Establishment of SAP model
Healthy wild-type male Sprague Dawley (SD) rats weighing 200~220 g purchased from Chengdu Dossy Experimental Animal Co., Ltd. (Chengdu, China) and fed in a suitable environment with 25°C and 12 h dark/light cycle, given free access to water and food. Experimental procedures were approved by the Institutional Animal Care and Use Committee at the General Hospital of Western Theater Command and carried out in accordance with the established International Guiding Principles for Animal Research. The rats were fasted for 12 h before all surgical procedures. All experimental animals were anesthetized with isoflurane (RWD Life Science, Shenzhen, China) during the operation. The SAP models were induced by retrograde injection of 4% sodium taurocholate (TCA, 1 ml/kg body weight, Sigma, USA) into the common biliopancreatic duct as previously described [23].
Isolation, expansion and identification of CP-MSCs
CP-MSCs derived from human placental chorionic plate and were cultured in MSC Serum Free Media (Yocon, China). The specific experimental methods of CP-MSCs isolation, expansion and identification are detailed in our previous research [13]. Immunophenotypic analysis and osteogenic and adipogenic differentiation experiments confirmed that CP-MSCs isolated from human placental chorionic plate MSCs meet the criteria of MSCs proposed by the International Society for Cellular Therapies (Fig. S1).
Bioluminescence imaging
First, CP-MSCs were infected with lentivirus carrying a luciferase gene, and then CP-MSCs stably expressing luciferase were selected. Kunming mice were anesthetized, and 4% sodium taurocholate was injected through the pancreaticobiliary duct to prepare the SAP model. At 6 h after the operation, 1 × 106 CP-MSCs expressing luciferase were infused through the tail vein. Observe the survival and distribution of CP-MSCs in SAP mice at 1 h, 24 h, 72 h, 5 d, 7 d after exogenous CP-MSCs transplantation. D-Luciferin (150 mg/kg body weight) was administered i.p. to each mouse 10 min prior to imaging. Mice were then placed in an In Vivo Imaging System (IVIS) and the photons/sec emitted from the tissues were quantified using LivingImage software v3.2 (Caliper Life Sciences, Alameda, CA).
CP-MSCs in vivo tracking
Ten SD rats were randomly divided into 2 groups: Control group and SAP group (5 per group). CP-MSCs labeled with CM-Dil (1 × 106 cells/100g) were transplanted into rats via the tail vein at 6 h and 30 h after the operation. All rats were sacrificed 72 h after the first CP-MSCs transplantation, lung, heart, liver, pancreas, spleen, kidney, duodenum and colon were collected, and then fixed in 4% paraformaldehyde for 24 h and dehydrated in 30% sucrose solution. Subsequently, the tissues were embedded in Tissue Freezing Medium and cut into 8 μm thick sections. The slides were washed with PBS and stained with DAPI to visualize the nuclei. The distribution of CP-MSCs in different organs was observed under a fluorescence microscope.
CP-MSCs transplantation in SAP rats
Thirty-two SD rats were randomly divided into 4 groups: Control group, Control + CP-MSCs group, SAP group and SAP + CP-MSCs group (8 per group). In the CP-MSCs intervention group, CP-MSCs (1×106 cells/100g) were delivered through tail vein 6 h and 30 h after the operation. All rats were sacrificed 72 h after the first CP-MSCs transplantation, serum, liver and pancreas tissues were collected (Fig. 2a).
Histopathology
Pancreas samples were fixed in 10% buffered formaldehyde, embedded in paraffin, and sectioned. The 4 μm thick deparaffinized sections were stained with H&E for routine histology. According to the scoring criteria reported by Schmidt et al. [24], the degree of pancreatic edema, acinar cell necrosis, hemorrhage, and inflammatory infiltrate were scored. Five different fields were randomly observed under the microscope each slide.
Cell Counting Kit-8 (CCK-8) assay
The CCK-8 assay was used to detect the effect of CM-Dil on the proliferation of CP-MSCs. The detailed operating steps were seen in supplementary materials.
Enzyme-Linked Immunosorbent Assay (ELISA)
Inflammatory factors (IL-1β, IL-6, TNF-α, TGF-β, IL-4 and IL-10), amylase and lipase in rat serum were detected by ELISA kits (Shanghai Jianglai Biotech, China). In addition, the human tumor necrosis factor-α-induced gene/protein 6 (TSG-6) ELISA kit (Shanghai Jianglai Biotech, China) was used to detect the concentration of TSG-6 in the culture supernatant of CP-MSCs. Detailed operating steps were according to the products’ instructions.
Detection of myeloperoxidase (MPO) activity in pancreatic tissue
Accurately weigh pancreatic tissue of the same quality, then grind it into a homogenate, and follow the products’ instructions in the kit to detect MPO activity in the pancreatic tissue of each group.
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen Inc., USA), according to the products’ instructions. The RNA was quantified by measuring the absorbance at 260nm and 280nm using a spectrophotometer (NanoDrop Technologies, USA). RT-qPCR was performed with a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) using one step SYBR PrimeScript RT-PCR Kit (TaKaRa, Japan). The sequences of primers are listed in supplementary Table S1.
Immunofluorescence Staining
Immunofluorescence staining is used to detect the polarizing phenotype of macrophages in pancreas and liver tissues,and the detailed method was seen in supplementary materials.
Preparation and polarization induction of bone marrow-derived macrophages
Bone marrow (BM)-derived macrophages were isolated from SD rats by flushing the BM with DMEM (Hyclone, USA) as previously described [25, 26]. Bone marrow macrophage induction medium was used to induce differentiation of precursor cells into macrophages. After 7-10 days in culture, nonadherent cells were removed and adherent cells were ready for experiment. Macrophages were induced with M1 or M2 polarization induction medium for 24 hours, and then collected for subsequent experiments.
Bone marrow macrophage induction medium: DMEM + 10% FBS (Gibico, USA), 10 ng/ml M-CSF (Peprotech, USA).
Macrophage M1 polarization induction medium: DMEM + 10% FBS, 100 ng/ml LPS (Sigma, USA), 50 ng/ml IFN-γ (Peprotech, USA).
Macrophage M2 polarization induction medium: DMEM + 10% FBS, 10 ng/ml IL-10 (Peprotech, USA), 10 ng/ml IL-13 (Peprotech, USA).
Selective depletion of M1 or M2 macrophages
In vitro experiment Unpolarized-induced macrophages (M0), M1 polarization-induced macrophages (M1), and M2 polarization-induced macrophages (M2) were inoculated into six-well plates at 1 × 106 cells/well. After attachment of macrophages, GdCl3 (100 μM, Sigma, USA) or mannosylated clodronate-encapsulated liposomes (MCLs, Encapsula Nano Sciences, USA) were added to the macrophage medium. The volume ratio of MCLs to culture medium is 1:100. Macrophages were collected for apoptosis analysis after 48 h of intervention.
In vivo experiment Thirty-six SD rats were randomly divided into 6 groups: SAP group, SAP + CP-MSCs group, SAP + GdCl3 group, SAP + GdCl3 + CP-MSCs group, SAP + MCLs group and SAP + MCLs + CP-MSCs group (6 per group). In the GdCl3 intervention group, the GdCl3 solution (0.5%, 20 mg/kg) was infused via the tail vein immediately after the operation. In the MCLs intervention group, 1 ml of MCLs solution was infused via the tail vein immediately after the operation. In the CP-MSCs intervention group, CP-MSCs (1× 106 cells/100g) were delivered through the tail vein 6 h and 30 h after the operation. All rats were sacrificed 72 h after the first CP-MSCs transplantation, serum, liver and pancreas tissues were collected (Fig. 5a).
Flow cytometric analysis of macrophage apoptosis
Macrophage apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit (Beijing Solarbio Science & Technology Co., Ltd., China), and the detailed operating steps was seen in supplementary materials.
TSG-6 shRNA transfection
CP-MSCs were transfected with TSG-6 specific or nonspecific control short hairpin A (shRNA, Shanghai Genechem Co., Ltd., China) using transfection reagent in shRNA transfection media according to the manufacturer’s protocol. Puromycin (Sigma, USA) was employed to select stable knockdown cells for at least three passages.
CP-MSC intervened with SAP rat serum
To simulate the microenvironment of CP-MSCs in SAP rats, and CP-MSCs were cultured in MSC Serum Free Media containing 0%, 25% and 50% SAP rat serum. After 12 h of cultivation, CP-MSCs were collected for RT-qPCR assay.
Macrophages and CP-MSCs noncontact co-culture
To explore the effects of different polarized phenotype macrophages on CP-MSCs. First CP-MSCs were inoculated in six-well plates, and then M1 or M2 macrophages were inoculated in the upper layer of the transwell chamber (pore diameter: 0.4 μm). After 24 h of co-cultivation, CP-MSCs were collected for RT-qPCR assay.
To explore the mechanism of CP-MSCs regulating macrophage polarization. First, M1 macrophages were inoculated in six-well plates, and then CP-MSCs (TSG-6 shRNA) or CP-MSCs (scr shRNA) were inoculated in the upper layer of the transwell chamber (pore diameter: 0.4 μm). After 24 h of co-cultivation, macrophages were collected for flow cytometry analysis and RT-qPCR assay.
Flow cytometry analysis
Polarized phenotype of macrophages was analyzed using the following antibodies: FITC-conjugated CD163 (Bio-Rad, USA), PE-conjugated CD86 (BD Biosciences, USA), Alexa-Flour647-conjugated CD68 (Bio-Rad, USA). Non-specific isotype-matched antibodies served as controls. The cells were analyzed using a flow cytometry instrument (BD CantoⅡ, USA) and the data were analyzed using FlowJo V10.
Statistical analysis
Statistics as well as graphical representations were performed using GraphPad Prism™ 7.0 (GraphPad Software Inc., USA). All data are expressed as the means ± SEM. Comparisons between two groups were performed using Student's t-test. Comparisons between more than two groups were analyzed by one-way ANOVA test. Results were considered statistically significant when P < 0.05.