NOD/ShiLtJ (NOD; Stock No: 001976) mice were purchased from the Jackson Laboratory and bred in the local animal facility. Mice were maintained under specific pathogen-free conditions in a controlled environment with a 12/12-h light/dark cycle, 21 °C and 55–60% humidity, and had access to chow and water ad libitum.
MSC isolation and labelling
The cells were isolated from the bone marrow of 6 to 8-week-old male NOD mice using the method previously described22. Cell characterization confirmed the presence of MSC specific markers (Sca-1, CD105, and CD44), the absence of hematopoietic markers (CD45 and CD11b) and the trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages, as previously reported50,51,52. Cells were used between the 7th and 10th passages. To track the cells in vivo after transplantation, cells were labelled by transduction with a third-generation lentiviral system to stably express Luc transgene50. Briefly, the lentiviral particles were assembled by using the packaging plasmids pRSV-Rev, pMDLg/pRRE, pMD2.G (gifts from Didier Trono, Addgene plasmids # 12253, # 12251, and # 12259)53, and the transfer plasmid for Luc (a gift from Eric Campeau, Addgene plasmid # 21471)40. Transduced cells preserved their properties, being able to differentiate into osteocytes, adipocytes and chondrocytes, when cultured under appropriate conditions, as previously reported50. In some experiments, Luc+ cells were additionally labelled with VivoTrack 680 (VT680) just prior to transplantation. VT680 labelling of MSC was performed according to the manufacturer’s recommendations (PerkinElmer, NEV12000). Briefly, cell suspension (106 cells/ml) in phosphate-buffered saline (PBS) was incubated with 50 μg/ml VT680 for 15 min, at room temperature, in the dark, and washed three times with PBS containing 1% FBS before use.
Double-labelled MSC (that expressed Luc and VT680) were transplanted by different routes, i.e., intravenous, intrapancreatic, intrasplenic and subcutaneous, in pre-diabetic 12-week-old NOD females. Twenty-four hours before transplant, labelled MSC were plated at 20,000 cells/cm2 so that to form an 80% confluent culture of actively dividing cells on the transplant day. Mice were anaesthetized with a mixture of ketamine-xylazine-acepromazine (80-10-2 mg/kg bodyweight) and cell suspension prepared by trypsinization (using 0.25% Trypsin-EDTA, ThermoFisher) was resuspended in ice-cold PBS, pH 7.4, and kept on ice until injection. For subcutaneous transplantation, aliquots of 5 × 105 cells in 50 μl PBS/site were injected in both the interscapular and inguinal regions. For intravenous transplantation, a same number of cells was resuspended in 250 μl PBS and slowly injected (in the course of 10–15 min per animal) in the lateral tail vein. For intrasplenic and intrapancreatic transplantation, small laparotomies were performed under sterile conditions to expose the tail of the spleen and the pancreas, respectively. A number of 5 × 105 cells (resuspended in 50 or 100 μl PBS for intra-pancreatic or intrasplenic transplantation, respectively) were transplanted using a 1700 Series Hamilton syringe with a 28 G needle. After transplant, the incisions were sutured with a 6/0 Optilene polypropylene monofilament (BBraun) and Baneocin® 250 UI/5000 UI antibiotic ointment was applied on the surgical suture. Complete healing was observed in all animals, with complete epithelization by 7 days post-transplant. For analgesia, intrasplenic and intrapancreatic transplanted animals were subcutaneously injected with buprenorphine hydrochloride (0.1 mg/kg, Temgesic) after laparotomy.
In vivo imaging
The survival and migration of MSC was monitored up to 7 days post- transplant, using an IVIS Spectrum in vivo imaging system (PerkinElmer). For FLI, mice were anesthetized with 1.5% isoflurane (Isoflutek, 710004), placed in the IVIS imaging box and imaged dorsally or laterally. For BLI, anesthetized mice were injected intraperitoneally with D-Luciferin Potassium Salt (PerkinElmer, 122799) (150 mg/kg bodyweight) and 12 min later, the total signal produced by transplanted cells was imaged. In experiments for in vivo apoptosis imaging, a second injection with Z-DEVD-aminoluciferin (VivoGlo Caspase 3/7 Substrate, Promega, P1781) 100 mg/kg bodyweight was applied at 4–6 h after D-luciferin injection, at a time when BLI signal produced by D-luciferin was totally extinct. Fluorescence and bioluminescence images of live animals or isolated organs were analysed using Living Image 4.5 software (PerkinElmer) by manually defining the regions of interest. Imaging data were normalized and expressed as radiance (p/s/cm²/sr) for bioluminescence or Radiant Efficiency ([p/s/cm²/sr]/[µW/cm²]) for fluorescence, and the colour scale was adjusted according to the strength of signal. To quantify the fluorescent signal, spectral unmixing analysis was performed to extract the autofluorescence from the specific signal. For quantification, the relative signal intensity was calculated as a percentage from the signal intensity at day 0 (30 min post-transplantation).
In vivo hypoxia visualization was performed as previously described22. Briefly, 24 h prior to transplantation, MSC were transiently transfected with HRE-luciferase plasmid (Addgene # 26731, a gift from Navdeep Chandel)54 by electroporation (NEPA21; Nepagene). This Luciferase reporter construct contains tree hypoxia response elements (HRE) from the Pgk-1 gene upstream of firefly luciferase. For hypoxia visualization in vitro, HRE-luc transfected MSC were seeded at pre-confluence (20,000 cells/cm2) and, 24 h later, the cells were incubated in 2% O2 atmosphere (hypoxia) for another 24 h. Hypoxic condition was achieved using a dedicated hypoxia station (Whitley H35 Hypoxystation, Don Whitley Scientific Limited, U.K.). As control, transfected cells were maintained in 21% O2 (normoxia). BLI signal was measured with IVIS Spectrum by imaging the cells immediately after the addition of 150 μg/ml D-Luciferin. To mimic the proinflammatory environment in vitro, MSC were stimulated with TNFα (R&D Systems, 410-MT) and IFNγ (R&D Systems, 485-MI) at 20 ng/ml each, under either normoxia or hypoxia for 24 h.
Cell proliferation assay
MSC were plated in two six-well cell culture plates (Eppendorf, 0030720113) at a density of 50,000 cells/well and grown for 5 days in either normoxia or hypoxia. Cell proliferation was determined after 1, 3, and 5 by counting the cells (after trypsinization). The experiment was performed three times with biological duplicates for each time point.
The apoptosis assays were employed to estimate the effect of double labelling of MSC with Luc and VT680, as well as to quantify the cell death induced in MSC after the treatment with TNFa and IFNγ (20 ng/ml, each) for 48 h. To estimate the effect of double labelling, 105 MSC were incubated with 5 µl APC Annexin V (Biolegend, 640920) and 5 μg/ml Propidium Iodide (Sigma-Aldrich, P4170) in 100 μl, for 15 min in the dark. To determine apoptosis of MSC in pro-inflammatory conditions, 105 cells (collected as both floating and attached cells) were stained with CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent (ThermoFisher Scientific, R37111), according to the manufacturer’s instructions. Cells stained with Annexin V and PI or CellEvent™ Caspase-3/7 were analysed by flow-cytometry. At least 100,000 events were recorded for each sample, using a CytoFLEX Flow Cytometer (Beckman Coulter, U.S.A.) and the acquired data were analysed using CytExpert version 2.1 software.
Furthermore, apoptosis induced by hypoxia, in the presence and absence of the pro-inflammatory cytokines was also evidenced by time-lapse fluorescence microscopy using a PAULA Smart Cell Imager (Leica Microsystems, Germany). After 24 h of treatment, CellEvent™ Caspase-3/7 reagent was added onto the cells and the cells were imaged for the next 24 h at a time interval of 20 min.
In vitro phagocytosis assay
Peritoneal macrophages were obtained from 8 to 12-week-old male NOD mice by lavage with 5 ml of ice-cold PBS, followed by macrophage purification using EasySep™ Mouse Monocyte Isolation Kit (Stem Cells Technologies, #19861). The purified cells were cultured in four-well plate wells at 2.5 × 105 cells/well for 24 h in RPMI medium supplemented with 10%FBS before being washed and further incubated with VT680-labelled MSC (treated or not-treated in inflammatory conditions) at a cell ratio of 1:5 (macrophages/MSC). After one hour of co-incubation, the not-adhered MSC were removed by three washes with PBS, and the adhered cells (MSC and macrophages) were collected with trypsin. A cell suspension containing 105 cells was then incubated in 100 µl FACS buffer with FITC-labelled anti-CD45 antibody and the presence of VT680 within macrophages was analysed by flow cytometry to estimate the extent to which peritoneal macrophages engulfed normal or apoptotic MSC.
Histology and image acquisition
The pancreas was isolated and processed by overnight fixation into PBS with 1.5% paraformaldehyde and 0.1% glutaraldehyde, followed by 24-h cryoprotection in PBS with 30% sucrose. Fixed samples were soaked in OCT (Optimal Cutting Temperature) compound and frozen on a metal block immersed in liquid nitrogen, before being sliced in seven-μm cryosections. For apoptosis assay, sections were incubated with anti-Cleaved Caspase-3 (Asp175) antibody (Cell Signaling Technology, #9661) for 2 h at room temperature followed by incubation with NL557-conjugated Anti-Rabbit IgG secondary antibody (R&D Systems, NL004), for one hour in the dark. For macrophage identification, the slides were incubated with anti-CD68 monoclonal antibody (BioLegend, 137001) overnight at 4 °C, followed by incubation with AF488-labelled anti-CD45 monoclonal antibody (BioLegend, 103122), 2 h at room temperature and dark and then by incubation with TRITC-conjugated Anti-Rat IgG secondary antibody (Sigma-Aldrich, T5778) for one hour in the dark. Two-three sections at 150 μm apart were imaged per each animal. Image acquisition was performed using a Leica DMi8 inverted fluorescent microscope equipped with HC PL APO 10x/0.45 NA dry, and HC PL APO 40x/1.3 NA oil objectives. Fluorophores were excited with a multi-LED Spectra-X light source (Lumencor) and images were captured with a sCMOS camera Leica DFC9000 and subsequently processed with Leica LAS X software. Tile scan z-stack images were acquired with LAS X Navigator module and mosaic merged with smooth overlap blending and then digitally processed for extended depth of field.
Data were expressed as mean ± SEM and analysed with GraphPad Prism 7.0. Longitudinal comparisons between different groups were performed with one-way or two-way analysis of variance (ANOVA) with Tukey’s post-test analysis. Statistical significance was defined as p < 0.05.