gives a schematic of our experimental design. We first confirmed that low dose irradiation (<1 Gy) of hESCs was capable of upregulating known stress-responsive genes: Gadd45, which mediates activation of the p38/JNK pathway via MTK1/MEKK4 kinase, and Cxcl10, a chemokine for receptor CXCR3 that is involved in recruitment of inflammatory cells (Fig. S1
). At a higher dose of 4 Gy, we observed massive cell death that was concurrent with the development of holes and patchy regions in hESC colonies at 48 hours (); hole formation has also been reported in colonies six hours after 5 Gy irradiation (14
). However, the surviving hESCs continued to express common pluripotency markers such as TRA-1-81, SSEA4, TRA-1-60, and embryonic transcription factors such as Oct4, Sox2, and Nanog that are key regulators of pluripotency and self-renewal ().
Figure 1 (A) Schematic of experimental setup. (B,C) Immunostaining of pluripotency markers in control (B) and irradiated (C) hESCs shows maintenance of marker expression 48 hours after irradiation. (D) Flow cytometry of FITC Annexin V and propidium iodide (PI) (more ...)
We were curious about the relative extent of apoptosis and cell death after irradiation at the different dosages, and so double-stained hESCs with Annexin V (early apoptosis) and PI (cell death) 48 hours after irradiation, and analyzed the cells with flow cytometry (). Clearly, there is a trend towards increasing apoptosis and cell death at the higher radiation doses (2 and 4 Gy) compared to low dose (0.4 Gy) and control. The majority (>70%) of hESCs are dead after 4 Gy irradiation, though an apoptotic minority (<30%) appears to survive at 48 hrs. This latter population likely represents the surviving cells that continue to express pluripotency markers, as seen in . However, the definitive test of pluripotency of human cells is the ability to form a teratoma, which we performed next.
To confirm that surviving hESCs are pluripotent, we injected irradiated cells into immuno-compromised mice and monitored for teratoma formation. We tracked their growth kinetics in vivo
by using hESCs that constitutively express a Fluc-eGFP double fusion reporter gene (), enabling longitudinal monitoring of cellular photon emission, and by extension their proliferation, as described (20
). After irradiation and injection of Fluc+/eGFP+
hESCs, we found that photon emission from the 2 and 4 Gy groups reached a statistical minimum at seven days that was less than photon emission from cells exposed to 0 and 0.4 Gy, suggesting massive cell death (P
<0.05, n=8 per group, see ). Based on the photon intensities, we estimated that 38±30%, 63±20%, 80±9%, and 83±7% (mean±SEM) of the 0, 0.4, 2, and 4 Gy-irradiated cells, respectively, had died at day 7.
Figure 2 Bioluminescence reporter gene imaging of irradiated hESCs in living animals. (A) The double fusion reporter gene construct carrying firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP). (B) Diagram of the subcutaneous injection sites (more ...)
We expected that the 2 and 4 Gy groups would continue to die, but surprisingly all four groups emitted similar levels of photons by day 21, indicating that surviving hESCs had recovered from high dose irradiation. We confirmed this result by studying long-term in vitro
cultures of irradiated hESCs, and found that cell proliferation was inhibited in the first week after high dose irradiation, but thereafter all groups exhibited similar growth kinetics (Fig. S2
). Note that after the post-irradiation “recovery period”, we did not observe any compensatory increase
in cell proliferation in the high dose groups. Finally, of the eight mice used in this study, five developed teratomas in the 4 Gy group by the sixth week (see for representative H&E images, and Fig. S3
for a representative gross image of four teratomas from a single mouse). The three mice that failed to form teratomas in the 4 Gy group likely experienced significant apoptosis and cell death, and not loss of pluripotency. To confirm this, we performed a careful microarray study of the core set of pluripotency genes to determine whether there are any detectable changes in pluripotency programs, however subtle, in response to ionizing radiation.
For the transcriptomic analysis of irradiated hESCs, RNA was isolated from cells 24 hours after irradiation, then labeled and hybridized to microarrays (raw data files have been uploaded to GEO under accession number GSE20951). When analyzing microarray data, it is often informative to start from a system-wide rather than individual-gene view of the resulting data, especially when the overall gene fold changes are no more than seven-fold (Table S1). An overview of the gene profiles can be seen in the heatmap of . Most apparent is the co-clustering of the control and low dose samples (0 and 0.4 Gy), which were distinct from the co-clustering of the high dose samples (2 and 4 Gy). This pattern is also evident in , in which global Pearson correlation shows 95% correlation between the 2 and 4 Gy groups, but only 86% correlation with the low-dose 0.4 Gy group. A Venn diagram of the entities that are significantly different (P<0.05, fold change ≥1.4) between radiation dosage and control further illustrates this pattern (, note that microarrays often contain multiple probes, or “entities”, for a given gene). Again, we observe the same grouping as in the Pearson correlation: the 2 and 4 Gy-irradiated samples exhibit a higher degree of overlap between themselves than they do with the 0.4 Gy group.
Figure 3 Microarray analysis of hESCs 24 hours after irradiation. (A) Pearson clustering of the data for 0, 0.4, 2, and 4 Gy irradiated hESCs (n=3 biological replicates per group). Note that one replicate from the 0.4 Gy group was lost due to poor array hybridization. (more ...)
We next used Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com
) and Gene Set Enrichment Analysis (GSEA) (19
) to further analyze the microarray data. Selected canonical pathways and functions that are disrupted after 4 Gy irradiation (vs. control) are summarized in ; full data sets can be found in Tables S2 and S3. After 4 Gy irradiation, canonical pathways such as VDR/RXR activation, p53 signaling, aryl hydrocarbon signaling, and functions such as cancer, cell death, cell cycle, growth and proliferation, and embryonic development are significantly affected in hESCs. Specifically, several tumor protein p53 associated genes such as TP53Inp1 (up 2.6-fold) and target genes such as Cdkn1A (up 2-fold) and Mdm2 (up 1.7-fold) (23
), as well as several tumor necrosis factor receptor superfamily members, were disregulated after irradiation. A small group of genes associated with development also exhibited differential expression, including Hes1 (down 1.8-fold, (24
)), Runx1 (up 1.5 fold), and Pbx1 (down 1.8-fold); note that many of these genes are also associated with cancer (). Supporting the observation that genes related to cancer are disregulated with radiation, GSEA, a method for analyzing a priori
gene sets within microarray data, revealed upregulation of gene sets that have also been reported in cells after treatment with chemotherapeutic drugs (25
Selected genes and biological processes affected by 4 Gy irradiation of hESCs
Gene Set Enrichment Analysis (GSEA) of 4 vs. 0 Gy irradiated hESCs
We have also analyzed the progression of gene and pathway changes that occur in hESCs at each increasing radiation dose: between 0 and 0.4 Gy (Tables S4-S6), 0.4 and 2 Gy (Tables S7-S9), and 2 and 4 Gy (Tables S10-S12). Similar to 4 Gy radiation, 0.4 Gy irradiation affects cellular functions such as cell death, cancer, and signaling pathways such as p53, though not important p53 downstream target genes such as Cdkn1A and Mdm2. Because Cdkn1A is an important negative regulator of cell cycling (28
), the lack of upregulation of Cdkn1A by 0.4 Gy irradiation could partly explain why we did not observe a similar reduction in cell proliferation as in the 2 and 4 Gy groups. Relative to 0.4 Gy irradiation, 2 Gy irradiation affects canonical TFG-β and Wnt/β-catenin signaling, including the genes Tgfbr2 (up 1.4-fold), Wnt1 (up 1.4-fold), Wnt10A (up 2.1-fold) and Wnt9a (up 1.8-fold); notably, Wnt proteins play important and diverse roles in embryonic stem cells (29
). 2 Gy irradiation also induces Cdkn1A upregulation by 2.3-fold, but not Mdm2. Interestingly, many genes involved in functions such as cellular compromise, amino acid metabolism, molecular transport, and cell morphology, in addition to cancer and cell death, were significantly disrupted by 2 Gy of radiation, including a number of solute carrier family proteins such as Slc6a13 (up 2-fold) and Slc25a13 (down 2.2-fold). Clearly there is an overall significant increase in cellular dysfunction after 2 Gy irradiation, which explains the results from our in vivo
and in vitro
studies ( and ). Finally, in the 2 vs. 4 Gy group, the overall gene changes were not large, but a small group of genes related to organ and tissue development did have altered expression, such as Tnfsf11 (up 1.6-fold), Otx1 (down 1.6-fold), B4galt1 (down 1.4-fold), and Mef2C (up 1.9-fold). Presumably there are subtle development and differentiation processes that are activated with 4 Gy irradiation, but are not robust enough to cause loss of pluripotency, as evidenced by successful formation of teratomas from 4 Gy-irradiated hESCs. Furthermore, the gene expression profiles of irradiated cells showed no increased correlation with previously reported data for differentiated hESCs and primary cell types (20
), giving additional evidence that differentiation is not significantly increased with ionizing radiation (Fig. S4
With these observations, we were particularly interested to understand whether core pluripotency genes were also affected by radiation. Importantly, well-known embryonic transcription factors such as Oct4 (Pou5f1), Sox2, and Nanog, which are expressed exclusively or predominantly in hESCs and are critical for maintaining pluripotency and self-renewal, were not present in any of our significant gene lists across all radiation dosages. Moving beyond individual genes, we created a gene set of 26 known factors that are well-known to be specific to hESCs (bottom row of ). GSEA revealed no significant up- or down-regulation of this set in the microarray data for the highest radiation dose, 4 Gy (P
=-1.0). This finding agrees with a previous report that showed normalization of Oct4 and Nanog after only 24 hours in 2 Gy-irradiated hESCs (14
). We therefore conclude that pluripotency gene programs are not significantly affected by high dose radiation, and this accounts for the observation that surviving hESCs are still capable of forming all three embryonic germ layers.