Laminin ln521

Migrating cells were analyzed by using live-cell analyzer JuLI™ Br as well as xCELLigence Real-Time cell Analysis instrument. Therefore, O4⁺ RRMS and control hiOL were purified by flow cytometry at day 12 of differentiation and seeded onto Laminin LN521 (Biolamina) coated 12-well plates (for JuLI™ Br) or CIM-Plate 16 (for xCELLigence). For JuLI™ Br recording, cells were allowed to sit for 4 h followed by recordings of undirected moving for 24 h. Cells were tracked and analyzed using MTRACKJ plugin. For xCELLigence analysis, O4⁺ hiOL were seeded and impedance-based measurement was performed immediately for 16 h.

Since boundary cap NCSCs are known to migrate toward islets, the migration behavior of the CD271+ cells was evaluated. CD271+ cells were co-cultured with either human islets or human ICC derived from pluripotent stem cells. A drop of approximately 50,000 cells and 2–3 islets were placed on opposite sides of a coverslip placed in a 4-well plate at a distance of approximately 1000 µm. The cells were cultured in medium suitable for islet culture. Coverslips were pretreated with Laminin LN521 (BioLamina AB, Sundbyberg, Sweden) for 2 h at 37 °C in 5% CO₂ and 95% air. To visualize the cells, they were stained with CellTracker Fluorescent Probes (1:1000; Green CMFDA; Thermo Fisher Scientific). Images were taken on days 1–7 of co-culture, and the distance between cells and islets or ICC was measured. Experiments were repeated with three biological replicates, and the migration capacity toward human islets and ICC was compared and evaluated by measuring the distance to the CD271+ cells.

The use of iPSC lines for this study was approved by the ethics committee of the medical faculty of Heinrich-Heine University under the number 5013. iPSCs were cultured on laminin (LN) 521 (Biolamina) coated plates in StemMACS iPSC brew medium (Miltenyi). Differentiation into HLCs was performed as described previously (Graffmann et al., 2016 (link)) with minor changes. To start the differentiation, iPSCs were passaged as single cells onto plates coated with a 3:1 mixture of LN111 and LN521. The next day, the medium was changed to definitive endoderm (DE) medium: 96% RPMI 1640, 2% B27 (without retinoic acid), 1% Glutamax (Glx), 1% Penicillin/Streptomycin (P/S) (all Gibco), 100 ng/ml Activin A (Peprotech), which was replaced daily. On the first day an additional 2.5 µM Chir 99021 (Stemgent) was included. Afterwards the cells were cultivated for 4 days in hepatic endoderm (HE) medium with daily medium changes: 78% Knockout DMEM, 20% Knockout serum replacement, 0.5% Glx, 1% P/S, 0.01% 2-Mercaptoethanol (all Gibco) and 1% DMSO (Sigma-Aldrich). In the last step, hepatocyte-like medium was used for up to 10 days with medium change every other day: 82% Leibovitz 15 medium, 8% fetal calf serum, 8% Tryptose Phosphate Broth, 1% Glx, 1% P/S (all Gibco) with 1 µM Insulin (Sigma-Aldrich), 10 ng/ml hepatocyte growth factor (HGF) (Peprotech), 25 ng/ml Dexamethasone (DEX) (Sigma-Aldrich).