Transforming growth factor β1 tgf β1

L-ascorbic acid (LAA), ethylenediaminetetraacetic acid (EDTA), heparin, laminin from Engelbreth-Holm-Swarm murine sarcoma, Lowry assay kit (Peterson’s modification), poly-D-lysine hydrobromide, protease inhibitor cocktail (Cat#P8340), sodium selenite, and Terg-A-Zyme detergent were purchased from Millipore Sigma (Oakville, ON, Canada). Transforming growth factor-β1 (TGF-β1; cat #100–21) and fibroblast growth factor 2 (FGF2; cat#100-18B) were purchased from Peprotech (Cranbury, NJ, United States) was purchased from Peprotech (Cranbury, NJ, United States). Recombinant human transferrin (cat#777TRF029) was purchased from InVitria (Fort Collins, CO, United States). Radioimmunoprecipitation assay (RIPA) 10x buffer and the ROCK inhibitor Y-27632 (cat# 13624S) were purchased from Cell Signaling Technologies (Whitby, ON, Canada). BrainPhys™ Neuronal Medium, EasySep™ Release Human CD45 Positive Selection Kit, and the STEMdiff™ Cerebral Organoid Kit (cat#08570) were obtained from STEMCELL Technologies (Vancouver, BC, Canada). A full list of antibodies and their suppliers is provided in Table 2. All other reagents were sourced from Fisher Scientific (Ottawa, ON, Canada).

Human HSCs were obtained from biopsies of healthy liver parenchyma from three different patients undergoing partial hepatectomy. The protocol was approved by the local research ethics committee of the Department of Surgery of the Geneva University Hospital. All donors provided their written informed consent for use of the samples in the present study. HSCs were isolated as previously described [57 (link),58 (link)]. Cells were cultured in 24-well plates (100,000 cells/well) in IMDM medium (Invitrogen, Basel, Switzerland) containing 10% FCS, penicillin, and streptomycin (Invitrogen, Basel, Switzerland) at 37 °C with 5% CO₂. Cells were used for experiments between passages 3 to 6. HSCs were either left untreated, or treated with recombinant IL-1β (10 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h, or IL-1β (10 ng/mL) and IL-1Ra (15 μg/mL, Kineret, Amgen Europe B.V, Breda, The Netherlands) together, or Transforming growth factor-β 1 (TGF-β1) (50 ng/mL, PreproTech, UK) for 48h. For α-SMA protein detection, HSCs were treated and cultured for 5 days.

Cells expanded separately in monolayer from those used in clonal analysis were cultured in both environments during differentiation (hypoxia → hypoxia (HH), hypoxia → normoxia (HN), normoxia → hypoxia (NH), normoxia → normoxia (NN)). The micromass differentiation protocol was modified from the procedure described by Ahrens et al. [40] (link). This method was shown to induce DIAS cell chondrogenesis in a manner similar to the aggrecan method described previously [18] (link) in an unpublished study from our laboratory. Coated surfaces were prepared in 24-well TCP plates. A sterile 0.08% chondroitin sulfate (Sigma) solution was prepared and 20 µl was dropped into each well and allowed to dry overnight.
DIAS cells were suspended in chondrogenic medium consisting of base medium with 50 µg/ml ascorbic acid-2-phosphate (Acros Organics, Geel, Belgium), 0.4 mM proline (Acros), 50 mg/ml ITS+ Premix (BD Biosciences, Bedford, MA), 10⁻⁷ M dexamethasone (Sigma), 10 ng/ml transforming growth factor β1 (TGF-β1) (Peprotech, Rocky Hill, NJ), 100 ng/ml recombinant human insulin-like growth factor (Peprotech), and 1% FBS. 2×10⁵ cells were seeded in a 20 µl droplet on the dried surface. After 4 hours, 500 µl of chondrogenic medium was carefully added around the condensed cell mass, and 250 µl of media was exchanged every other day for 14 days.