Department of Internal Medicine, Innovative Research Institute for Cell Therapy, Seoul National University Hospital, Korea.
Abstract
Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed 'hypoxic-priming') efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation.
Figure 1 Hypoxia is a natural signal during spontaneous differentiation of ESCs, and hypoxic priming of EBs reduced pluripotency genes whereas induced genes of meso-endoderm and vascular-lineage Embryoid bodies (EBs) were formed using a hanging drop method. The round droplet (500 cells per 20 µl) gathers ESCs and induces cell aggregation (EB). After 3 days, EBs was further incubated in hypoxic or normoxic conditions for immunofluorescence staining.Hypoxic region detected by hypoxic marker, anti-pimonidazole adduct antibody in EBs. EBs was collected on day 5 (a,b) and on day 10 (c). Positive immunoreactivity was detected as red fluorescence (b,c). Negative control treated with mouse isotype-IgG for hypoxia marker in EB. No positive staining was found (d). Magnification = 100×. Scale bar = 100 µM.EBs were made for 3 days in normoxic conditions and then further cultured in normoxia or hypoxic conditions for indicated day(s), respectively. EBs collected for total RNA isolation.EBs made using hanging drop method were exposed to hypoxia for 2 days. The real-time PCR was used to assess the expression of genes associated with pluripotency (Oct4, Nanog), ectoderm (NCAM, Nestin), mesoderm (SMA, Desmin), endoderm (Troma-1) and hypoxia responsive gene (VEGF) (n = 4, *p < 0.05).Hypoxia response gene (VEGF) and vascular marker genes (VEGFR2, PECAM, SMA, VE-cadherin) were upregulated in 1% oxygen environments. Oct4 was significantly downregulated compared with normoxia. Graphs show relative percent change over day 4 normoxic-EB (n = 4, *p < 0.05).Figure 2 HIF-1α, not HIF-2α, transcriptionally repressed Oct4 EBs formed by hanging drop for 3 days, and cultured under normoxic or hypoxic condition for 16 h. Western blotting of HIF-1α, HIF-2α and Oct4 (left). Quantification graph (right, n = 4, *p < 0.05).Western blotting of HIF-1α, HIF-2α and Oct4 for 16 h under 21 and 1% O2 (top left). ESCs were transfected with pEGFP-HIF-1α/pEGFP-HIF-1β or pEGFP-HIF-2α/pEGFP-HIF-1β for 24 h and formed EBs. Quantification graph (top right, n = 3, *p < 0.05 versus Nor). RT-PCR and real-time PCR for Oct4 mRNA (bottom, n = 4, *p < 0.001).Quantitative real-time RT-PCR for VEGF, VEGFR1 and bFGF. ESCs were transfected with pEGFP-HIF-1α or pEGFP-HIF-2α for 24 h, formed EBs and further incubated for 16 h under 21 and 1% O2 (n = 3, *p < 0.01, **p < 0.05).HIF-1α specific knockdown (shHIF1α) significantly reversed the hypoxia-mediated Oct4 reduction in mRNA and protein levels. Western blotting (top, n = 3, *p < 0.05 versus Hyp), RT-PCR and real-time PCR (bottom, n = 4, *p < 0.05).Figure 3 HIF-1α transcriptionally repressed Oct4 by binding to rHREs in the Oct4 promoter under hypoxia A. Upstream sequence of the mouse Oct4 promoter (GenBank accession no. S58422) (Nordhoff et al, 2001). Nucleotides are numbered relative to the translation start site, and 4 potential rHREs (5′-TGCA(C/G)-3′) are marked by squares.B,C. Differentiating E14 ESCs cultured in the absence of LIF and feeder cells were co-transfected with 1 µg pGL3-Oct4-rHRE (B), pGL3-EPO-HRE (C), 0.2 µg pCMV-β-gal, 0.5 µg pEGFP-HIF-1α or pEGFP-HIF-2α and 0.5 µg pEGFP-HIF-1β plasmid for 24 h and incubated for 16 h under 21 and 1% O2 (n = 4).D. HIF-1 decreases Oct4 expression by binding to rHRE site of Oct4 promoter.E. ChIP analysis shows the HIF-1α binding to the Oct4 promoter region. Lysates of differentiating E14- or C57-ESCs exposed to hypoxia for 16 h were immunoprecipitated with antibody for HIF-1α. The precipitated DNAs was evaluated by PCR using specific primers for rHREs (rHRE1 to rHRE4) on Oct4 promoter (n = 3 each).F. HIF-1α binds to three of four rHRE HIF-binding sites. Schematic diagram of the four rHRE HIF-binding motifs in full-sequence Oct4 promoter and the promoter serial deletion mutants (left). Differentiating E14 ESCs were transfected with various combinations of full-length Oct4 promoter, Oct4 promoter deletion mutants and HIF-1α/β, and promoter luciferase activity was measured at 16 h later (right, n = 3 each, p < 0.05).Figure 4 Vascular differentiation is enhanced in hypoxia-primed EBs A. EBs formed by hanging drop was cultured under normoxic or hypoxic condition for 2 days, respectively, and attached onto 0.3% gelatin-coated 24-well plate (2–3 EBs per well) in DMEM medium with 10% FBS. On the next day, the culture medium was replaced with vascular differentiation medium, and the cells were further incubated up to 14 days.B. Morphology of differentiated EBs on 1 day and 7 days after reattachment. Cells spreading to the surroundings of the reattached aggregates (left and center) showed the appearance of differentiated cells, and the hypoxia-primed EBs (HyEB) spread out far more than normoxic-EBs (NorEB). Quantitative graph of migrating distance from aggregate center on 7 days after reattachment (right). Measurement of three-different points per one EB and 10-EBs were used (p < 0.05). Magnification = 40×, scale bar = 300 µm.C,D. Immunofluorescence staining against PECAM (red) on day 7 (C) and PECAM (red)/α-SMA (green) on day 14 after EB reattachment (D). PECAM and α-SMA were very strong in hypoxia-primed EBs. Nucleus for SytoxBlue (blue). NorEB, normoxic-EBs; HyEB, hypoxia-primed EBs. Representative confocal microscopic photographs were shown and no fluorescence signal in isotype IgG control and in negative control. Scale bar = 30 µm. Magnification = 400×.E,F. Quantitative graph of PECAM+ area (E) and SMA+ area (F) using ImageJ program.Figure 5 The endothelial differentiation of hypoxia-primed EBs is dependent on HIF-1α and VEGF A. HIF-1α protein markedly reduced after the transfection of shHIF-1α. ESCs, Mock or shHIF-1α stable transfectant formed EBs and incubated for 16 h under normoxic or hypoxic conditions. Western blotting of HIF-1α (left). Quantitative graph (right) (n = 4, *p < 0.05).B. Hypoxia responsive gene (VEGF) and vascular marker genes (PECAM, VEGFR2, VE-cadherin) were investigated with real-time PCR (n = 4, *p < 0.05).C. EBs were cultured under normoxic or hypoxic condition for 2 days, respectively, and attached onto 0.3% gelatin-coated 24-well plate (2–3 EBs per well) in DMEM medium with 10% FBS. On the next day, the culture medium was replaced with vascular differentiation EGM medium and the cells were further incubated for 3 days. Neutralizing antibody against mouse VEGF (50 µg/ml) or control istotype IgG antibody was added to the culture medium every day.D,E. E14 or C57 cells were analysed by FACS for PECAM, VE-cadherin (endothelial cells) and α-SMA (smooth muscle cells) on day 3 after EB reattachment. N, normoxic-EBs; H, hypoxia-primed EBs; nVEGF Ab, neutralizing anti-mVEGF antibody; IgG, istotype IgG antibody. Quantitative graph of FACS analysis from four-different experiments was shown. The percentage was normalized to normoxic group and shown as percent fold (n = 4 each).F,G. E14 ESCs were transfected with Mock or Oct4 plasmid for 24 h, formed EBs and incubated for 2 days under normoxic or hypoxic conditions. Oct4, VEGF, mesoderm (vasculogenic marker genes; PECAM, VEGFR2, VE-cadherin) and endoderm gene (Troma-1) were investigated with real-time PCR (n = 3).Figure 6In vivo vasculogenic potential of hypoxia-primed EBs is confirmed in mice hind limb ischemic model A. BALB/c-nude mice underwent hindlimb ischemic surgery, and then normoxic-EBs (NorEB), hypoxia-primed EBs (HyEB) or hypoxia-primed shHIF1-EBs (shHIF1-HyEB) was intramuscularly injected. To trace differentiating EBs, E14 cells were labelled with DiI (red) before EB formation.B. LDPI images. In the HyEB-injection group, the blood flow of ischemic limbs was recovered (arrow).C. LDPI was sequentially evaluated after transplantation of each EBs into hindlimb ischemic BALB/c-nude mice (n = 5 each, p < 0.01).D. The adductor muscle on day 7 post-transplantation was longitudinally sectioned and analysed by confocal microscopy for PECAM (green). Capillary endothelial cells stained with PECAM are detected adjacent to skeletal myocytes (phase bright) and are co-localized with DiI immunofluorescence (red). No fluorescence signal in the IgG control group. Magnification = 400×, scale bar = 20 µm.E,F. Number of DiI−/PECAM+ capillaries (E) or DiI+/PECAM+ cells (F) incorporating into ischemic limb muscle were presented (numbers/mm2, n = 5 each, *p < 0.001, **p < 0.05).G. Proposed model for enhancement of vascular differentiation of hypoxia-primed differentiating ESCs.
