Pancreatic islet and progenitor cell surface markers with cell sorting potential
The aim of the study was to identify surface bio-markers and corresponding antibody tools that can be used for the imaging and immunoisolation of the pancreatic beta cell and its progenitors. This may prove essential to obtain therapeutic grade human beta cells via stem cell differentiation.
Using bioinformatics-driven data mining, we generated a gene list encoding putative plasma membrane proteins specifically expressed at distinct stages of the developing pancreas and islet beta cells. In situ hybridisation and immunohistochemistry were used to further prioritise and identify candidates.
In the developing pancreas seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor-related protein 11 (LRP11), dispatched homologue 2 (Drosophila) (DISP2) and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) were found to be expressed in early islet cells, whereas discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch-like EGF repeat containing (DNER) were expressed in early pancreatic progenitors. The expression pattern of DDR1 overlaps with the early pancreatic and duodenal homeobox 1 (PDX1)+/NK6 homeobox 1 (NKX6-1)+ multipotent progenitor cells from embryonic day 11, whereas DNER expression in part overlaps with neurogenin 3 (NEUROG3)+ cells. In the adult pancreas SEZ6L2, LRP11, DISP2 and SLC30A8, but also FXYD domain containing ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7) and transmembrane protein 27 (TMEM27), retain an islet-specific expression, whereas DDR1 is undetectable. In contrast, DNER is expressed at low levels in peripheral mouse and human islet cells. Re-expression of DDR1 and upregulation of DNER is observed in duct-ligated pancreas. Antibodies to DNER and DISP2 have been successfully used in cell sorting.
Extracellular epitopes of SEZ6L2, LRP11, DISP2, DDR1 and DNER have been identified as useful tags by applying specific antibodies to visualise pancreatic cell types at specific stages of development. Furthermore, antibodies recognising DISP2 and DNER are suitable for FACS-mediated cell purification.
KeywordsBeta cell DDR1 DNER DISP2 LRP11 Pancreas Progenitor SEZ6L2 Surface bio-marker
Discoidin domain receptor tyrosine kinase 1
Dispatched homologue 2 (Drosophila)
Delta/notch-like EGF repeat containing
Embryonic day 18.5 in the mouse
Endocrine pancreas consortium
Expressed sequence tag
FXYD domain containing ion transport regulator 2
Freestyle suspension human embryonic kidney 293 cells
HEPACAM family member 2
Human embryonic stem cells
In situ hybridisation
Keyhole limpet haemocyanin
Low density lipoprotein receptor-related protein 11
NK6 homeobox 1
Pancreatic duct ligation
Pancreatic and duodenal homeobox 1
Pancreas specific transcription factor, 1a
Seizure related 6 homologue like
Solute carrier family 30 (zinc transporter), member 8
Transmembrane protein 27
Weeks of gestation
Yellow fluorescent protein
As diabetes is caused by reduction of functional beta cell mass (absolute vs relative in type 1 diabetes vs type 2 diabetes [1, 2, 3]) much emphasis is focused on beta cell regeneration and replacement. Neogenesis of functional beta cells occurs in mice following partial pancreatic duct ligation (PDL) . Proof-of-principle transplantation studies in type 1 diabetes patients have demonstrated that reconstitution of functional beta cell mass can re-establish normoglycaemia [5, 6]. Limited access to human donor islet material has stimulated activities with the aim of generating therapeutic beta cells from pluripotent human embryonic stem cells (hESCs) and induced pluripotent stem cells [7, 8, 9, 10, 11]. hESCs have currently been differentiated towards heterogeneous cultures containing beta cell-like cells that are often multihormonal [8, 9].
Antibody-mediated cell sorting has been instrumental in dissecting the complexity of the haematopoietic system . An expanding collection of surface marker antibodies have been generated towards mature pancreatic cells through cellular immunisation , autoimmune hybridoma formation [14, 15], islet-specific panning of phage-libraries [16, 17, 18] and by bioinformatics approaches . Stage-specific surface markers may serve as purification tags to better characterise and purify relevant cell populations during pancreatic differentiation, regeneration and directed differentiation in vitro.
Chemokine (C-X-C motif) receptor 4 (CXCR4) has been identified as a definitive endoderm-specific marker during differentiation of ESC cells towards endoderm SRY (sex determining region Y)-box 17-positive (SOX17+) . It has been applied with success by Cai et al.  where the CXCR4+ vs CXCR4– sorted cells yielded up to 95% vs <10% pancreatic and duodenal homeobox 1-positive (PDX1+) cell population upon further differentiation .
The cell surface antigen CD133 (also known as prominin 1 [PROM1]) has been identified as a marker of a cell population that may contain neurogenin 3 (NEUROG3)-positive pancreatic endocrine progenitor cells . Enrichment of NEUROG3-positive cells in this PROM1-positive fraction was, however, only calculated to be 1.7-fold and most cells isolated by PROM1 were carboxypeptidase A (CPA1)-positive.
Surface markers for mature islet cell types have been published and include FXYD domain containing ion transport regulator 2 (FXYD2) , tetraspanin 7 (TSPAN7) , transmembrane protein 27 (TMEM27) [24, 25, 26, 27] and delta- , alpha- [18, 28] and beta cell-specific antibodies , as well as pan-islet  antibodies recognising unknown structures.
In this study we present a bioinformatics approach combined with in situ hybridisation/immunohistochemistry (IHC) screen allowing the identification of novel surface bio-markers of endocrine progenitors (discoidin domain receptor tyrosine kinase 1 [DDR1] and delta/notch-like EGF repeat containing [DNER]) and mature islet of Langerhans cells (dispatched homologue 2 (Drosophila) [DISP2], seizure related 6 homologue like [SEZ6L2], low density lipoprotein receptor-related protein 11 [LRP11] and HEPACAM family member 2 [HEPACAM2]). We also confirmed TSPAN7 and TMEM27 to be on our list. Furthermore, we show that DISP2 and DNER can serve as tags for cell purification using specific monoclonal and polyclonal antibodies.
Generation of gene lists
Genes on the Neurog3-knockout list (array list 1 in electronic supplementray material [ESM] Table 1) were based on array data from mouse pancreas isolated from CD1 Neurog3−/− and wild-type embryos at embryonic day 18.5 (e18.5) . On this list we expect to find genes characterising mature endocrine cells.
Genes on the Neurog3-yellow fluorescent protein (YFP) list (array list 2 in ESM Table 1) were based on array data from e15.5 Neurog3eYFP/+-positive and -negative FACS-sorted pancreatic cells . On this list we could possibly identify genes regulated similarly to Neurog3 in the endocrine progenitors.
The unbiased list (array list 3 in ESM Table 1) was generated by identifying assemblies in the Database of Transcribed Sequences (DoTS) contained in the Endocrine Pancreas Consortium (EPCon) libraries and then sorting for relevant surface proteins by bioinformatics. This list would be expected to contain any pancreatic surface marker gene. For further details, see ESM Detailed research design and methods.
Prioritisation of candidates
Candidates were prioritised according to: existence of human orthologues, transmembrane properties, plasma membrane localisation and degree of differential expression in the whole body. For further details, see ESM Detailed research design and methods.
In situ hybridisation
Probes of approximately 500 bp were designed and cloned in pCR4-TOPO TA cloning vector from e15.5 mouse gut RNA. Digoxigenin (DIG)-labelled RNA probes were generated and hybridisation was carried out as described . Briefly, slides were thawed at room temperature, probe added at 1 ng/μl overnight at 65°C, washed and anti-DIG antibody was added overnight at room temperature. In situ signal was developed with 5-bromo-4-chloro-3′-indolyphosphate/ nitro-blue tetrazolium (BCIP/NBT) in NaCl, Tris-HCl, MgCl, Tween-20 (NTMT) buffer.
Peptide selection for immunisation
Candidates were evaluated towards post-translational modifications and with respect to linear B cell epitopes by using CBS Prediction Servers (www.cbs.dtu.dk/services/). Furthermore, potential peptide areas were compared between mouse/human species to identify conserved versus species-specific epitopes. Using BLASTp, selected peptides with potential cross-reactivity were eliminated. Finally, a cysteine was added to facilitate subsequent coupling to a carrier-protein keyhole limpet haemocyanin (KLH). Selected peptides mapping to the extra-cellular side were synthesised (Schafer-N, Copenhagen, Denmark) for immunisation.
Peptides were conjugated to KLH using Imject Maleimide Activated mcKLH (mcKLH) Kit (Pierce #77611; Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. The conjugates were purified by dialysis in PBS overnight.
Immunisations and antiserum generation
The KLH-conjugated peptide was injected into RBF (Robertsonian POSF) mice or into rabbits. For further details, see ESM Detailed research design and methods.
Production of monoclonal antibodies
A mouse with high-titred serum was chosen for production of monoclonal DISP2 antibodies and was boosted intravenously with 10 μg antigen. Spleen cells were fused and hybridoma supernatant fractions were screened by indirect ELISA for specific binding to the immunising antigen. The secondary screening of positive hits from the primary screen was performed by IHC staining of sections of adult mouse pancreas.
Whole mouse embryos, dissected guts and adult pancreas were isolated, fixed in 4% paraformaldehyde (PFA), embedded in optimum cutting temperature Tissue-Tech (OCT) and sectioned as frozen (10 μm section) or stored in methanol. Tissue from one human adult non-pathological pancreas from a heart-beating, cadaveric, non-diabetic donor was procured at a European hospital associated with the Eurotransplant Foundation (Leiden, the Netherlands) and with the beta cell bank of the JDRF Center for Beta Cell Therapy of Diabetes, as approved by the ethical committee of the Free University of Brussels (‘Commissie Medische Ethiek–VUB’, reference #2002/VS).
AlphaTC cells were grown in DMEM low (1,000 mg/l d-glucose, sodium pyruvate), 10% FCS, and 100 U of penicillin and 50–100 μg of streptomycin. Freestyle suspension human embryonic kidney 293 (HEK-F) cells were grown in freestyle 293 medium (Gibco, #12338; Invitrogen A/S, Hellerup, Denmark), with 100 U of penicillin and 100 μg of streptomycin added. AlphaTC and HEK-F cells were harvested with cell dissociation solution C5789-100 M (Sigma-Aldrich, Brøndby, Denmark) or 0.05% Trypsin-EDTA (Invitrogen, Hellerup, Denmark), washed and fixed for 1 h at room temperature, subsequently washed in PBS and stored at +5°C until use.
IHC on sections and data collection was carried out as described previously . Whole mount IHC and data collection were performed essentially as described previously . Primary antibodies were used and detected as listed in ESM Table 2.
In vitro differentiation of human embryonic pancreas
Human fetal pancreases from tissue fragments were obtained immediately after elective termination of pregnancy as previously described [34, 35]. For culture, pancreases were laid on Millicell culture plate inserts (Millipore, Billerica, MA, USA) in 60 mm sterile Petri dishes containing 5 ml of RPMI 1640 (Invitrogen) supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), Hepes (10 mmol/l), l-glutamine (2 mmol/l), non-essential amino acids (1×, Gibco) and 10% heat-inactivated calf serum (Hyclone, Logan, UT, USA). Under such culture conditions, the pancreases grew at the air/medium interface as described for rodent pancreases [36, 37]. Cultures were maintained at 37°C in humidified 95% air/5% CO2. The medium was changed every other day.
The pancreatic duct of 8-week-old Balb/C mice was ligated as described . Ligated and sham-operated animals were killed at day 7 after surgery and preserved for IHC as above.
Preparation of harvested alphaTC and HEK-F cells for immunocytochemistry was done by spinning cells on to a microscope slide in a StatSpin Cytofuge2 for 4 min at 850 rpm (40 g). Cells were stained like IHC tissue sections; however, peroxidase quenching by H2O2 block was omitted.
Cells were blocked in 5%, 0.5% Tris-NaCl-blocking reagent buffer (TNB) (PerkinElmer, Skovlunde, Denmark) and 5% donkey serum at room temperature for 1 h. Primary antibody was added (mouse immunoglobulin [Ig]G1 anti-DISP2, goat IgG anti-DNER, isotype mouse IgG1 and non-immune goat IgG) and incubated for 2 h at room temperature with occasional gentle mixing. After washing in PBS, cells were incubated with secondary antibody for 1 h. Finally, cells were washed in PBS. Stained cells were analysed on a FACSAria flow cytometer (BD Biosciences [Becton, Dickinson, Brøndby, Denmark]) in at least three independent experiments. Graphs were made in FSC Express (De Novo Software, Los Angeles, CA, USA).
The optical stacks of the whole mount stainings were trimmed and equidistant subsets of sections were used to generate visually informative projections. For DNER/paired box 6 (PAX6), DNER/NEUROG3, DNER/PDX1 and DNER/insulin/glucagon every second, third, third and eighth optical section was used respectively. Double and triple overlays were done in LSM Image Browser (http://www.zeiss.de) or Adobe Photoshop (ATEA, Ballerup, Denmark).
Bioinformatics ranking of candidates
To identify bio-surface markers selectively expressed in the islet of Langerhans and in the endocrine progenitors we took advantage of Neurog3 null  as well as of Neurog3-YFP  array data. An additional unbiased list of potential pancreas bio-surface markers (the EpconDB list) was generated as a shared resource within the Beta Cell Biology Consortium.
ISH and IHC screen
The detailed IHC screen identified DDR1 and DNER as potential endocrine progenitor bio-surface markers; DISP2, LRP11, SEZ6L2 and solute carrier family 30 (zinc transporter), member 8 (SLC30A8) as potential endocrine markers and HEPACAM2 as a potential beta cell selective marker (HEPACAM2 to be described elsewhere by J. Jensen) (Fig. 1). So far, we have been unable to make monoclonal antibodies to the small extracellular domains of SLC30A8 (polyclonal antisera are commercially available, Mellitech [Grenoble, France], PZ8 and RZ8). Here, we describe the expression patterns of the five novel surface proteins in more detail: DDR1 (GeneID: 25678) is a single-span transmembrane receptor tyrosine kinase with 414 extracellular amino acids (aa). DDR1 is activated by ectodomain binding to fibrillar collagen . DDR1-ectodomain shedding has been described . Ddr1−/− mice are viable but female mice are unable to lactate . DNER (GeneID: 227325) is a single-span transmembrane Notch ligand, with 638 aa being extracellular. Dner−/− mice are viable but display motor discoordination in the fixed bar and rota-rod tests . DISP2 (GeneID: 214240) is a 12 transmembrane 1345 aa protein of which two-thirds are extracellular. Its function is unknown, but DISP1 is involved in release of bioactive hedgehog (cholesterol-modified) in mice [43, 44]. LRP11 (GeneID: 237253) is a 483 aa single-span transmembrane protein with 433 aa on the extracellular side. LRP proteins have a multitude of ligands and functions [45, 46]. SEZ6L2 (GeneID: 233878) is a 923 aa single-span transmembrane protein with 857 aa on the extracellular side. SEZ6L2 is known as a prognostic marker for lung cancer . Sez6l2−/− mice show no particular abnormalities .
All markers identified have been targeted with antibodies directed towards predicted extracellular epitopes. Surface labelling is documented for all antibodies in the ESM (ESM Figs 2 and 3). In addition, these antibodies also displayed cytoplasmic staining (e.g. active protein biosynthesis, vesicular storage etc.) in IHC, which was useful to document the tissue-specific expression pattern of the different markers.
DDR1 is a bio-surface marker of central pancreatic endocrine and ductal progenitors
DNER is a bio-surface marker of pancreatic endocrine progenitors
In cultured human WG11 embryonic pancreas, DNER immunoreactivity is mainly observed in single cells (Fig. 4l) confirming what is observed in the mouse. DNER has a general weak islet-specific expression in the adult mouse pancreas but with a few strong DNER-positive islet cells that tend to co-express glucagon (Fig. 4i–k). In the human adult pancreas there is a strong patchy signal for DNER in the islets. As in mouse, DNER overlaps partially with glucagon-positive cells (Fig. 4m–o), but interestingly also with somatostatin-positive cells (Fig. 4p–r). DNER is only very weakly co-expressed with insulin-positive cells (Fig. 4s–u) as in the mouse.
SEZ6L2, DISP2 and LRP11 are bio-surface markers of hormone-positive cells
DNER and DDR1 are upregulated in PDL pancreas
Since we defined DDR1 and DNER as fetal endocrine pancreas progenitor markers we tested for an upregulation/re-expression in the adult pancreas that was injured by PDL to activate beta cell progenitors . The PDL pancreas displays a strong continuous expression of DDR1 immunoreactivity in the PDX1-positive progenitor area (ESM Fig. 1h) reminiscent of the e15.5 embryonic mouse pancreas and in the human embryonic pancreas (Fig. 3). Strong upregulation of DNER is observed in the islets (ESM Fig. 1n) and a weaker expression in a ‘salt and pepper’ pattern is evident in the PDX1 progenitor area (ESM Fig. 1m–o). This expression pattern corresponds to what is observed in the embryonic mouse and human pancreas.
Progenitor and mature cell markers can be used to sort cells
Using a comprehensive bioinformatics approach combined with ISH and IHC screens we present an accumulating panel of surface marker antigens. We show that these are expressed during pancreatic development at various levels of endocrine cell maturation: either before endocrine specification (DDR1), around endocrine cell specification (DNER) or after the progenitor state (DISP2, LRP11, SEZ6L2). These markers therefore provide novel candidates for future purification of cells at various stages of endocrine maturation.
We find DDR1 to specifically label the centralised field of multipotent pancreatic progenitors at the secondary transition from which NEUROG3-positive cells emerge. The DDR1-positive cells that are NEUROG3-negative are likely to contain the pre-NEUROG3 cell as well as ductal committed cells. The DDR1-positive cell population also contain proliferating cells (data not shown). Strikingly, the DDR1 expression area in the ductal-trunk domain sharply demarks the border between the pancreatic ductal junctions to the duodenal epithelium. Within the early duodenal domain the DDR1-positive cells thus mark the committed pancreatic progenitor cells that not only harbour the endocrine cell differentiation potential but also cells with a proliferation potential. Similar cells could potentially be isolated from the hESC culture and expanded before final differentiation.
Of interest to the dynamics within the central trunk-domain is the DNER-labelling of non-dividing (data not shown) cell populations partially overlapping with NEUROG3+ cells but also cells that are hormone−/NEUROG3−. DNER does not follow any known marker and we therefore speculate that DNER marks cells prior to, as well as at, the early NEUROG3-expressing stage. Furthermore, DNER is exclusively expressed in the pancreas within the endoderm making it a very attractive surface marker for the pancreatic endocrine progenitor cell pool at the NEUROG3-stage. The progenitor nature of DDR1- and DNER-expression was also confirmed in the PDL model where we observed a re-expression of DDR1 in the regenerating tissue and an upregulation of DNER in the pre-existing islets. In the human system, the DDR1 expression recapitulates that of the mouse, making DDR1 an attractive surface tag for isolating both mouse and human pancreatic progenitor cells from mixed populations. DNER is expressed in the human embryonic pancreas in a pattern that also mirrors that observed in the mouse. In the adult human pancreas DNER tends to co-localise with the glucagon-positive cells, but also with other islet cell types albeit at lower expression level.
Compared with the published PROM1 surface marker that has also been proposed to be of use in purification/isolation, we find that PROM1 has low expression levels in the DDR1/DNER central pancreas area when compared with its expression levels in the acinar cells. We have now shown that a polyclonal antiserum to DNER also works in FACS, thus providing a potential useful tool to purify endocrine progenitor cells from fetal pancreatic tissue as well as from stem cell-derived cultures. So far, commercially available monoclonal DNER-antibodies have not worked in FACS purification but future monoclonal antibodies may recognise available surface epitopes to serve such a purpose.
We characterised DISP2, SEZ6L2 and LRP11 as pan-endocrine islet markers. Peptide designs (to optimise for human cross-reaction) from all three proteins representing putative extracellular accessible epitopes were successfully used in generating polyclonal as well as monoclonal (DISP2) antibodies. This was demonstrated by our FACS data, with DISP2 resulting in a clean separation between DISP2-positive and -negative cells. The DISP2 monoclonal antibody F66 fully cross-reacted to human DISP2 as verified by IHC. Interestingly, our DISP2 antibody works equally well in FACS after use of trypsin compared with cell dissociation buffer. We predict the three surface markers to be useful in the purification and/or imaging of hormone-positive islet cells. Our list of membrane proteins also contained TSPAN7 and TMEM27 [23, 24] recently identified by others but, for example FXYD2  was not on any of our three lists, thus predicting that additional beta cell- or progenitor-specific surface markers are yet to be identified.
In summary, we have identified novel surface markers for pancreatic endocrine progenitors as well as of mature endocrine cells. Antibodies to such extracellular epitopes could facilitate cell purification steps in protocols involving differentiation of pluripotent stem cells towards glucose-sensitive beta cells for diabetes therapy. The described approach is based on bioinformatics leading to identification of putative candidate antigens that can result in useful antibody generation. This approach complements a recently published strategy using whole cellular immunisation to identify monoclonal antibodies against cell stage-specific epitopes [13, 28], which together have resulted in a panel of novel immunoisolation tools that will prove to be instrumental to further characterise and dissect the successive differentiation states characterising the formation of mature insulin-producing beta cells.
This work was supported by the National Institute of Diabetes & Kidney Diseases of the National Institutes of Health, Bethesda, MD (U01DK072473) as part of the Antibody Core of the Beta Cell Biology Consortium. J. Hald was supported by ‘MADBETA-Grant’ (U01DK072473). O.D. Madsen was supported by the European Union 6th Framework Programme (BetaCellTherapy, 512145) as part of the Juvenile Diabetes Research Foundation Center for Beta Cell Therapy of Diabetes. The authors thank S. Refsgaard Lindskog, A. Bjerregaard, M. Lauritzen and G. Leuckx for expert technical assistance and P. Serup for the interpretation of DDR1 data sets. R Gorski (Gorski Consulting, Philadelphia, PA, USA) contributed to the unbiased EPCon gene list
JH planned and designed the experiments, obtained and analysed the data, performed the experiment in Fig. 6, drew the figures and wrote the paper. TG carried out the experimental analysis and interpretation of data leading to Fig. 5. CR did the experiment on FXYD2 and captured and interpreted the data. LR and AES designed and carried out the experimental analysis leading to the production of the DISP2 monoclonal antibody as well as the polyclonal antisera to LRP11 and SEZ6L2 and participated in their characterisation. HH designed the PDL experiment including interpretation of data. JA-R did the whole mount stainings and acquired/analysed the whole mount data in Fig. 4. JJ provided the Neurog3 knockout array data including thorough bioinformatic input and prioritization of candidates, and contributed to the generation of Fig. 1. RS designed experiments and provided the embryonic human tissue and participated in evaluating human antigen expression. GG participated in the design of overall strategy and experiments and provided the Neurog3-YFP array data and participated in the ISH screen. CS Jr and KK participated in the original conceptual design of the experimental approach, provided the unbiased EPCon gene list and participated in analysis and interpretation of data. JNJ gave critical input and focused the experiments. ODM conceived the studies and provided critical input in data analysis and interpretation and co-wrote the paper. All authors have read and given critical input during revisions of the paper and all have approved the final version.
Duality of interest
G. Gradwohl, J. Hald and O.D. Madsen have filed a patent on the use of DNER as a surface tag for immunoisolation. J. Hald and O.D. Madsen have filed a patent on the use of DDR1 as a surface tag for immunoisolation. The remaining authors declare that there is no duality of interest associated with this manuscript.