Mem non essential amino acids

Cardiac differentiation of the isolated cells was induced following the protocol described by Smits and colleagues [12 (link)]. Briefly, cells were seeded with a density of 10⁵ cells per 6-well in hCSC medium. After 24 h, differentiation was induced with a cardiac differentiation medium consisting of a 1:1-mixture of IMDM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and Ham’s F12 nutrient mixture with GlutaMAX-I (Gibco), containing 10% horse serum (Dianova, Hamburg, Germany), 1x MEM nonessential amino acids (Bio Whittaker, Lonza, Basel, Switzerland) and 1x insulin-transferrin-selenium (Gibco). Then, 5 µM 5-azacytidine was added in three consecutive days and differentiation medium was refreshed at day 4. Six days after the start of the differentiation, ascorbic acid (Sigma Aldrich) was added every two days and 1 ng/mL transforming growth factor β (TGF-β) (Peprotech, Hamburg, Germany) was added twice weekly. Medium was refreshed every two to three days. After 28 days, the protein expression was analyzed by immunocytochemical staining for α-actinin as described above. As undifferentiated control, cells were cultured in hCSC medium.

Human bladder biopsies were obtained from men undergoing surgical resection. All the studies carried out on patient’s samples were approved by the Institutional Ethical Committee (Ospedale San Raffaele, Milan, protocol URBBAN, Rev. February 2nd, 2014, approval date March 3rd, 2014) and the specific informed consent was obtained. All the experimental procedures involving human biologic material were carried out in compliance with relevant guidelines and regulations. Cells were obtained through a primary explant technique. Briefly, the tissue was minced with a scalpel into small pieces (1–2 mm³) and washed in fresh medium to eliminate the excess of mucus or blood proteins. Tissue pieces were then placed on a dish for primary culture and incubated in MEM-Non-essential amino acids (100× , without L-Glutamine) (Lonza), supplemented with 1% FCS, antibiotics (100 U/mL penicillin and 100 μg/mL streptomycine-sulphate), 200 nM Hydrocortisone (Sigma), 10 nM Triiodothyronine (Sigma), 5 ng/ml Epidermal growth factor (Gibco, Life Technologies). The incubation was carried out for 3–5 days. Then the medium was changed at weekly intervals until a substantial outgrowth of cells was observed. Thus, the explants were removed and the cells transferred to a fresh dish. Cells were cultured for a maximum of 20 days.