Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.
Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.
Cells were dissociated using 0.5mM Ultrapure™ EDTA (Invitrogen, #15575-638) in PBS at 37°C for 3-5 minutes. This submission contains pluripotent embronic stem cell and differentiated samples. The human embronic stem cell line MAN13 is a clinical grade hESC line which has been well characterised (PMID: 28583200) (https://hpscreg.eu/cell-line/UMANe002-A) and was derived under local ethical approval (Central Manchester LREC favourable opinion 03/CM/684) and UK Human Fertilisation and Embryology Authority license (HFEA licence R0171) with fully informed parental consent. For pluripotent RNA-seq samples (Time 0), MAN13 hESCs were expanded in 6-well cell culture plates previously coated with 5µg/ml Human Recombinant Vitronectin (Gibco, #A27940) in Dulbecco's Phosphate Buffered Saline (PBS) modified without calcium chloride and magnesium chloride (Sigma-Aldrich, #D8537). Cells were cultured in mTeSR™1 basal medium (StemCell Technologies, #85851) with 20% O2 and 5% CO2 at 37°C. Medium was replaced every 48 hours, and cells passaged when 80-90% confluent. Cells were dissociated using 0.5mM Ultrapure™ EDTA (Invitrogen, #15575-638) in PBS at 37°C for 3-5 minutes. HiPSCs were grown in 6-well plates pre-coated with 5µg/ml human recombinant Vitronectin in PBS. The cells were maintained in TeSR™-E8™ medium (E8) (StemCell Technologies, #05990) at 20% O2 and 5% CO2 at 37°C, E8 medium was replaced every other day. Cells were passaged every 4-7 days as described previously for hESCs. For 2D Chondrogenic samples: MAN13 hESCs were differentiated towards chondroprogenitors using the RAPID-E protocol (https://www.biorxiv.org/content/10.1101/2023.06.20.545738v1 Mancini, F. E., Humphreys, P. E. A., Woods, S., Bates, N., Cuvertino, S., O'Flaherty, J., Biant, L., Domingos, M. A. N. & Kimber, S. J. Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis. bioRxiv 2023.06.20.545738 (2023). doi:10.1101/2023.06.20.545738). When MAN13 cells were 50-60% confluent, pluripotency maintenance medium was removed and replaced with Advanced Differentiation Basal Medium (ADBM), consisting of Advanced DMEM F-12 (Thermo Fisher Scientific, #12634010) supplemented with 1% (v/v) L-Glutamine (Gibco, #25030081), 2% (v/v) B27 Supplement (Gibco, #17504001), 0.1mM β-mercaptoethanol (Gibco, #31350010). Cells were sub-cultured on days 4 and 8 of differentiation onto human fibronectin (Merck Millipore, #FC010) coated plates at a ratio of 1:4. Samples for RNA-seq analysis were cultured with addition of 100 nM of TTNPB, as standard for the RAPID-E protocol, or in complete absence of TTNPB throughout the protocol. Total RNA was extracted using Monarch® Total RNA Miniprep Kit (New England Biolabs inc. #T2010S) following manufacturer protocol. Stranded mRNA protocol