Rhfgf basic

Exosomes were extracted from iPSCs using total exosome isolation reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s instructions. Briefly, iPSCs were cultured in 6-well plates, until 80% confluence, in serum-free iPSC growth medium containing Dulbecco’s modified eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham 1:1 (Biological Industries, Beit Haemek, Israel) containing 20% KnockOut SR (Life Technologies, Waltham, MA, USA), 1% nonessential amino acid, 1% L-glutamine (Biological Industries, Beit Haemek, Israel), 0.2% 2-mercaptoethanol (Life Technologies, Waltham, MA, USA), and rhFGF basic (4 ng/mL) (R and D Systems, Minneapolis, MN, USA), at 37 °C, 95% air, and 5% CO₂ in an incubator. Culture medium was then collected and centrifuged at 2000× g for 30 min. The supernatant was then transferred to a new tube and mixed well with 5 mL of total exosome isolation reagent. The homogenous solution was incubated at 4 °C overnight and then centrifuged at 4 °C at 10,000× g for 1 h. The supernatant was then aspirated, and the exosomes were suspended in 500 µL PBS and stored at −80 °C until use. Extracted exosomes were analyzed for particle visualization and rapid, automated analysis of size distribution and concentration by nanoparticle tracking system (NanoSight NS300, Weizmann institution, Rehovot, Israel). Exosomes were saved at −80 °C for following experiments.

First, hPSCs were induced to NKX2.1⁺ lung progenitors as described earlier (‘ALO differentiation’ section). The NKX2.1⁺ lung progenitors were then resuspended in growth factor reduced Matrigel (Corning) at a density of 100,000 cells ml⁻¹ and pipetted (50 μl droplets) onto the base of tissue-culture plates. The three-dimensional culture was maintained in alveolar medium composed of cSFDM containing 250 ng ml⁻¹ FGF2 (rhFGFbasic; R&D Systems), 100 ng ml⁻¹ FGF10, 50 nM dexamethasone, 100 nM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (Millipore-Sigma), 100 nM 3-isobutyl-1-methylxan-thine (Millipore-Sigma) and 10 μM Y-27632 (Tocris).

Partially digested tumor minces were cultured in Advanced DMEM-F12 medium supplemented with 6 mg/mL d-Glucose, 2% B27-supplement (1×), 2 mM L-glutamine, 5 mM HEPES buffer and 6 μg/mL heparin sodium salt. Fibroblast growth factor (rhFGF-basic, 10 ng/mL, R&D Systems, Wiesbaden, Germany), rhFGF10 (20 ng/mL, R&D Systems, Wiesbaden, Germany) and rhNodal (20 ng/mL, R&D Systems, Wiesbaden, Germany) were added to the culture medium and renewed every 3–4 days. Medium was changed twice per week or when beginning to turn orange. When they reached 80–90% confluency, cells were detached by accutase (PAA) and split 1:1 to 1:10. Cultures were tested for authenticity and contamination, utilizing Multiplex Cell Line Authentication (MCA) and Cell Contamination Test Analyses (McCT; Multiplexion, Heidelberg, Germany).

Patient-derived pancreatic cancer cultures PC9 and PC18 were generated and cultivated as described previously [22 ]. Cultures were subjected to SNP typing and Multiplex Cell Contamination Testing (Multiplexion, Heidelberg, Germany). PC9 and PC18 cultures were grown in DMEM Advanced F12 medium supplemented with 0.6% (w/v) glucose, 2 mM L-glutamine, 2% B27 supplement (1×) (all Thermo Fisher Scientific), 12 μg/mL heparin and 5 mM HEPES buffer (both Sigma Aldrich), referred to as CSCN medium. Cytokines, i.e., 10 ng/mL rhFGF-basic, 20 ng/mL rhFGF-10, and 20 ng/mL rhNodal (all R&D Systems) were added to the cultures and renewed twice a week.

For hematopoietic differentiation, the StemDiff™ Hematopoietic Kit (Stem Cell Technologies) was used in accordance with the manufacturer’s instructions. For neural differentiation, iPSCs were dissociated into single cells using Accutase™ (Stem Cell Technologies), transferred at 2,000 cells/well into 96-multiwell ultra-low attachment (ULA) round-bottom plates (BD Falcon), and centrifuged for 5 min at 1,500 rpm for embryoid body formation. Next, neural induction was performed in three different types of media. Embryoid bodies were incubated for 3 days in Dulbecco’s modified Eagle medium (DMEM)/F12 (Thermo Fisher) supplemented with N2 (Themo Fisher), B27 (Thermo Fisher), 100 nM LDN193189 (Tocris) and 10 μM SB431542 (Tocris). Subsequently, 20 ng/ml rhFGF basic (R&D Systems) and 20 ng/ml rhEGF (R&D Systems) were added. After incubation for a few days, EBs were plated onto poly-L-ornithine (Sigma) and L-laminin (Thermo Fisher)-coated plates and then neural rosettes were manually collected and maintained in DMEM/F12 supplemented with N2, B27, 20 ng/ml rhFGF basic, 20 ng/ml rhEGF, and 3 μM CHIR99021 (Tocris).