Insulin like growth factor igf 1

For manually scraped embryoid bodies (EBs), hiPSC were harvested from T25 flasks at 70–80% confluency, by manual scraping. Prior to detachment the spent medium was removed and cells were incubated for 30 min with 3 ml EB formation medium which consisted of knockout-DMEM, 10% knockout serum-replacement (KOSR), 2 mM l-glutamine, 2 mM non-essential amino acids supplemented with 1 ng dickkopf-related protein-1 (dkk1)/ml (Cambridge Biosciences), 1 ng Noggin/ml, 5 ng insulin-like growth factor (IGF-1)/ml (both from R&D Systems). Aggregates were formed by scraping a Fine Tip Mini Pastette (Alpha Laboratories) across the growth area. These were pelleted and resuspended in retinal induction medium (DMEM/F12 (Invitrogen), supplemented with 1 ng Noggin/ml, 5 ng IGF-1/ml, 1 ng dkk1/ml, 10% (w/v) KOSR, 1% chemically-defined, serum-free supplement based on Bottenstein’s N-1 formulation (N2) supplement (Invitrogen), and 2 mM l-glutamine (Lamba et al. 2006 (link)). Suspended aggregates were transferred to low attachment bacterial-grade culture dishes (Sterilin, 3 cm diam.) and incubated at 37 °C with 5% CO₂ for 3 days; cultures were manually agitated once daily to avoid mass clumping.

The fetal kidney cells were cultured in serum supplemented culture media in 48 well plate at seeding density of 2×10⁴ cells/well at 37°C in 5% CO2. After 24 hours, this culture media of the cells was replaced with serum free media and further cultured for 48 hours under same culture conditions. The cell free culture supernatant collected after 48 hours was analyzed for different growth factors using commercial immunoassay kits for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP)-7 (all from Abcam, MA, USA) and insulin-like growth factor (IGF)-1 (R&D Systems, MN, USA). Fresh serum free media was used as control medium.