Tgfbi

The H1 hESC line was acquired from the WiCell Research Institute (Madison, WI), propagated on Matrigel-coated vessels and cultured in mTeSR (both from StemCell Tech). The H7 hESC cell line (WiCell Research Institute) was propagated and maintained on Matrigel coated vessels in X-Vivo10 Medium (Lonza), supplemented with 80ng/ml FGF2 and 0.5ng/ml TGFBI (R&D Systems). hESC were cultured in feeder-free, serum-free conditions and cells were passaged at approximately 80% confluence by treatment with 5mg/ml Collagenase IV for 5 min, followed by wash with PBS and reconstitution in an appropriate volume of medium. If an estimation of an accurate cell number of starting material was required prior to the differentiation process, after collagen treatment one representative cell culture vessel was sacrificed and hESC cells disaggregated with 0.25% Trypsin-EDTA (all from Life Technologies). 10% FBS (PAA) in RPMI 1640 medium (Life Technologies) was then used to stop trypsinization and the number of total and viable cells determined using a NucleoCounter YC-100 (Chemometec). The cells were then washed with PBS, scraped in medium and passaged into new Matrigel coated vessels at a cell density of 0.6x10⁵cells/cm². hESC culture media were changed daily.

To analyze the secretome, CM was recovered from cells cultured under FBS-free BM during the last 24 hours of culture. For functional experiments with CM, normal hMSCs (passages 6–8) were used.
Recombinant human IGFBP7, SerpinE1, Fibronectin and TGFBI (R&D Systems, Cat N°: 1334-B7, 1786-PI, 4305-FN and 3409-GB respectively) were independently added to cell culture at the indicated final concentrations and incubated for 6 days. Media and factors were replaced at day 3.