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. Author manuscript; available in PMC: 2020 Mar 5.
Published in final edited form as: Cell Metab. 2018 Nov 15;29(3):745–754.e4. doi: 10.1016/j.cmet.2018.10.007

Ectonucleoside triphosphate diphosphohydrolase-3 antibody targets adult human pancreatic β-cells for in vitro and in vivo analysis

Diane C Saunders 1,13, Marcela Brissova 2,13, Neil Phillips 2,13, Shristi Shrestha 3, John T Walker 1, Radhika Aramandla 2, Greg Poffenberger 2, David K Flaherty 4, Kevin P Weller 4, Julie Pelletier 5, Tracy Cooper 6, Matt T Goff 6, John Virostko 7, Alena Shostak 2, E Danielle Dean 2, Dale L Greiner 8, Leonard D Shultz 9, Nripesh Prasad 3, Shawn E Levy 3, Robert H Carnahan 6,10, Chunhua Dai 2, Jean Sévigny 5,11, Alvin C Powers 1,2,12,14
PMCID: PMC6402969  NIHMSID: NIHMS1510722  PMID: 30449685

SUMMARY

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 (T1D, T2D). 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 emerges 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.

Graphic abstract

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eTOC BLURB

Identification of human pancreatic β-cell-specific markers is important to advance our knowledge of human β-cell development and physiology. XXX et al identify NTPDase3 as a highly enriched biomarker expressed on adult human β-cells and show that an NTPDase3 antibody can be used for in vivo live β-cell sorting and imaging.

INTRODUCTION

Diabetes is a major health concern worldwide, and its growing prevalence poses an increasing demand for effective therapeutic and preventative approaches. The disease is broadly classified as type 1 or type 2, with both types characterized by dysfunction or destruction of insulinsecreting β-cells of the Islets of Langerhans. The islet functions as a mini-organ, and its heterogeneous composition and anatomical location pose challenges particularly to identifying characteristics of individual cell subtypes in normal or disease states. Because of this, several groups have developed systems to isolate subpopulations of islet cells for transcriptional, metabolic, and functional analyses. These isolations are generally accomplished by dispersing and sorting cells using antibodies that target either cell surface antigens on live cells or intracellular proteins in fixed, permeabilized cells (Blodgett et al., 2015; Bramswig et al., 2013; Dorrell et al., 2011b). Other groups have alternatively sorted β-cells based on zinc content, using the zinc-binding fluorochrome Newport Green (Parnaud et al., 2008). Each approach has advantages and drawbacks, but one critical limitation is the lack of a cell surface antibody that specifically targets adult human β-cells.

In addition to analyzing human β-cells ex vivo, there is great need to identify and visualize β-cells non-invasively in vivo. Although reduced β-cell mass is an accepted feature of diabetes progression, current knowledge has originated from post-mortem analysis since there are no effective methods to quantify β-cell mass non-invasively in humans (Alavi and Werner, 2018; Laurent et al., 2015). This limitation has greatly hindered our understanding of disease risk and progression and prevents the evaluation of interventions designed to preserve or increase β-cell mass. Many traditional imaging modalities lack necessary sensitivity for the small size and sparse distribution of islets, and new reagents are needed that distinguish β-cells from other endocrine cells and neighboring exocrine tissue (Sweet et al., 2004). Antibodies typically have greater specificity and affinity than other molecules such as peptides; however, multiple antibodies targeting islet cell surface antigens have unsuccessfully been tried for islet imaging applications in vivo (Laurent et al., 2015; Olafsen and Wu, 2010).

In efforts to identify cell surface markers of human β-cells, we examined expression of nucleoside triphosphate diphosphoyhydrolase-3 (NTPDase3), an enzyme localized to the endocrine islet in the human pancreas, using a monoclonal antibody (Lavoie et al., 2010; Munkonda et al., 2009). NTPDase3 (encoded by gene ENTPD3) is a member of the ENTPDase family, which is comprised of eight members. The function of NTPDase3 is to modulate local extracellular nucleotide levels, thereby affecting availability of ligands that mediate nucleotide signaling (Antonioli et al., 2015; Robson et al., 2006; Zimmermann et al., 2012). The ectonucleotidase NTPDase3 has been mainly studied in the rodent brain, where it is proposed to help regulate synaptic function (Belcher et al., 2006). There is very limited information about NTPDase3 protein distribution in humans, but the NIH Genotype-Tissue Expression (GTEx) data set shows that in addition to the pancreas, NTPDase3 mRNA is primarily expressed in the brain, salivary gland, and urinary bladder. Of particular interest to the current study, NTPDase3 is detectable by immunohistochemistry in pancreatic islets of both human and rodents, and recent work has suggested activity in regulating insulin secretion (Lavoie et al., 2010; Petit et al., 2009; Syed et al., 2013). Our work shows that NTPDase3 is a cell surface biomarker of adult human β-cells and the antibody directed to this protein could be developed for multiple applications including β-cell sorting, in vivo imaging, and targeting.

RESULTS AND DISCUSSION

NTPDase3 is highly expressed in adult pancreatic β-cells

To characterize expression of NTPDase3 (Figure 1A), we performed immunohistochemistry on pancreatic tissue sections from human donors (n=32, age range of 0 – 64 years; see Table S1) and found NTPDase3 expression on the membrane of most adult β-cells, but importantly, not in adult α-cells (Figure 1B and 1C). No colocalization of NTPDase3 with pancreatic polypeptide (PP) hormone (Figure 1E) or amylase (exocrine enzyme; Figure 1F) was observed, though a small number of somatostatin-expressing δ-cells did express NTPDase3 (Figure 1D). These results differ from a prior report in mice where NTPDase3 was found to colocalize with markers of all islet cells (α-, β-, δ-, and PP cells) by immunohistochemistry (Lavoie et al., 2010). Significantly, NTPDase3 expression was preserved in β-cells from individuals with T2D and in residual β-cells from individuals with T1D (Figures 2A and S1B-D). Though glucose-stimulated insulin secretion was reduced in T2D islets (Figure S1D), this reduction in β-cell activity did not affect NTPDase3 expression (Figure S1C), in agreement with transcriptome data from a previous study of T2D islets (Segerstolpe et al., 2016). The identity and role of the few NTPDase3-negative β-cells will be of future interest.

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

Figure 1.

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(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.

Figure 2.

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(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).

Interestingly, although the NTPDase3 antibody labeled adult human β-cells with high specificity, its expression during early pancreas development, especially the first few years of life, is quite dynamic. We did not detect NTPDase3 expression in human β-cells at 15 or 18 weeks of gestation (G18w, Figure 2B; see also Figure S1A and S1C), but NTPDase3 was expressed in developing pancreatic epithelium. During the neonatal period (birth – 3 months), human acinar cells, but only a few β-cells, expressed NTPDase3 (4d and 2mo, Figure 2B; see also Figure S1A and S1C). In infancy, between 7 months and 2 years of age, NTPDase3 expression began to decline in acinar cells while concurrently emerging in an increasing proportion of β-cells; this transition was heterogeneous among individuals and even in islets in the same pancreas.

During this age range, both acinar cells and β-cells expressed NTPDase3 (Figure S1A and S1C). By early childhood, around 5 years of age, acinar expression was completely absent and

NTPDase3 was only expressed in β-cells (5y – 35y, Figure 2B; see also Figure S1A and S1C).

Based on the expression pattern of NTPDase3 and current knowledge of postnatal β-cell development, we postulate that NTPDase3 could play an important role in the acquisition of β-cell functional maturity. As modulators of extracellular ATP, NTPDases directly impact purinergic signaling pathways controlling processes like glucose-stimulated insulin secretion (Khan et al., 2014; Lavoie et al., 2010; Petit et al., 2009; Silva et al., 2008; Wuttke et al., 2013). Additionally, NTPDase3 could be shifting an ATP-driven proinflammatory islet environment to a more anti-inflammatory milieu by modulating the purinergic signals delivered to immune cells through the conversion of β-cell-derived ATP/ADP to AMP (Antonioli et al., 2013; Deaglio and Robson, 2011). Furthermore, the cellular expression of specific purinergic receptor subtypes appears to change during rodent pancreatic development and also under diabetic conditions, suggesting that NTPDase expression may be important for both β-cell function and islet inflammation (Antonioli et al., 2013; Coutinho-Silva et al., 2001; 2003; Deaglio and Robson, 2011). Future studies are necessary to explore the relationship between NTPDase3 expression and β-cell functional maturity.

NTPDase3 antibody enables sorting of live human β-cells

To investigate the utility of NTPDase3 as a biomarker for mature β-cells, we developed a cell sorting strategy to label live human islet cells (Figure 3A). Use of the NTPDase3 antibody in combination with previously characterized cell surface markers (Bramswig et al., 2013; Dorrell et al., 2008) enabled effective separation of α- and β-cell subpopulations (Figure 3B). Importantly, this method was applicable to islet cells from a variety of physiologic states, including adolescence (Figure S2B), early-onset T1D (Figure S2C), T2D (Figure S2D), and a form of monogenic diabetes (MODY3) (Figure S2E) (Haliyur et al., 2018). We validated the purity of our subpopulations using two complementary approaches, immunocytochemistry and RNA sequencing (RNA-seq). Based on hormone expression (insulin, glucagon, somatostatin), β- and α- cell populations were enriched to 96% and 98% purity, respectively, from a dispersed islet preparation composed of many disparate cell types (Figure 3C). Transcripts of α-cell-specific genes (e.g. ARX, HNF4A) were highly expressed in α-cell samples and minimally expressed in β-cell samples; the inverse was true of β-cell-specific genes (e.g. GLP1R, PDX1), which were detected at high levels only in β-cell samples (Figure 3D and 3E). Genes common to both α- and β-cells (e.g. ISL1, PAX6) showed similar abundance in both cell types. NTPDase3 transcript (ENTPD3) was approximately 10-fold higher in β-cells than α-cells, in agreement with previously published data sets (Bramswig et al., 2013; Segerstolpe et al., 2016). NTPDase3 expression levels were similar in all four β-cell subtypes previously identified by Dorrell et al., 2016 (Figure 3F), indicating that the small NTPDase3-negative β-cell population identified by immunohistochemistry does not correlate with an existing β-cell subtype.

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

Figure 3.

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(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).

Prior studies have relied on isolating live β-cells based on cell surface markers through exclusion of other cell types (Bramswig et al., 2013; Dorrell et al., 2016) or they used paraformaldehyde-fixed and permeabilized islet cell preparations with intracellular insulin as a β-cell marker (Blodgett et al., 2015) to achieve a higher cell purity. Consistent with immunohistochemical analysis, there were a small number of δ-cells found in our sorted NTPDase3+ population (<1%; Figure 3C), indicating that this method of β-cell isolation should be used with caution in instances where the δ-cell population may be elevated. Nonetheless, the addition of NTPDase3 as a positive selection marker for β-cells importantly enhances the current cell sorting strategy for human islets. Combined with highly specific cell surface markers for endocrine and α-cells (Dorrell et al., 2016; 2011b; 2008), it offers a new and improved method to yield highly pure β-cells which should considerably improve the accuracy of their transcriptional profiling without compromising membrane integrity as required for sorting with intracellular insulin.

Targeting NTPDase3 in live human β-cells opens several new avenues for investigation and discovery. Isolation of viable β-cell populations with greater purity should enable physiological experiments requiring live β-cells, including electrophysiology, high-resolution imaging, and formation of β-cell-enriched pseudoislets. This method should also allow chromatin immunoprecipitation (CHIP)-seq and assay for transposase-accessible chromatin sequencing (ATAC-seq) experiments on human β-cells.

NTPDase3 antibody detects human β-cells in vivo

Since the NTPDase3 antibody allowed the sorting of live human β-cells, we then tested whether it could detect human β-cells in vivo. To accomplish this, we utilized immunodeficient mice bearing human islets engrafted either under the kidney capsule or in the anterior chamber of the eye (ACE). When mice received an intravenous injection of unlabeled NTPDase3 antibody and grafts were removed twenty-four hours later, visualization with a secondary antibody revealed that NTPDase3 bound β-cells in vivo with high specificity (Figure 4). We next conjugated the NTPDase3 antibody to DyLight550, injected the labeled antibody, and imaged the mice twenty-four hours post-injection (Figures 5A and S3). We observed fluorescent signal in the human islet grafts that was not present with an isotype control antibody (Figure 5B). Immunohistochemistry on removed islet grafts confirmed this highly specific binding of the conjugated NTPDase3 antibody to human β-cells (Figure 5C). These data indicate that the antibody circulates, exits the vascular space, and reaches engrafted islet cells, where the NTPDase3 epitope is exposed on the β-cell surface in vivo. Such results suggest that the epitope would be accessible in the native pancreas, which is important for imaging applications.

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

Figure 4.

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(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.

Figure 5.

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(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.

Our use of transplanted human islets notably expands the relevance of NTPDase3 as a potential in vivo imaging tool using radiolabeled ligands targeting NTPDase3. The ability to quantify β-cell mass in vivo would also improve evaluation of clinical outcomes, ranging from assessment of islet transplantation to recovery of endogenous β-cell mass through pharmacological intervention. In determining the ability of NTPDase3-directed imaging to assess β-cell mass in endogenous islets, whole-body analysis of NTPDase3 expression will be important. Since there is limited information about NTPDase3 expression in humans and expression in mouse and human tissues may differ, addressing this question will require future human investigation. The antibody used in this study is highly specific to the human NTPDase3 isoform and does not cross-react with the mouse protein (Munkonda et al., 2009), which is why we elected to use the human islet transplant model. We note that limited expression in other tissues does not prevent use for in vivo β-cell imaging since there are approaches to co-register a NTPDase3 signal with other imaging modalities such as magnetic resonance imaging (MRI) or computed tomography (CT) to determine a pancreas-specific signal and differentiate between signals coming from multiple locations. Such a co-registration approach has been used for positron emission tomography (PET) imaging of brain, adrenal, and thyroid and for pancreatic islet imaging (Brom et al., 2018; Virostko et al., 2011). The current results provide key evidence of in vivo imaging characteristics lacked by other imaging agents (Eriksson et al., 2016) and may enhance the understanding of β-cell mass dynamics, which has not been possible to study in humans. Additionally, in vitro analysis of T1D and T2D tissues suggests that NTPDase3 expression is not altered under pathophysiological conditions, a desirable quality of agents aimed at quantifying β-cell mass.

Thus, these findings of NTPDase3 expression in mature human β-cells should provide opportunities to advance the knowledge of human β-cell development, physiology, and regulation of β-cell mass.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Alvin C. Powers (al.powers@vanderbilt.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

Immunodeficient 10–12 week old NOD-scid-IL2rγnull (NSG) male mice (Jackson Laboratory, Bar Harbor, ME) were used for human islet transplantation studies. Animals were maintained by Vanderbilt Division of Animal Care in group-housing in sterile containers within a pathogen-free barrier facility housed with a 12hr light/12hr dark cycle and access to free water and standard rodent chow. All animal procedures were approved from by the Vanderbilt Institutional Animal Care and Use Committees.

Primary cell cultures

Primary human islets were cultured in CMRL 1066 media (5.5 mM glucose, 10% FBS, 1% Pen/Strep, 2 mM L-glutamine) in 5% CO2 at 37°C for 24–72 hours prior to reported studies. Function of islets from normal adult controls and T2D donors (Table S1) was assessed using a dynamic cell perifusion system and radioimmunoassay as described previously (Kayton et al., 2015).

Human subjects

Pancreata and islets from juvenile, adult, T1D, T2D, and MODY donors were obtained through partnerships with the Alberta Diabetes Institute (ADI), International Institute for Advancement of Medicine (IIAM), National Disease Research Interchange (NDRI), Integrated Islet Distribution Program (IIDP), Network for Pancreatic Organ Donors with Diabetes (nPOD), and local organ procurement organizations. Most pancreata from normal donors were processed either for islet isolation (Balamurugan et al., 2003) or histological analysis (described below). In some normal and T2D pancreatic organs, islets and tissue specimens were procured from the same organ (Table S1). The Vanderbilt University Institutional Review Board declared studies on deidentified human pancreatic specimens does not qualify as human subject research.

METHOD DETAILS

Human pancreas procurement, islet isolation, and preparation of tissue for histological analysis

Pancreata and islets from juvenile, adult, T1D, T2D, and MODY3 donors were obtained through partnerships with the Alberta Diabetes Institute (ADI), International Institute for Advancement of Medicine (IIAM), National Disease Research Interchange (NDRI), Integrated Islet Distribution Program (IIDP), Network for Pancreatic Organ Donors with Diabetes (nPOD), and local organ procurement organizations within 18 hours from cold clamp as described previously (Balamurugan et al., 2003; Brissova et al., 2018). Most pancreata from normal donors were processed either for islet isolation (Balamurugan et al., 2003) or histological analysis as described previously (Balamurugan et al., 2003; Brissova et al., 2018). Donor demographic information is summarized in Table S1. The Vanderbilt University Institutional Review Board does not classify de-identified human pancreatic specimens as human subject research. Pancreatic organs were processed for islet isolation in Pittsburgh using an approach previously described (Balamurugan et al., 2003; Brissova et al., 2018) and shipped to Vanderbilt University for further analysis following shipping protocols developed by the IIDP. Assays using isolated islets were performed at Vanderbilt within 48 hours of islet arrival.

Immunohistochemical analysis

Immunohistochemical analysis of pancreas was performed on serial 5-µm cryosections as previously described (Brissova et al., 2014). Primary antibodies to all antigens and their working dilutions are listed in the Key Resources Table. The antigens were visualized using appropriate secondary antibodies listed in the Key Resources Table. Digital images were acquired with Zeiss LSM510 META and LSM880 laser scanning confocal microscopes (Zeiss Microscopy Ltd, Cambridge, UK).

KEY RESOURCES TABLE.
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Goat anti-Somatostatin (1:500) Santa Cruz Cat# sc-7819
RRID:AB_2302603
Guinea pig anti-Insulin (1:500) Dako Cat# A0564
RRID:AB_2617169
Rabbit anti-α-Amylase (1:1000) Sigma Cat# A8273
RRID:AB_258380
Rabbit anti-Glucagon (1:100) Cell Signaling Cat# 2760
RRID:AB_659831
Rabbit anti-Pancreatic Polypeptide (1:1000) Peninsula Laboratories Cat# T-4088
RRID:AB_518533
Mouse anti-CD39L3 IgG2b (NTPDase3; 1:50) http://ectonucleotidases-ab.com and Vanderbilt Antibody and Protein Resource Cat# hN3-B3S and
hN3-H10S
Mouse anti-HIC3–2D12 (HPa3; 1:200; flow cytometry) Gift from Drs. Philip Streeter and Markus Grompe N/A
Mouse anti-HIC0–4F9-Biotin (HPi1; 1:100; flow cytometry) Novus Cat# NBP1–18872B
RRID:AB_2126328
Mouse IgG2b Isotype Control-DyLight550 Novus Cat# NBP2–27231R
Donkey anti-goat-Cy5 (1:200) Jackson ImmunoResearch Cat# 705–175-147
RRID:AB_2340415
Donkey anti-Guinea pig-Cy2 (1:500) Jackson ImmunoResearch Cat# 706–225-148
RRID:AB_2340467
Donkey anti-Guinea pig-Cy3 (1:500) Jackson ImmunoResearch Cat# 706–165-148
RRID:AB_2340461
Donkey anti-Guinea pig-Cy5 (1:200) Jackson ImmunoResearch Cat# 706–175-148
RRID:AB_2340462
Donkey anti-mouse-Alexa488 (1:500) Jackson ImmunoResearch Cat# 715–545-150
RRID:AB_2340846
Donkey anti-mouse-Cy2 (1:500) Jackson ImmunoResearch Cat# 715–225-150
RRID:AB_2340826
Donkey anti-mouse-Cy3 (1:500) Jackson ImmunoResearch Cat# 715–165-150
RRID:AB_2340813
Donkey anti-mouse-Cy5 (1:200) Jackson ImmunoResearch Cat# 715–175-151
RRID:AB_2340820
Donkey anti-rabbit-Cy2 (1:500) Jackson ImmunoResearch Cat# 711–225-152
RRID:AB_2340612
Donkey anti-rabbit-Cy5 (1:200) Jackson ImmunoResearch Cat# 711–175-152 RRID:AB_2340607
Goat anti-mouse-PE (1:1000; flow cytometry) BD Biosciences Cat# 550589
RRID:AB_393768
Goat anti-mouse-APC (1:500; flow cytometry) BD Biosciences Cat# 550826
RRID:AB_398465
Streptavidin BV421 (1:500; flow cytometry) BD Biosciences Cat# 563259
Biological Samples
Human pancreatic islets Alberta Diabetes Institute http://www.ualberta.ca/alberta-diabetes/facilities/core-services/isletcore
Human pancreatic islets Integrated Islet Distribution Program (IIDP) http://iidp.coh.org; RRID:SCR_014387
Human pancreatic islets Network for Pancreatic Organ Donors with Diabetes (nPOD) www.jdrfnpod.org; RRID:SCR_014641
Human pancreatic tissue International Institute for the Advancement of Medicine (IIAM) http://www.iiam.org; RRID:SCR_016172
Human pancreatic tissue National Disease Research Interchange (NDRI) http://ndriresource.org; RRID:SCR_000550
Chemicals, Peptides, and Recombinant Proteins
Propidium Iodide BD Biosciences Cat# 556463
D-(+)-Glucose Sigma Cat# G7528
Collagenase NB1 Premium Grade Crescent Chemical Cat# 17455
Neutral Protease NB1 Premium Grade Crescent Chemical Cat# 30301
DNase I Worthington Biochemical Corporation Cat# LS006333
RPMI 1640 with L-glutamine Mediatech Cat# 99–595-CM
Biocoll-Separating Solution, Density 1.10 g/mL Cedarlane Cat# L6155
Biocoll-Separating Solution, Density 1.077 g/mL Cedarlane Cat# L6113
Fetal Bovine Serum Gibco Cat# 16–140-071
Dithizone Sigma Cat# D5130
CMRL 1066 Medium Mediatech Cat# 99–663-CV
Penicillin/Streptomycin mix Gibco Cat# 15140163
L-Glutamine Gibco Cat# 2030–081
16% Paraformaldehyde Electron Microscopy Sciences Cat# 15710
O.C.T. compound Fisher Scientific Cat# 4585
Critical Commercial Assays
Insulin Radioimmunoassay Millipore Cat# RI-13K
RNAqueous®-Micro Kit Fisher Scientific (Invitrogen) Cat# AM1931
Deposited Data
RNA-seq data for FACS purified human beta cell subsets (Dorrell et al., 2016) NCBI Gene Expression Omnibus GEO:GSE80780
RNA-seq data for FACS purified human control and T1D alpha cells NCBI Gene Expression Omnibus GEO:GSE106148
RNA-seq data for FACS purified human beta cells NCBI Gene Expression Omnibus GEO:GSE116559
Experimental Models: Organisms/Strains
Mouse: NOD-scid-IL2rγnull (NSG) Jackson Laboratory http://www.jax.org/strain/005557
Software and Algorithms
TopHat (v2.1) Trapnell et al., 2009 http://tophat.cbcb.umd.edu;
RRID:SCR_013035
Avadis NGS analysis Platform Strand life Sciences, Bengaluru http://www.avadis-ngs.com; RRID:SCR_000644
Trimmed Mean of M-values (TMM) algorithm Dillies et al., 2013
Robinson et al., 2010
Prism v.7.0 Graphpad Software http://www.graphpad.com;
RRID:SCR_002798
Zeiss LSM Imaging Software (confocal) Carl Zeiss http://www.zeiss.com;
RRID:SCR_014344
cellSens Olympus http://www.olympus-lifescience.com/en/software/cellsens; RRID:SCR_016238
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α- and β-cell sorting by flow cytometry

Human islets from five normal donors (ages 26–55 years, BMI 24–36) were dispersed using a modified protocol as described previously (Aamodt et al., 2016; Brissova et al., 2018). Briefly, 0.025% trypsin was used to disperse cells and reaction was quenched with modified RPMI medium (10% FBS, 1% Pen/Strep, 5 mM glucose). Cells were washed in the same medium and counted on a hemocytometer, then transferred to FACS buffer (2 mM EDTA, 2% FBS, 1X PBS). Indirect antibody labeling was completed via two sequential incubation periods at 4°C, with one wash in FACS buffer following each incubation. Primary (HPi1, HPa3, NTPDase3) and secondary antibodies, listed in the Key Resources Table, have been characterized previously and used to isolate high-quality RNA from α- and β-cells (Bramswig et al., 2013; Dorrell et al., 2011b; 2011a). Appropriate single color compensation controls were run alongside samples. Prior to sorting, propidium iodide (0.05 ug/100,000 cells; BD Biosciences, San Jose, CA) was added to samples for non-viable cell exclusion. Flow analysis was performed using an LSRFortessa cell analyzer (BD Biosciences), and a FACSAria III cell sorter (BD Biosciences) was used for FACS. Analysis of flow cytometry data was completed using FlowJo 10.1.5 (FlowJo LLC, Ashland, OR). Gating strategy is shown in Figure S3. Where indicated, a subset of collected cells was washed 2X in PBS and collected onto a Plus Gold slide (Fisher Scientific, Waltham, MA) using a Cytospin 4 cytocentrifuge (Thermo Fisher, Waltham, MA), then fixed in 4% PFA for 10 minutes prior to immunocytochemical analysis.

RNA-sequencing

Sorted α- and β-cells (9,000–123,000) were stored in 200 µL lysis/binding solution (Ambion, Foster City, CA) at −80°C prior to RNA isolation us ing the RNAqueous micro-scale phenol-free total RNA isolation kit (Ambion). Trace DNA was removed with TURBO DNA-free (Ambion), RNA integrity was evaluated (Agilent 2100 Bioanalyzer, Santa Clara, CA; 8.11±0.34 RIN, n=10), and high-integrity total RNA was amplified (Ovation system; NuGEN Technologies, San Carlos, CA) per standard protocol as described previously (Brissova et al., 2014). Amplified cDNA was sheared to target 200bp fragment size and libraries were prepared using NEBNext DNA Library Prep (New England Biolabs, Ipswich, MA). 50bp Paired End (PE) sequencing was performed on an Illumina HiSeq 2500 (Illumina, San Diego, CA) using traditional Illumina methods (Malone and Oliver, 2011) to generate approximately 50 million reads per sample. Raw reads were mapped to the reference human genome hg19 using TopHat v2.1 (Trapnell et al., 2009). Aligned reads were then imported onto the Avadis NGS analysis platform (Strand Life Sciences, Bengaluru, India) and filtered based on read quality followed by read statistics to remove duplicates. Transcript abundance was quantified using the TMM (Trimmed Mean of M-values) algorithm (Dillies et al., 2013; Robinson and Oshlack, 2010) as the normalization method.

Islet transplantation into immunodeficient mice

Immunodeficient 10–12 week old NOD.Cg-Prkdcscid IL2rgtm1Wjl/ SzJ (NSG) male mice (Jackson Laboratory) were used for human islet transplantation studies. Animals were maintained by Vanderbilt Division of Animal Care in group housing in sterile containers within a pathogen-free barrier facility housed with a 12hr light/12hr dark cycle and access to free water and standard rodent chow. All animal procedures were approved from by the Vanderbilt Institutional Animal Care and Use Committees. Human islets from four normal donors (ages 10–61 years, BMI 2535) were obtained through ADI, IIDP, NDRI, and nPOD (Table S1). For each set of islets, 3–5 mice were transplanted with 100 islet equivalents each into the anterior chamber of the eye (ACE) or with 2,000–4,000 islet equivalents beneath the kidney capsule as described previously (Abdulreda et al., 2013; Brissova et al., 2004; Speier et al., 2008a; 2008b). Islets were allowed to engraft for 1 month prior to imaging.

In vivo imaging

Mice were anesthetized using a standard dose of isoflurane and injected with 100 µL of saline containing 30 µg of DyLight550-conjugated NTPDase3 antibody (Vanderbilt Antibody and Protein Resource) or isotype control (Novus International, St. Louis, MO) into the right retroorbital space. Twenty four hours post-antibody injection, mice were anesthetized and placed on the microscope stage using stereotactic instruments (Narishige Co., Tokyo, Japan) with left eye facing up and oriented to reveal islet graft (visible under white light). Images showing the ACE bearing grafted islets were obtained using an Olympus SZX12 stereo zoom microscope equipped with a DP80 camera and fluorescence (Olympus, Tokyo, Japan). DyLight550 fluorescence intensity of grafts was quantified by digital image analysis using cellSens software (Olympus). Following imaging, mice were sacrificed and grafts were removed and fixed according to the standard protocol for pancreas fixation (described above).

QUANTIFICATION AND STATISTICAL ANALYSIS

Measurement of fluorescence intensity

Fluorescence intensity in grafts of mice receiving injections of DyLight550-conjugated NTPDase3 antibody or isotype control was analyzed relative to background using cellSens software (Olympus). The cellSens software was also utilized to assess relative fluorescence intensity of NTPDase3 in islets versus exocrine tissue in order to follow NTPDase3 distribution in islets and exocrine tissue during postanal development and in T1D and T2D.

Statistical analysis

Data were expressed as mean ± standard error of mean. A p-value less than 0.05 was considered significant. Statistical analysis (unpaired t-test for two groups; one-way ANVOA and Dunnett’s multiple comparisons test for three or more groups) was performed using GraphPad Prism software. Statistical details of experiments are described in the Figure Legends and Results.

DATA AND SOFTWARE AVAILABILITY

Sequencing datasets

The sequencing data from NTPDase3-based sorting methods is available under GEO accession numbers GSE106148 (α-cells) and GSE116559 (β-cells). Sequencing data using existing cell surface antibodies (Dorrell et al., 2016) is available under GEO accession number GSE80780.

Supplementary Material

1

Limitations of Study.

  • There is a small percentage of β-cells that do not express NTPDase3. Additionally, NTPDase3 antibody labels a subset of δ-cells, so other reagents may be necessary to exclude these cells from sorted subpopulations.

  • Since β-cells from very young donors (less than 2 years of age) do not express NTPDase3, the antibody will not be useful in sorting islet cells at this age. However, the dynamic pattern of NTPDase3 expression in acinar cells during the early neonatal period warrants future investigation.

  • Additional work is required to determine whether the NTPDase3 antibody will have sufficient signal-to-noise ratio to achieve the resolution necessary for β-cell imaging in vivo in the native human pancreas.

HIGHLIGHTS.

  • NTPDase3 is a marker highly expressed in adult human pancreatic β-cells

  • NTPDase3 expression is maintained in β-cells from individuals with diabetes

  • Use of an NTPDase3 antibody enables isolation of live β-cells from human islets

  • NTPDase3 antibody also allows detection of human β-cells by in vivo imaging

ACKNOWLEDGEMENTS

This research was performed using resources and/or funding provided by the NIDDK-supported Human Islet Research Network (HIRN, RRID:SCR_014393; http://hirnetwork.org; UC4 DK104211, DK108120, DK104218, DK112232), by DK94199, DK106755, DK72473, DK89572, DK97829, F30DK118830, and the Vanderbilt Diabetes Research and Training Center (DK20593), and by grants from the JDRF (15–68-Q-R, 17–2013-321, 17–2013-324), The Leona M. and Harry B. Helmsley Charitable Trust (2017PG-T1D017), and the Department of Veterans Affairs (BX000666). This research was also performed with the assistance of the NIDDK-funded Integrated Islet Distribution Program (IIDP, RRID:SCR_014387; http://iidp.coh.org; 2UC4DK098085) and the Network for Pancreatic Organ Donors with Diabetes (nPOD, RRID:SCR_014641; www.jdrfnpod.org), a collaborative type 1 diabetes research project sponsored by JDRF (5-SRA-2018–557-Q-R) and The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053). We acknowledge the use of tissues procured by the NIH-supported National Disease Research Interchange (NDRI, RRID:SCR_000550; U42OD11158) and the International Institute for Advancement of Medicine (IIAM, RRID:SCR_016172), who partner with Organ Procurement Organizations in the United States. We are especially thankful to organ donors and their families.

Imaging was performed through use of the Vanderbilt Imaging Shared Resource (CA68485, DK20593, DK58404, DK59637, EY08126) and the Islet Procurement and Analysis Core of the Vanderbilt Diabetes Research and Training Center (DK20593). Flow Cytometry experiments were performed in the VMC Flow Cytometry Shared Resource, supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404). NTPDase3 antibody from J.S. (Munkonda et al., 2009) was produced, purified, and labeled by the Vanderbilt Antibody and Protein Resource, supported by the Vanderbilt Institute of Chemical Biology and the Vanderbilt Ingram Cancer Center (P30 CA68485). J.S. received support from the Canadian Institutes of Health Research (CIHR) and was the recipient of a “Chercheur National” research award from the Fonds de recherche du Québec – Santé (FRQS). We thank Drs. Alejandro Caicedo, Rayner Rodriguez-Diaz, and Midhat H. Abdulreda at the University of Miami for education and training on transplantation into the anterior chamber of the eye.

Footnotes

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DECLARATION OF INTERESTS

The NTPDase3 antibody used in the manuscript is available for purchase (http://ectonucleotidases-ab.com) by an entity controlled by one of the co-authors (J.S.). Any funds generated from sale of this reagent are used to pay for the cost of antibody production; any remaining funds are reinvested to generate and characterize new antibodies. J.S. declares no personal conflict of interest. The other authors declare no competing interests.

REFERENCES

  1. Aamodt KI, Aramandla R, Brown JJ, Fiaschi-Taesch N, Wang P, Stewart AF, Brissova M, and Powers AC (2016). Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration. Am. J. Physiol. Endocrinol. Metab 311, E859–E868. doi: 10.1152/ajpendo.00515.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abdulreda MH, Caicedo A, and Berggren P-O (2013). Transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time. J. Vis. Exp 73, e50466. doi: 10.3791/50466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alavi A, and Werner TJ (2018). Futility of attempts to detect and quantify beta cells by PET imaging in the pancreas: why it is time to abandon the approach. Diabetologia 29, 1639–4. doi: 10.1007/s00125-018-4676-1 [DOI] [PubMed] [Google Scholar]
  4. Antonioli L, Blandizzi C, Csóka B, Pacher P, and Haskó G (2015). Adenosine signalling in diabetes mellitus--pathophysiology and therapeutic considerations. Nat. Rev. Endocrinol 11, 228–241. doi: 10.1038/nrendo.2015.10 [DOI] [PubMed] [Google Scholar]
  5. Antonioli L, Pacher P, Vizi ES, and Haskó G (2013). CD39 and CD73 in immunity and inflammation. Trends Mol. Med 19, 355–367. doi: 10.1016/j.molmed.2013.03.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Balamurugan AN, Chang Y, Fung JJ, Trucco M, and Bottino R (2003). Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata. Am. J. Transplant 3, 1135–1142. [DOI] [PubMed] [Google Scholar]
  7. Belcher SM, Zsarnovszky A, Crawford PA, Hemani H, Spurling L, and Kirley TL (2006). Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain: implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors. Neuroscience 137, 1331–1346. doi: 10.1016/j.neuroscience.2005.08.086 [DOI] [PubMed] [Google Scholar]
  8. Blodgett DM, Nowosielska A, Afik S, Pechhold S, Cura AJ, Kennedy NJ, Kim S, Kucukural A, Davis RJ, Kent SC, Greiner DL, Garber MG, Harlan DM, and diIorio P (2015). Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets. Diabetes 64, 3172–3181. doi: 10.2337/db15-0039 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bramswig NC, Everett LJ, Schug J, Dorrell C, Liu C, Luo Y, Streeter PR, Naji A, Grompe M, and Kaestner KH (2013). Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J. Clin. Invest 123, 1275–1284. doi: 10.1172/JCI66514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brissova M, Aamodt K, Brahmachary P, Prasad N, Hong J-Y, Dai C, Mellati M, Shostak A, Poffenberger G, Aramandla R, Levy SE, and Powers AC (2014). Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes β cell regeneration. Cell Metab 19, 498–511. doi: 10.1016/j.cmet.2014.02.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Brissova M, Fowler M, Wiebe P, Shostak A, Shiota M, Radhika A, Lin PC, Gannon M, and Powers AC (2004). Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets. Diabetes 53, 1318–1325. doi: 10.2337/diabetes.53.5.1318 [DOI] [PubMed] [Google Scholar]
  12. Brissova M, Haliyur R, Saunders D, Shrestha S, Dai C, Blodgett DM, Bottino R, Campbell-Thompson M, Aramandla R, Poffenberger G, Lindner J, Pan FC, Herrath von, M.G., Greiner DL, Shultz LD, Sanyoura M, Philipson LH, Atkinson M, Harlan DM, Levy SE, Prasad N, Stein R, and Powers AC (2018). α Cell Function and Gene Expression Are Compromised in Type 1 Diabetes. Cell Rep 22, 2667–2676. doi: 10.1016/j.celrep.2018.02.032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brom M, Joosten L, Frielink C, Peeters H, Bos D, van Zanten M, Boerman O, and Gotthardt M (2018). Validation of 111In-Exendin SPECT for the Determination of the β-Cell Mass in BioBreeding Diabetes-Prone Rats. Diabetes 67, 2012–2018. doi: 10.2337/db17-1312 [DOI] [PubMed] [Google Scholar]
  14. Coutinho-Silva R, Parsons M, Robson T, and Burnstock G (2001). Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing. Cell Tissue Res 306, 373–383. doi: 10.1007/s004410100458 [DOI] [PubMed] [Google Scholar]
  15. Coutinho-Silva R, Parsons M, Robson T, Lincoln J, and Burnstock G (2003). P2X and P2Y purinoceptor expression in pancreas from streptozotocin-diabetic rats. Mol. Cell. Endocrinol 204, 141–154. doi: 10.1016/S0303-7207(03)00003-0 [DOI] [PubMed] [Google Scholar]
  16. Deaglio S, and Robson SC (2011). Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity. Adv. Pharmacol 61, 301–332. doi: 10.1016/B978-0-12385526-8.00010-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dillies M-A, Rau A, Aubert J, Hennequet-Antier C, Jeanmougin M, Servant N, Keime C, Marot G, Castel D, Estelle J, Guernec G, Jagla B, Jouneau L, Laloë D, Le Gall C, Schaëffer B, Le Crom S, Guedj M, Jaffrézic F, and French StatOmique Consortium. (2013). A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Brief. Bioinformatics 14, 671–683. doi: 10.1093/bib/bbs046 [DOI] [PubMed] [Google Scholar]
  18. Dorrell C, Abraham SL, Lanxon-Cookson KM, Canaday PS, Streeter PR, and Grompe M (2008). Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers. Stem Cell Res 1, 183–194. doi: 10.1016/j.scr.2008.04.001 [DOI] [PubMed] [Google Scholar]
  19. Dorrell C, Grompe MT, Pan FC, Zhong Y, Canaday PS, Shultz LD, Greiner DL, Wright CV, Streeter PR, and Grompe M (2011a). Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers. Mol. Cell. Endocrinol 339, 144–150. doi: 10.1016/j.mce.2011.04.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dorrell C, Schug J, Canaday PS, Russ HA, Tarlow BD, Grompe MT, Horton T, Hebrok M, Streeter PR, Kaestner KH, and Grompe M (2016). Human islets contain four distinct subtypes of β cells. Nat. Commun 7, 11756. doi: 10.1038/ncomms11756 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dorrell C, Schug J, Lin CF, Canaday PS, Fox AJ, Smirnova O, Bonnah R, Streeter PR, Stoeckert CJ, Kaestner KH, and Grompe M (2011b). Transcriptomes of the major human pancreatic cell types. Diabetologia 54, 2832–2844. doi: 10.1007/s00125-011-2283-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Eriksson O, Laughlin M, Brom M, Nuutila P, Roden M, Hwa A, Bonadonna R, and Gotthardt M (2016). In vivo imaging of beta cells with radiotracers: state of the art, prospects and recommendations for development and use. Diabetologia 59, 1340–1349. doi: 10.1007/s00125-016-3959-7 [DOI] [PubMed] [Google Scholar]
  23. Haliyur R, Tong X, Sanyoura M, Shrestha S, Lindner J, Saunders DC, Aramandla R, Poffenberger G, Redick SD, Bottino R, Prasad N, Levy SE, Blind RD, Harlan DM, Philipson LH, Stein RW, Brissova M, and Powers AC (2018). Human Islets Expressing HNF1A Variant Have Defective β-cell Transcriptional Regulatory Networks. J. Clin. Invest doi:pending [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kayton NS, Poffenberger G, Henske J, Dai C, Thompson C, Aramandla R, Shostak A, Nicholson W, Brissova M, Bush WS, and Powers AC (2015). Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles. Am. J. Physiol. Endocrinol. Metab 308, E592–602. doi: 10.1152/ajpendo.00437.2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Khan S, Yan-Do R, Duong E, Wu X, Bautista A, Cheley S, MacDonald PE, and Braun M (2014). Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells. Diabetologia 57, 2535–2545. doi: 10.1007/s00125-014-3368-8 [DOI] [PubMed] [Google Scholar]
  26. Laurent D, Vinet L, Lamprianou S, Daval M, Filhoulaud G, Ktorza A, Wang H, Sewing S, Juretschke HP, Glombik H, Meda P, Boisgard R, Nguyen DL, Stasiuk GJ, Long NJ, Montet X, Hecht P, Kramer W, Rutter GA, and Hecksher-Sørensen J (2015). Pancreatic β-cell imaging in humans: fiction or option? Diabetes Obes. Metab 18, 6–15. doi: 10.1111/dom.12544 [DOI] [PubMed] [Google Scholar]
  27. Lavoie EG, Fausther M, Kauffenstein G, Kukulski F, Künzli BM, Friess H, and Sévigny J (2010). Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion. Am. J. Physiol. Endocrinol. Metab 299, E647–56. doi: 10.1152/ajpendo.00126.2010 [DOI] [PubMed] [Google Scholar]
  28. Malone JH, and Oliver B (2011). Microarrays, deep sequencing and the true measure of the transcriptome. BMC Biol 9, 34. doi: 10.1186/1741-7007-9-34 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Munkonda MN, Pelletier J, Ivanenkov VV, Fausther M, Tremblay A, Künzli B, Kirley TL, and Sévigny J (2009). Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3. FEBS Journal 276, 479–496. doi: 10.1111/j.1742-4658.2008.06797.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olafsen T, and Wu AM (2010). Antibody vectors for imaging. Semin. Nucl. Med 40, 167–181. doi: 10.1053/j.semnuclmed.2009.12.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parnaud G, Bosco D, Berney T, Pattou F, Kerr-Conte J, Donath MY, Bruun C, Mandrup-Poulsen T, Billestrup N, and Halban PA (2008). Proliferation of sorted human and rat beta cells. Diabetologia 51, 91–100. doi: 10.1007/s00125-007-0855-1 [DOI] [PubMed] [Google Scholar]
  32. Petit P, Lajoix A-D, and Gross R (2009). P2 purinergic signalling in the pancreatic beta-cell: control of insulin secretion and pharmacology. Eur. J. Pharm. Sci 37, 67–75. doi: 10.1016/j.ejps.2009.01.007 [DOI] [PubMed] [Google Scholar]
  33. Robinson MD, and Oshlack A (2010). A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11, R25. doi: 10.1186/gb-2010-11-3-r25 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robson SC, Sévigny J, and Zimmermann H (2006). The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance. Purinergic Signal 2, 409–430. doi: 10.1007/s11302-006-9003-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Segerstolpe Å, Palasantza A, Eliasson P, Andersson E-M, Andréasson A-C, Sun X, Picelli S, Sabirsh A, Clausen M, Bjursell MK, Smith DM, Kasper M, Ämmälä C, and Sandberg R (2016). Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes. Cell Metab 24, 593–607. doi: 10.1016/j.cmet.2016.08.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Silva AM, Rodrigues RJ, Tomé ÂR, Cunha RA, Misler S, Rosário LM, and Santos RM (2008). Electrophysiological and immunocytochemical evidence for P2X purinergic receptors in pancreatic beta cells. Pancreas 36, 279–283. doi: 10.1097/MPA.0b013e31815a8473 [DOI] [PubMed] [Google Scholar]
  37. Speier S, Nyqvist D, Cabrera O, Yu J, Molano RD, Pileggi A, Moede T, Köhler M, Wilbertz J, Leibiger B, Ricordi C, Leibiger IB, Caicedo A, and Berggren P-O (2008a). Noninvasive in vivo imaging of pancreatic islet cell biology. Nat. Med 14, 574–578. doi: 10.1038/nm1701 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Speier S, Nyqvist D, Köhler M, Caicedo A, Leibiger IB, and Berggren P-O (2008b). Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye. Nat. Protoc 3, 1278–1286. doi: 10.1038/nprot.2008.118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sweet IR, Cook DL, Lernmark Å, Greenbaum CJ, Wallen AR, Marcum ES, Stekhova SA, and Krohn KA (2004). Systematic screening of potential beta-cell imaging agents. Biochem. Biophys. Res. Commun 314, 976–983. doi: 10.1016/j.bbrc.2003.12.182 [DOI] [PubMed] [Google Scholar]
  40. Syed SK, Kauffman AL, Beavers LS, Alston JT, Farb TB, Ficorilli J, Marcelo MC, Brenner MB, Bokvist K, Barrett DG, and Efanov AM (2013). Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion. Am. J. Physiol. Endocrinol. Metab 305, E1319–26. doi: 10.1152/ajpendo.00328.2013 [DOI] [PubMed] [Google Scholar]
  41. Trapnell C, Pachter L, and Salzberg SL (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111. doi: 10.1093/bioinformatics/btp120 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Virostko J, Henske J, Vinet L, Lamprianou S, Dai C, Radhika A, Baldwin RM, Ansari MS, Hefti F, Skovronsky D, Kung HF, Herrera PL, Peterson TE, Meda P, and Powers AC (2011). Multimodal image coregistration and inducible selective cell ablation to evaluate imaging ligands. Proc. Natl. Acad. Sci 108, 20719–20724. doi: 10.1073/pnas.1109480108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wuttke A, Idevall-Hagren O, and Tengholm A (2013). P2Y₁ receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion. FASEB J 27, 1610–1620. doi: 10.1096/fj.12-221499 [DOI] [PubMed] [Google Scholar]
  44. Zimmermann H, Zebisch M, and Sträter N (2012). Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8, 437–502. doi: 10.1007/s11302-012-9309-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1510722-supplement-1.pdf (22.8MB, pdf)

Data Availability Statement

Sequencing datasets

The sequencing data from NTPDase3-based sorting methods is available under GEO accession numbers GSE106148 (α-cells) and GSE116559 (β-cells). Sequencing data using existing cell surface antibodies (Dorrell et al., 2016) is available under GEO accession number GSE80780.

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