Nutristem xf medium

hESC line VUB02 was derived and characterized as previously described^{35 (link)}. Cells were routinely cultured at 37 °C at 5% CO2 on dishes coated with Laminin521® at 10 µg/cm² (Biolamina) in Nutristem® XF medium (Biological Industries) supplemented with 0.1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were passaged as single cells using TrypLE™ Express (Thermo Fisher Scientific) when 80–90% confluent in a ratio between 1:10 and 1:40. Initial karyotype was determined by aCGH (4 × 44 K, Agilent Technologies) as previously described^{7 (link)}.

hMSCs (approximately 10,000 cells/cm²) were seeded in MSC media containing human serum for 24 h to allow cell attachment to the bottom of the flasks. The MSC media were removed, and the culture flask washed twice with phosphate buffer saline (PBS) prior to adding serum-free media (MSC NutriStem XF medium, Biological Industries, Kibbutz Beit-Haemek, Israel). Conditioned media (CM) were collected every 48 h, and the cells were passaged at 90% confluency. CM collected were centrifuged (300×g, 4 °C, 5 min) and stored at − 80 °C until use. The hMSCs used in the study were in passage 4–7. The isolation of extracellular vesicles (EVs) was performed by a series of differential centrifugation and filtration of the CM as described in [14 (link)]. Briefly, CM were thawed on water bath and centrifuged at 16,500×g for 20 min followed by filtration through 0.22-μm filters to deplete cell debris and large EVs. The sEV/exosomes were then pelleted by ultracentrifugation at 120,000×g for 70 min in a T-645.5 rotor (Sorvall wx Ultra series, Thermo Scientific, Rockford, IL, USA). The sEV pellets were re-suspended in cold PBS and stored at − 80 °C until use. The whole procedure was performed at 4 °C.

Human iPSCs were obtained as previously described [14 (link)]. Briefly, 1 × 10⁵ skin fibroblasts were nucleofected with three episomal plasmids: pCXLE-hUL (Addgene #27080), pCXLE-hSK (Addgene #27078) and pCXLE-hOCT4-shp53 (Addgene #27077), and plated in fibroblast medium for 1 week. From day 8, the cells were cultured in NutriStem XF Medium (Biological Industries, Kibbutz Beit Haemek, Israel). iPSC colonies were manually cut and passaged for expansion. Absence of mycoplasma contamination was verified by PCR analysis using EZ-PCR kit (Biological Industries, Kibbutz Beit Haemek, Israel).

The parental hiPSC line, registered in the Human Pluripotent Stem Cell Registry (hPSCreg^®) as MUSIi012-A (accessible at https://hpscreg.eu/cell-line/MUSIi012-A), was established from CD34⁺ hematopoietic stem/progenitor cells (HSPCs) collected from G-CSF mobilized peripheral blood of a healthy female donor using non-integrating episomal reprogramming plasmids under ethics approval by Siriraj Institutional Review Board (COA No. Si 248/2011) after informed consent. Cells were maintained on Matrigel-coated plates (Corning, Corning, New York, United States) in NutriStem XF medium (Sartorius, Göttingen, Germany), passaged every 2–3 days using Versene solution (Gibco, Waltham, MA, United States), and cultured in a humidified atmosphere of 5% CO₂ at 37°C.

Human iPSCs, MUSIi013-A single-cell clone, reprogrammed from UCB-derived NK cells (NK/iPSC) [15 (link)] were routinely cultivated in NutriStem XF medium (Sartorius, Göttingen, Germany) on Matrigel-coated plates (Corning, Corning, New York, NY, USA) and mechanically passaged every two to three days. HEK293FT cells were cultured in high-glucose DMEM (DMEM-HG) medium while hematologic cancer cell lines, including K562, REH, and Raji cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium. DMEM-HG and RPMI1640 medium were supplemented with 10% FBS, L-glutamine, and antibiotics (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). All cells were culture at 37 °C and 5% CO₂.