the increased appearance of islets budding from the ducts at the same times (shortly after birth and just before weaning), confirming the predicted waves of neogenesis. Using these data to estimate the number of β-cells at each time point, we estimated that ~70% of the β-cells seen at day 31 could be accounted for by replication of preexisting β-cells, while the remaining 30% were from neogenesis (22
). This estimate is consistent with the findings from our duct-specific lineage tracing experiments described next (20
Carbonic anhydrase II (CAII), which only starts to be expressed in ducts at the very end of gestation (23
), has been considered a marker for ducts to distinguish the mature ductal phenotype from the embryonic tubular structures often called embryonic ducts. Thus, bigenic CAIICre:ROSA26R mice can provide duct-specific lineage tracing with only genetically marked ducts expressing the reporter at birth with no CAII or Cre expressed in β-cells (20
). We showed that in the 4 weeks after birth, both islets and acini were formed from cells that once expressed CAII: 38% of the islets were marked (17% of all insulin+ cells) as well as a number of acinar cells; some lobes were marked and others not. Since the pancreatic weight increases fourfold between day 17 and day 31 (6
), we interpret the lobular pattern of marked islets and acini as evidence of new lobe formation in the neonatal period. It should be noted that two recent lineage-tracing studies using inducible Cre-ER driven by either the hepatocyte nuclear factor (Hnf) 1β (24
) or mucin1 promoter (25
) found no marked islet nor acinar tissue in neonates when tamoxifen was administered at the end of gestation (24
) or birth (25
). These studies found only marked ducts during this time of rapid pancreatic expansion, but their negative data may be due to 1
) their low efficiency of marking ducts (20 and 7.6%, respectively) and only ~1,000 insulin+ cells counted per animal and 2
) a marked heterogeneity within the ducts of the expressed product of their driver gene.
Recently, Peng et al. (26
) published studies that showed an increasing number of islets from 1 week to 2 months of age. The obvious caveat for enumeration of islets over time in that small clusters of islet cells, which initially may have been below the measurable limit, may increase in size due to proliferation and be counted as newly formed. Interestingly, they also report that Ki67 mRNA and telomere length were significantly correlated in single islets at either 2 weeks or 4 months but differed for islets within the same animal, suggesting islets of differing ages in adult mice.
If new islets were generated from preexisting ductal tissue, one might expect expression of residual duct markers for a short period of time after hormone expression. Indeed, transient expression of markers of duct cells has been demonstrated in β-cells of newborn rats (27
) as well as in regenerated islets after partial pancreatectomy (22
), in grafts of purified human duct cells (29
), and in islets of mice conditionally expressing Pax4 in glucagon-producing cells (POE::GluCre) (30
), suggesting their recent passage through the ductal phenotype.