ABSTRACT
DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids ("mini-brains") from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.
Subject(s)
Brain/cytology , DNA Damage , Organoids/cytology , Brain/metabolism , Brain/radiation effects , Cells, Cultured , Histones/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/radiation effects , Organ Culture Techniques , Organoids/metabolism , Organoids/radiation effects , Radiation Dosage , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
Terahertz (THz) irradiation has been exploited in biomedical applications involving non-invasive manipulation of living cells. We developed an apparatus for studying the effects of THz pulse irradiation on living human induced pluripotent stem cells. The THz pulse of the maximum electric field reached 0.5 MV/cm and was applied for one hour with 1 kHz repetition to the entire cell-culture area, a diameter of 1 mm. RNA sequencing of global gene-expression revealed that many THz-regulated genes were driven by zinc-finger transcription factors. Combined with a consideration of the interactions of metal ions and a THz electric field, these results imply that the local intracellular concentration of metal ions, such as Zn2+, was changed by the effective electrical force of our THz pulse.
Subject(s)
Gene Regulatory Networks/radiation effects , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/radiation effects , Terahertz Radiation , Cell Survival , Electricity , Humans , Induced Pluripotent Stem Cells/cytology , Transcription Factors/metabolismABSTRACT
Human induced pluripotent stem cells (hiPSCs) are considered to be one of the most promising cell resources for regenerative medicine. HiPSCs usually maintain their pluripotency when they are cultured on feeder cell layers or are attached to a cell-adhesive extracellular matrix. In this study, we developed a culture system based on UV/ozone modification for conventional cell culture plastics to generate a suitable surface condition for hiPSCs. Time of flight secondary ion mass spectrometry (ToF-SIMS) was carried out to elucidate the relationship between hiPSC adhesion and UV/ozone irradiation-induced changes to surface chemistry of cell culture plastics. Cell culture plastics with modified surfaces enabled growth of a feeder-free hiPSC culture with markedly reduced cell-adhesive matrix coating. Our cell culture system using UV/ozone-modified cell culture plastics may produce clinically relevant hiPSCs at low costs, and can be easily scaled up in culture systems to produce a large number of hiPSCs.
Subject(s)
Cell Culture Techniques/economics , Cost-Benefit Analysis , Induced Pluripotent Stem Cells/cytology , Ozone/pharmacology , Plastics/pharmacology , Ultraviolet Rays , Alkaline Phosphatase/metabolism , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cells, Cultured , Collagen/pharmacology , Drug Combinations , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/radiation effects , Karyotype , Laminin/pharmacology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Polystyrenes , Proteoglycans/pharmacology , Surface PropertiesABSTRACT
A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions.
Subject(s)
G1 Phase Cell Cycle Checkpoints/genetics , Induced Pluripotent Stem Cells/metabolism , S Phase Cell Cycle Checkpoints/genetics , Animals , Cell Division , Cellular Reprogramming , Erythroblasts , G1 Phase Cell Cycle Checkpoints/radiation effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/radiation effects , Neoplasms/genetics , Open Reading Frames , Point Mutation , S Phase Cell Cycle Checkpoints/radiation effects , X-RaysABSTRACT
Neurological diseases and disorders are leading causes of death and disability worldwide. Many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. As the global burden of these pathologies continues to rise there is a significant need for the development of novel therapeutics. Due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. It is now believed that paracrine factors, such as extracellular vesicles (EVs), play a critical role in the functionality associated with stem cells. The diverse biological cargo contained within EVs are proposed to mediate these effects and, to date, the reparative and regenerative effects of stem cell EVs have been demonstrated in a wide range of cell types. While a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the CNS. Here, we report the isolation, characterization, and functional assessment of EVs from two sources of human stem cells, mesenchymal stem cells and induced pluripotent stem cells. We demonstrate the ability of these EVs to enhance the processes of cellular migration and angiogenesis, which are critical for both normal cellular development as well as cellular repair. Furthermore, we investigate their reparative effects on damaged cells, specifically those with relevance to the central nervous system. Collectively, our data highlight the similarities and differences among these EV populations and support the view that stem cells EV can be used to repair or partially reverse cellular damage. Graphical Abstract Stem cell-derived Extracellular Vesicles (EVs) for repair of damaged cells. EVs isolated from human induced pluripotent stem cells and mesenchymal stem cells contribute to the partial reversal of phenotypes induced by different sources of cellular damage.
Subject(s)
Extracellular Vesicles , Induced Pluripotent Stem Cells/ultrastructure , Nervous System Diseases/therapy , A549 Cells , Cell Line , Cell Movement , Cell Survival , Cytokines/biosynthesis , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , Extracellular Vesicles/radiation effects , Humans , Immunity, Innate , Induced Pluripotent Stem Cells/radiation effects , Neovascularization, Pathologic/therapy , Nervous System Diseases/pathology , Proteomics , RNA/genetics , Radiation, IonizingABSTRACT
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a dual capability to self-renew and differentiate into all cell types necessary to develop an entire organism. Differentiation is associated with dynamic epigenetic alteration and transcriptional change, while self-renewal depends on maintaining the genome DNA accurately. Genome stability of PSCs is strictly regulated to maintain pluripotency. However, the DNA damage response (DDR) mechanism in PSCs is still unclear. There is accumulating evidence that genome stability and pluripotency are regulated by a transcriptional change in undifferentiated and differentiated states. iPSCs are ideal for analyzing transcriptional regulation during reprogramming and differentiation. This study aimed to elucidate the transcriptional alteration surrounding genome stability maintenance, including DNA repair, cell cycle checkpoints and apoptosis in fibroblasts, iPSCs and neural progenitor cells (NPCs) derived from iPSCs as differentiated cells. After ionizing radiation exposure, foci for the DNA double-stranded break marker γ-H2AX increased, peaking at 0.5 h in all cells (>90%), decreasing after 4 h in fibroblasts (32.3%) and NPCs (22.3%), but still remaining at 52.5% (NB1RGB C2 clone) and 54.7% (201B7 cells) in iPSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were detected, indicating that iPSCs' apoptosis increases. In addition, RNA sequencing (RNA-Seq) analysis showed high expression of apoptosis genes (TP53, CASP3 and BID) in iPSCs. Results suggested that increased apoptosis activity maintains accurate, undifferentiated genome DNA in the cell population.
Subject(s)
Apoptosis/genetics , Cell Differentiation/genetics , Cellular Reprogramming/genetics , DNA Damage/genetics , Gene Expression Regulation , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Transcription, Genetic , Apoptosis/radiation effects , Cell Differentiation/radiation effects , Cell Line , Cellular Reprogramming/radiation effects , DNA Repair/genetics , DNA Repair/radiation effects , Fibroblasts/cytology , Fibroblasts/radiation effects , Gene Expression Regulation/radiation effects , Humans , Induced Pluripotent Stem Cells/radiation effects , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neural Stem Cells/radiation effects , Radiation, Ionizing , Skin/cytologyABSTRACT
Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1 Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48 h and 9 days post IR (pIR). All but one of the analyzed marker genes were reduced 48 h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.
Subject(s)
Gene Expression Regulation/radiation effects , Induced Pluripotent Stem Cells/radiation effects , Neural Stem Cells/radiation effects , Radiation, Ionizing , Cell Differentiation , Humans , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Radiation Dosage , Time FactorsABSTRACT
Stem cells are one of the most important sources to develope a new strategy for repairing bone lesions through tissue engineering. Osteogenic differentiation of stem cells can be affected by various factors such as biological, chemical, physiological, and physical ones. The application of ELF-EMFs has been the subject of many research in bone tissue engineering and evidence suggests that this exogenous physical stimulus can promote osteogenic differentiation in several types of cells. The purpose of this paper is to review the current knowledge on the effects of EMFs on stem cells in bone tissue engineering studies. We recapitulated and analyzed 39 articles that were focused on the application of EMFs for bone tissue engineering purposes. We tabulated scattered information from these articles for easy use and tried to provide an overview of conducted research and identify the knowledge gaps in the field.
Subject(s)
Bone and Bones/radiation effects , Electromagnetic Fields , Regenerative Medicine/methods , Animals , Bone and Bones/cytology , Electromagnetic Fields/adverse effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/radiation effects , Tissue EngineeringABSTRACT
To investigate the role of Wnt/ß-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of ß-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/ß-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by ß-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed ß-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/ß-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/ß-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/ß-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.
Subject(s)
Induced Pluripotent Stem Cells/cytology , Laser Coagulation , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/radiation effects , Wnt Signaling Pathway , Cell Differentiation/radiation effects , Cell Proliferation/radiation effects , Cell Shape/radiation effects , Fluorescence , Gene Expression Regulation/radiation effects , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/radiation effects , Intercellular Signaling Peptides and Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Retinal Pigment Epithelium/metabolism , Time Factors , Wnt Signaling Pathway/genetics , Wnt Signaling Pathway/radiation effects , Zonula Occludens-1 Protein/metabolism , beta Catenin/metabolismABSTRACT
PURPOSE: Data on the response of chondrocytes differentiated from hiPSCs (hiPSC-DCHs) to ionizing radiation (IR) are lacking. The aim of present study was to assess DNA damage response (DDR) mechanisms of IR-treated hiPSC-DCHs. METHODS AND MATERIALS: The following IR-response characteristics in irradiated hiPSC-DCHs were assessed: 1) the kinetics of DNA DSB formation; 2) activation of major DNA repair mechanisms; 3) cell cycle changes and 4) reactive oxygen species (ROS), level of key markers of apoptosis and senescence. RESULTS: DNA DSBs were observed in 30% of the hiPSC-DCHs overall, and in 60% after high-dose (> 2 Gy) IR. Nevertheless, these cells displayed efficient DNA repair mechanisms, which reduced the DSBs over time until it reached 30% by activating key genes involved in homologous recombination and non-homologous end joining mechanisms. As similar to mature chondrocytes, irradiated hiPSC-DCH cells revealed accumulation of cells in G2 phase. Overall, the hiPSC-DCH cells were characterized by low levels of ROS, cPARP and high levels of senescence. CONCLUSIONS: The chondrocyte-like cells derived from hiPSC demonstrated features characteristic of both mature chondrocytes and "parental" hiPSCs. The main difference between hiPSC-derived chondrocytes and hiPSCs and mature chondrocytes appears to be the more efficient DDR mechanism of hiPSC-DCHs. The unique properties of these cells suggest that they could potentially be used safely in regenerative medicine if these preliminary findings are confirmed in future studies.
Subject(s)
Cell Differentiation/radiation effects , Chondrocytes/physiology , Chondrogenesis/radiation effects , Gamma Rays , Induced Pluripotent Stem Cells/physiology , Cell Line , Chondrocytes/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA End-Joining Repair/radiation effects , G2 Phase/radiation effects , Humans , Induced Pluripotent Stem Cells/radiation effects , Reactive Oxygen Species/metabolism , Regenerative Medicine/methodsABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/cytology , Trophoblasts/cytology , Animals , Cell Death/radiation effects , Cell Differentiation , Cell Line , Coculture Techniques/methods , Humans , Induced Pluripotent Stem Cells/radiation effects , Infrared Rays/adverse effects , Lasers/adverse effects , Mice , Pluripotent Stem Cells/radiation effects , Regenerative Medicine , Trophoblasts/radiation effectsABSTRACT
BACKGROUND: Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) share many common features, including similar morphology, gene expression and in vitro differentiation profiles. However, genomic stability is much lower in iPSCs than in ESCs. In the current study, we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis. METHODS: Mouse iPSCs, ESCs and embryonic fibroblasts were exposed to ionizing radiation (4 Gy) to introduce double-strand DNA breaks. At 4 h later, fidelity of DNA damage repair was assessed using whole-genome re-sequencing. We also analyzed genomic stability in mice derived from iPSCs versus ESCs. RESULTS: In comparison to ESCs and embryonic fibroblasts, iPSCs had lower DNA damage repair capacity, more somatic mutations and short indels after irradiation. iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair. Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice. CONCLUSIONS: The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due, at least in part, to low fidelity of DNA damage repair.
Subject(s)
DNA Damage , DNA End-Joining Repair/genetics , Genomic Instability/genetics , Induced Pluripotent Stem Cells/metabolism , Animals , Cells, Cultured , DNA Breaks, Double-Stranded/radiation effects , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Expression/radiation effects , Genomic Instability/radiation effects , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/radiation effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/radiation effects , Radiation, IonizingABSTRACT
Stem cells and their paracrine factors have emerged as a resource for regenerative medicine. Many studies have shown the beneficial effects of paracrine factors secreted from adult stem cells, such as exosomes, on skin aging. However, to date, few reports have demonstrated the use of exosomes derived from human pluripotent stem cells for the treatment of skin aging. In this study, we collected exosomes from the conditioned medium of human induced pluripotent stem cells (iPSCs) and investigated the effect on aged human dermal fibroblasts (HDFs). Cell proliferation and viability were determined by an MTT assay and cell migration capacity was shown by a scratch wound assay and a transwell migration assay. To induce photoaging and natural senescence, HDFs were irradiated by UVB (315 nm) and subcultured for over 30 passages, respectively. The expression level of certain mRNAs was evaluated by quantitative real-time PCR (qPCR). Senescence-associated-ß-galactosidase (SA-ß-Gal) activity was assessed as a marker of natural senescence. As a result, we found that exosomes derived from human iPSCs (iPSCs-Exo) stimulated the proliferation and migration of HDFs under normal conditions. Pretreatment with iPSCs-Exo inhibited the damages of HDFs and overexpression of matrix-degrading enzymes (MMP-1/3) caused by UVB irradiation. The iPSCs-Exo also increased the expression level of collagen type I in the photo-aged HDFs. In addition, we demonstrated that iPSCs-Exo significantly reduced the expression level of SA-ß-Gal and MMP-1/3 and restored the collagen type I expression in senescent HDFs. Taken together, it is anticipated that these results suggest a therapeutic potential of iPSCs-Exo for the treatment of skin aging.
Subject(s)
Cellular Senescence , Exosomes/metabolism , Fibroblasts/cytology , Induced Pluripotent Stem Cells/metabolism , Skin Aging/physiology , Biomarkers/metabolism , Cell Movement/radiation effects , Cell Proliferation/radiation effects , Cellular Senescence/radiation effects , Collagen Type I/genetics , Collagen Type I/metabolism , Dermis/cytology , Exosomes/radiation effects , Exosomes/ultrastructure , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Expression Regulation/radiation effects , Humans , Induced Pluripotent Stem Cells/radiation effects , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Skin Aging/radiation effects , Ultraviolet RaysABSTRACT
Cardiac arrhythmia presumably induced through cardiac fibrosis is a recurrent long-term consequence of exposure to ionizing radiation. However, there is also evidence that cardiac arrhythmia can occur in patients shortly after irradiation. In this study, the authors employed multielectrode arrays to investigate the short-term effects of x-ray radiation on the electrophysiological behavior of cardiomyocytes derived from human-induced pluripotent stem cells. These cardiomyocytes with spontaneous pacemaker activity were cultured on single-well multielectrode arrays. After exposure to 0, 0.5, 1, 2, 5, 10 Gy x-ray radiation, electrical activity was measured at time points ranging from 10 min to 96 h. RNA sequencing was employed to verify the expression of genes specifically involved in cardiomyocyte differentiation and function. A decrease in beating rate was observed after irradiation with 5 and 10 Gy starting 48 h after exposure. Cells exposed to higher doses of radiation were more prone to show changes in electrophysiological spatial distribution. No radiation-induced effects with respect to the corrected QT interval were detectable. Gene expression analysis showed up regulation of typical cardiac features like ACTC1 or HCN4. In this study, early dose-dependent changes in electrophysiological behavior were determined after x-ray irradiation. Results point towards a dose-dependent effect on pacemaker function of cardiomyocytes and indicate a possible connection between irradiation and short-term changes in electrophysiological cardiac function. Cardiomyocytes derived from human-induced pluripotent stem cells on multielectrode arrays represent a promising in vitro cardiac-modeling system for preclinical studies.
Subject(s)
Arrhythmias, Cardiac/pathology , Electrophysiological Phenomena/radiation effects , Gene Expression Regulation/radiation effects , Induced Pluripotent Stem Cells/physiology , Myocytes, Cardiac/physiology , Radiation, Ionizing , Arrhythmias, Cardiac/etiology , Cell Differentiation/radiation effects , Cell Proliferation/radiation effects , Cells, Cultured , Dose-Response Relationship, Radiation , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/radiation effects , Myocytes, Cardiac/cytology , Myocytes, Cardiac/radiation effectsABSTRACT
According to the current therapies failure for bone fractures and lesions, tissue engineering showed a great potential to help solve these challenges. Because the use of growth factors is very limited in the clinic, it could be very useful that could be introducing an alternative to it. Extremely low frequency pulsed electromagnetic fields (PEMF, 1 mT, 50 Hz) were used for achieving this aim. The PEMF potential in combination with electrospun polycaprolactone (PCL) nanofibers was used to investigate the osteogenic potential of human induced pluripotent stem cells (iPSCs). Several relevant osteogenic markers, such as Alizarin red staining, alkaline phosphatase activity, calcium content, gene expression, and immunocytochemistry, were used to evaluate osteoinductivity of PEMF. Results were shown that PEMF alone can induce osteogenic differentiation, but this capability increased when used in combination with PCL nanofibers significantly. In addition, simultaneous use of osteogenic medium, PEMF and PCL surprisingly increased osteogenic differentiation potential of iPSCs. According to the results, PEMF alone, iPSCs-seeded PCL, and both of them could be considered as a promising candidate for use in bone tissue engineering applications.
Subject(s)
Induced Pluripotent Stem Cells/radiation effects , Nanofibers , Osteogenesis/radiation effects , Tissue Engineering/methods , Tissue Scaffolds , Cell Differentiation/radiation effects , Cells, Cultured , Electromagnetic Fields , Humans , PolyestersABSTRACT
AIMS: Electrical stimulation (EleS) can promote cardiac differentiation, but the underlying mechanism is not well known. This study investigated the effect of EleS on cardiomyocyte (CM) differentiation of human induced pluripotent stem cells (hiPSCs) and evaluated the therapeutic effects for the treatment of myocardial infarction (MI). RESULTS: Cardiac differentiation of hiPSCs was induced with EleS after embryoid body formation. Spontaneously beating hiPSCs were observed as early at 2 days when treated with EleS compared with control treatment. The cardiac differentiation efficiency of hiPSCs was significantly enhanced by EleS. In addition, the functional maturation of hiPSC-CMs under EleS was confirmed by calcium indicators, intracellular Ca2+ levels, and expression of structural genes. Mechanistically, EleS mediated cardiac differentiation of hiPSCs through activation of Ca2+/PKC/ERK pathways, as revealed by RNA sequencing, quantitative polymerase chain reaction, and Western blotting. After transplantation in immunodeficient MI mice, EleS-preconditioned hiPSC-derived cells significantly improved cardiac function and attenuated expansion of infarct size. The preconditioned hiPSC-derived CMs were functionally integrated with the host heart. INNOVATION: We show EleS as an efficacious time-saving approach for CM generation. The global RNA profiling shows that EleS can accelerate cardiac differentiation of hiPSCs through activation of multiple pathways. The cardiac-mimetic electrical signals will provide a novel approach to generate functional CMs and facilitate cardiac tissue engineering for successful heart regeneration. CONCLUSION: EleS can enhance efficiency of cardiac differentiation in hiPSCs and promote CM maturation. The EleS-preconditioned CMs emerge as a promising approach for clinical application in MI treatment. Antioxid. Redox Signal. 28, 371-384.
Subject(s)
Cell Differentiation/radiation effects , Induced Pluripotent Stem Cells/transplantation , Myocardial Infarction/therapy , Myocytes, Cardiac/transplantation , Animals , Cells, Cultured , Disease Models, Animal , Electric Stimulation , Heart/physiopathology , Humans , Induced Pluripotent Stem Cells/radiation effects , Mice , Myocardial Infarction/pathology , Myocytes, Cardiac/radiation effects , Regeneration/radiation effects , Tissue EngineeringABSTRACT
Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatmentassociated purposes. Therefore, the aim of the present study was to examine and compare early IRinduced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, nonhomologous endjoining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SCbased clinical protocols.
Subject(s)
DNA Breaks, Double-Stranded/radiation effects , Human Embryonic Stem Cells/radiation effects , Induced Pluripotent Stem Cells/radiation effects , Transcriptome/radiation effects , BRCA2 Protein/genetics , Cells, Cultured , DNA-Activated Protein Kinase/genetics , DNA-Binding Proteins/genetics , Gene Expression/radiation effects , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Nuclear Proteins/genetics , Rad51 Recombinase/genetics , Radiation, IonizingABSTRACT
BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.
Subject(s)
Cellular Senescence/radiation effects , DNA Breaks, Double-Stranded , DNA Repair/genetics , Fibroblasts/radiation effects , Human Embryonic Stem Cells/radiation effects , Induced Pluripotent Stem Cells/radiation effects , Cell Line , Cellular Reprogramming , Cellular Senescence/genetics , DNA/genetics , DNA/metabolism , DNA Breaks, Double-Stranded/radiation effects , Fibroblasts/cytology , Fibroblasts/metabolism , G1 Phase Cell Cycle Checkpoints/genetics , G2 Phase Cell Cycle Checkpoints/genetics , Gamma Rays , Gene Expression , Histones/genetics , Histones/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Phosphorylation/radiation effects , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
Induced pluripotent stem (iPS) cells have demonstrated they can undergo self-renewal, attain pluripotency, and differentiate into various types of functional cells. In clinical transplantation of iPS cells, however, a major problem is the prevention of tumorigenesis. We speculated that tumor formation could be inhibited by means of irradiation. Since the main purpose of this study was to explore the prevention of tumor formation in human iPS (hiPS) cells, we tested the effects of irradiation on tumor-associated factors such as radiosensitivity, pluripotency and cell death in hiPS cells. The irradiated hiPS cells showed much higher radiosensitivity, because the survival fraction of hiPS cells irradiated with 2 Gy was < 10%, and there was no change of pluripotency. Irradiation with 2 and 4 Gy caused substantial cell death, which was mostly the result of apoptosis. Irradiation with 2 Gy was detrimental enough to cause loss of proliferation capability and trigger substantial cell death in vitro. The hiPS cells irradiated with 2 Gy were injected into NOG mice (NOD/Shi-scid, IL-2 Rγnull) for the analysis of tumor formation. The group of mice into which hiPS cells irradiated with 2 Gy was transplanted showed significant suppression of tumor formation in comparison with that of the group into which non-irradiated hiPS cells were transplanted. It can be presumed that this diminished rate of tumor formation was due to loss of proliferation and cell death caused by irradiation. Our findings suggest that tumor formation following cell therapy or organ transplantation induced by hiPS cells may be prevented by irradiation.
Subject(s)
Carcinogenesis/pathology , Carcinogenesis/radiation effects , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/radiation effects , Animals , Biomarkers/metabolism , Cell Death/radiation effects , Cell Differentiation/radiation effects , Cell Line , Gamma Rays , Humans , Mice, Inbred NOD , Radiation Tolerance/radiation effects , Stem Cell TransplantationABSTRACT
Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.