Treatment of mice with Calpeptin
The ethics committee for animal welfare of the University of Burgundy and the French ministry of higher education and research approved all animal experiments (under reference APAFIS #36855-2022041914584260). Calpeptin (C8999, Merck) was reconstituted in DMSO at 10 mg/mL. C57BL/6J (Ly.2) (Envigo) or C57BL/6SJL (Ly.1) mice (Charles River Laboratories) were intravenously (i.v.) injected with Calpeptin at 10 mg/kg, following four administrations, every 5 days. Male and female mice, 7-16-week-old, were randomly allocated to experimental groups and no blinding method was used for experiments. There were no animal exclusion criteria. Five days after the last injection, bones (tibias and femurs) from the two bottom legs were crushed in a mortar in 2 mL of PBS1×. After centrifugation at 500 g for 5 min, the supernatant was recovered to purify EVs following the procedure described below. Cells were washed in 10 mL PBS1× and filtrated with a sterile cell strainer at 70 µm, after centrifugation, Lin− cells (130-110-470, Miltenyi Biotec) or Sca1+ cells (130-123-124, Miltenyi Biotec) were magnetically purified using LS columns (Miltenyi Biotec), counted with Trypan blue (Thermo Fisher Scientific) and processed with flow cytometry procedure, transplantation or CFU assay.
Purification Of Evs From Mice
The supernatant was recovered for another centrifugation (10,000 g, 5 min) to remove cell debris. The supernatant was next recuperated, and then we pulled-down EVs by using the total EVs isolation kit (15254394, Invitrogen) used at 10% (v/v), mixed by inverting the tubes several times and incubated for 15 min at 4°C followed by a centrifugation (10,000 g, 10 min). The pellet of EVs was reconstituted in 100 µl of 0.1 µm-filtrated PBS1×. EVs were evaluated for their size and concentration by nanoparticle tracking analysis (NTA) using a NanoSight NS300 Instrument (Malvern Instruments) and conserved at -80°C. EVs were suspended in lysis buffer (Cell Signaling Technology) and characterized by enzyme-linked immunosorbent assay (ELISA) for p-Smad2 (S465/S467) / p-Smad3 (S423/S425) (12001C, Cell Signaling Technology) or active TGF-β1 (437707, Biolegend).
Purification Of Evs From Murine Ms-5 Cells
Murine MS-5 mesenchymal stromal cells (ACC-441, DSMZ) were cultured in Iscove's Modified Dulbecco's Media (IMDM) (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin-Amphotericin (PSA) (Pan Biotech, Aidenbach, Germany). When cells confluence in T75 flask reached 80%, we administrated IMDM supplemented with 20% of EVs-depleted FBS and 1% PSA. To deplete EVs from FBS, we ultra-centrifuged at 120,000 g (Optima XE-90 ultracentrifuge, Beckman Coulter), overnight in Ultra-Clear centrifuge tubes (Beckman Coulter). MS-5 cells were treated with the TGF-β-RI inhibitor SB431542 (S4317, Merck), used at 2 µM each day, during four days. Then, we recovered the supernatant (30 mL), cells were removed after centrifugation (500 g, 5 min) and the supernatant was recovered for another centrifugation (10,000 g, 10 min) to remove cell debris. The supernatant was recovered and we pulled-down EVs by ultra-centrifugation at 120,000 g during 90 min. For microscopy, 20 µl of PKH67 green fluorescent cell linker kit for general cell membrane labeling (Merck) was administrated for 30 min in the supernatant and we pulled-down EVs by ultra-centrifugation at 120,000 g during 90 min. EVs in the pellet were reconstituted in 0.1 µm-filtrated PBS1× and evaluated for their size and concentration by NTA using a NanoSight NS300 Instrument (Malvern Instruments, Malvern, England) and conserved at -80°C. For western blot and ELISA, EVs were directly reconstituted in lysis buffer (Cell Signaling Technology).
MS-5 EVs tested on primitive hematopoietic cells ex vivo
Bones (tibias and femurs) from the two bottom legs of C57BL/6J mice (Envigo) were crushed in a mortar in 10 mL of PBS1×. Total BM cells were then filtered with a sterile cell strainer at 70 µm and Lin− cells (130-110-470, Miltenyi Biotec) or Sca1+ cells (130-123-124, Miltenyi Biotec) were magnetically purified using LS columns (Miltenyi Biotec). Sca1+ cells were divided into four wells (fresh, w/o EVs, EVs-DMSO and EVs-SB431542) and treated with 109 particles per 4×105 cells. Lin− cells were divided in three conditions (w/o EVs, EVs-DMSO and EVs-SB431542) and treated with 109 particles per 106 cells. To determine EVs cells’ uptake, PKH67+ EVs were applied during 4 hours on cells. Using flow cytometry or microscopy, we determined the percentage of Lin−, Sca1+ or SLAM cells that have uptaken fluorescent PKH67+ EVs. EVs and cells were co-cultured for 48 hours in 96-well round bottom plates with 200 µL of the StemMACS media (Miltenyi Biotec), supplemented with PSA, murine Stem cell factor (SCF, 25 ng/mL, 130-101-741, Miltenyi Biotec), murine Interleukin 3 (IL3, 10 ng/mL, 130-096-687, Miltenyi Biotec), and murine Interleukin 6 (IL6, 10 ng/mL, 130-096-682, Miltenyi Biotec). Using flow cytometry, we tested the effect of 48 hours exposure of Sca1+ cells to EVs, on the cell cycle activity of SLAM cells following Ki67 staining, as well as on the TGF-β pathway by p-Smad2/3 staining, as well as on HSC maintenance ex vivo following transplantation studies. To assure that Calpeptin was not an inhibitor of the TGF-β pathway, we treated 2.5×105 Lin− cells ex vivo with 0.5 µM of SB431542 or Calpeptin. Following 18 hours, p-Smad2/3 was assessed by flow cytometry on SLAM cells.
Transplantation of primitive hematopoietic cells
Sca1+ cells, either freshly isolated from the BM of C57BL/6.SJL (Ly.1) mice (Charles River Laboratories), or after their exposure with EVs for 48 hours, were quantified by blue Trypan. Then, 4×105 cells were i.v. transplanted in the tail vein of C57BL/6J (Ly.2) recipient mice (Envigo), pretreated, one and two days before the transplantation, with intraperitoneal injections of 20 mg/kg of Busulfan (Merck). Percentages of donor cells were quantified in PB, after hemolysis, as well as in BM, by flow cytometry, 16-weeks following the transplantation. Donor Sca1+ cells (4×105 cells), isolated from Ly.1 mice treated with Calpeptin were also transplanted into Ly.2 recipient mice and hematopoiesis reconstitution was monitored, by flow cytometry, 16-weeks after the transplantation, in BM and PB of recipient mice.
Hematopoietic Colony-forming Unit (Cfu) Assay
Sca1+ cells (130-110-470, Miltenyi Biotec) were isolated from mice treated with Calpeptin, counted and 105 cells were added to semisolid methylcellulose media supplemented with growth factors (Methocult M3434; Stem Cell Technologies), at 37°C in 98% humidity and 5% CO2. Hematopoietic CFUs were counted after height days. Total CFU and distribution among CFU-GEMM, CFU-GM, CFU-G and CFU-M were measured on the microscope.
Fluorescent Microscopy
Four hours following the incubation of Sca1+ cells with PKH67+ EVs (109 particles per 4×105 cells) in the StemMACS media (Miltenyi Biotec), cells were stained with cell surface markers (SLAM). Cells were fixed and permeabilized, using BD Cytofix/Cytoperm Plus Fixation/ Permeabilization Kit and the Permeabilization Buffer Plus (BD Biosciences). We used the p-Smad2 (S465/467) antibody (#18338, Cell Signaling Technology) and the secondary anti-Rabbit-AF568 antibody (Thermo Fisher Scientific). Then, SLAM cells were purified by FACS and applied on a glass slide (Superfrost plus, Thermo Fisher Scientific) for up to 10 min, fixed with ProLong Gold Antifade reagent containing DAPI (P36931, Thermo Fisher Scientific). Fluorescent images were acquired with an Axio Imager M2 (Zeiss). Fluorescent optical sections of cells were obtained under magnification ×63, using an Axio Imager M2 (Zeiss) coupled with an Apotome.2 (Zeiss). Fluorescent images were processed for study (Fiji, NIH software).
Western Blot
Cells and EVs were suspended in western blot lysis buffer (Cell Signaling Technology). On lysates, OD 620 nm was measured to normalize the amount of the loaded sample. 50 µg of proteins were supplemented with the Laemmli buffer (Cell Signaling Technology). Targeted proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. We used the anti-phospho-Smad2 (S465/467) / Smad3 (S423/425) antibody (1:1,000, #8828, Cell Signaling Technology), mix of the anti-Smad2 and anti-Smad3 antibodies (1:1,000 each, #5339, #9523, Cell Signaling Technology), the anti-Smad4 antibody (1: 1,000, #46535, Cell Signaling Technology), anti-Grp94 antibody (1:1,000, ADISPA-850, Enzo) and the anti-Flot1 antibody (1:1,000, 610821, BD Biosciences) used as a loading control. We also used anti-TGF-β-RI (ABF17-I, Merck) and anti-TGF-β-RII (MAB532, R&D Systems) antibodies. Appropriate secondary anti-rat, anti-mouse or anti-rabbit antibodies, conjugated with Horseradish Peroxidase were used (1:5,000, Cell Signaling Technology). Chemiluminescence was performed (Chemidoc, Bio-Rad), after applying ultra-sensitive enhanced chemiluminescent (ECL) substrate (SuperSignal West Femto Maximum Sensitivity, Thermo Fisher Scientific). Protein sizes were controlled with a protein ladder (Page Ruler Plus Prestained Protein Ladder, Thermo Fisher Scientific), and protein expression levels were assessed by using ImageJ (NIH Software).
Flow Cytometry And Fluorescent-activated Cell Sorting (Facs)
To stain hematopoietic populations on BM cells, we used the following antibodies on Lin− purified cells (130-110-470, Miltenyi Biotec); Sca1-APC-Cy7 (1:100, 108126, Biolegend), c-Kit-BV650 (1:100, 135125, Biolegend), CD48-PE-Cy7 (1:200, 560731, BD Biosciences), CD150-PerCP-Cy5.5 (1:100, 115921, Biolegend). For PB reconstitution in vivo, we used the following antibodies; anti-CD45.1-APC antibody (1:100, 130-121-215, Miltenyi Biotec), anti-CD45.2-PE antibody (1:100, 130-124-080, Miltenyi Biotec), anti-CD49b-PE-CF594 (1:100, 562453, BD Biosciences), anti-CD11b-PE-Cy7 (1:100, 552850, BD Biosciences), anti-CD19-FITC (130-102-494, Miltenyi Biotec) and anti-CD3ɛ-Vioblue (1:100, 130-102-441, Miltenyi Biotec). Viability was assessed with Fixable Viability Stain FVS440UV, FVS450 or FVS780 (1:1,000, BD Biosciences). After staining with cell surface markers and FVS for viability, cells were fixed and permeabilized, using BD Cytofix/Cytoperm Plus Fixation/ Permeabilization Kit and the Permeabilization Buffer Plus (BD Biosciences). For studies on cell cycle, we used anti-Ki67-BV421 antibody (1:50, 562899, BD Biosciences) and 7-AAD (1:20, 559925, BD Biosciences). Anti-p-Smad2 (S465/S467) / p-Smad3 (S423/S425)-PE-CF594 antibody (1:100, 562697, BD Biosciences) was used after cell surface staining and fixation/permeabilization. Cells subsets were analyzed using Aurora (Cytek) or LSR-Fortessa (BD Biosciences). For fluorescent microscopy, SLAM cells were sorted on a FACS Aria cell sorter (BD Biosciences). Data were analyzed using FlowJo software (v10, TreeStar Inc, Ashland, USA).
Statistic
All data were expressed as means ± standard deviation (SD). Differences between two groups were assessed with the two-tailed Student’s unpaired t test. The one-way ANOVA with Tukey’s multiple comparison test was used to assess differences between more than two groups. No statistical methods were used to predetermine the sample size. Mice were randomly allocated to experimental groups. No blinding method was used for injection. There was no animal exclusion criteria. The variance was similar between the groups that were being statistically compared. Statistics were performed using Prism 6 (GraphPad, San Diego, USA).