Effects of ionizing radiation on self-renewal and pluripotency of human embryonic stem cells

Abstract

Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far, no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs, nor has any study assessed their ability to form teratomas, the definitive test of pluripotency. In this study, we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy), medium-dose (2 Gy), and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death, p53 signaling, cell cycling, cancer, embryonic and organ development, and others. Using Gene Set Enrichment Analysis, we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses, definitive proof of pluripotency. Further, using a bioluminescence imaging technique, we have found that irradiation causes hESCs to initially die after transplantation, but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude, we show that similar to somatic cells, irradiated hESCs suffer significant death and apoptosis after irradiation. However, they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies.

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) double-stained hESCs 48 hours after the indicated radiation dose (one representative experiment of three is shown).

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) double-stained hESCs 48 hours after the indicated radiation dose (one representative experiment of three is shown).

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) double-stained hESCs 48 hours after the indicated radiation dose (one representative experiment of three is shown).

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) double-stained hESCs 48 hours after the indicated radiation dose (one representative experiment of three is shown).

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 for each radiation group plus control, as well as representative bioluminescent images for three mice through day 42. (C) Plot of longitudinal bioluminescent signal intensities for each group (*0 and 0.4 Gy vs. 2 and 4 Gy, P<0.05). Data presented as mean±SEM. (D) H&E staining of teratoma section from representative 4 Gy group demonstrating three embryonic germ layers.

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. Each gene is represented by a single row, and each sample by a single column. Red = upregulated, Green = downregulated. (B) Global Pearson correlation of microarray data. (C) Venn diagram of the significant entities (P<0.05, fold change ≥1.4) between each radiation group and control (0 Gy).