Human fgf10

Fresh tumor samples were collected from the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University. In brief, the samples were digested using Collagenase Type IV (Stemcell, 07909) for 50 min and then filtered through a 100-µm sieve (Falcon, 352360) to remove incompletely digested material. The cell suspension was embedded into the ground substance mixed with DMEM/F12 and Matrigel matrix at a proportion of 1:1. The mixture was seeded into a 24-well plate and cultured in self-configured medium containing DMEM/F12, 1 × B27 supplement, 1 μmol/L prostaglandin E2 (MCE, HY-101952), 10 mmol/L nicotinamide (Sigma, N0636), 1.25 mmol/L N-acetyl-l-cysteine (Sigma, A7250), 50 ng/ml human EFG, 500 nmol/L A83-01 (PeproTech, 9094360), 5 ng/ml human FGF2, 10 ng/ml human FGF 10 (PeproTech, 100-26-5), 0.3 μmol/L CHIR 99021 (Sigma, SML1046), 1 μmol/L forskolin (Abcam, ab120058), 50 ng/ml R-spondin (R&D Systems, 3266-RS), 10 μmol/L Rho-associated kinase (ROCK) inhibitor Y-27632 (TargetMol, T1725), and 25 ng/ml Noggin (PeproTech, 120-10C). The diameter of the organoids was measured after approximately 10 days.

Organoids were grown in airway organoid medium, previously described (Sachs et al, 2019 (link)), which consists of AdDF+++ supplemented with 1× B27 supplement (Life Technologies; 17504‐044), 1.25 mM N‐acetyl‐l‐cysteine (Sigma‐Aldrich; A9165), 10 mM nicotinamide (Sigma‐Aldrich; N0636), 500 nM A83‐01 (Tocris; 2939), 5 µM Y‐27632 (Abmole; Y‐27632), 1 µM SB202190 (Sigma‐Aldrich; S7067), 100 ng/ml human FGF10 (PeproTech; 100‐26), 25 ng/ml FGF7 (PeproTech; 100‐19), 1% (vol/vol) RSPO3, and Noggin (produced via the r‐PEX protein expression platform at U‐Protein Express BV). This medium was termed airway medium (AO). For passaging, organoids were collected, washed with DMEM (Life Technologies; 10566016) supplemented with penicillin (10,000 IU/ml) and streptomycin (10,000 IU/ml) (DMEM+P/S) and disrupted either by mechanical shearing or by digestion with TrypLE Express (Life Technologies; 12605‐010). After passaging, organoid fragments were replated in fresh BME. During expansion, medium was replaced twice a week.

For cell aggregation, 2.75 × 10⁶ cCICs were plated in 5 mL EB medium (KnockOut DMEM [Gibco 10829‐018] supplemented with 15% KnockOut Serum Replacement [Gibco 10828‐028], 0.1 mM MEM Non‐Essential Amino Acids Solution [Gibco 11140‐050], 1X GlutaMAX‐I [Gibco 35050‐079]) in low‐attachment petri dish for 4 days at 37°C, 5% CO₂. For mesoderm induction, cCIC‐spheres were transferred to AF‐coated tissue culture dish in EB medium supplemented with 10% ES‐FBS to allow attachment overnight, followed by mesodermal induction media ([Gibco, 31980030] and Ham's F12 [HyClone, SH30026.01] supplemented with 5 ng/mL Activin A [Peprotech, 120‐14E], 0.5 ng/mL BMP4 [Peprotech, 120‐05ET], 5 ng/mL human vascular endothelial growth factor—VEGF [Peprotech, 100‐20], and 1X Pen/Strep [Gibco, 15140163]) for 24 hours, cardiac induction media (StemPro‐34 SFM medium [Gibco, 10639011] supplemented with 2 mM l‐glutamine [Gibco, 25030081], 0.5 mM Ascorbic acid [Sigma‐Aldrich, A4403‐100MG], 5 ng/mL human VEGF, 10 ng/mL human basic FGF, and 50 ng/mL human FGF10 [Peprotech, 100‐26]) for 7 days. Subsequently, cells were washed twice in cold PBS and fixed in 1% paraformaldehyde (PFA) for immunocytochemistry. For protein lysates, cell pellets were collected before mesodermal induction and at the end of cardiac induction.

The unsorted or sorted lung epithelial cells were resuspended in Matrigel and plated into a Matrigel-coated 24-well plate in domes (1–2 × 10⁴ cells in 50 μl Matrigel/well). The plate was incubated at 37°C for 30–45 min before adding 1 ml of medium per well. The cells were incubated in a humidified atmosphere of a cell culture incubator (37°C, 5% CO₂). The medium was changed every 3 days. The media used were the following: Lungosphere medium [1 × B-27 without vitamin A, 100 U/ml penicillin, 100 μg/ml streptomycin, in phenol red-free DMEM/F12], supplemented with EGF (20 ng/ml) and/or FGFs [1 or 2 nM FGF2-STAB, FGF2-wt, murine FGF7 (#450-60, Peprotech), murine FGF9 (#450-30, Peprotech), or human FGF10 (#100-26, Peprotech)] as needed according to experiment, and 10 μM Y-27632 (only for the first 3 days of culture); a WNT lung organoid medium (Lee et al., 2017 (link)): 50 ng/ml murine WNT3A (#315-20, Peprotech) or 50% WNT3A-conditioned medium,100 ng/ml murine Noggin (#250-38, Peprotech), 500 ng/ml human R-spondin 1 (#120-44, Peprotech), and 40 ng/ml EGF in lungosphere medium, with or without 40 ng/ml FGF10. Organoid forming efficiency (OFE,%) was calculated as (number of organoids formed)/(number of cells seeded) × 100. Organoid size was measured from organoid photographs using ImageJ (NIH) as the area occupied by the organoid.

Ectocervical cells were cultured in ADF medium supplemented with 12 mM HEPES, 1% GlutaMax, 1% B27, 1% N2, 0.5 µg ml⁻¹ hydrocortisone (Sigma, H0888-1G), 10 ng ml⁻¹ human EGF (Invitrogen, PHG0311), 100 ng ml⁻¹ human noggin, 100 ng ml⁻¹ human FGF10 (Peprotech, 120-10 C, 100-26-25), 1.25 mM N-acetyl-l-cysteine, 10 mM nicotinamide, 2 µM TGF-β receptor kinase Inhibitor IV, 10 µM Y-27632, 10 µM forskolin (Sigma, F6886) and 1% penicillin–streptomycin.