ERC000011
ENA-CHECKLIST
2022-08-16T12:15:53Z
ENA-FIRST-PUBLIC
2022-08-16T12:15:53Z
ENA-LAST-UPDATE
SAMEA14414239
External Id
UNIVERSITY OF MANCHESTER
INSDC center name
2022-08-16T12:15:53Z
INSDC first public
2022-08-16T12:15:53Z
INSDC last update
public
INSDC status
ERS12019247
SRA accession
E-MTAB-11758:Sample 2
Submitter Id
ArrayExpress
broker name
MAN-13
cell_line
human
common name
Protocols: Cell samples were taken as described previously [PMID: 20967028, Smith CA, Humphreys PA, Naven MA, Mancini FE, Kimber SJ. Directed differentiation of hPSCs through lateral plate mesoderm for generation of articular cartilage progenitors. BioRxiv. 2021:2021.03.24.436807.]. hESCs were differentiated as previously described [Smith CA, Humphreys PA, Naven MA, Mancini FE, Kimber SJ. Directed differentiation of hPSCs through lateral plate mesoderm for generation of articular cartilage progenitors. BioRxiv. 2021:2021.03.24.436807.]. Briefly, 100,000 hESCs/ cm2 were split into fibronectin coated 16.6 µg/mL (Millipore) 6-well plates and cultured until 70% confluency to start the protocol. To start, mTSeR1 medium was removed and directed differentiation basal medium (DDBM) was added for chondrogenic differentiation. Chondrogenic pellets were generated as described previously [Smith CA, Humphreys PA, Naven MA, Mancini FE, Kimber SJ. Directed differentiation of hPSCs through lateral plate mesoderm for generation of articular cartilage progenitors. BioRxiv. 2021:2021.03.24.436807.]. Briefly, day 11 chondroprogenitors were detached from adherent culture by TryplE express (Thermo) and centrifuged at 300x RCF in day 11 culture medium for 5 minutes to compact cells. The cells were cultured for a further three days at 37 °C in 5% CO2. Day 11 medium was changed at day 11 + 3 for chondrogenic-basal medium (CM) containing GDF5 (40 ng/ml), TGFβ3 (10 ng/ml) (both Peprotech) and BMP2 (0.5 X EC 66) (R&D systems). Pellets were cultured for a further 7 days (i.e. a total of 21 days) with medium changed every 3 days. hESCs line MAN13 [PMID: 28583200] was cultured in feeder-free culture conditions in 6 well plates coated in vitronectin (5 µg/mL) (Thermo) in mTSeR1 medium (Stem Cell Technologies). Cells were passaged at 80% confluence by detachment with 0.5 mM EDTA in PBS. The culture medium was changed every two days. Samples were lysed using RLT buffer and RNA extracted using RNeasy Qiagen kit. TruSeq Stranded mRNA assay (Illumina)
description
directed chondrogenic differentiation through lateral plate mesoderm
growth condition
Homo sapiens
organism
Homo sapiens
organism
embryonic stem cell
progenitor cell type
Homo sapiens
scientific_name
Sample 2
title