Biolaminin

Biolaminin (Biolamina) was applied to 35-mm dishes and the coated dishes were pre-incubated overnight at 4 °C. After seeding approximately 25 × 10³ cells per cm², cells are incubated for 24 h in respective cell maintenance medium. Subsequently, the tissue was washed with PBS and supplemented with 1 ml of medium containing either Ad.MyoD. After 30 min incubation (37 °C, 5% CO₂), adenoviral supernatant was removed and 3 ml of differentiation medium supplemented with 7.5 µM CHIR99021 (Tocris) and 0.5 µM LDN193189 (Tocris) added. After 72 h, cells were washed with PBS and the differentiation medium was renewed supplemented with 20 ng/µl FGF-2 (Tocris). Every second day until day 14 of the experiment, differentiation medium was refreshed, and from day 7 to 14 electric pulse stimulation (EPS) was applied to the cells. See Supplemental Information Table S1 for the differentiation medium composition.

Experiments with mice (C57Bl/6J strain, The Jackson Laboratory, Bar Harbor, MN) were approved by the Animal Care and Use Committee (Inst. Molecular Genetics, ASCR) in accordance with the European Union Directive 2010/63/EU for animal experiments, U.K. Animals (Scientific Procedures) Act, 1986, and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and the ARRIVE guidelines. Mice were anesthetized using a mixed solution of Zoletil (40 mg/kg, Virbac SA, Carros, France) and 2% Rometar (10 mg/kg, Spofa, Czech Republic). Pancreases were perfused with collagenase IX (Sigma-Aldrich) solution in HBSS buffer and trimmed with surgical scissors. The pancreases were subsequently digested with collagenase for 10 min at 37°C. To remove exogenous tissue, samples were washed with HBSS two times. The tissue was then filtered through a 500 μm cell strainer, and islets were separated on a Ficoll gradient (Sigma-Aldrich) by centrifugation. The islets were placed in CMRL medium (PAN-Biotech, Aidenbach, Germany) and kept overnight at 37°C. The next day, the islets were seeded on wells coated with Biolaminin (BioLamina, Sundyberg, Sweden) and incubated overnight at 37°C. Experiments were carried out on the third day.

The plasmid Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin was packaged into lentivirus (Genechem Technologies Inc., Shanghai, China) to introduce the green fluorescent protein (GFP) into NSCs. Neurospheres were dissociated into single cells with accutase (Stemcell Technologies Inc., Vancouver, BC, Canada.) and seeded to 6 cm diameter culture dishes at a concentration of 1 × 10⁶ cell/dish. Dishes were coated with biolaminin (10 μg/mL, BioLamina, Sundbyberg, Sweden). After 24 h, NSCs were infected with the lentivirus according to the manufacturer’s instructions. Two days after lentiviral transfection, GFP-positive NSCs were screened in medium containing puromycin (1 μg/mL) and sorted with a FACS Arial cell sorter (BD Biosciences, San Jose, CA, USA). GFP-NSCs were then propagated for at least 5 passages in NSC medium and transplanted into mice hippocampus.