Rock inhibitor y 27632

Human dermal fibroblasts and iPS-fibro were maintained in F3 medium containing DMEM medium (PanEco) supplemented with 10% FBS (Gibco), and 5 ng/ml bFGF (Miltenyi Biotec). The culture medium was replaced twice a week. A detailed description of fibroblast isolation procedure from skin biopsy is provided in Additional File 1.
Human iPSCs were generated from dermal fibroblasts of two healthy donors using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific). The protocol for the generation of iPSCs is provided in Additional File 1. iPSCs were routinely maintained on Matrigel-coated (Corning) plates in Essential 8™ medium (Gibco). Cells were passaged with 0.05% Trypsin–EDTA solution (Gibco) and plated at 10–15% confluency in fresh Essential 8™ medium supplemented with 5 μM of ROCK inhibitor Y-27632 (Miltenyi Biotec). The culture medium was replaced daily.

Fibroblasts isolated from CTRL and patient donors were grown and expanded in vitro in DMEM 10% fetal bovine serum, 1% penicillin streptomycin and 1% Glutamax. Cells were then reprogrammed with Sendai virus, transduced with the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher) containing Oct4, Sox2, Klf4, and c-Myc reprogramming factors. Virus was washed after 24 h. At 7 days after transduction cells were plated on Vitronectin and medium was changed to Essential 8 Flex Medium (ThermoFisher). Passaging of iPSCs was done every 3–4 days or at a maximum of 80% confluency by using EDTA 0,5mM for 3–5 min at 37 °C. Cells were split in 1:3 ratio on Geltrex-coated plates in the presence of 10µM ROCK inhibitor Y-27632 (Miltenyi Biotec). Complete virus elimination was corroborated in iPSCs at passage 10 by RT-PCR with specific SeV genome detection primers (TaqMan Mr04269880-mr, ThermoFisher). Each iPSC line was subjected to rigorous quality control at the Norwegian Core Facility for Human Pluripotent Stem Cell Research Centre, including phenotyping, regular monitoring of morphology and pluripotency marker expressions.

To establish iPSC lines, BM-MNCs or MSCs from three GS-2 patients and one healthy donor were transduced for 2 consecutive days with bicystronic (pSIN4-CMV-K2M, Addgene, 21164; pSIN4-EF1α-O2S, Addgene, 21162) or polycystronic lentiviral vectors (pRRL-PPT-SF-OSKM-IRES-idTomPRE), kindly provided by Prof. Dr. Axel Schambach, Hannover Medical School, Germany [17 (link)], carrying the reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc. After transduction, MSCs were cultured in DMF10 and MNCs in StemMACS HSC expansion medium (Miltenyi, 130-100-463) supplemented with StemMACS HSC cytokine cocktail (STF, Stem Cell Factor/Thrombopoietin/Flt3-ligand, Miltenyi, 130-100-843) for 7 days. Transduced cells were plated on Matrigel-coated dishes (Corning, 354277), and the culture medium was replaced by StemMACS iPS-Brew XF culture medium (iPS-Brew, Miltenyi, 130-107-086) with 20 ng/mL basic fibroblast growth factor (bFGF, Immunotools, 11343627). Colonies were picked around day 18 after reprogramming, disrupted by microdissection, and re-plated in iPS-Brew/bFGF supplemented with 10 μM Rock inhibitor Y27632 (Miltenyi, 130-103-922).

iPSCs colonies were cultivated in Essential 8 medium (Thermo Fisher) on vitronectin-coated (Thermo Fisher) B35 plates (Falcon)⁷⁷ . For standard passaging, cells were washed once with D-PBS, incubated for 2 min in a solution of D-PBS with 0.5 mM EDTA, detached in 1 mL of fresh medium and transferred in a new coated plate. For procedures requiring the single cell condition, after D-PBS washing, cells were incubated with 1 mL of Accutase (Thermo Fisher) for 5 min at 37 °C. Once detached, Accutase was diluted with DMEM-F12 medium (Gibco) and centrifuged for 5 min at 300×g in a 15 mL tube. Cells were then resuspended in the final medium supplemented with ROCK inhibitor Y27632 10 µM (Miltenyi) according to the different protocols. iPSCs were frozen in KnockOut Serum (Thermo Fisher) with 10% DMSO. Cells were periodically tested for Mycoplasma.
To mimic GBA loss, a healthy donor-derived iPSC line was treated with the GCase inhibitor CBE at 100 µM concentration for 3 to 9 days, once differentiated into neural precursors or neurons.

For the experiments two cell lines have been used H9 and hiPSCs derived from BJ fibroblasts. Cell culture in microfluidics is similar to traditional multiwells, concerning the type of medium and reagents used; the unique variables is the volume handling. In the microfluidic platform used in this work, the effective channel volume is 5.4 μl. For the media change, a total volume of 12 μl was perfused to guarantee an extensive medium refreshment and waste products removal.
Prior to cell seeding, microfluidic channels within the chip were filled with 4°C cold Matrigel Reduced Factor® (MRF, BD) 0.5% v/v in DMEM, incubated at room temperature for at least 1 h and washed with StemMACSTM iPS-Brew XF medium (Miltenyi Biotech).
hPSCs were then dissociated in single cell using Tryple (Thermo Fisher Scientific) and injected at 700 cells/mm2 density into the channels in medium supplemented with 10 μM ROCK inhibitor (Y-27632, Miltenyi Biotech) to preserve cell viability.
Because of the reduced medium volume in the microchannels, to prevent a significant evaporation, the microfluidic platform was placed in a 100 mm Petri dish and maintained in an isotonic bath with PBS for an optimal humidity of the chamber.

For generating enteroids, the small intestine or colon were cut longitudinally and incubated for 15 min at room temperature in DPBS containing 2.5 μg/mL amphotericin B, 25 μg/mL gentamicin and 50 μg/mL normocin. The intestines were incubated in 10 mM DTT for 15 min at room temperature and followed by gentle rotation in 8 mM EDTA at 4 °C for 75 min. After three washes with DPBS, the crypts were separated by snap shaking. The crypts were washed with DPBS containing antibiotics and then pelleted by spinning at 40 × g for 2 min at 4 °C. The pellet was suspended in a solution of 66% Matrigel (Corning, Corning, NY), 33% LWRN medium, and 10 μM Rock inhibitor (Y27632; Miltenyi, San Diego, CA) at a concentration of 2 crypts/μL. A volume of 250 μL of crypt suspension were plated in a 6-well plate. On the fourth day of culture, enteroids were treated with either vehicle or 100 ng/mL human recombinant RANKL (ProSpec, East Brunswick, NJ), 100 ng/mL Tweak (PeproTech Inc., Rocky Hill, NJ), 10 ng/mL Ltα/β for 24- or 72-h and then processed for RNA or protein or for histological analysis. For UEA staining, frozen enteroid sections were stained with FITC-conjugated-UEA1 for 1-h in dark at room temperature and then washed with PBST thrice before imaging.