Y27632

Following spheroid encapsulation, hydrogel disks were transferred to 24‐well plates where they were immersed in 1 ml of corresponding complete growth medium for each cell type and cultured for up to 5 days. To assess the effect of biochemical cues on MSC migration for spheroid fusion, mouse (rmPDGF‐BB) or human (rhPDGF‐BB) platelet‐derived growth factors were included in the cell culture media at a concentration of 10 ng/ml. The growth factor supplemented media were refreshed every 48 h.
To elucidate the cellular mechanism involved in 3D MSC migration for spheroid fusion, small‐molecule inhibitors were employed following previous literature.¹⁴ For these experiments, 10 μM of Y‐27632 (ATCC) was used to inhibit ROCK and block actomyosin contraction, and 50 μM of NSC‐23766 (Selleckchem) was used to inhibit Rac1 and block actin polymerization. These inhibitors were included in the media used for the culture of encapsulated spheroids, which were refreshed every 48 h.

Cell samples were plated on glass coverslips coated with Matrigel on the day of capture and cultured for 24 h at 37 °C in RPMI + with Y-27632 (10 uM, ATCC). After 24 h, coverslips were washed with PBS and fixed using 4% PFA in PBS for 10 min at room temperature. Coverslips were then washed 3 times with PBS before staining.

Jejunum and colon tissues (∼2–3 cm in length) were opened longitudinally and washed with cold PBS. Tissues were transferred to a Petri dish filled with cold PBS and gently scraped with a microscope slide to remove mucus and jejunum villi before transfer to a dissociation solution [PBS containing 9 mM EDTA (Fisher Scientific, cat# AM9260G), 3 mM 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, cat# 10197777001), 10 μM ROCK inhibitor Y27632 (ATCC, cat#ACS-3030] and 1% penicillin/streptomycin). After 30 min of incubation at room temperature on a rotating shaker, tissues were transferred in a Petri dish filled with cold PBS. Crypts were isolated from the tissue by firm scraping with a microscope slide and filtered with a 100 μm cell strainer. After centrifugation (500 g, 5 min, 4°C) the pellet was re-suspended in cold Dulbecco’s Modified Eagle Medium (DMEM, Fisher Scientific, cat#31966047) supplemented with 10 µM Y27632 and crypts were counted with an hemocytometer. An aliquot was centrifuged (500 g, 5 min, 4°C) and the crypt pellet was lyzed in TRI Reagent (Zymo Research, cat# R2050) and stored at −80°C until RNA purification.

Iscove’s Modified Dulbecco’s Medium (IMDM), EpiLife™ Medium, Defined Keratinocytes SFM (K-SFM), mouse EGF recombinant protein (EGF), type IV collagenase, dispase, trypsin-EDTA, antibiotic-antimycotic and gentamicin were obtained from Life Technologies/Gibco (Grand Island, NY, USA). Pierce BCA Protein Assay Kit, TRIzol reagents, M-MLV reverse transcriptase, Fluo-4 AM, Pluronic F-127 and Pierce Peptide Desalting Spin Columns were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was bought from Hyclone (Pasching, Austria). Y-27632 was obtained from ATCC (Manassas, VA, USA). Taq DNA polymerase was purchased from TaKaRa (Tokyo, Japan). FASP Protein Digestion Kit (ab270519) and Phalloidin-iFluor 488 Reagent (ab176753) were purchased from abcam (Cambridge, UK). In Situ Cell Death Detection Kit (11684795910) was obtained from Roche Diagnostics (Indianapolis, IN, USA). DNAse I was from Amersham Biosciences (Piscataway, NJ, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

The method used was adapted from previously published protocols^{23 (link)}. iPS cells were seeded on Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (SLS, 356231) at a density of 1.6 × 10⁵ cells per 6 well in StemMACS iPS-Brew XF (Milteney Biotech, 130-104-368) supplemented with 10 μM Y-27632 (ATCC ACS-3030) for 24 h. The following day (day 1), the medium was changed to N2B27 medium, a 1:1 ratio of Neurobasal medium (Thermo, 21103049) and DMEM/F12 (Gibco, 11330-032), with N2 (Thermo, 17502048), B27 (Thermo, 12587010), Glutamax (Thermo, 350050061) and freshly supplemented with 8 μM of CHIR (Sigma, SML1046) and 25 ng/ml of BMP4 (ThermoFisher, PHC9534). After 72 h (day 4), the medium was replaced with StemPro-34 SFM (Gibco, 10639011) supplemented with 200 ng/ml human VEGFA (Peprotech, 100-20) and 2 μM Forskolin (R&D system, 1099). On day 6, cells were selected by Magnetic Activated Cell Sorting (MACS) for iPS-EC expressing CD144 using Microbeads Kit (Miltenyi Biotec, 130-097-857). Positive cells were seeded on mouse Collagen IV (BioTechne, 3410-010-02) coated plate in EGM2-MV medium (Promocell, C-22011) supplemented with 20% FBS (Thermo, 10500064) and 50 ng/ml VEGFA. Cells were used for experiments up to 3 passages.