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In vitro, cells was infected with lentiviral particles of the LV-STI-1-shRNA (sc-153893-V, Santa Cruz), LV-HIF-1α-shRNA (sc-35562-V, Santa Cruz) or scramble LV-control-shRNA (sc-108080-V, Santa Cruz) under manufacture's instruction (Lukacs et al, 2010).

To induce Lgl1 deletion in vitro, cells were incubated with adenovirus-cre-GFP (Vector BioLabs) and adenovirus-GFP2.

To induce hepatic differentiation of hMSG-EpiPCs in vitro, cells were cultured in 24-well plates containing thick Matrigel with conditioned medium for 16 days.

For further TSA testing in vitro cells were plated in 12 well plates with 2.5×105 cells per well.

At 5 d in vitro, cells were passaged and plated out at 0.8 1.0×106 cells per precoated plate and expanded as above for an additional 3 4 d (expansion, ∼300%).

On day 7 in vitro, cells were pulse-labelled with BrdU (0.5 µM) and 1 µl (4 M) NaOH in DMEM-F12+Glutamax for 4 hours.

For maturation of CD11c+ DC in vitro, cells were incubated for 24 hrs in complete IMDM with LPS from Salmonella abortus equi (1 µg/ml) (Sigma).

After 72 hr in vitro, cells were shifted to control medium containing 1% fetal bovine serum (FBS) and 5 days later fixed in 4% paraformaldehyde pH 7.4 and processed for immune-fluorescence.

In order to determine whether incubation with TGFβ1 had a similar impact on primary DSC to hESC incubated in vitro, cells were obtained from decidua recovered from pregnancies of <10 wks gestation.

In vitro, cells were coincubated with cartilage for 1 week.

To obtain cell extracts from culture in vitro, cells were harvested by trypsinisation and counted.