Virgin Beta Cells Persist throughout Life at a Neogenic Niche within Pancreatic Islets

Abstract

Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.

Keywords: GCaMP6; Urocortin3; alpha cell; beta cell maturation; beta cell neogenesis; diabetes; insulin; islet architecture; stem cell; transdifferentiation.

Figure 1.. The absence of Ucn3 marks beta cells in the neogenic niche.

(A) Gene expression of Ucn3, Nkx6.1, Mafa and Mafb by RNAseq on FACS sorted beta cells during perinatal maturation. Gene structure and chromosome number are indicated for each panel. (B) Detection of insulin, glucagon and Ucn3 in a 3-month old islet. Insets show Ucn3-negative beta cells. (C) Fraction of Ucn3-negative beta cells at different ages (counted n=3 animals per time point, 10 islets each). Error bars reflect SEM, * P <0.05, ** P < 0.01. (D) Ki67-positive beta cells maintain Ucn3 expression indicating that proliferating beta cells and Ucn3-negative beta cells are not the same. (E-I) Image analysis to detect the islet outline and center of mass to compute the position of a cell relative to the center and nearest edge. Cells are manually classified as (F) mature beta cell (insulin and Ucn3 co-positive), (G) alpha cell (glucagon positive), (H) immature beta cell (insulin-positive, Ucn3-negative) or (I) Ucn3 low beta cell (insulin-positive, Ucn3-low). (J) Normalized cumulative distribution of Ucn3-negative beta cells compared to mature beta cells of all ages combined (3, 6 and 9 weeks; 3, 8 and 14 months; 3 animals per age, 16,896 cells). See Figure S1 for distributions by age and Table S1A for the P values and D statistics for each pairwise comparison. (K) Ucn3-negative, insulin positive beta cells (white, but not blue, indicated by the arrow) at the islet periphery of Ucn3-Cre x mT/mG mice are Ucn3-lineage negative (they express mTomato, instead of mGFP). These cells make up 0.75% ± 0.56% (n = 3) of all insulin-positive beta cells. See also Figure S2.

Figure 2.. Comparison of mature and immature beta cells from the same islets.

(A) View from two angles of a 3D reconstructed islet expressing mIns1-H2b-mCherry (all beta cells) and Ucn3-EGFP (mature beta cells only). Arrows indicate immature beta cells. See also Figure S2. (B) Immature beta cells (arrows) in the neogenic niche revealed by virtual slicing of a 3D reconstructed islet. See also Movie S1. (C) Imaging cytometer analysis of individual immature and mature beta cells. (D) FACS strategy to obtain Ucn3-eGFP mIns1-H2b-mCherry co-positive mature and mCherry single positive immature beta cells from the same islets. Immature beta cells start expressing Ucn3-eGFP in culture 12 hours after sorting (inset). (E) Correlation matrix of the 200 top differentially expressed (100 enriched, 100 depleted) genes between mature and immature cells. (F) Venn diagram comparing gene expression (RPKM>1 in either population) between mature and immature beta cells. Expression was considered different when the absolute log₂FC > 1 and FDR < 0.001. (G) Heat map of the most differentially expressed genes between mature and immature beta cells. (H) Expression of key genes in mature and immature beta cells by RNAseq. See also Figure S3. (I-L) Colocalization of insulin, Ucn3 and Mafa (I), Mafb (J), Pdx1 (K), or Nkx6–1 (L) in an adult mouse islet. Arrows indicate immature beta cells.

Figure 3.. Ucn3 negative beta cells are transcriptionally immature.

(A) Gene expression of select genes involved in insulin secretion in mature and immature beta cells by RNAseq. (B) Visualization of differential expression of the Kegg pathway analysis for insulin secretion (FDR < 0.001). (C) Heat map of the differential expression of tricarboxylic acid (TCA) cycle genes between immature and mature beta cells. (D) Heat map of the differential expression of genes involved in oxidative phosphorylation between immature and mature beta cells. (E) Gene expression of ‘disallowed’ genes in mature and immature beta cells by RNAseq. (F) Immunofluorescence detection of G6pc2 (white), insulin (red) and Ucn3 (green) in a mouse islet. Arrow indicates an immature beta cell. (G) Distribution of 2329 G6pc2-positive and negative beta cells within mouse islets (2329 cells). See Table S1B for the P values and D statistics for each pairwise comparison. (H) Immunofluorescence detection of Ero1lb (white), insulin (red) and Ucn3 (green) in a mouse islet. Arrows indicate examples of beta cells with Ero1lb but not Ucn3 (top) and with Ucn3 but not Ero1lb (bottom). (I) Distribution of 2043 Ero1lb-positive and negative beta cells within mouse islets (2073 cells).

Figure 4.. Ucn3 negative beta cells are functionally immature.

(A) Ucn3-negative beta cells at the periphery (arrows) do not express cell-surface Glut2. (B) Distribution of Glut2-negative beta cells at the periphery of the islet (6783 cells total). See Table S1B for the P values and D statistics for each pairwise comparison and Figure S4 for a comparison of immature beta cells with other heterogeneous populations of beta cells. (C) Uptake of the glucose analog 6-NBDG over time by all beta cells except for immature beta cells at the periphery of intact islets. (D) Quantification of 6-NBDG uptake. Numbered lines corresponds to panel C. Data represent mean ± SEM for 3 immature and 7 mature beta cells. * P < 0.05, *** P < 0.001. See also Movie S2. (E) Treatment with a high dose of the Glut2-dependent beta cell toxin streptozotocin ablates a majority of beta cells. Remaining Ucn3 lineage-positive beta cells are often insulin negative, while immature beta cells (arrows) survive owing to the lack of cell surface Glut2. (F) Ucn3 lineage-negative beta cells at the islet edge (arrows) in citrate controls. (G) STZ-induced death of Ucn3-eGFP x mIns1-H2b-mCherry mature beta cells in intact islets in real time. The death of beta cells is marked by the acute loss of eGFP protein and the nuclear uptake of the dead cell marker Sytox Blue. In contrast, mCherry single positive immature beta cells (numbered) do not take up Sytox Blue. Individual immature beta cells are labeled for clarity. Note that immature beta cells #4–6 disappear from the Z-stack as the islet volume expands due to the extensive cell death. See also Movie S3. (H) Immature beta cells cannot support calcium influx following depolarization. Representative traces of depolarization-induced inward calcium currents (circled) from mIns1-mCherry+ immature beta cells and Ucn3-eGFP positive mature beta cells from the same preparations. (I) Full Current-Voltage (I-V) plot contrasting the voltage-dependent inward calcium current in Ucn3-eGFP positive mature beta cells with the lack thereof in mIns1-mCherry+ immature beta cells from the same preparations. Data represent mean ± SEM for 4 immature and 8 mature beta cells from four individual animals. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5.. Ucn3-negative beta cells are present in human islets of young and adult donors and donors with T1D.

Expression of insulin, glucagon and Ucn3. Arrows indicate Ucn3-negative beta cells. (A) Neonate, female, no diabetes, nPOD #6200. (B) Infant, 5 months old, male, no diabetes, nPOD #6115. (C) Adult, 20 years old, male, no diabetes, nPOD #6238. (D) Adult, 26 years old, female, 15 years with diagnosed T1D, nPOD #6196. (E) Adult, 79 years old, female, 56 years with diagnosed T1D (medalist), nPOD #6065.

Figure 6.. Immature beta cells reflect a transient stage in the transdifferentiation between alpha and beta cells at the islet edge.

(A) A pair of cells at the islet periphery with an alpha lineage mark (arrows) that now express Ucn3 instead of glucagon. (B) Three potential scenarios that could account for the detection of beta cells with an alpha cell lineage-label and their predicted distribution across the cross-sectional islet area: 1) random labeling of beta cells by the ‘leaky’ expression of Cre recombinase in beta cells, 2) labeling of bi-hormonal progenitors during development is predicted to lead to randomly localized clusters of lineage-labeled beta cells that expanded from a single bi-hormonal progenitor, 3) transdifferentiation of alpha cells into beta cells at the periphery of adult islets. See also Figure S5. (C) Observed distribution of alpha lineage-labeled beta cells (3459 cells). See Table S1C for P values and D statistics for each pairwise comparison. (D) Alpha lineage-labeled islet, visualized by lsl-YFP, that features two transdifferentiated cells. One of these co-expresses Ucn3 and is mature (bottom, arrow), the other cell is an immature beta cell of alpha cell-descent that expresses insulin but not yet Ucn3 (top, arrow). (E) Lineage-labeling all alpha cells in 2-month old mice via Gcg-CreER followed by a 4-month chase demonstrates that alpha cells continue to transdifferentiate into beta cells. (F) The fraction of beta cells of alpha cell descent increased significantly four months after lineage-labeling all alpha cells, as measured relative to either total beta cell or alpha cell number. * P < 0.05, ** P < 0.01. (G) Conversely, the alpha cell fraction, which is nearly completely lineage-labeled immediately after tamoxifen administration, is notably diluted 4 months later by alpha cells without a lineage-label. (H) Islets from triple transgenic offspring of a cross between mIns1-mCherry x Gcg-Cre x lsl-YFP reveal the presence of mCherry positive beta cells that carry the YFP alpha cell lineage-label at the edge of the intact islet. Inset shows detail of the same cluster from different angle to emphasize the nuclear mCherry in transdifferentiated cells. See also Movie S4. (I) FACS strategy to isolate transdifferentiated cells along with alpha and beta cells from dissociated islets of mIns1-mCherry x Gcg-Cre x lsl-YFP triple transgenic islets. See also Figure S6. (J) Venn diagram of genes that are detectably expressed (RPKM > 1) among alpha, beta and transdifferentiated cells. Expression was considered different when the absolute log₂FC > 1 and FDR < 0.001. (K) Heat map of alpha, beta and transdifferentiated cells based on the most differentially expressed genes from panel 6J. (L) Expression of key genes in alpha, beta and transdifferentiated cells by RNAseq. Gene structure and chromosome number are indicated for each panel.

Figure 7.. Transdifferentiated cells are functionally mature

(A) Expression of arginine vasopressin (AVP) receptor Avp1rb in alpha, beta and delta cells. (B) Glucagon secretion in response to AVP. Data represent mean ± SEM; n = 4. *** P < 0.001. (C) Alpha cell calcium activity in response to brief stimulation with increasing doses of AVP, indicated by arrows. Depolarization (30 mM KCl) serves as positive control. See also Movie S5. (D) Coordinated beta cell calcium activity in response to continuous stimulation with 16.8 mM glucose. Arrow indicates the start of continuous stimulation. See also Movie S5. (E) Consecutive stimulation with a brief pulse of AVP (arrow) followed by continuous stimulation with 16.8 mM glucose in an islet where GCaMP6 expression is restricted to the alpha cell lineage and nuclear mCherry marks current beta cells. See also Movie S6. (F) Still images of key frames in at the indicated times in (E). Yellow ellipsoid contains conventional alpha cells; blue ellipsoid contains alpha-to-beta transdifferentiated cells. (G) Beta-to-alpha transdifferentiated cells revealed by the absence of Ucn3 and expression of glucagon in Ucn3-Cre lineage cells at the islet periphery. (H) Responses of two islets where expression of GCaMP6 is restricted to the alpha cell lineage (Gcg-Cre, top) or mature beta cell lineage (Ucn3-Cre; bottom). Stimulation protocol as in Figure 6E. Both islets were imaged simultaneously in a single recording. See also Movie S7. (I) Still images of key frames in (H).