Rhil 6

Eight-week-old male C57BL/6 mice were routinely bred in the animal facility of the Zhongshan Hospital at Fudan University (Shanghai, China). The mice were housed at 20–25 °C with a relative humidity of 50–70% and were provided free access to water and food. The experimental procedures were approved by the Animal Care and Use Committee of Zhongshan Hospital at Fudan University. Intratracheal BLM (2.5 mg/kg) was instilled in mice to induce ALI, and mice in the control group received an equal volume of PBS. MSC SLP (50 mg/kg) was dissolved in PBS and administered intratracheally 1 h after BLM instillation. The mice were sacrificed to collect peripheral blood, bronchoalveolar lavage fluids (BALFs), and lung tissues on day 7. To evaluate the role of IL-6 in ALI, recombinant human (rh) IL-6 (PeproTech, #200-06, NJ, USA) was administrated intratracheally at a dose of 1000 ng per mouse. MSC SLP (50 mg/kg), dissolved in PBS, was also administered intratracheally 1 h after rh IL-6 instillation, and the mice were sacrificed on day 2.

CD9⁺Lin⁻CD34⁺CD45RA⁻ and CD9⁻Lin⁻CD34⁺CD45RA⁻ HSPCs were cultured in 96-well plates with StemSpan SFEM II medium (09655, STEMCELL Technologies, Vancouver, BC, Canada) supplemented with recombinant human stem-cell factor (rhSCF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-3 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rhIL-9 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–G colony-stimulating factor (G-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh–GM colony-stimulating factor (GM-CSF; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), rh-thrombopoietin (TPO; 50 ng/mL; PeproTech, Rocky Hill, NJ, USA), and 20% BIT 9500 Serum Substitute (09500, STEMCELL Technologies, Vancouver, BC, Canada). Besides, 24-well Transwells (3470, Corning Incorporated, Corning, NY, USA) were used for the indirect co-culture of immune cell progenitors and CD9⁺Lin⁻CD34⁺CD45RA⁻ HSPCs, and additional rhIL-7 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added in the transwell co-culture system. Lin⁻CD34⁺CD45RA⁺CD38⁺CD9⁺CD10⁺ and Lin⁻CD34⁺CD45RA⁺CD38⁺CD161⁺ were used for pre-B cells and NK/Tp enrichment, respectively (Supplemental Fig. 6d).^{68 (link)} All cultures were incubated at 37 °C in a humidified chamber under 5% carbon dioxide.

Mononuclear cells (MNCs) were isolated from bone marrow samples using density gradient centrifugation. The CD138⁺ and CD138⁻ fractions were then isolated from the MNCs using CD138 microbeads (Miltenyi Biotec) and an AutoMACS magnetic cell sorter (Miltenyi Biotec). The CD138⁻ fraction was further depleted of normal hematopoietic progenitors using CD34, CD3, CD4 and CD8 microbeads (Miltenyi Biotec). The resulting two fractions (CD138⁺CD34⁻CD3⁻CD4⁻CD8⁻ and CD138⁻ CD34⁻CD3⁻CD4⁻CD8⁻ cells; 2.5 × 10⁵/mL) were plated with or without different concentrations of chloramphenicol in round-bottom 96-well plates for colorimetric assays or in a methylcellulose culture system containing rhIL-6 (10 ng/mL, PeproTech) for clonogenic assays (1 × 10⁵ cells per well). Tumor cell colonies were counted after 2–3 weeks of culture. As a control for MM cells, CD34⁺ progenitor cells from one donor's peripheral blood were cultured in a methylcellulose culture system with 50 ng/ml GM-CSF (cells: 100,000/well). The phenotype of the cells in these colonies was confirmed by flow cytometry.

HCAEC and human pulmonary artery endothelial cells (HPAEC) purchased from Clonetics (Lonza, USA) were cultured in EBM-2 medium (Clonetics, Lonza, USA) supplemented with EGM-2MV Single Quots and 5% FBS (Clonetics, Lonza) as described previously [23 (link)]. For experiments, cells at passages three to six were used and the serum in culture medium was decreased to 2% FBS. Macrophages derived from human PBMC were cultured as described [24 (link)]. Cells were cultured under standard conditions (37°C, 5% CO₂, 80% humidity) in a Class 100 HEPA air filtered system (SteriCult, Fisher Scientific, Switzerland). Culture medium without antibiotics was used to prevent masked low-level contamination in cell cultures. Treatments were carried out using recombinant human (rh) IL-4 (4 ng/mL; purity >98%; PeproTech, USA), rh IL-6 (20 U/mL; purity >98%; PeproTech, USA), rh soluble Frizzled-related peptide-1 (sFRP1; 10 μg/mL; purity > 95%; R&D systems, USA), rh/mouse Wnt5A (250 ng/mL; CHO-derived Gln38-Lys380, purity >80%, endotoxin level <1.0 EU/μg of protein; R&D systems, USA) and rh Wnt inhibitory factor 1 (WIF1; 15 μg/mL; purity >97%; R&D systems, USA). Sterile biopure ep Dualfilter T.I.P.S. sterile filter tips (Eppendorf, Germany) were used throughout the study.

Liquid culture of CD34+ cells and CFC assay were previously described^{32 (link)}. Briefly, CD34+ were cultured in StemSpan or IMDM 5% fetal bovine serum medium (StemCell Technologies) in presence of rhSCF (100ng/ml), rhIL6 (20ng/ml), rhFlt3-L (100ng/ml) and rhTPO (20ng/ml) (all from Peprotech) for liquid culture, while for CFC assay were plated at 10³ cells/ml in semi-solid medium (MethoCult H4434 Classic - StemCell technologies) and scored by microscope 14 days later. Proliferation and mortality of liquid cultures were assessed at 3, 7, 11 and 14 days post-transduction. OP9-ΔL1 stromal cell line was kindly provided by I. Schmutz and M. Cavazzana and co-culture was performed as described for 21 days^{14 (link)}. In vitro proliferation of T cells was assessed by cell proliferation dye efluor670 (eBioscience) staining of total splenocytes or magnetically purified CD4+ and CD4+CD25- cells, where indicated, upon stimulation by antiCD3/CD28/CD2 beads (Treg suppression inspector beads – Miltenyi Biotec) following manufacturer’s instructions.