Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters

Abstract

Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

Keywords: Glut2; differentiation; duct; islet; pancreas; plasticity; progenitor cell; β-cell.

Figure 1.

In vitro dedifferentiation of neonatal mouse islets. Photomicrographs depicting neonatal (P7) mouse islets immediately following isolation (A), 4 d after plating on collagen under dedifferentiation culture conditions (B), and after 1–4 weeks in vitro (C). The total proportion of cytokeratin-19 (Ck19⁺)-expressing cells significantly increased after islets (D, white bar) were cultured in ductal epithelial promoting conditions (D, hatched bar = 1 week; black bar = 4 weeks) and which was maintained. The cell proliferation index (total EdU⁺/DAPI⁺ cells, E) increased after islets (E, white bars) were cultured for ductal dedifferentiation for 1 week (E, hatched bar), and decreased thereafter (E, black bar, 4 weeks). Size bars denote 50 μm, n > 10 experiments, data are represented as % mean ± SEM, **p < 0.01, *** p < 0.001.

Figure 2.

Specificity of HPAP to the β-cell using RIPCre;Z/AP^+/+ mice and lineage tracing of the β-cell during in vitro islet dedifferentiation. RIPCre;Z/AP^+/+ transgenic mouse model showing insulin⁺ β-cells (green, A) tracked by the reporter protein HPAP (red, A), and quantified in (C). The proportion of insulin-expressing β-cells (B, green) co-labeled by HPAP (B, red) in β-cell clusters containing fewer than 5 β-cells was significantly lower than in islets (B, C, black bar). After islets were cultured in dedifferentiation media for 1 week, all insulin expression was lost (D, red), and only a minority of cells retained HPAP expression (D, green). The majority of cells present in culture after 1 week immunostained for Ck19 (E, red), as did the rare remaining HPAP⁺ cells (green). These HPAP⁺Ck19⁺ were proliferative at 1 week in culture (F, hatched bar) but proliferation decreased thereafter (F, 4 weeks, black bar). There was a significant increase in β-cell apoptosis after 5 d in dedifferentiation conditions (G, insulin, green; TUNEL, red). After replating the resultant epithelial-like monolayer of cells in redifferentiation medium, the majority of the rare, remaining HPAP⁺ cells (H, green) re-expressed insulin (H, red). Scale bar denotes 50 μm, n > 6, data are represented as % mean ± SEM, **p < 0.01, *** p < 0.001.

Figure 3.

Pancreatic β-cells sorted by flow-cytometry using Glut2 and GPM6a presence. Insulin-expressing β-cells (A, green) could be identified by GPM6a (A, red) using immunofluorescence. Whole mouse P7 pancreas (B, left) was dispersed and sorted into distinct populations of insulin⁺ (GPM6a⁺) cells according to differential Glut2 expression: GPM6a⁺Glut2⁺ (B, red) and GPM6a⁺ Glut2^MID/LO(green), further split into GPM6a⁺Glut2^LO (B, green) and GPM6a⁺Glut2^MID (B, left, darker green, upper). Cells recovered after sorting (C) were immunostained for Glut2 (green) and insulin (red). Insulin staining (C, red) was more prominent in proto-typical β-cells (GPM6a⁺Glut2^HI, top panel) as compared to GPM6a⁺Glut2^MID/LO cells. Glut2 immunostaining (C, green) was diminished in the GPM6a⁺Glut2^MIDcells (C, middle), and was absent in the GPM6a⁺Glut2^LO cells (C, green, bottom). After flow cytometry, cell recovery, and 1 week in dedifferentiation culture, the majority of Ins⁺Glut2^LO cells co-expressed Ck19 (red) and HPAP (green) (D, arrows and circle). Redifferentiation of dedifferentiated ductal-like cells after 4 weeks in culture demonstrated the β-cell lineage reporter HPAP (E, green) co-localizing with insulin (E, red, arrow). Scale bar denotes 50 μm, n = 5.

Figure 4.

Confirmation of the β-cell origin of GPM6a⁺Glut2^LO cells using the reporter strain ROSA-eYFP. (A) insulin (red) and GFP (green) immunostained β-cells are shown within RIPCre;ROSA-eYFP^+/+ P7 mouse pancreas sections (n = 5). After sorting live cells by flow cytometry from whole pancreas using GFP and Glut2, the GFP⁺Glut2^LO cells dedifferentiated in vitro to ductal-lineage cells (B, Ck19, red and GFP, green, n = 4). These cells were able to redifferentiate back into insulin-expressing cells in vitro (C, insulin, red; GFP, green, n = 4).

Figure 5.

Lineage plasticity of pancreatic Glut2^LO β-cells. GPM6a⁺ cells recovered from whole mouse RIPCre;Z/AP^+/+ P7 pancreas after flow cytometry and cell sorting based on Glut2 presence were placed in neural proliferation conditions. Neurospheres (A) were generated from GPM6a⁺Glut2^LO cells, compared to non-proliferative GPM6a⁺Glut2^HI cells (B). After culture in neural differentiation conditions, the neurospheres generated multiple morphologies, including neuron-like processes (C, D). When subjected to immunofluorescent staining, cells with a neuronal appearance were visualized by β-III tubulin (E, green), and astrocytes by GFAP (F, red). These neural-lineage cells co-stained with HPAP (red, E; green, F; n = 5). Neurospheres (G, GFP, green, and insulin, red) were similarly generated from sorted GFP⁺Glut2^LO β-cells derived from RIPCre;ROSA-eYFP^+/+ P7 pancreas, when cultured under neural proliferation conditions, and demonstrated an immature β-cell phenotype by quantitative PCR with decreased expression of Ins and Glut2, and increased expression of Ngn3 and MafB (H, *p < 0.05, neurospheres vs P7 islets, n = 4). Scale bar denotes 100 μm (A, B), 50 μm (C-G).

Figure 6.

Ins⁺Glut2^LO β-cells are primarily found outside of islets. FAC-sorted GPM6a⁺Glut2^LO cells isolated from P7 islets represented 0.3 ± 0.1% of live cells (A), a lower proportion relative to that found in whole pancreas (see Fig. 3B). (B) Mouse P7 pancreas section immunostained for insulin (i, red), Glut2 (ii, green), EdU (iii, yellow), and merged images (iv), demonstrating an Ins⁺Glut2^LOEdU⁺ cell (arrow) located in an extra-islet β-cell cluster. Quantification of Ins⁺Glut2^LO cells by β-cell grouping indicated their primary location in clusters as compared to islets (C, ***p < 0.001 cluster vs total and islet). The proliferation rate of Ins⁺ cells at P7 was higher in extra-islet clusters than in islets (D, **p < 0.01), and there was a high occurrence of Ins⁺Glut2^LO cell proliferation within islets and clusters at P7 (E, **p < 0.01). The proportions of total extra-islet, insulin-expressing clusters (F, **p < 0.01), total Ins⁺Glut2^LO cells (G, **p < 0.01), and total β-cell proliferation by Ki67 (H, ***p < 0.001) all decreased from 7 d to 3 months of age in the mouse. Scale bar denotes 50 μm, n > 5. Data are represented as % mean ± SEM.

Figure 7.

Alternate cell fates of HPAP⁺ cells in vivo. HPAP⁺ cells from RIPCre/Z/AP^+/+ neonatal (<14 d, n = 5) mouse pancreas sections rarely immunostained for non-insulin markers, including glucagon (Gcg) or somatostatin (Sst) (Fig. 7A and B, white bars) whether quantified by proportion of marker/HPAP (A) or HPAP/marker (B). In mice older than 1 y (n = 4), the proportion of HPAP⁺ (green) labeled non-insulin-expressing cells increased, including those containing glucagon (Fig. 7A and B, black bars, and C, red) or somatostatin (Fig. 7A and B, black bars, and D, red) as compared to young mice. Arrows indicate dual stained cells. Scale bar denotes 50 μm. Data are represented as % mean ± SEM, C and D, >1 y vs <14 d, t-test, * p < 0.05, ** p < 0.01.