A versatile system to introduce clusters of genomic double-strand breaks in large cell populations.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids.

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.

Laser-assisted cell removing (LACR) technology contributes to the purification process of the undifferentiated cell fraction during pluripotent stem cell culture.

Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine.

Low-dose irradiation of mouse embryos increases Smad-p21 pathway activity and preserves pluripotency.

PURPOSE: To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. METHODS: Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0-1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. RESULTS: More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. CONCLUSIONS: Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency.

The Effects of Photobiomodulation Delivered by Light-Emitting Diode on Stem Cells from Human Exfoliated Deciduous Teeth: A Study on the Relevance to Pluripotent Stem Cell Viability and Proliferation.

OBJECTIVE: Photobiomodulation (PBM) can modulate the proliferation of some types of stem cells. However, few reports have addressed the effects of PBM delivered by light-emitting diode (LED) on stem cells obtained from the pulp tissue of deciduous teeth. The aim of the present study was to investigate the effect of PBM delivered by red LED (630 nm, 75 mW, 37 mW/cm2) with different radiant exposures on the cell cycle, mitochondrial membrane potential, and senescence of stem cells from human exfoliated deciduous teeth (SHED). MATERIALS AND METHODS: Cultures were irradiated with LED (2, 4, 8, 16, and 32 J/cm2). After 24 h, the cell cycle and mitochondrial membrane potential of the cultures were evaluated using flow cytometry. Nonirradiated cultures served as control. RESULTS: Cultures irradiated with 16 J/cm2 had higher percentages of cells in the synthesis phase than control cultures (p < 0.05), and no significant differences were found regarding the percentage of cells with viable mitochondria between irradiated and control cultures. No significant difference in cell senescence was found between control cultures and cultures irradiated with 2 or 16 J/cm2. CONCLUSIONS: LED irradiation at 630 nm (37 mW/cm2, 75 mW) with radiant exposure of 16 J/cm2 was capable of inducing a proliferative response in stem cells from the pulp tissue of deciduous teeth without affecting mitochondrial function or inducing senescence.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians.

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo.

The expression of pluripotency and neuronal differentiation markers under the influence of electromagnetic field and nitric oxide.

Nitric oxide (NO) is a diatomic free radical compound that as a secondary messenger contributes to cell physiological functions and its variations influence proteins activity and triggering intracellular signaling cascades. Low frequency electromagnetic field (EMF) alters the cell biology such as cell differentiation by targeting the plasma membrane and entering force to the ions and small electrical ligands. The effect of these chemical (NO) and physical (EMF) factors on the expression of the stemness and neuronal differentiation markers in rat bone marrow mesenchymal stem cells (BMSC) was investigated. The cells were treated with low (50micromolar) and high (1mM) concentrations of Deta-NO as a NO donor molecule and 50Hz low frequency EMF. The expression of pluripotency and neuronal differentiation genes and proteins was investigated using real time qPCR and Immunocytochemistry techniques. The simultaneous treatment of EMF with NO (1mM) led to the down-regulation of stemness markers expression and up-regulation of neuronal differentiation markers expression. Cell proliferation decreased and cell morphology changed which caused the majority of cells obtains neuronal protein markers in their cytoplasm. The decrease in the expression of neuronal differentiation Nestin and DCX markers without any change in the expression of pluripotency Oct4 marker (treated with low concentration of NO) indicates protection of stemness state in these cells. Treatment with NO demonstrated a double behavior. NO low concentration helped the cells protect the stemness state but NO high concentration plus EMF pushed cells into differentiation pathway.

Ronin influences the DNA damage response in pluripotent stem cells.

Early mammalian embryonic cells must maintain a particularly robust DNA repair system, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4 and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes.

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.

Pluripotent Stem Cells and DNA Damage Response to Ionizing Radiations.

Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences.

Analysis of Stem Cell Motility In Vivo Based on Immunodetection of Planarian Neoblasts and Tracing of BrdU-Labeled Cells After Partial Irradiation.

Planarian flatworms have become an important system for the study of stem cell behavior and regulation in vivo. These organisms are able to regenerate any part of their body upon damage or amputation. A crucial cellular event in the process of planarian regeneration is the migration of pluripotent stem cells (known as neoblasts) to the site of injury. Here we describe two approaches for analyzing migration of planarian stem cells to an area where these have been ablated by localized X-ray irradiation. The first approach involves immunolabeling of mitotic neoblasts, while the second is based on tracing stem cells and their progeny after BrdU incorporation. The use of planarians in studies of cell motility is suitable for the identification of factors that influence stem cell migration in vivo and is amenable to RNA interference or pharmacological screening.

Repair of Ischemic Injury by Pluripotent Stem Cell Based Cell Therapy without Teratoma through Selective Photosensitivity.

Stem-toxic small molecules have been developed to induce selective cell death of pluripotent stem cells (PSCs) to lower the risk of teratoma formation. However, despite their high efficacies, chemical-based approaches may carry unexpected toxicities on specific differentiated cell types. Herein, we took advantage of KillerRed (KR) as a suicide gene, to selectively induce phototoxicity using visible light via the production of reactive oxygen species. PSCs in an undifferentiated state that exclusively expressed KR (KR-PSCs) were eliminated by a single exposure to visible light. This highly selective cell death in KR-PSCs was exploited to successfully inhibit teratoma formation. In particular, endothelial cells from KR-mPSCs remained fully functional in vitro and sufficient to repair ischemic injury in vivo regardless of light exposure, suggesting that a genetic approach in which KR is expressed in a tightly controlled manner would be a viable strategy to inhibit teratoma formation for future safe PSC-based therapies.

Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.

UNLABELLED: Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. SIGNIFICANCE: The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells.

Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells.

Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harvested immediately after irradiation, and at 1 and 5 days after irradiation. Cell cycle analysis, colony forming ability (CFU-F), differentiation ability, and expression of osteogenic-specific runt-related transcription factor 2 (RUNX2), adipogenic peroxisome proliferator-activated receptor gamma (PPARγ), oxidative stress-specific dismutase-1 (SOD1) and Glutathione peroxidase (GPX1) were analyzed. Irradiation arrested cell cycle progression in BMMSCs and hESMSCs. Colony formation ability of irradiated MSCs decreased in a dose-dependent manner. Irradiated hESMSCs showed higher adipogenic differentiation compared with BMMSCs, together with an increase in the adipogenic PPARγ expression. PPARγ expression was upregulated as early as 4 h after irradiation, along with the expression of SOD1. More than 70% downregulation was found in Wnt3A, Wnt4, Wnt 7A, Wnt10A and Wnt11 in BMMSCs, but not in hESMSCs. hESMSCs are highly proliferative but radiosensitive compared with BMMSCs. Increased PPARγ expression relative to RUNX2 and downregulation of Wnt ligands in irradiated MSCs suggest Wnt mediated the fate determination of irradiated MSCs.

mESC-based in vitro differentiation models to study vascular response and functionality following genotoxic insults.

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear γH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells.

Fate of D3 mouse embryonic stem cells exposed to X-rays or carbon ions.

The risk of radiation exposure during embryonic development is still a major problem in radiotoxicology. In this study we investigated the response of the murine embryonic stem cell (mESC) line D3 to two radiation qualities: sparsely ionizing X-rays and densely ionizing carbon ions. We analyzed clonogenic cell survival, proliferation, induction of chromosome aberrations as well as the capability of cells to differentiate to beating cardiomyocytes up to 3 days after exposure. Our results show that, for all endpoints investigated, carbon ions are more effective than X-rays at the same radiation dose. Additionally, in long term studies (≥8 days post-irradiation) chromosomal damage and the pluripotency state were investigated. These studies reveal that pluripotency markers are present in the progeny of cells surviving the exposure to both radiation types. However, only in the progeny of X-ray exposed cells the aberration frequency was comparable to that of the control population, while the progeny of carbon ion irradiated cells harbored significantly more aberrations than the control, generally translocations. We conclude that cells surviving the radiation exposure maintain pluripotency but may carry stable chromosomal rearrangements after densely ionizing radiation.

Mouse embryonic stem cells irradiated with γ-rays differentiate into cardiomyocytes but with altered contractile properties.

Embryonic stem cells (ESCs) for their derivation from the inner cell mass of a blastocyst represent a valuable in vitro model to investigate the effects of ionizing radiation on early embryonic cellular response. Following irradiation, both human and mouse ESCs (mESCs) maintain their pluripotent status and the capacity to differentiate into embryoid bodies and to form teratomas. Although informative of the maintenance of a pluripotent status, these studies never investigated the capability of irradiated ESCs to form specific differentiated phenotypes. Here, for the first time, 5Gy-irradiated mESCs were differentiated into cardiomyocytes, thus allowing the analysis of the long-term effects of ionizing radiations on the differentiation potential of a pluripotent stem cell population. On treated mESCs, 96h after irradiation, a genome-wide expression analysis was first performed in order to determine whether the treatment influenced gene expression of the surviving mESCs. Microarrays analysis showed that only 186 genes were differentially expressed in treated mESCs compared to control cells; a quarter of these genes were involved in cellular differentiation, with three main gene networks emerging, including cardiogenesis. Based on these results, we differentiated irradiated mESCs into cardiomyocytes. On day 5, 8 and 12 of differentiation, treated cells showed a significant alteration (qRT-PCR) of the expression of marker genes (Gata-4, Nkx-2.5, Tnnc1 and Alpk3) when compared to control cells. At day 15 of differentiation, although the organization of sarcomeric α-actinin and troponin T proteins appeared similar in cardiomyocytes differentiated from either mock or treated cells, the video evaluation of the kinematics and dynamics of the beating cardiac syncytium evidenced altered contractile properties of cardiomyocytes derived from irradiated mESCs. This alteration correlated with significant reduction of Connexin 43 foci. Our results indicate that mESCs populations that survive an ionizing irradiation treatment are capable to differentiate into cardiomyocytes, but they have altered contractile properties.

Pluripotent stem cells escape from senescence-associated DNA methylation changes.

Pluripotent stem cells evade replicative senescence, whereas other primary cells lose their proliferation and differentiation potential after a limited number of cell divisions, and this is accompanied by specific senescence-associated DNA methylation (SA-DNAm) changes. Here, we investigate SA-DNAm changes in mesenchymal stromal cells (MSC) upon long-term culture, irradiation-induced senescence, immortalization, and reprogramming into induced pluripotent stem cells (iPSC) using high-density HumanMethylation450 BeadChips. SA-DNAm changes are highly reproducible and they are enriched in intergenic and nonpromoter regions of developmental genes. Furthermore, SA-hypomethylation in particular appears to be associated with H3K9me3, H3K27me3, and Polycomb-group 2 target genes. We demonstrate that ionizing irradiation, although associated with a senescence phenotype, does not affect SA-DNAm. Furthermore, overexpression of the catalytic subunit of the human telomerase (TERT) or conditional immortalization with a doxycycline-inducible system (TERT and SV40-TAg) result in telomere extension, but do not prevent SA-DNAm. In contrast, we demonstrate that reprogramming into iPSC prevents almost the entire set of SA-DNAm changes. Our results indicate that long-term culture is associated with an epigenetically controlled process that stalls cells in a particular functional state, whereas irradiation-induced senescence and immortalization are not causally related to this process. Absence of SA-DNAm in pluripotent cells may play a central role for their escape from cellular senescence.

Sourcing and using stem cell lines for radiation research: Potential, challenges and good stem cell culture practice.

PURPOSE: Exposition of best practice in management and experimental use of human stem cell lines in radiobiological research. This paper outlines the key challenges to be addressed by radiobiologists wishing to use human pluripotent stem cell (hPSC) lines in their research including human embryonic stem cell (hESC) lines and human induced pluirpotency stem (hiPSC) lines. It emphasises the importance of guidance already established for cell culture in general and outlines some further considerations specific to the culture of human pluripotent stem cell lines which may impact on the interpretation of data from radiobiological studies using these cells. Fundamental standards include obtaining cells from bona fide suppliers with suitable quality controls, screening cell lines to ensure absence of mycoplasma and authentication of cell lines by DNA profiling. For hESC and hiPSC lines, it is particularly important to recognise the significance of phenotypic and genetic stability and this paper will address approaches to reduce their impact. Quality assured banking of these two types of stem cell lines will facilitate reliable supply of quality controlled cells that can provide standardisation between laboratories and in the same laboratory over time. CONCLUSIONS: hPSC lines could play an important role in future radiobiological research providing certain fundamental principles of good stem cell culture practice are adopted at the outset of such work.

Effect of 5-[(125)I]iodo-2'-deoxyuridine uptake on the proliferation and pluripotency of human embryonic stem cells.

PURPOSE: Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because, in principle, they can differentiate into any cell type found in the human body. In addition, studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state, as well as the consequences of IR exposure on the development of organisms. However, the effect of IR, in particular radionuclide uptake, on the pluripotency, proliferation and survival of hESC has not been extensively studied. METHODS: In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU), a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC. RESULTS: We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However, treatment with 0.1 µCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 µCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls. CONCLUSIONS: Our results provide important initial insights into the sensitivity of hESC to IR, and especially that produced by the decay of an internalized radionuclide.