Human embryonic stem cells (hESCs)
UCSF4 hESCs were used for this study17
. Cells were maintained in feeder-free conditions (Suppl Fig. 1a, b
) using Cell Start matrix (Invitrogen), mTeSR medium (Stem Cell Technologies), and passaged every 5 days. Colonies were regularly stained for markers typically used for hESCs (Nanog, Sox2, Oct4, Tra1-60, Tra-1-81, SSEA4) (Suppl Fig. 1, c–h
Embryoid body (EB) formation
For EB formation, clumps of hESC were grown in suspension in EB medium (knock out DMEM, 20% FBS, non essential amino acids, L-glutamine 2 mM, 0.1 mM β-mercapto ethanol) for 7 days during which EB spheroids formed. For spontaneous differentiation experiments, EBs were transferred to culture-treated plates coated with Cell Start. EBs adhered and were allowed to differentiate for 2 weeks, and samples were used for qPCR at 1 and 2 weeks post attachment. Immunocytochemistry was performed at 2 weeks for germ layer markers: beta III tubulin (ectoderm), smooth muscle actin (mesoderm), and alpha fetoprotein (endoderm). Some EBs were maintained in suspension for the duration of the experiment, and samples were used for qPCR after 1, 2, and 3 weeks in suspension.
Briefly, cells were fixed in the plates with 4% paraformaldehyde for 15 min at room temperature (RT), then permeabilized with 0.2% Triton X-100 for 15 min at RT. Samples were blocked with 5% BSA for 1 h at RT, primary antibodies were added and incubated overnight at +4, washed with 0.1% Triton X-100 3 times, secondary antibodies were added and incubated overnight at +4, then washed again with 0.1% Triton X100 and DAPI was added. Cells were observed under an inverted fluorescent microscope (Leica), and pictures were taken with a Leica DFC 500 camera. Primary antibodies were used at 1/100 as follows: goat anti-human Nanog, goat anti-human Sox2, mouse anti-beta II tubulin, mouse anti-alpha feto protein (all from R&D), rabbit anti-Oct3/4 and mouse anti-smooth muscle actin (both from Santa Cruz) and mouse anti-Tra-1-60, mouse anti-Tra-1-81, and mouse anti stage-specific embryonic antigen 4 (all from Millipore); secondary antibodies were used at 1/500 as follows: donkey anti-goat alexa 568, goat anti-rabbit alexa 555, goat anti-mouse alexa 555.
mRNA levels were assessed by qPCR (Eppendorf) using the following cycle: 95° 5 min, 95° 15 sec, 58° 30 sec, 72° 15 sec, with 40 cycles. The primers were as follows (L, left, R, right): actin: L: 5′- GAT GAG ATT GGC ATG GCT TT-3′ ; R: 5′-CAC CTT CAC CGT TCC AGT TT-3′; SPRY1.1: l, 5′-GGG ATT GTC CGA AAA GGA TT-3′; R, 5′-TTG ATT TTG GGG ATC CAT GT-3′; SPRY1.2: L, 5′-CGC TGT TAA ATG TGC CTG AA-3′; R, 5′- TTG ATT TTG GGG ATC CAT GT-3′; SPRY2: L, 5′-TTG CAC ATC GCA GAA AGA AG-3′; R, 5′-GGT CAC TCC AGC AGG CTT AG-3′; SPRY3: L, 5′-CCT TGC TGG AAT AGG GAT CA-3′; R, 5′ TAA GGC CAT GTT GTG GTT GA-3′; SPRY4: L, 5′-GGG AGC CAC TGA GAA CAG AG-3′; R, 5′-TGG CTC CTA AAT CCA TCC TG-3′; Nestin: L, 5′-TCC AGG AAC GGA AAA TCA AG-3′; R, 5′-GCC TCC TCA TCC CCT ACT TC-3′; SMA: L, 5′-CAG GGA AAA GAT GAC CCA GA-3′; R, 5′-AGG CAT AGA GGG AGA GCA CA-3′; AFP: L, 5′-CTT GTG AAG CAA AAG CCA CA-3′; R, CCC TCT TCA GGA AAG CAG AC-3′
siRNA transfection reagents were obtained from Invitrogen, and the transfection protocol was adopted from Ma et al with slight modification18
and no toxicity (Suppl Fig. 2
). Briefly, colonies were dissociated into single cells, centrifuged at 800 rpm 4 min RT, and resuspended in mTeSR without antibiotics. 105
cells were added to the transfection mix for 1–2 min, then mTeSR without antibiotics and containing Rock inhibitor (Y-27632) was added to a total of 500 μl each and plated in a 12 well plate previously coated with Cell Start. Lipofectamine 2000 transfection mix was prepared. Three siRNA sets were used for each gene, as follows: SPRY2:
SET 1: primer 1, UGG AAG GUA ACA CCA UAA ACA AGG C; primer 2, GCC UUG UUU AUG GUG UUA CCU UCC A; SET 2: primer 1, UUG AGC UCA GAU UUG GGU UGC ACC C, primer 2, GGG UGC AAC CCA AAU CUG AGC UCA A; SET 3, primer 1: AGA CCG UGG AGU CUC UCG UGU UUG U, primer 2, ACA AAC ACG AGA GAC UCC ACG GUC U. SPRY4
: set 1, primer 1: CCC UAG AAG CCU GUU UCU CCG UAC A; primer 2, UGU ACG GAG AAA CAG GCU UCU AGG G; Set 2, primer 1, GAG GCC UGU GGG AAG UGU AAA UGC A; primer 2, UGC AUU UAC ACU UCC CAC AGG CCU C; Set 3, primer 1: CAG CCA UGU GGA GAA UGA CUA CAU A, primer 2: UAU GUA GUC AUU CUC CAC AUG GCU G.
Cells were seeded at the same number and counted every 24 h for 4 days after transfection.
Cell death was evaluated by flow cytometry with Annexin V/PI (Annexin V Fluor staining kit, Roche). Briefly, cells were incubated with Annexin V/PI mix for 15 min at RT in incubation buffer, then collected with Accutase and resuspended in incubation buffer and processed for flow cytometry (LSR from BD).
Proliferation was assessed by BrdU labeling coupled to cell cycle analysis by flow cytometry using the EZ BrdU Kit (Phoenix Flow Systems).
Cell morphology was observed by phase contrast using inverted light microscope from Leica, and pictures were taken with a Leica DFC 500 camera.
Cells were fixed with 3.7% formaldehyde for 10 min at RT, permeabilized with 0.1% Triton X-100 for 4 min, and blocked with 1% BSA for 20 min. Alexa 488 Phalloidin (Molecular Probes) was then added and cells incubated for 20 min RT in the dark, then washed and DAPI added. Cells were imaged as described above.
Transmission electron microscopy imaging (TEM)
Cells were grown on Thermanox cover slips (Ted Pella Inc.), fixed for 2 h at RT with 2.0% electron microscopy grade glutaraldehyde in 0.1 M sodium cacodylate buffer, rinsed and dehydrated. Cells were then embedded in resin (1 part epoxy resin to 2 parts 100% propylene oxide) for 2 h at RT, then in 2 parts epoxy resin to 1 part propylene oxide for another 2 h, and finally in pure resin overnight. Samples were then baked for 12–14 hours at 60°C and separated from the coverslips, then baked again for another 24 h, glued to blocks and processed for sectioning at 100 μm.
Growth factor stimulation
siRNA-treated and control cells were exposed to FGF2, IGF1 and EGF (300 ng/ml each) starting 48 h after siRNA treatment for a period of 48 h, then harvested and counted.