Identification of markers for newly formed beta-cells in the perinatal period: a time of recognized beta-cell immaturity

Abstract

Markers of beta-cell maturity would be useful in staging the differentiation of stem/progenitor cells to beta-cells whether in vivo or in vitro. We previously identified markers for newly formed beta-cells in regenerating rat pancreases after 90% partial pancreatectomy. To test the generality of these markers of newly formed beta-cells, we examined their expression during the perinatal period, a time of recognized beta-cell immaturity. We show by semiquantitative RT-PCR and immunostaining over the time course from embryonic day 18/20 to birth, 1 day, 2 days, 3 days, 7 days, and adult that MMP-2, CK-19, and SPD are truly markers of new and immature beta-cells and that their expression transiently peaks in the perinatal period and is not entirely synchronous. The shared expression of these markers among fetal, newborn, and newly regenerated beta-cells, but not adult, strongly supports their use as potential markers for new beta-cells in the assessment of both the maturity of stem cell-derived insulin-producing cells and the presence of newly formed islets (neogenesis) in the adult pancreas.

Figure 1

Differential expression of marker mRNAs in neonatal (1-day-old) islets, adult ducts, and adult islets by RT-PCR. (A) In this representative gel, CK-19 and MMP-2 are differentially expressed in newborn islets compared with adult islets; they are also expressed in adult pancreatic ducts. (B) Gene expression of markers is higher in 1-day-old neonatal islets (gray) than in adult islets (black) normalized to the internal control ribosomal S25. n=4–5 samples for each time point. Error bars represent standard error of the mean.

Figure 2

Each candidate marker had a distinct pattern of gene expression, as seen in this representative graph from one of two complete time courses of samples from islets pooled from multiple pups. α-Tubulin was used as internal control for normalization. RT-PCR, 32 cycles each. All expression values were normalized to the housekeeping gene, α-tubulin, and then shown relative to day 1 expression level.

Figure 3

Time course (E20 to adult) of the protein expression of the markers by immunostaining. For each marker (green) and insulin (red), immunostaining and confocal microscopy were done in parallel at the same settings for all samples, so the relative intensity reflects the protein expression. Colocalization of the marker and insulin is seen as yellow. For each marker, the top panel of images is that of the marker alone and the bottom panel has the merged image of both channels. Bar = 50 μm.

Figure 4

Expression of MMP-2 (green) in neonatal islets is seen in insulin-positive (red) β-cells (overlap is seen as yellow) (A). In the pancreas at birth, there is almost complete colocalization of insulin and MMP-2 expression. B shows only MMP2 expression (green). Bar = 50 μm.

Figure 5

MMP-2 (green) (A) and CK-19 (green) (B) are localized in cells one to two cells away from the lumen of the main ducts and in insulin-positive cells (red) budding from these ducts at day 1. Coexpression is seen as yellow. Bar = 50 μm.