Growth factor reduced (gfr)

The HBL-100 cell line was cultured in DMEM/F12 supplemented with 10% FBS. Conditioned medium (CM) was collected when the cultured HBL-100 cells reached 70% confluence HBL-100 after 24 hours. After centrifugation and filtration through a 0.22 μm filter to remove the cells, the medium was stored at -80°C. The CM was thawed at 4°C overnight and was supplemented with 2% matrigel (Growth Factor Reduced; BD Biosciences, CA, USA) before used. DMEM/F12 containing 10% FBS was used as a control.

Human iPSC lines (UC01 and UC15) and ESC line (H1) cultured in chemically defined mTeSR1 medium (Stem Cell Technologies) on a Matrigel matrix (Growth Factor Reduced, BD Bioscience). The human iPSC colonies were passaged using Accutase (life).
For the HB differentiation, we started the definitive endoderm (DE) stage induction first. Briefly, when human iPSCs reached nearly 70% confluence, mTeSR1 medium was replaced with differentiation medium (RPMI1640 [Gibco], supply with 1 × B27[minus insulin, Invitrogen]), containing 100 ng/mL Activin A (R&D Systems) and 3 μM CHIR99021 (CHIR) for 1 day, and on the following 2 days, CHIR was omitted from the medium. Then, DE population was cultured in differentiation medium, containing 20 ng/mL BMP2, 20 ng/mL BMP4, and 30 ng/mL FGF4 for 4 days to specify hepatic endoderm (HE), and then subsequently differentiated into HBs by treatment with differentiation medium containing 20 ng/mL BMP4 and 20 ng/mL HGF for 3 days. The medium was changed daily during the differentiation period. All grow factors were purchased from PeproTech except that indicate.

All animal work was approved by the OHSU Institutional Animal Use and Care Committee. WT male and female C57Bl/6 N and Ddx58 (−/−) 8–10-week-old mice were purchased from Jackson Labs and injected subcutaneously with Growth factor reduced (Matrigel BD) with 400 ng mL⁻¹ recombinant human bFGF (Millipore). One-week later Matrigel plugs, as well as lung, liver, and heart, were harvested from mice and RNA was isolated using the Eurx RNA purification kit according to manufacturer’s instructions. Matrigel plugs were homogenized and analyzed for hemoglobin content using a colorimetric assay kit (Sigma). In addition, RNA from tissue was used to analyze endothelial activity using the Qiagen endothelial cell activity qRT-PCR array according to manufacturer’s instructions.