P s f

Cells at the Cp stage with the treatment of BMP4, a combination of BMP4 and WNT3A, or a combination of BMP4 and C59 were dissociated and resuspended in FACS Buffer (PBS^−/− with 1% FBS and 1% penicillin/streptomycin/fungizone (P/S/F; Gibco) at approximately 40 × 10⁶ cells per ml. The cells were treated with Human Tru Stain FC X^TM (BioLegend, #422302) for 10 min at room temperature. Approximately, 10,000 cells in 100 µl were used for each compensation. Cells were labeled with appropriate antibodies including their associated isotype control (FITC-CD45, #304006; PE/Cy7-CD146, #361008; PE-CD166, #343904, all from BioLegend). Cells were incubated for 30 min at 4 °C and washed with FACS buffer twice. Samples were resuspended in a sorting medium consisting of DMEM/F12 with 2% FBS, 2% P/S/F, 2% HEPES (Gibco), and DAPI (BioLegend, #422801) at 4 × 10⁶ cells per ml and filtered through a 40 µm cell strainer. Cells were stored on ice prior to sorting. Five microliters of all antibodies were used per million cells in 100 µl staining volume; 10 µl of Tru Stain FC X^TM was used per million cells in 100 µl staining volume. DAPI was used at 3 µM. An Aria-II FACS machine was used to compensate for the color overlapping and to gate the samples. Data were analyzed using FlowJo software (version 10.5.3).

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS; order number 10270–06), penicillin-streptomycin-fungizone (P/S/F), nonessential amino acids (NEAA), basic fibroblast growth factor (bFGF), β-glycerolphosphate (βGP), ascorbic acid (AA), dexamethasone (Dex), alamarBlue® solution and Quant-iT^TM PicoGreen^® double stranded DNA (dsDNA) reagent kit were from Gibco (Zug, Switzerland). 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was from abcr GmbH & Co. (Karlsruhe, Germany). Methanol (MeOH) was from Merck (Zug, Switzerland) and Lithium Bromide (LiBr) from Thermo Fisher Scientific (Reinach, Switzerland). All other substances were of analytical grade and were purchased from Sigma (Buchs, Switzerland). Silkworm cocoons were kindly supplied by Trudel Silk Inc (Zurich, Switzerland).

Human embryonic kidney cell lines 293 (#CRL-1573) and 293T expressing the SV40 T-antigen (#CRL-3216) were obtained from American Type Culture Collection (ATCC, Manassas, USA). HEK293 cells stably over expressing recombinant human ACE2 (293/ hACE2) or RaACE2 (293/RaACE2) were established in the lab and maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, USA) with 10% fetal bovine serum (FBS, Gibco) and 100 units of penicillin, 100 μg of streptomycin, and 0.25 μg of fungizone (PSF, Gibco) per milliliter.