Human fc blocking reagent

For isolation of cells for scRNAseq, freshly isolated PBMC were FC block- treated (2 ul FC blocking reagent human (Miltenyi Biotech) and 48 ul staining buffer (1% BSA in PBS, filtered through a 40 um filter)/1x10⁶ cells) and incubated 10’ on ice. Cells were washed and stained in 50 ul final/1x10⁶ cells of antibody cocktail (CD3-APC (HIT3α, BD), CD19-APC (SJ25C1, BD), CD14-PE-Cy7 (M5E2, BD), CD56-PE-Cy7 (B159, BD), HLA-DR-APC-H7 (B159, BD), CD11c-AF700 (3.9, eBioscience), CD123-BV650 (6H6, Biolegends), CD16-BV605 (3G8, Biolegends)). Life- dead staining with 7AAD (BD) was done shortly before sorting on a FACS (ARIAII, BD) with the 100 uM nozzle using gating strategy shown in Supplemental Figure 1. For isolation of cells for qPCR, the same panel was used including also EMP1-FITC (Biozol) (Supplemental Figure 4A).

Surface immunostainings were performed at day 6 post-isolation of the CD14 + cells from PBMCs. After a 15 min-incubation step with human Fc blocking reagent (MACS Miltenyi Biotec), cell surface markers were detected by a 30 min-incubation at 4 °C with 4 µg/mL Pacific Blue-conjugated mouse anti-CD14 (clone 63D3, Biolegend), a 1:20 dilution of APC-conjugated mouse anti-CD163 (clone REA812, MACS Miltenyi Biotec), 0.14 µg/mL PE-conjugated mouse anti-HLA-DR (clone LN3, Invitrogen) and a 1:20 dilution of PE-Cy7-conjugated mouse anti-CD11b (clone ICRF44, BD Biosciences), diluted in staining buffer (PBS - 1% FBS). Flow cytometric analysis was performed using a BD FACS Canto II and the data were analyzed with Flow Jo software (Tree Star).

To evaluate intracellular cytokine production, 1 x 10⁶ PBMCs or BACs were stimulated with Phorbol-12-Myristate-13-Acetate (PMA, 25 ng/ml, Sigma Chemical, St. Louis, MO) plus ionomycin (1μM, Sigma Chemical) in the presence of brefeldin A (5 μg/ml, Sigma Chemical) for 4 h at 37°C and in 5% CO₂. After activation, the cells were washed with 1ml of wash solution and incubated with human Fc-blocking reagent (Miltenyi Biotec, Auburn, CA) for 15 min at 4°C. Monoclonal antibodies (anti-CD161-FITC, CD4-APC-Cy7, CD8-PE-Cy7, or CD3-PE-TexRed) or their respective IgG isotype controls were then added and incubated for 15 min at room temperature in the dark. Then, the cells were washed with 1ml of wash solution and permeabilized with 500μl of 1X permeabilizing solution (BD) for 10 min at room temperature in the dark. The cells were then washed, and monoclonal antibodies (anti-IFN-γ-APC, IL-17A-Alexa700 [Biolegend], IL-4-PE, or TNF-α-PE [BD]) or their corresponding IgG isotype controls were added and incubated for 30 min at room temperature. The cells were washed, and 100,000 events were immediately read using a FACSCanto II flow cytometer (BD). Data analysis was performed using FlowJo research software version OS 10.2 (Tree Star, Inc. Ashland, OR).

Day 10 hPSC-derived HLF+ HOXA+ hematopoietic progenitors were collected, counted, and seeded in StemPro-34 base media (Thermo Fisher) supplemented with the following factors:•

Day 0-5: SCF (50 ng/mL [Peprotech, 300-07]), TPO (10 ng/mL [Peprotech, 300-18]), IL-3 (50 ng/mL [Peprotech, 200-03]), FLT3L (50 ng/mL [Peprotech, 300-19]), M-CSF (50 ng/mL [Peprotech, 300-25]), and ITS-X (Thermo Fisher, 51500-056])

•

Day 6-10: FLT3L (50 ng/mL), M-CSF (50 ng/mL), GM-CSF (25 ng/mL [Peprotech, 300-03]), and ITS-X

•

Day 10-17: M-CSF (100 ng/mL), GM-CSF (50 ng/mL), and ITS-X

At day 17, cells were dissociated using TrypLE (Thermo Fisher), stained with CD11b APC antibody (BioLegend, 101212) and CD68 PE Cy7 antibody (Thermo Fisher, 25-0689-42) in the presence of human Fc blocking reagent (Miltenyi Biotec, 130-059-901), and analyzed for flow cytometry.

Cell suspensions were pre-incubated with 5 μL human Fc Blocking Reagent (Miltenyi Biotech, Bisley, UK) or normal rat serum (in dilution 1:100) for 10 minutes at 4 °C. Aqua Live/Dead™ 405 nm cell stain (Invitrogen, Paisley, UK) or Zombie UV Aqua Fixable Viability Stain (Biolegend, London, UK) was added in dilution 1:200 in PBS and incubated for 5 minutes, followed by the surface antibody cocktail for 30 mins in the dark at 4 °C. All rat (anti-mouse) and mouse (anti-human) antibodies were used in dilution 1:100 and 1:25, respectively. All antibodies used in this study are listed in Supplementary Table 1. Intranuclear Ki-67 staining was performed using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific, Waltham, USA).
Following three washes, samples were measured with BD LSRFortessa using BD FACSDiva 6.2 software (Becton Dickinson, Basel, Switzerland) and data subsequently analysed with FlowJo 10.4 software (TreeStar, USA). Each FACS tube was run until its exhaustion. Cell sorting of murine kidney and bladder MNPs and B cells was done on unfixed cells using Aria-Fusion III (Becton Dickinson, Basel, Switzerland) or iCyt Synergy (Sony Biotechnology Inc.).