Bsa fraction 5

NMP differentiation was based on the published protocols^{20 (link),56 (link)} with minor modifications. In preparation for differentiation, ESCs were feeder-depleted and plated in gelatin-coated (0.1% Sigma, G1890-100G) 6-well plates (Falcon, 353046) at a density of 8×10³ cells/cm² in ES medium. On D0 of differentiation media was changed to N2B27 media (Composition: 49.5% Advanced Dulbecco’s Modified Medium F-12 (Gibco, 12634028); 49% Neurobasal medium (Gibco, 21103049); 0.5% N2-supplement (Gibco, 17502001); 1% B27-supplement (Gibco, 17504044)) supplied with 1x Glutamax (Gibco, 17504044), 40 µg/ml BSA Fraction V (Gibco, 15260037) and 100 mM 2-Mercaptoethanol (Gibco, 21985-023)) supplemented with 10 ng/ml hFGF-2 (Miltenyi Biotec, 130-104-925). The media was further supplemented with 5 µM CHIR99021 (StemMACS, 130-103-926) on D2 and with or without 50 ng/ml hGdf11 (130-105-776, Miltenyi Biotec) on D3. Throughout differentiation, the medium was refreshed every 24 h. NMP identity at D3 was routinely confirmed by co-expression analysis of Sox2 and T/Bra using immunofluorescence.

Confluent normal and tumor cells were set up in a 6-well plate. Plates were washed with PBS, then infected with NDV at multiplicity of infection (MOI) 0.001 for 60 minutes at 37 °C. Excess virus was removed and cells were washed. Cultures were then overlaid with maintenance medium (similar to growth medium except that FBS was replaced with 0.3% BSA Fraction V (Gibco). A hundred microliter culture supernatants were collected at 24, 48, and 72 hours post-infection and stored at −80 °C upon use for virus growth assay and apoptosis assay. For virus growth assay_, supernatants from each time point were ten-fold serially diluted and titred for virus in A549 cells with end point dilution assay. After 5 days incubation, plates were formalin-inactivated and incubated with mouse monoclonal anti-NDV immunoglobulin (IgG) (Abcam, Cambridge, UK) 1:200 dilution, followed by incubation with goat anti-mouse IgG alexa fluor 488-conjugated (Invitrogen, Carlsbad, CA, USA). Fluorescence was measured in Varioskan Lux microplate reader (Thermo Fisher Scientific, Singapore). TCID₅₀ value was calculated with Reed and Muench method^{48 (link)}.

The H1N1 influenza A virus strain A/RomaISS/2/08 H1N1, kindly provided by Dr. Isabella Donatelli (National Institute of Health, Rome), was prepared by infecting monolayers with 1 plaque-forming unit (p.f.u.)/cell, as already described by us [19 (link),20 (link)]. Briefly, after 90 min incubation at 35 °C, cells were layered with culture medium supplemented with 4% bovine serum albumin (BSA, fraction V, Gibco; Paisley, UK) and 0.5 µg of N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Sigma Chemical Co., St. Louis, MO, USA) and incubated at the same temperature until extensive cytopathic effect (c.p.e.) was observed. Then, after at least three freezing and thawing cycles, cell debris was pelleted (10 min at 3000 rpm) and supernatants were stored at −80 °C. Viral HA activity was revealed by hemagglutination test, whereas viral infectious particles were quantified by plaque assay [22 (link)].
Virus purification was carried out as already described by us and reported in the European patent number EP 2,780,365 B1 [23 ]. Briefly, after differential centrifugation, clarified infected supernatants were layered onto a discontinuous sucrose gradient (from 0% to 60%) and centrifuged at 85,000× g for 2 h. Purified viral particles were collected from the 20/40% sucrose interface and stored at −80 °C [23 ].

Xenograft tissue was minced using sterile scalpels and dissociated for an average of 45 min in HBSS, 1 mg/ml collagenase (Roche), 25% BSA fraction V (GIBCO) and 100 U/mL penicillin and streptomycin. This was followed by further dissociation using trypsin (GIBCO). Red blood cell lysis was performed with ammonium chloride-potassium (ACK) buffer (Invitrogen). Cells were filtered through a 40 μm filter and resuspended in a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA). Cells were then labeled with MicroBeads (Miltenyi Biotec) and incubated at 4 °C for 15 min. After incubation, the cell suspension was separated using a magnetic separator. After FACS qualification, human cells were resuspended in DMEM with 10% fetal bovine serum for other in vitro assays.