Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

Abstract

Identification of cell-surface markers specific to human pancreatic β cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human β cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet β cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human β cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human β cells. Thus, NTPDase3 is a cell-surface biomarker of adult human β cells, and the antibody directed to this protein should be a useful new reagent for β cell sorting, in vivo imaging, and targeting.

Keywords: development; diabetes; glucagon; imaging; insulin; islet; pancreas.

Figure 1.. NTPDase3 is expressed specifically in adult human β-cells.

(A) Overview of NTPDase3 analysis and experimental applications. (B) Representative image of an islet in an adult pancreas (18y; donor N1 in Table S1) showing NTPDase3 expression in β-cells (insulin, INS). NTPDase3 is not expressed in adult α-cells (labeled for glucagon, GCG; C), PP cells (pancreatic polypeptide, PP; E), or acinar cells (amylase, AMY; E). A small percentage of δ-cells (somatostatin, SST; D) were also labeled by the NTPDase3 antibody, as indicated by the white arrowhead. Scale bars in panels B-F are 50 µm. Human pancreatic donor information is available in Table S1 (panels B-D: N1; panels E-F: N2).

Figure 2.. Pattern of NTPDase expression within human pancreas in disease and during postnatal development.

(A) NTPDase3 expression (red) is retained in β-cells (INS, green) from individuals with type 1 (T1D) and type 2 (T2D) diabetes. See also Figures S1B and S1D. (B) NTPDase3 has a different pattern of expression in the human pancreas at different stages of development; in top row, NTPDase3 is initially restricted to pancreatic epithelium and acinar cells, but by 5 years of age, acinar cell expression has ceased and only β-cells specifically express NTPDase3 (bottom row). See also Figures S1A and S1C. Scale bars in panels A and B are 50 µm unless otherwise indicated. Human pancreatic donor information is available in Table S1 (panel A: N1, N10, 1A, 1B, 2D, 2F; panel B: J2, J3, J5, J8, J15, J17, N1, N6).

Figure 3.. NTPDase3 antibody effectively and efficiently allows isolation of β-cells from live dispersed human islet cells.

(A) Experimental overview of islet dispersion and sorting. (B) Separation of α- and β-cell subpopulations by flow cytometry. Indirect antibody labeling was used to preselect endocrine cells (HPi1⁺) and subsequently identify α-cells (HPa3⁺) and β-cells (NTPDase3⁺). See also Figure S2 and Key Resources Table. (C) FACS-collected islet cells were transferred on glass slides by cytospin and assessed by immunocytochemistry. Two independent islet preparations are shown; scale bar is 50 µm. (D-E) RNA-sequencing analysis of purified human α- and β-cells from normal adult donors (n=5; ages 26–55 years). (D) Principal component analysis (PCA) plot shows clustering of α- and β-cell samples. (E) Heat map of a selected gene subset shows relative gene expression in individual α- and β-cell samples. (F) NTPDase3 expression is similar across four β-cell subsets (β1-β4) identified by Dorrell et al., 2016. Relative expression values are from associated dataset (GEO:GSE80780) and represent five individual donors (D1-D5). Human pancreatic donor information from this study is available in Table S1 (panel B: N3; panel C: N3, N9; panels D-E: N4, N5, N8, N9, N13).

Figure 4.. Detection of intravenously-injected NTPDase3 antibody in human islet graft beneath the kidney capsule of NSG mice.

(A) Experimental overview of human islet transplantation, antibody administration, and immunohistochemical visualization. (B) Macro view of islet graft (outlined in dashed white line) and surrounding kidney tissue. Anti-mouse-Cy3conjugated secondary antibody (red) shows NTPDase3 bound to human β-cells (INS, green). (C) Immunohistochemical detail of islet graft, as denoted by white box in (B). Scale bars in panels B and C are 50 µm. Transplanted islets are from a normal adult donor (N2; see Table S1).

Figure 5.. Targeting NTPDase3 detects human β-cells in vivo.

(A) Experimental overview of human islet transplantation, antibody administration, and imaging. (B) Bright-field images of islet grafts (outlined in white dashed lines) in the anterior chamber of the eye (ACE) of NSG mice receiving injections of DyLight550-conjugated antibody (left: isotype control, IgG2b-DyLight550; right: NTPDase3-DyLight550). Scale bar is 200 µm. (C) Fluorescence intensity in grafts of mice receiving injections of DyLight550-conjugated NTPDase3 antibody (n=7) or isotype control (n=5) was analyzed relative to background, ***, p=0.006 (t-test). (D) Immunohistochemistry on sections of islet grafts after removal from NSG mice. DyLight550 signal (first column from left, red) remained intact following graft removal and fixation. Secondary anti-mouse-Alexa488 antibody (second column from left; green) recognized bound NTPDase3 antibody. DyLight550 and Alexa488 signals co-localized with insulin labeling (INS, blue). Sections were counterstained with nuclear dye DAPI (white). Scale bars are 50 µm. Transplanted islets shown in panels B and D are from a normal adult donor (n=2 mice each, IgG2b-DyL550 and NTPDase3-DyL550). Complete donor information is available in Table S1 (panels B, D: N14; panel C: J18, N7, N14). See also Figure S3.