Human islets contain four distinct subtypes of β cells

Abstract

Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing β cells causes diabetes mellitus, a disease that harms millions. Until now, β cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human β cells, which we refer to as β1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the β cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined β cell heterogeneity is functionally and likely medically relevant.

Figure 1. β cells are antigenically heterogeneous in normal and pathological islets.

Human islet samples were enzymatically dispersed and antibody labelled for flow cytometric analysis, and β cells were isolated by FACS with the sorting scheme illustrated in Supplementary Fig. 1. (a,b) Representative examples of CD9 versus ST8SIA1 expression on the purified β cell populations from two healthy islet donors. (c–j) CD9 versus ST8SIA1 expression on cells from eight islet samples collected from T2D patients. (k) Mean β cell subtype distribution (±s.d.) from 17 normal islet preps. Significant frequency variance (P<1 × 10⁻²⁴, single factor analysis of variance) was observed between these populations and β1 is significantly more frequent (P=8 × 10⁻⁴, 1.2 × 10⁻¹², 5 × 10⁻¹³ by t-test (unequal variance)) than any of the others and β2 was significantly more frequent than β3 or β4 (1 × 10⁻⁸, 2 × 10⁻⁹ by t-test (unequal variance)) as indicated with asterisks. (l) The mean percentage (±s.d.) of ST8SIA1⁺ β cells is abnormally high (P=0.028, t-test (unequal variance)) in T2D islets (n=8) compared with normal islets (n=17).

Figure 2. Clinical parameters and subset frequencies of human islet specimens.

(a) Donor information corresponding to islets obtained from 17 healthy and 8 diabetic donors. HbA1c, glycated haemoglobin; T2D, type 2 diabetes. Duration of cold ischaemia refers to the time of pancreatic cold storage in University of Wisconsin solution before islet isolation. (b) Unsupervised clustering of a subset of clinical parameters and the percentages of ST8SIA1⁺ β cells measured by FACS were visualized using Hierarchical Clustering Explorer 3.5.

Figure 3. β cell heterogeneity in normal pancreatic tissue sections.

(a) Human pancreas co-labelled with antibodies recognizing CD9 and proinsulin. Most proinsulin⁺ β cells were CD9⁻ (hollow arrows) but a subset was CD9⁺ (solid arrows). (b) Tissue co-labelled with antibodies recognizing ST8SIA1 and proinsulin. Most proinsulin⁺ β cells were ST8SIA1⁻ (hollow arrows) but a subset was ST8SIA1⁺ (solid arrows). (c) Compound labelling of proinsulin, CD9 and ST8SIA1 reveals proinsulin⁺ cells expressing every combination of the other markers; examples of β1 (CD9⁻ST8SIA1⁻), β2 (CD9⁺ST8SIA1⁻), β3 (CD9⁻ST8SIA1⁺) and β4 (CD9⁺ST8SIA1⁺) cells are indicated. Note that both CD9 and ST8SIA1 are found on proinsulin⁻ (non-β) cell types as well, as shown in Supplementary Fig. 1. (d) HCN1 was detected in a subset of proinsulin⁺ β cells; examples of high/positive and low/negative cells are indicated with solid and hollow arrows, respectively. Tissues illustrated are 5 μm cryosections of normal human pancreas. Scale bar, 25 μm.

Figure 4. Comparative gene expression in β cell subtypes.

RNA-seq data derived from β cells isolated from five different normal donors were normalized and compared with determine genes, which were differentially expressed. (a) All 23,292 transcripts for all samples arrayed as a heat map. (b) Known genes associated with insulin signalling/processing/secretion and β cell identity. (c) Genes differentially expressed between the ST8SIA1⁺ β3/β4 and ST8SIA1⁻ β1/β2 subtypes. (d) Genes differentially expressed between the CD9⁺ β2/β4 and CD9⁻ β1/β3 subtypes.

Figure 5. Functional characterization of β cell subtypes and islet cultures.

No significant differences in the mean levels of insulin RNA (a) and insulin protein (b) were found in the four β cell subtypes (n=5 and n=3 specimens, respectively; all values reported as mean±s.d.). (c) Insulin secretion (pmol of insulin per β cell per hour) were measured by incubation of reaggregated cells under basal (2.8 mM glucose) and stimulating (22.2 mM glucose) conditions. Basal levels were significantly different only between β1 and β4 (P=0.037, t-test (equal variance)), but glucose-stimulated insulin secretion (GSIS) was significantly higher in the β1 subtype than in any of the other β cell subtypes (β1 versus β2: P=0.045, β1 versus β3: P=0.042, β1 versus β4: P=0.005, t-test (equal variance)). The significant basal secretion difference is indicated with a double-asterisk (**) and the significant GSIS differences are indicated with a single asterisk (*). (d) Fold-change representation of the GSIS values reported in c. (e) Insulin secretion (pmol of insulin per islet per hour) is shown for intact human islets exposed to basal and stimulating glucose concentrations with or without HCN inhibition by ivabradine (30 mM). Basal insulin secretion was significantly increased (P=0.039, paired t-test) in the presence of ivabradine, and GSIS was significantly (P=0.003, paired t-test) reduced. The significant basal secretion difference is indicated with a double-asterisk (**) and the significant GSIS differences are indicated with a single asterisk (*). (f) Fold-change representation of the GSIS values reported in e. (g) Basal human c-peptide secretion (percentage of total content) from hES derived β-like cells. Six such lines were analysed in three experiments each. Basal insulin secretion was significantly (P=0.01, t-test (equal variance)) increased by exposure to ivabradine. (h) GSIS of hES-derived β-like cells (n=6). No significant change (P=0.12, t-test (equal variance)) was found with ivabradine.