Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets

Abstract

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and β-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase β-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, β-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and β-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal β-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and β-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

Figure 1

Schema of islet cell subset separation and frequencies of donor islet cell subsets. A: Islets are composed of a heterogeneous mixture of α-cells (GCG⁺), β-cells (INS⁺), and δ-cells (SST⁺). Islets are dissociated into a single-cell suspension (B), fixed and permeabilized to stain for intracellular hormone content (C), and sorted using a FACS (D) into distinct populations. E: Composition of endocrine islet populations of α- and β-cells differs from donor to donor.

Figure 2

β-Cell/α-cell ratios do not differ with age, sex, or BMI. The percentage of α-, β-, and δ-cells were calculated for 34 human islet samples, as in Fig. 1. The relative ratios of β-cells to α-cells were calculated for each donor (average 1.42) and plotted against age (A), sex (B), and BMI (C). Linear regression analysis was performed for plots A and C (dashed line). No difference in β-cell/α-cell ratio can be attributed to age (Pearson r = 0.17 [95% CI −0.17 to 0.48]) or sex (unpaired P value = 0.95 [95% CI for SEM −0.55 to 0.52]). There is a trend toward a negative correlation of β-cell/α-cell ratio with increased BMI, but the values are not significant (Pearson r −0.27 [95% CI −0.56 to 0.07]).

Figure 3

The α- and β-cell gene expression patterns: heat map analysis. The cluster and heat map diagram shows the relative gene expression (blue represents minimal expression; white shows neutral expression; red indicates high gene expression) for sorted fetal human β-cells (n = 6; INS⁺), adult human β-cells (n = 7; INS⁺), adult human α-cells (n = 6; GCG⁺), and fetal human α-cells (n = 5; GCG⁺) separated by population. The sample dendrogram (columns) was calculated using hierarchical clustering, while the genes (rows) were clustered using the k-means algorithm. The specific genes listed to the right of each cluster demonstrate high relative gene expression in the cluster-specific samples compared with expression levels in all other samples.

Figure 4

Positive isolation of islet cell populations from adult islets increases cell type purity. Dissociated islets were analyzed as in Supplementary Fig. 1. Scatter plots for each population are shown (INS⁺ cells, red; GCG⁺ cells, blue; SST⁺ cells, green; unlabeled cells, purple). Transcriptome sequencing and gene expression were computed in TPM as described. Hormone staining for INS and GCG only (A) showed increased amounts of other hormones in the purified populations (B). Triple hormone staining that removes SST⁺ cells from the INS⁺ and GCG⁺ populations (C) provides a more specific overall gene expression profile for each cell type (D). The gene rank for each hormone is number 1 in each respective cell type (INS number 1 in INS⁺ cells; GCG number 1 in GCG⁺ cells). When SST protein is gated out of the sorted cells, the gene rank drops from 22 (B) to 2,765 (D) in INS⁺ cells (red), and from 161 (B) to 4,184 (D) in GCG⁺ cells (blue).

Figure 5

α-Cells express INS mRNA. Dissociated islets were stained with an antibody to INS and with a PNA probe to INS mRNA. Two islet clusters are shown. A: Arrows indicate INS mRNA⁺ cells that are negative for INS protein staining. B: As an internal control, islet cells show INS mRNA and GCG protein (*), GCG protein without INS mRNA (arrow), and two cells without GCG protein or INS mRNA message (arrowheads). Dissociated islets were stained with antibodies to INS (C–G) and GCG (H–L), and with a PNA probe to INS mRNA. A representative β-cell is shown that is positive for INS protein and message, but negative for GCG protein (C and D). A representative GCG⁺ α-cell is shown that is negative for INS protein (H), but positive for INS mRNA (I). Control RIN-5F cells all stain positive for INS mRNA (M), negative for the control probe (N), and with markedly diminished intensity following RNase treatment (O).

Figure 6

Hormone protein expression in purified islet cell populations. A total of 3,000 cells were used for protein expression (INS, GCG, SST, β-actin, and GLUT1) determination via dot blot (representative image of three different replicates) as described. Staining was examined in whole islets; the three purified α-, β-, and δ-cell populations; and unlabeled cells, which did not stain for any of the three hormones. Each hormone is detected in the whole islet preparation, and its intensity increased within each respective purified cell population (INS in β-cells, GCG in α-cells, and SST in δ-cells).