Matrigel

An in vitro angiogenesis assay was conducted to evaluate the formation of three-dimensional tubular structures by endothelial cells cultured on Matrigel (BD, Franklin Lakes, NJ, USA). HPMEC suspensions (1 × 10⁵ cells/mL) were seeded into μ-Slide angiogenesis plates (ibidi GmbH, Lochhamer Schlag, Germany), which were precoated with Matrigel. After the corresponding interventions, the cells were cultured at 37°C with 5% CO₂ for 6 hours, and the development of tubular structures was examined using an inverted microscope. Tube formation was quantified by measuring the lengths of tubes from each well using ImageJ (Media Cybernetics, Atlanta, GA, USA). A Matrigel plug angiogenesis assay was conducted to detect the newly formed blood vessels in the transplanted gel plugs in mice. After mixing high concentration Matrigel (Corning Incorporated Life Sciences, Tewksbury, MA, USA) with an endothelial cell suspension subjected to particular treatments, the mixture was injected into mice subcutaneously. After 14 days of inoculation, the Matrigel plug was excised, fixed with formalin, and stained with endothelial cell marker the CD31(1 : 2,000, Cell Signaling Technology) to detect blood vessels via immunohistochemistry. CD31staining-positive endothelial markers indicated the presence of newly formed capillaries in the sectioned gel plugs.

iPSC colonies were maintained in mTesR medium (StemCell Technologies) and cultured on Matrigel (Corning, 354277). Cells were typically passaged once a week using GCDR (StemCell Technologies). iPSCs were differentiated into NCCs with the STEMDiff Neural Crest Differentiation Kit (StemCell Technologies) according to the company instructions. Briefly, one well of iPSCs was detached with Accutase (ThermoFisher) into single cells. Cells were resuspended in provided medium containing 10 µM Y-27632 (Cell Signaling, 13624S) and plated at 8.6×10⁴ cells/cm² on Matrigel-coated 12-well plates. Cells were cultured with daily medium changes without Y-27632. On day 6, cells were passaged with Accutase into single cells (NCC P1) and maintained in Mesencult-ACF Plus medium (StemCell Technologies) supplemented with 2 mM L-glutamine until confluent. Cells were expanded for three passages before osteoblast differentiation. Briefly, cells were passaged at 5×10³ cells/cm² on uncoated 24-well plates and cultured for 4-12 days in osteogenic medium [DMEM/F-12 with GlutaMAX (Gibco) supplemented with 10% FBS, 0.1 µM dexamethasone, 10 mM β-glycerophosphate and 200 µM ascorbic acid] with medium changes every other day. Cells were fixed with 4% PFA and stained with 2% Alizarin Red (pH 4.8).

Patient and control iPSC lines used in this study were generated by the Cincinnati Children’s Pluripotent Stem Cell Facility. iPSC colonies were maintained in mTesR medium (StemCell Technologies) and cultured on Matrigel (Corning, 354277). Cells were typically passaged once a week using GCDR (StemCell Technologies). iPSCs were differentiated into NCCs with the STEMDiff Neural Crest Differentiation Kit (StemCell Technologies) according to the company instructions. Briefly, one well of iPSCs was detached with Accutase (ThermoFisher) into single cells. Cells were resuspended in provided medium containing 10 µM Y-27632 (Cell Signaling, 13624S) and plated at 8.6 × 10⁴ cells/cm² on Matrigel-coated 12-well plates. Cells were cultured with daily medium changes without Y-27632. On day 6, cells were passaged with Accutase into single cells (NCC P1).