CRISPR/Cas9 Directed Reprogramming of iPSC for Accelerated Motor Neuron Differentiation Leads to Dysregulation of Neuronal Fate Patterning and Function.

Neurodegeneration causes a significant disease burden and there are few therapeutic interventions available for reversing or slowing the disease progression. Induced pluripotent stem cells (iPSCs) hold significant potential since they are sourced from adult tissue and have the capacity to be differentiated into numerous cell lineages, including motor neurons. This differentiation process traditionally relies on cell lineage patterning factors to be supplied in the differentiation media. Genetic engineering of iPSC with the introduction of recombinant master regulators of motor neuron (MN) differentiation has the potential to shorten and streamline cell developmental programs. We have established stable iPSC cell lines with transient induction of exogenous LHX3 and ISL1 from the Tet-activator regulatory region and have demonstrated that induction of the transgenes is not sufficient for the development of mature MNs in the absence of neuron patterning factors. Comparative global transcriptome analysis of MN development from native and Lhx-ISL1 modified iPSC cultures demonstrated that the genetic manipulation helped to streamline the neuronal patterning process. However, leaky gene expression of the exogenous MN master regulators in iPSC resulted in the premature activation of genetic pathways characteristic of the mature MN function. Dysregulation of metabolic and regulatory pathways within the developmental process affected the MN electrophysiological responses.

Optimization of Application-Driven Development of In Vitro Neuromuscular Junction Models.

Neuromuscular junctions (NMJs) are specialized synapses responsible for signal transduction between motor neurons (MNs) and skeletal muscle tissue. Malfunction at this site can result from developmental disorders, toxic environmental exposures, and neurodegenerative diseases leading to severe neurological dysfunction. Exploring these conditions in human or animal subjects is restricted by ethical concerns and confounding environmental factors. Therefore, in vitro NMJ models provide exciting opportunities for advancements in tissue engineering. In the last two decades, multiple NMJ prototypes and platforms have been reported, and each model system design is strongly tied to a specific application: exploring developmental physiology, disease modeling, or high-throughput screening. Directing the differentiation of stem cells into mature MNs and/or skeletal muscle for NMJ modeling has provided critical cues to recapitulate early-stage development. Patient-derived inducible pluripotent stem cells provide a personalized approach to investigating NMJ disease, especially when disease etiology cannot be resolved down to a specific gene mutation. Having reproducible NMJ culture replicates is useful for high-throughput screening to evaluate drug toxicity and determine the impact of environmental threat exposures. Cutting-edge bioengineering techniques have propelled this field forward with innovative microfabrication and design approaches allowing both two-dimensional and three-dimensional NMJ culture models. Many of these NMJ systems require further validation for broader application by regulatory agencies, pharmaceutical companies, and the general research community. In this summary, we present a comprehensive review on the current state-of-art research in NMJ models and discuss their ability to provide valuable insight into cell and tissue interactions. Impact statement In vitro neuromuscular junction (NMJ) models reveal the specialized mechanisms of communication between neurons and muscle tissue. This site can be disrupted by developmental disorders, toxic environmental exposures, or neurodegenerative diseases, which often lead to fatal outcomes and is therefore of critical importance to the medical community. Many bioengineering approaches for in vitro NMJ modeling have been designed to mimic development and disease; other approaches include in vitro NMJ models for high-throughput toxicology screening, providing a platform to limit or replace animal testing. This review describes various NMJ applications and the bioengineering advancements allowing for human NMJ characteristics to be more accurately recapitulated. While the extensive range of NMJ device structures has hindered standardization attempts, there is still a need to harmonize these devices for broader application and to continue advancing the field of NMJ modeling.

Neuromuscular Junction Model Optimized for Electrical Platforms.

Neuromuscular junctions (NMJs), specialized synapses between motor neurons and muscle fibers, are essential for muscle activity. A simple and reproducible cell-based in vitro NMJ platform is needed to test the impact of chemicals on the neuron-muscle communication. Our platform utilizes genetically modified neurons and muscle cells, optimized culture conditions, and commercially available multielectrode array system for recording action potentials. Neuronal cells (NSC34) were optogenetically modified with channelrhodopsin chimera to allow for simultaneous, light-mediated, millisecond-precise activation of neuronal population. This signal is propagated through functional synapses to the muscle fibers. Muscle cells (C2C12) were modified by incorporating gap junction protein (Connexin-43) to improve intracellular communication without affecting muscle differentiation. This communication between muscle fibers resulted in better signal propagation and signal strength. Optimized culture medium facilitated the growth and differentiation of both cell types together. Our system was validated using vecuronium, a muscle relaxant, which abolished the muscle response. This in vitro model provides a unique tool for establishing a NMJ platform that is easy to record and analyze. Potential applications include nondestructive long-term screening of drugs affecting the NMJ.

Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation.

BACKGROUND: Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. RESULTS: Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-ß signaling pathway and consistent activation of sonic hedgehog, Wnt/ß-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. CONCLUSIONS: Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions.

The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p < 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p < 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p < 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.

Micromachining of Polyurethane Membranes for Tissue Engineering Applications.

Engineered tissue barrier models offer in vitro alternatives in toxicology and disease research. To mimic barrier-tissue microenvironment, a porous membrane that can approach the stiffness of physiological basement membranes is required. While several biocompatible membranes with micrometer range thickness (10 µm) and a stiffness less than polystyrene (3 GPa) or polyethylene (PET, 2 GPa), have been developed, there has been little effort to optimize the process to enable rapid and reproducible pore production in these membranes. Here, we investigate the use of laser irradiation with femtosecond (fs) pulses because the combination of high-precision and cold-ablation causes minimal damage to polymeric membranes. This process enables automated, high-throughput and reproducible fabrication of thin, microporous membranes that can be utilized to culture cells at air-liquid interface (ALI), a unique culture technique that simulates the tissue-barrier microenvironment. We show the optimization of laser parameters on a thin polyurethane membrane and patterned pores with an average diameter of 5 µm. Tissue was cultured at ALI for 28 days to demonstrate the membrane's utility in constructing a tissue barrier model. These results confirm the utilization of fs laser machining as a viable method for creating a porous barrier substrate in tissue engineering platforms.

A microfluidic method to measure bulging heights for bulge testing of polydimethylsiloxane (PDMS) and polyurethane (PU) elastomeric membranes.

Thin and flexible elastomeric membranes are frequently used in many microfluidic applications including microfluidic valves and organs-on-a-chip. The elastic properties of these membranes play an important role in the design of such microfluidic devices. Bulge testing, which is a common method to characterize the elastic behavior of these membranes, involves direct observation of the changes in the bulge height in response to a range of applied pressures. Here, we report a microfluidic approach to measure the bulging height of elastic membranes to replace direct observation of the bulge height under a microscope. Bulging height is measured by tracking the displacement of a fluid inside a microfluidic channel, where the fluid in the channel was designed to be directly in contact with the elastomeric membrane. Polydimethylsiloxane (PDMS) and polyurethane (PU) membranes with thickness 12-35 µm were fabricated by spin coating for bulge testing using both direct optical observation and the microfluidic method. Bulging height determined from the optical method was subject to interpretation by the user, whereas the microfluidic approach provided a simple but sensitive method for determining the bulging height of membranes down to a few micrometers. This work validates the proof of principle that uses microfluidics to accurately measure bulging height in conventional bulge testing for polydimethylsiloxane (PDMS) and polyurethane (PU)eElastomeric membranes.

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.

BACKGROUND: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. RESULTS: The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. CONCLUSIONS: This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs.

Hollow fiber integrated microfluidic platforms for in vitro Co-culture of multiple cell types.

This study demonstrates a rapid prototyping approach for fabricating and integrating porous hollow fibers (HFs) into microfluidic device. Integration of HF can enhance mass transfer and recapitulate tubular shapes for tissue-engineered environments. We demonstrate the integration of single or multiple HFs, which can give the users the flexibility to control the total surface area for tissue development. We also present three microfluidic designs to enable different co-culture conditions such as the ability to co-culture multiple cell types simultaneously on a flat and tubular surface, or inside the lumen of multiple HFs. Additionally, we introduce a pressurized cell seeding process that can allow the cells to uniformly adhere on the inner surface of HFs without losing their viabilities. Co-cultures of lung epithelial cells and microvascular endothelial cells were demonstrated on the different platforms for at least five days. Overall, these platforms provide new opportunities for co-culturing of multiple cell types in a single device to reconstruct native tissue micro-environment for biomedical and tissue engineering research.

Advances and Challenges in Recapitulating Human Pulmonary Systems: At the Cusp of Biology and Materials.

The aim of this review is to provide an overview of physiologically relevant microengineered lung-on-a-chip (LoC) platforms for a variety of different biomedical applications with emphasis on drug screening. First, a brief outline of lung anatomy and physiology is presented followed by discussion of the lung parenchyma and its extracellular matrix. Next, we point out the technical challenges in recapitulating the complexity of lung in conventional static two-dimensional microenvironments and the need for alternate lung platforms. The importance of scaling laws is also emphasized in designing these in vitro microengineered lung platforms. The review then discusses current LoC platforms that have been used for testing the efficacy of drugs or as model systems for investigating disorders of the lung parenchyma. Finally, the design parameters in developing an ideal physiologically relevant LoC platform are presented. As this emerging field of organ-on-a-chip can serve an alternative platform for animal testing of drugs or modeling human diseases in vitro, it has significant potential to impact the future of pharmaceutical research.

Tyrosine-derived stimuli responsive, fluorescent amino acids.

A series of fluorescent unnatural amino acids (UAAs) bearing stilbene and meta-phenylenevinylene (m-PPV) backbone have been synthesized and their optical properties were studied. These novel UAAs were derived from protected diiodo-l-tyrosine using palladium-catalyzed Heck couplings with a series of styrene analogs. Unlike the other fluorescent UAAs, whose emissions are restricted to a narrow range of wavelengths, these new amino acids display the emission peaks at broad range wavelengths (from 400-800 nm); including NIR with QY of 4% in HEPES buffer. The incorporation of both pyridine and phenol functional groups leads to distinct red, green, and blue (RGB) emission, in its basic, acidic and neutral states, respectively. More importantly, these amino acids showed reversible pH and redox response showing their promise as stimuli responsive fluorescent probes. To further demonstrate the utility of these UAAs in peptide synthesis, one of the amino acids was incorporated into a cell penetrating peptide (CPP) sequence through standard solid phase peptide synthesis. Resultant CPP was treated with two different cell lines and the internalization was monitored by confocal fluorescence microscopy.

Plasmonic giant quantum dots: hybrid nanostructures for truly simultaneous optical imaging, photothermal effect and thermometry.

Hybrid semiconductor-metal nanoscale constructs are of both fundamental and practical interest. Semiconductor nanocrystals are active emitters of photons when stimulated optically, while the interaction of light with nanosized metal objects results in scattering and ohmic damping due to absorption. In a combined structure, the properties of both components can be realized together. At the same time, metal-semiconductor coupling may intervene to modify absorption and/or emission processes taking place in the semiconductor, resulting in a range of effects from photoluminescence quenching to enhancement. We show here that photostable 'giant' quantum dots when placed at the center of an ultrathin gold shell retain their key optical property of bright and blinking-free photoluminescence, while the metal shell imparts efficient photothermal transduction. The latter is despite the highly compact total particle size (40-60 nm "inorganic" diameter and <100 nm hydrodynamic diameter) and the very thin nature of the optically transparent Au shell. Importantly, the sensitivity of the quantum dot emission to local temperature provides a novel internal thermometer for recording temperature during infrared irradiation-induced photothermal heating.

Ionic liquids as a class of materials for transdermal delivery and pathogen neutralization.

Biofilm-protected microbial infections in skin are a serious health risk that remains to be adequately addressed. The lack of progress in developing effective treatment strategies is largely due to the transport barriers posed by the stratum corneum of the skin and the biofilm. In this work, we report on the use of Ionic Liquids (ILs) for biofilm disruption and enhanced antibiotic delivery across skin layers. We outline the syntheses of ILs, analysis of relevant physicochemical properties, and subsequent neutralization effects on two biofilm-forming pathogens: Pseudomonas aeruginosa and Salmonella enterica. Further, the ILs were also examined for cytotoxicity, skin irritation, delivery of antibiotics through the skin, and treatment of biofilms in a wound model. Of the materials examined, choline-geranate emerged as a multipurpose IL with excellent antimicrobial activity, minimal toxicity to epithelial cells as well as skin, and effective permeation enhancement for drug delivery. Specifically, choline-geranate was comparable with, or more effective than, bleach treatment against established biofilms of S. enterica and P. aeruginosa, respectively. In addition, choline-geranate increased delivery of cefadroxil, an antibiotic, by >16-fold into the deep tissue layers of the skin without inducing skin irritation. The in vivo efficacy of choline-geranate was validated using a biofilm-infected wound model (>95% bacterial death after 2-h treatment). This work establishes the use of ILs for simultaneous enhancement of topical drug delivery and antibiotic activity.

Functionalization-dependent induction of cellular survival pathways by CdSe quantum dots in primary normal human bronchial epithelial cells.

Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity. Our results indicate that, while negatively charged QDs are relatively noncytotoxic compared to positively charged QDs, the same does not hold true for their genotoxic potential. Keeping QD core composition and size constant, 3 nm CdSe QD cores were functionalized with mercaptopropionic acid (MPA) or cysteamine (CYST), resulting in negatively or positively charged surfaces, respectively. CYST-QDs were found to induce significant cytotoxicity accompanied by DNA strand breakage. However, MPA-QDs, even in the absence of cytotoxicity and reactive oxygen species formation, also induced a high number of DNA strand breaks. QD-induced DNA damage was confirmed by identifying the presence of p53 binding protein 1 (53BP1) in the nuclei of exposed cells and subsequent diminishment of p53 from cytoplasmic cellular extracts. Further, high-throughput real-time PCR analyses revealed upregulation of DNA damage and response genes and several proinflammatory cytokine genes. Most importantly, transcriptome sequencing revealed upregulation of the metallothionein family of genes in cells exposed to MPA-QDs but not CYST-QDs. These data indicate that cytotoxic assays must be supplemented with genotoxic analyses to better understand cellular responses and the full impact of nanoparticle exposure when making recommendations with regard to risk assessment.

Comprehensive analysis of the effects of CdSe quantum dot size, surface charge, and functionalization on primary human lung cells.

The growing potential of quantum dots (QDs) in applications as diverse as biomedicine and energy has provoked much dialogue about their conceivable impact on human health and the environment at large. Consequently, there has been an urgent need to understand their interaction with biological systems. Parameters such as size, composition, surface charge, and functionalization can be modified in ways to either enhance biocompatibility or reduce their deleterious effects. In the current study, we simultaneously compared the impact of size, charge, and functionalization alone or in combination on biological responses using primary normal human bronchial epithelial cells. Using a suite of cellular end points and gene expression analysis, we determined the biological impact of each of these properties. Our results suggest that positively charged QDs are significantly more cytotoxic compared to negative QDs. Furthermore, while QDs functionalized with long ligands were found to be more cytotoxic than those functionalized with short ligands, negative QDs functionalized with long ligands also demonstrated size-dependent cytotoxicity. We conclude that QD-elicited cytotoxicity is not a function of a single property but a combination of factors. The mechanism of toxicity was found to be independent of reactive oxygen species formation, as cellular viability could not be rescued in the presence of the antioxidant n-acetyl cysteine. Further exploring these responses at the molecular level, we found that the relatively benign negative QDs increased gene expression of proinflammatory cytokines and those associated with DNA damage, while the highly toxic positive QDs induced changes in genes associated with mitochondrial function. In an attempt to tentatively "rank" the contribution of each property in the observed QD-induced responses, we concluded that QD charge and ligand length, and to a lesser extent, size, are key factors that should be considered when engineering nanomaterials with minimal bioimpact (charge > functionalization > size).

Design, synthesis, and a novel application of quorum-sensing agonists as potential drug-delivery vehicles.

Surface adhered bacterial colonies or biofilms are an important problem in medical and food industries. Bacteria use a chemical language to monitor their quorum and to express virulence factors, which eventually help them in colonization and manifestation of an infection. The LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems of Pseudomonas aeruginosa control expression of virulence factors in a population density-dependent fashion. In this study we investigated the role of synthetic analogs to RhlR-RhlI system of P. aeruginosa strains (PAO-1; wild-type and mutants JP-1, PDO-100, and JP-2) responsible for production of acyl-homoserine lactones-2; butanol homoserine lactone (AHL-2; C(4)-HSL). We synthesized double (QS1207) and single (QS0108) sulfur analogs against (C(4)-HSL; AHL-2), an autoinducer of Pseudomonas QS system. Extensive biological investigation of these analogs suggested a growth promoting activity for these analogs in Pseudomonas controlling biofilm production and exo-protease secretion. We hypothesized that these thiolactone analogs could be potentially utilized as potent drug-delivery vehicles against biofilm-producing pathogens. As a proof of principle we conjugated the single sulfur analog QS0108 with the broad-spectrum antibiotic, ciprofloxacin (QS0108-Cip). The QS analog-antibiotic conjugate was significantly more effective at disrupting both the nascent and mature biofilms of P. aeruginosa than the free antibiotic.

Fullerene derivatives induce premature senescence: a new toxicity paradigm or novel biomedical applications.

Engineered fullerenes (C(60)) are extensively used for commercial and clinical applications based on their unique physicochemical properties. Such materials have also been recognized as byproducts of many industrial activities. Functionalization of C(60) may significantly influence the nature of its interactions with biological systems, impacting its applications and raising uncertainties about its health effects. In the present study, we compared the bioimpact of two chemically modified fullerene derivatives, hexa carboxyl fullerene adduct (Hexa-C(60)) and tris carboxyl fullerene adduct (tris-C(60)) to pristine fullerene C(60) encapsulated with gamma (gamma)-cyclodextrin C(60) (CD-C(60)), using human cutaneous epithelial cells (HEK) to simulate possible applications and occupational dermal exposure route. We report, for the first time, the discovery of premature senescence as a potential endpoint of nanomaterial elicited biological effects, providing a new paradigm for nanoparticle-induced toxicity in human cells. Moreover, this response appeared to be functionalization specific, in that, only tris-C(60) induced senescence. We investigated key biological responses, such as cellular viability, intracellular ROS generation, cell proliferation and cell cycle responses. Our results indicate that the often observed 'anti-apoptotic' function of fullerene derivatives may be independent of their 'ROS scavenging' role as previously reported. We discovered that the tris-C(60)-induced responses were associated with G(0)/G(1) cell cycle arrest and cellular senescence. On further evaluation of the molecular mechanisms underlying the senescent response, a significant decrease in the expression levels of HERC5 was noted. HERC5 is a ubiquitin ligase of the HERC family and is implicated to be involved in innate immune responses to viral and bacterial infections.

Low dose, low-LET ionizing radiation-induced radioadaptation and associated early responses in unirradiated cells.

Numerous investigators have reported that irradiation of cells with a low dose of ionizing radiation (IR) can induce a condition of enhanced radioresistance, i.e. a radioadaptive response. In this report, we investigated the hypothesis that a radioadaptive bystander effect may be induced in unirradiated cells by a transmissible factor(s) present in the supernatants of cells exposed to low dose gamma-rays. Normal human lung fibroblasts (HFL-1) were irradiated with a 1 cGy dose of gamma-rays and their supernatants were transferred to unirradiated HFL-1 as a bystander cell model. Compared with the directly irradiated cells, such treatment resulted in increased clonogenic survival following subsequent gamma-irradiation with 2 and 4 Gy. This radioadaptive bystander effect was found to be preceded by early decreases in cellular levels of TP53 protein, increase in intracellular ROS, and increase in the redox and DNA repair protein AP-endonuclease (APE). The demonstration that radioadaptation can occur in unirradiated cells via a fluid-phase, transferable factor(s) adds to the complexity of the current understanding of mechanisms by which radioadaptive responses can be induced by low dose, low-LET IR.

Alpha-particle-induced increases in the radioresistance of normal human bystander cells.

Numerous investigators have reported that direct exposure of cells to a low dose of ionizing radiation can induce a condition of enhanced radioresistance, i.e. a "radioadaptive" response. In this report, we investigated the hypothesis that a radioadaptive bystander effect may be induced in unirradiated cells by a transmissible factor(s) present in the supernatants of cells exposed to a low dose of alpha particles. Normal human lung fibroblasts (HFL-1) were irradiated with 1 cGy of alpha particles and their supernatants were transferred to unirradiated HFL-1 cells as a bystander cell model. Compared to directly irradiated cells that were not treated with supernatants from HFL-1 cells exposed to low-dose radiation, such treatment resulted in increased clonogenic survival after subsequent exposure to 10 and 19 cGy of alpha particles. Increases in protein levels of AP-endonuclease, a redox and DNA base excision repair protein, were found in the bystander cells, but not in directly irradiated cells. Supernatants from alpha-particle-irradiated cells were also found to increase the clonogenicity of unirradiated cells. These results, in conjunction with our earlier findings that supernatants from cells exposed to a low dose of alpha particles contain growth-promoting activity, suggest that this new bystander effect may be related to an increase in DNA repair and cell growth/cell cycle regulation.