Experimental design
We recently established a stem cell culture model of diabetes based on the use of cord blood (CB)-derived CD34+ HSPCs [14]. This method already provided a consistent and reproducible recapitulation of the major CD34+ HSPC dysfunction hallmarks in diabetes [14]. To assess the ability of GLP-1 RA to prevent CD34+ HSPC dysfunction induced by glucose overload the cells were expanded in high glucose (HG; 30mM) conditions along with 50 nM or 100 nM LIRA treatment (Figure 1A). In a different experimental setting, CD34+ HSPCs were expanded in HG condition and then treated with LIRA only after loss of glucose tolerance. (Figure 1B). Afterwards, we assessed the ability of the drug to recover a compromised phenotype. CD34+ HSPCs cultured in normoglycemic condition (NG; 30mM mannitol) were used as control. At the end of both experiments, the main dysfunctional hallmarks of the cells, namely proliferation and CXCR4/SDF-1α axis impairment, were evaluated (Figure 1).
Cell culture
Umbilical cord blood (UCB) was collected from the umbilical cord of full-term normal deliveries in collaboration with Milano Cord Blood Bank (IRCSS Ca’ Granda Foundation – Ospedale Maggiore Policlinico). The mononuclear cell fraction was obtained by density gradient centrifugation using Ficoll-Paque (Lymphoprep, Sentinel Diagnostics) and CD34+ HSPCs were immunomagnetically isolated using CD34 Microbead Kit (MiniMACS kit, Miltenyi Biotec). Isolated CD34+ HSPCs were cultured in Stem Span medium (StemCell Technologies) supplemented with 20 ng/mL of interleukin (IL)‐6 (PeproTech), 20 ng/mL of IL‐3 (PeproTech), 50 ng/mL of fms‐like tyrosine kinase 3 (FLT3, PeproTech), and 50 ng/mL of stem cell factor (SCF, PeproTech). Cells were cultured in HG (30 mM of glucose, Sigma-Aldrich) or NG (30 mM of mannitol, Sigma-Aldrich) conditions for up to 20 days and treated or not with increasing concentration of LIRA (50nM and 100nM; MedCHemExpress) ± selective GLP-1R antagonist exendin (9-39) (150nM EXE; MedCHemExpress)
Cell proliferation assay
CD34+ HSPCs were seeded at an initial density of 2.0×105 cells/well and cultured for up to 20 days in NG and HG ± LIRA conditions. Cells were counted on days 5, 10, 15 and 20. Doubling time was calculated with the following formula:
Migration assays
Cell migration was determined the use of Boyden modified chamber consisting of transwell culture inserts (5‐μm pore membrane; Corning Incorporated, Corning, NY). In brief, 1×105 cells were seeded onto the upper chamber and allowed to migrate toward the lower chamber containing, or not, stromal cell-derived factor 1 (SDF‐1α 50 ng/mL; PeproTech EC Ltd.). The transwells were incubated at 37°C, 5% CO2, for 4 hours. Migrated cells in the lower chamber were counted and migration index were calculated with the following formula:
Cyclic adenosine monophosphate (cAMP) quantification
Intracellular cAMP was quantified by cAMP ELISA kit (Enzo Life Science) according to manufacturer’s instructions. Briefly, 5 x 105 CD34+ HSPCs were stimulated with LIRA ± EXE and lysed in 0.1 M HCl and 0,1% Triton X-100. Sample absorbance was spectrophotometrically evaluated at 405 nm by Tecan (Infinite M200 Pro, TECAN).
Intracellular Ca2+ handling
The measurement of intracellular Ca2+ has been assessed through single cell and population analysis by means of confocal Nikon A1R microscope and FLUOstar Omega (BMG Labtech) multiplate reader respectively. CD34+ HSPCs were starved for 2 hours (with IMDM and albumin 0.1%) and incubated with the Ca2+-sensitive dye Fluo-4 AM (ThermoFisher, 2µM) in Tyrode’s solution (containing in mM: 154 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES/NaOH, and 5.5 d-glucose, adjusted to pH 7.35) for 1 hour.
Confocal single cell analysis was performed by plating CD34+ HSPCs on fibronectin/polylisin D coated glass coverslips; fluorescence (F) images (512*512 pxls) were acquired at x60 magnification every 5 seconds in basal condition and following 100 nM LIRA addition at 37°C and 5% CO2 thanks to Okolab incubator mounted on the microscope stage. Changes in single cell mean F during acquisition time were quantified through NIS-Elements analysis software following F backgroud subtraction. Population analysis was performed by plating cells in 96-well dark plates by means of the multiplate reader equipped with an automatic injection system to inject LIRA (100 nM). F was acquired in each well every 0.74 second for 20 seconds just prior to compound injection and for 100 seconds after injection. Mean F prior compound injection was used as reference (F0) for signal normalization (F/F0).
RNA extraction and RT-qPCR
Total RNA from CD34+ HSPCs was isolated by using the Direct‐zol RNA Kit (Zymo Research), following manufacturer's protocol. One µg of total RNA was converted to cDNA with the Superscript III kit (Life Technologies) and used to assess GLP-1R gene expression. qPCR reactions were performed with SYBR Green Supermix 2X (BIO‐RAD Laboratories) on CFX96 Real–Time System PCR (BIO‐RAD Laboratories). Specific GLP-1R primers (Fw: 5’-GTGTGGCGGCCAATTACTAC-3’; Rv: 5’-CTTGGCAAGTCTGCATTTGA-3’) were appositely designed to evaluated mRNA expression by amplifying a region of 347 bp. The qPCR products were loaded on a 1% agarose gel with an appropriate molecular marker (PCR Marker Solution, Sigma-Aldrich). Then, the 347 bp bands were excised and purified with QIAquick Gel extraction kit (Qiagen) for subsequent Sanger sequencing analysis.
Sanger sequencing
The RT-qPCR products, appropriately purified from agarose, were sequenced with the help of an external service (Microsynth Biotech) by Sanger method with the use of GLP-1R Fw primer. Sequencing results were analysed by a Multiple sequence ClustalW alignment (BioEdit software) throughout the comparison of the published GLP-1R cDNA sequence (NCBI Reference sequence: NM_002062.5).
Western Blot
CD34+ stem cells and capan-1 cells were lysed in lysis buffer (50 mM TRIS-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton) added with protease inhibitors (1:10, Halt Protease Inhibitor Cocktail, Thermo Scientific). Protein lysate was then quantified by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Fourty μg and 20 μg of protein from CD34+ stem cells and capan-1 cells respectively were resolved on 10% SDS‐PAGE in denaturing conditions. Proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore) at 400 mA, 4 °C for 90 minutes. To prevent aspecific binding, the membrane was blocked with 5% bovin serum albumin (BSA) in PBS + 0,1% Tween-20 (PBST) for 1 hour. The membranes were then incubated with the primary antibodies, appropriately diluted in 3% BSA-PBST, at 4 °C O/N and with the appropriate secondary antibody linked to horseradish peroxidase (HRP) the day after for 1 hour. Specific information about antibodies and appropriate dilutions are reported in Table 1. The signal was detected by Enhanced chemiluminescence (ECL) system and quantified by Chemidoc MP Imaging System (BIO-RAD Laboratories).
Flow cytometric assays
CD34+ HSPCs were incubated for 30 minutes with allophycocyanin‐conjugated monoclonal antihuman CXCR4 antibody (BD Biosciences) or with CellROX Green Flow Cytometry Assay Kit (Life Technologies) for the detection of CXCR4 and reactive oxygen species (ROS) respectively. The Gallios Flow Cytometer platform (Beckman Coulter Life Sciences) was used to analyze the samples after appropriate physical gating. At least 204 events in the indicated gates were acquired
Statistical analysis
Results are given as mean±SEM. All experiments were performed at least in triplicate, unless stated otherwise. The data were tested for the normality by using the Shapiro–Wilk normality test. Differences between data were evaluated by 1‐way, 2‐way repeated‐measures ANOVA followed by the post‐hoc Newman–Keuls multiple comparison test, as appropriate. A value of P≤0.05 was considered significant. All statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc.).