Hypoxia c chamber

hRCs were isolated and cultured using the established protocol in our laboratory (George et al., 2016 (link); Yim et al., 2019 (link)). The hRCs were cultured in GM containing a mixture of two types of media (1:1). One is DMEM/High glucose containing 10% FBS and 1% PS, and the other is keratinocyte medium (Thermo Fisher Scientific, Rochester, NY) supplemented with bovine pituitary extract, epidermal growth factor, 1% FBS, 0.08% insulin-transferrin-selenium (ITS; Lonza, Basel, Switzerland), and 1% PS. hRCs were seeded in 48-well plates (5,000 cells per well) and incubated in hypoxia (1% O₂) or normoxia (21% O₂) for 3 days with GM (positive control), SFM (negative control), CM (4×), single protein (ANGPTL4, follistatin, HGF, uPAR, or VEGF) or RPC (ANGPTL4 + follistatin + HGF + uPAR + VEGF) (Figure 1). For the normoxic culture, cells were cultured in a regular cell culture incubator with 21% O₂ and 5% CO₂. For the hypoxic culture, seeded cells were transferred in a hypoxia C-chamber (BioSpherix, Parish, NY) inside a standard CO₂ incubator (Revco Ultima II; Thermo Fisher Scientific) with a compact gas oxygen controller (ProOx P110; BioSpherix) to maintain oxygen concentration at 1 and 5% of CO₂ (Yim et al., 2019 (link)).

hPSCs (10,000 cells/cm²) were plated in a 15 cm culture dish and cultured overnight in hPSC-GM. The cells were re-fed with serum-free hPSC medium (hPSC-SFM; hPSC-GM without FBS and Amniomax Supplement) and incubated in normoxia (21% O₂) and hypoxia (1% O₂) (Hypoxia C-chamber, Biospherix, Parish, NY, United States) conditions for 3 days, respectively. hPSC-CM obtained from normoxia (hPSC-CM-N) and hypoxia (hPSC-CM-H) were collected and centrifuged at 1500 rpm for 5 min. The supernatant was collected to eliminate cellular debris. The hPSC-CM was concentrated 4-fold (4×) using Amnicon Ultra-15 Centrifugal Filter Units with a molecular cutoff of 3 kDa (Millipore Sigma, Germany). The concentrated hPSC-CM was frozen and stored at −80°C until use. For proteomics, hPSC-CM was collected from 3 different donors. The 4-fold (4×) concentrated hPSC-CM was analyzed for 200 human protein content using Quantibody Human Cytokine Array Q4000 (Quantibody quantitative multiplex ELISA array, catalog # QAH-CAA-4000: Raybiotech, Norcross, GA) (Figure 1A).