Collagenase digestion

Human chondrocyte line C28/I2 was bought from Sigma–Aldrich (St. Louis, MO, USA) and HEK293T cell line (ATCC: CRL‐1573) was bought from the American Type Culture Collection (ATCC, Manassas, VA, USA). Primary mouse chondrocytes (MCs) were isolated from the tibial plateau and femoral condyle at postnatal day 5 by 0.2% collagenase digestion (Sigma–Aldrich).²⁶ Chondrocytes and HEK293T cells were cultured in the Dulbecco's modified Eagle's medium (DMEM, BasalMedia, Shanghai, China) supplemented with 10% Fetal bovine serum (FBS, Procell, Wuhan, China), 1% penicillin/streptomycin (1%, Procell) at 37°C and under 5% CO₂ incubation.

AGM explant (AGM^ex) culture was performed as previously described^{2 (link)}. In brief, AGMs were deposited on a nylon membrane (Millipore) placed on metallic supports and cultured in MyeloCult M5300 or H5100 (Stem Cell Technologies) supplemented with 10 µM hydrocortisone (Sigma-Aldrich). After 6 or 12 hours or 1-4 days culture, explants were collected and dissociated into single cells by 0.125% collagenase digestion (Sigma-Aldrich) for flow cytometry analysis and further culture. In some conditions, 3-methyladenine (3-MA, Merck, 1 mM), chloroquine (CQ, Selleck, 2.5 or 5 µM), bafilomycin A1 (Baf, Selleck, 50 or 100 nM), and AS1411 (the aptamer of nucleolin, 5’-GGTGGT GGTGGTTGTGGTGGTGGTGG-3’, 10 µM, from Ruibiotech) were added into the explant cultures.
Endothelial cells, pre-HSC I and pre-HSC II cells were sorted from AGM region and were co-cultured with OP9-DL1 cells (stem cell factor, 100 ng/mL; IL-3, 100 ng/mL; and Flt3-ligand, 100 ng/mL; PeproTech) or OP9 cells (stem cell factor, 20 ng/mL; IL-7, 10 ng/mL; and Flt3-ligand, 10 ng/mL; PeproTech) for 7 days as previous report^{62 (link)} and cells were harvested by mechanical pipetting for flow cytometric analysis.

Spleens or lungs were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) or chopped into small pieces using scalpels, followed by 1 h of collagenase digestion (Sigma-Aldrich; catalog no. C5138) at 37°C and 5% CO₂. The lung homogenate and spleens were forced through 70- to 100-μm cell strainers (BD Biosciences) with the stopper from a 5-ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI 1640) by centrifuging 5 min at 1,800 rpm. A red blood cell lysis step was performed in between washes (Roche, catalog no. 11814389001). Cells were finally resuspended in enriched RPMI medium (RPMI 1640, 10% heat-inactivated fetal calf serum (FCS) (Biochrom GmbH), 10 mM HEPES (Invitrogen), 2 mM l-glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× nonessential amino acids (MP Biomedicals, LLC), 5 × 10⁵ M 2-mercaptoethanol (Sigma-Aldrich), and penicillin-streptomycin (Gibco). Cells were counted using an automatic Nucleocounter (Chemotec) and adjusted to 2 × 10⁵ cells/well for enzyme-linked immunosorbent assay (ELISA) and 1  × 10⁶ to 2 × 10⁶ cells/well for flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were collected from the heparinized blood using Percoll discontinuous density-gradient centrifugation at 400× g for 20 min. Primary cells were isolated from axillary lymph nodes (ALNs) and dorsal skin using collagenase digestion (1 mg/mL) (Sigma-Aldrich, Burlington, MA, USA) for 40 min incubation. The cell suspension was filtered using a 70 μm pore size nylon cell strainer (BD Falcon, Bedford, MA, USA) and then centrifuged at 450× g for 20 min. The cell pellet was washed twice with PBS. PBMCs and primary cells were stained with specific fluorescent-conjugated antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD23, anti-B220, anti-CD11b, and anti-chemokine–chemokine receptor 3) (BD Biosciences, San Jose, CA, USA) for 10 min on ice in a fluorescence-activated cell sorting buffer (PBS with 3% fetal bovine serum [FBS] and 0.1% sodium azide). After washing with the buffer three times, the stained cells were analyzed using a BD FACSCaliburTM two-color flow cytometer interfaced with the CellQuest software (643274, BD Biosciences). The absolute cell number was determined by dividing the number of cells of interest by the total cell number; the result is expressed as a percentage.

hAMSCs and oAMSCs (Figure 1: 1 and 4) were isolated by mixing adipose tissues with pre-warmed (37 °C) Dulbecco’s phosphate-buffered saline (DPBS; 1:1) and shaken thoroughly, followed by room temperature incubation for 30 min. The bottom fluid phase was then aspirated and DPBS was added to the upper phase (1:1). Vigorous shaking and collagenase digestion (0.15 U/mL; Sigma Aldrich, Darmstadt, Germany) followed for 60 minutes in a shaking water bath at 37 °C. Human and ovine FMSCs (Figure 1: 2 and 5) and BMSCs (Figure 1: 3 and 6) were isolated through gradient centrifugation (800× g for 30 min without brake) using Biocoll separating solution (Biochrom AG, Berlin, Germany). All human and ovine cells were plated in cell culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco by Life Technologies, Darmstadt, Germany) containing 10% serum, 1% L-glutamine, and 1% penicillin-streptomycin (Biochrom AG, Berlin, Germany). Incubation took place under standard conditions at 37 °C in a humidified atmosphere with 5% CO₂.

Spleens or lung were aseptically harvested from euthanized mice. Lungs were first homogenized in Gentle MACS tubes C (Miltenyi Biotec) followed by 1 h of collagenase-digestion (Sigma Aldrich; C5138) at 37 degrees, 5% CO₂. The lung homogenate and spleens were forced through 100 µm cell strainers (BD Biosciences) with the stopper from a 5 ml syringe (BD) and washed twice with cold RPMI medium (Gibco; RPMI-1640) by centrifuging 5 min at 1,800 rpm. Cells were finally re-suspended in enriched RPMI medium [RPMI-1640, 10% heat-inactivated FCS (Biochrom Gmbh), 10 mM Hepes (Invitrogen), 2 mM L-Glutamine (Invitrogen), 1 mM Natriumpyruvate (Invitrogen), 1× Non-essential amino acids (MP Biomedicals, LLC), 5×10^-5 M 2-mercaptoethanol (Sigma-Aldrich), and PenStrep (Gibco)]. Cells were counted using an automatic Nucleocounter™ (Chemotec) and adjusted to 2x10⁵ cells/well for ELISA and 1-2x10⁶ cells/well for flow cytometry.