The hESC line CLS1 (20) was cultured in 35 mm tissue culture dishes (Nunc, Roskilde, Denmark) on a confluent monolayer of γ-irradiated human foreskin fibroblasts (HFF) (American Type Culture Collection no. SCRC-1041). The culture medium consisted of the Knockout Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% Knockout Serum Replacement, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, penicillin/ streptomycin (all Invitrogen, Carlsbad, CA), and 4 ng/ml recombinant human bFGF (BioSource Europe, Nivelles, Belgium). Cells were sub-cultured in four-week intervals and transferred in a humidified hypoxic cell culture facility with an atmosphere of 5% O₂ and 5% CO₂ buffered with nitrogen (Xvivo System; BioSpherix, Redfield, NY). The medium was changed every other day. HeLa cells (ATCC no. CCL-2) was used as a control and cultured in Eagle’s Minimum Essential Medium, supplemented with 10% fetal bovine serum, glutamin, and antibiotics (Invitrogen) at 37°C in 5% CO₂.

To inhibit Notch and Hh signaling, γ-secretase inhibitor (γ-SI) (L1790; Sigma-Aldrich, Brøndby, Denmark) and cyclopamine (CP) (C4116; Sigma-Aldrich) were used, respectively. They were dissolved in DMSO and applied to achieve 1 μM final concentration for γ-SI and 10 μM concentration in the case of CP. The inhibitors were incubated with CLS1 cultures for a period of three weeks.

In order to visualize the nuclear DNA, Hoechst 33342 (Invitrogen) was added to the cultures to achieve a final concentration of 10 μg/ml and the incubation was continued at 37°C for 30 min. The cells were then washed and fixed with 4% buffered formaldehyde at 4°C for 20 min. After additional washing and blocking with 2% bovine serum albumin (BSA) at room temperature (RT) for 25 min, the cells were incubated with a 200-fold diluted SSEA1-specific mouse monoclonal antibody (sc-21702; Santa Cruz Biotechnology, Santa Cruz, CA) at RT for 1 h followed by a 200-fold diluted Alexa 488 goat anti-mouse antibody (Invitrogen) at RT for 35 min. To detect Oct4, the cells were first permeabilized with 0.2% Triton X-100 in 4% BSA at RT for 20 min, and then incubated with Oct4-specific antibody (ab19857; Abcam, Cambridge, United Kingdom) diluted 1:300 at RT for 1 h. The intra-nuclear complexes of primary antibody were reacted with Cy-5 donkey anti-goat conjugate (AP130S; Millipore, Bedford, MA) diluted 1:150 at RT for 35 min. Cells were finally washed and stored in PBS at 4°C. All washings and dilutions were done with phosphate buffered saline (PBS) and 1% BSA in PBS, respectively. The images were acquired using Axio Observer Z1 (Carl Zeiss, Göttingen, Germany) wide-field fluorescence system equipped with AxioCam MRm camera (Zeiss) and using three-channel full resolution tiled images based on 10-fold magnification. The histomorphometric analysis was carried out with the aid of AxioVision software package (Zeiss).

To monitor cell proliferation, the cultures were incubated with 10 μM bromodeoxyuridine (BrdU) (Sigma-Aldrich) at 37°C for 1 h in a CO₂ incubator, washed, and fixed with 4% buffered formaldehyde at RT for 20 min. After additional washing, the proteins were denatured with 1 N HCl at RT for 30 min, and to minimize un-specific antibody binding, a blocking with 2% BSA for 30 min was carried out. The cell membranes were then permeabilized with 0.2% Triton X-100 in 4% BSA for 20 min and subsequently washed, after which an incubation with anti-BrdU mouse monoclonal antibody (A-21300, Invitrogen) diluted 1:20 in 1% BSA followed at RT for 1 h. Binding sites of primary antibody were visualized with a 200-fold diluted Alexa 488 anti-mouse conjugate (A-11001, Invitrogen) at RT for 30 min. The cells were then counter-stained with Hoechst 33342 diluted to 10 μg/ml final concentration. After final washing, the cells were examined and images taken using Axio Observer. Two-channel full resolution tiled images were acquired based on 10-fold magnification.

The [³H]-thymidine incorporation experiments were carried out according to Chen et al. (21) with some modifications. Briefly, the medium was removed and replaced with fresh Knockout DMEM containing, in addition to all supplements, 825 μCi/mL thymidine [methyl-³ H]-tri-phosphate ammonium (TRK 242; Amersham Biosciences, Piscataway, NJ). The incubation proceeded at 37°C for further 4 h in a CO₂ incubator, after which, the cells were washed four times with PBS, released from the feeder layer, and solubilized in 100 μL of 0.5% SDS at 37°C during 30 min. The proteins were precipitated with an equal volume of trichloroacetic acid (TCA) at RT during 10 min incubation and recovered at 1000 rpm (110g RCF) for 10 min. The pellets were dissolved in 100 μL of 1 N sodium hydroxide, transferred to 3 mL of scintillation liquid (Ultima Gold; Perkin Elmer, Waltham, MA), and the levels of β-radiation were determined using a liquid scintillation counter (RackBeta; Perkin Elmer).

The total RNA was extracted using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA) according to manufacturers′ recommendations and the yield was determined spectrophotometrically (ND-1000; Nano-Drop Technologies, Wilmington, DE). cDNA synthesis was performed on 200 ng of RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories). For amplification reaction, an 8 μl cDNA aliquot was applied together with 13 μl of iQ SYBR Green Supermix (Bio-Rad Laboratories) reaction components, and 0.03 pmoles of 18S primers or 0.192 pmoles of gene-specific primers in a total volume of 25 μl in translucent microtiter plates (iCycler iQ PCR plates; Bio-Rad Laboratories). The samples were run on MyIQ single-color real-time PCR detection system (Bio-Rad Laboratories) using a two-step amplification cycle. The profile consisted of a single annealing/extension step of 30 sec at 60ºC and a denaturation step of 15 sec at 95ºC, for a total number of 40 cycles. All primers were designed with DNASTAR Software package (DNASTAR, Madison, WI) and were custom synthesized by DNA Technology A/S, Aarhus, Denmark (Table 1). Prior to the gene-specific analysis, the samples were evaluated for the contents of 18S rRNA that served as an internal endogenous standard. After normalization with respect to the 18S rRNA, the relative transcriptional levels were determined for each sample and gene in a semi-quantitative fashion by referring to a standard dilution series prepared from a pooled sample using MyIQ software (Bio-Rad Laboratories). To confirm the quality of each run, the occurrence of primer dimers was monitored by invoking a melting curve function of the program. All samples were analyzed in duplicates, with individual replicates being placed remotely from each other.

The designated colonies of CLS1 line were removed, washed, and lysed in 50 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.5% NP-40, 1 mM Na₃VO₄, 0.5 mM PMSF, 5 mM β-mercaptoethanol, 1x protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) on ice for 30 min and the lysates were subsequently cleared at 14,000 rpm (21.910 g RCF) at 4°C for 15 min. The protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories) and samples amounting to 20 μg were separated by SDS-PAGE using a 10% gel. The transfer to a nitrocellulose membrane (Millipore) was done by a dry electroblotting procedure (Invitrogen). After the transfer, the membranes were incubated with 5% (w/v) nonfat-milk in PBS at RT for 1 h to block non-specific binding. The primary antibodies were diluted in 1% (w/v) nonfat-milk in PBS to achieve optimal concentration, rabbit polyclonal anti-NF-κB (ab7970; Abcam) 1:1000, rabbit polyclonal anti-Oct4 (ab19857; Abcam) 1:500, and mouse monoclonal anti-β-actin (A1978; Sigma-Aldrich) 1:3000, and incubated with membranes at 4°C overnight. After washing, horseradish peroxidase-conjugated either anti-rabbit (sc-2004; Santa Cruz Biotechnology) diluted 1 :2000 or anti-mouse (P0260; DakoCytomation, Glostrup, Denmark) diluted 1:1000 were incubated with membranes at RT for 1 h, and the visualization of the antibody complexes was done on the basis of enhanced chemiluminiscence (ECL Plus; GE Healthcare Life Sciences, Hillerød, Denmark) using a Kodak Image Station 4,000 MM PRO (Carestream Molecular Imaging, Woodbridge, CT). Densitometric analysis was performed with the aid of an integrated Kodak MI software package (Carestream Molecular Imaging).

All experiments were performed at least twice, and the data are presented as means+standard error of mean (SEM). Comparison of independent samples was done with a Student’s t-test and statistical significance was assigned to differences with p<0.05.

We have previously reported that Notch signaling is necessary for the maintenance of pluripotency of hESCs exposed to hypoxic conditions (13). Therefore we sought to investigate whether inhibition of Notch and Hh pathways has the potential to promote differentiation alone or in combination at 5% oxygen concentration. To this end, we employed γ-SI to block formation of NICD and CP to inhibit Hh signaling. The differentiated hESCs were identified through their lack of Oct4 and presence of SSEA1. The immunofluorescence staining revealed a significant induction of SSEA1 in the centers of the colonies at the expense of Oct4-positive area irrespective of whether the inhibitors were used individually or simultaneously (Fig. 1A). Using histomorphometric analysis, it was established that the extent to which the hESCs self-renewal was inhibited, in terms of the proportion of undifferentiated colony areas, was similar when γ-SI and CP were used separately, 48% and 56%, respectively (Fig. 1B). However, their simultaneous application resulted in enhanced inhibition, with undifferentiated zones corresponding only to 36%. In all cases the inhibition was statistically significant.

The pattern of Oct4 distribution as identified by in-situ immunodetection was further confirmed by a real-time RT-PCR (Fig. 2A) and western blotting (Fig. 2B). These trials demonstrated that the transcriptional and translational expression resulting from inhibition closely resembled the overall reduction of undifferentiated colony areas. Confirming the loss of pluripotency in cells incubated with γ-SI and CP, another marker of pluripotency, the transcription factor Nanog, showed a practically identical decrease of mRNA expression (Fig. 2C).

As our initial experiments implicated Notch and Hh signaling in mediating differentiation of hESCs exposed to 5% oxygen, we next sought to investigate whether expression of the Hes1 and Hey1, and Gli1 and Ptc1 immediate downstream genes (22, 23), respectively, was altered in our system. The analysis of transcriptional activation by a real-time RT-PCR of colonies grown for three weeks in hypoxic conditions has interestingly shown a significant decrease of both Hes1 and Hey1 mRNA not only with γ-SI but also with CP and combined γ-SI+CP (Fig. 3A). Similarly, when analyzing the levels of Gli1 and Ptc1 transcripts, not only the Hh specific inhibitor CP demonstrated a significant effect but also the application of γ-SI, and γ-SI in combination with CP resulted in a significant suppression (Fig. 3B). It is noteworthy that the simultaneous action of γ-SI and CP seemed to have a significant additive effect.

Since the modulation of differentiation potential is associated with altered proliferative capacity, we investigated the effect of γ-SI and CP inhibitors on this parameter using BrdU and [³H]-thymidine incorporation assays, and NF-κB western blot analysis. BrdU was instrumental in identifying regions in the colonies, where the cell division was affected. As shown in Fig. 4A, the treatment with either γ-SI, CP or both resulted in a characteristic inhibition of proliferation in the central areas of the colonies. From comparison with Fig. 1A, it is obvious that these areas are coincident with the inhibition of self-renewal. Again, it is apparent that the simultaneous use of inhibitors produced a more pronounced effect than each of them individually. Quantitative assessment of the inhibition of proliferation was based on [³H]-thymidine incorporation (Fig. 4B). These results correspond well with the observed changes in colony morphology after BrdU visualization in panel A.

As for the expression of NF-κB, the protein levels increased as a result of inhibition with each of the individual inhibitors (2.6-fold on average) (Fig. 4C). Interestingly, however, the most significant increase occurred after the combined application (17.9-fold).