Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells (original) (raw)
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Antioxidant supplementation reduces genomic aberrations in human induced pluripotent stem cells
Stem cell reports, 2014
Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using oncogenic transcription factors. However, this method leads to genetic aberrations in iPSCs via unknown mechanisms, which may limit their clinical use. Here, we demonstrate that the supplementation of growth media with antioxidants reduces the genome instability of cells transduced with the reprogramming factors. Antioxidant supplementation did not affect transgene expression level or silencing kinetics. Importantly, iPSCs made with antioxidants had significantly fewer de novo copy number variations, but not fewer coding point mutations, than iPSCs made without antioxidants. Our results suggest that the quality and safety of human iPSCs might be enhanced by using antioxidants in the growth media during the generation and maintenance of iPSCs.
2014
Undoubtedly, the focus of the field of stem cell research is predominantly aimed at the artificial reprogramming of human somatic cells for the production of induced pluripotent stem (iPS) cells. This relatively new technology may circumvent the ethical issues of using human embryonic stem (hES) cells for the potential applications in cell replacement therapy. Besides such ethical issues, iPS cell technology offers the advantage of obtaining patient-derived tissues and/or cells, which may be utilized for autologous transplantation and tissue regeneration, investigation of a variety of human illnesses and for the screening of new drugs. The field of stem cell research has placed a major emphasis in understanding the genetic and epigenetic codes for pluripotency, in order to control and optimize autologous transplantation techniques and avoid teratoma formation.
Stem Cell Research, 2016
II is a healthy feeder-free and characterized human induced pluripotent stem (iPS) cell line. Cultured under xeno-free and defined conditions. The line is generated from healthy human fibroblasts with nonintegrating Sendai virus vectors encoding the four Yamanaka factors, OCT4, SOX2, KLF4 and cMYC. The generated iPS cells are free from reprogramming vectors and their purity, karyotypic stability and pluripotent capacity is confirmed.
Application of Stem Cells and iPS Cells in Toxicology
John Wiley & Sons, Ltd eBooks, 2016
Fertilization of an oocyte by a spermatozoid, or in general terms, fertilization of the female gamete by the male gamete results in the genesis of the basic unit in the development of an organism, the embryo. The primordial unicellular embryo will, after several rounds of division, develop into a multicellular organism and ultimately into an offspring resembling its progenitors in structure and function. At the molecular level, embryonic development is governed by the expression and interaction of a specific set of genes, certain proteins and peptides, and the signaling of growth factors. In other words, embryonic development is a complex process, which involves a vast but specific genetic network corresponding to its different stages. Early observations made in the sea urchin (Driesch, 1892, 1893, 1894; Gilbert, 2000; Roux, 1888; Roux, 1894) demonstrated that splitting a two-cell embryo into single cells resulted in the emergence of two fully developed organisms. Hans Spemann repeated these experiments in the salamander two-cell embryo, ultimately obtaining two fully developed "twins" (Gilbert, 2000; Spemann, 1921). These early experiments demonstrated the totipotency of the early embryonic cells and the retention of certain "information," which allows for the development of an organism. This information is progressively lost as the embryo develops and its cells differentiate and reach more specific roles (Gilbert, 2000; Spemann, 1921). However, the question remains, what is the stage at which cells lose this potential? Further experimentation revealed that cells derived from teratomas, or from mouse blastocysts inner cell mass (ICM) cultured upon a suitable fibroblast feeder layer, continue to proliferate without overt differentiation
Emerging Methods for Preparing iPS Cells
Japanese Journal of Clinical Oncology, 2012
In 1998, human embryonic stem cells were first generated and were expected to contribute greatly to regenerative medicine. However, when medical treatments were performed using human embryonic stem cells, there were problems, such as transplant rejection, as well as bioethical issues. Induced pluripotent stem cells were generated from mouse and human fibroblasts in 2006 and 2007 by introducing four transcription factors (Oct3/4, Sox2, c-Myc and Klf4). This process was defined as direct reprogramming, and induced pluripotent stem cells were better tolerated. Although induced pluripotent stem cells have contributed greatly to biomedical research and regenerative medicine, high tumorigenic potential is still a critical problem due to the introduction of the oncogene c-Myc and reprogramming with a virus vector. To address this, we reprogrammed somatic cells by transfection with microribonucleic acids to avoid using virus vectors for genomic integration into the host genome. We found that it was possible to reprogram mouse and human cells to pluripotency by direct transfection of three mature microribonucleic acids (mir-200c,-302s and-369s) with increased expression levels in embryonic stem cells and induced pluripotent stem cells. The microribonucleic acid-induced pluripotent stem cells have a reduced risk of mutations and tumorigenesis. Our laboratory also introduced four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) into cancer cells, generating induced pluripotent cancer cells that exhibited strikingly less malignant features, suggesting the possibility of a novel type of cancer therapy. However, the gene transduction method is not yet safe for clinical applications, due to a genomic integration that may cause tumor formation. We are currently investigating the reprogramming method using microribonucleic acids in cancer cells to develop a very safe, highly efficient and highly complete reprogramming for clinical applications.
Enhancement of Arsenic Trioxide-Mediated Changes in Human Induced Pluripotent Stem Cells (IPS)
International Journal of Environmental Research and Public Health, 2014
Induced pluripotent stem cells (IPS) are an artificially derived type of pluripotent stem cell, showing many of the same characteristics as natural pluripotent stem cells. IPS are a hopeful therapeutic model; however there is a critical need to determine their response to environmental toxins. Effects of arsenic on cells have been studied extensively; however, its effect on IPS is yet to be elucidated. Arsenic trioxide (ATO) has been shown to inhibit cell proliferation, induce apoptosis and genotoxicity in many cells. Based on ATOs action in other cells, we hypothesize that it will induce alterations in morphology, inhibit cell viability and induce a genotoxic effect on IPS. Cells were treated for 24 hours with ATO (0-9 µg/mL). Cell morphology, viability and DNA damage were documented. Results indicated sufficient changes in morphology of cell colonies mainly in cell ability to maintain grouping and ability to remain adherent. Cell viability decreased in
Cancers, 2013
We report here that the Jun dimerization protein 2 (JDP2) plays a critical role as a cofactor for the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and MafK in the regulation of the antioxidants and production of reactive oxygen species (ROS). JDP2 associates with Nrf2 and MafK (Nrf2-MafK) to increase the transcription of antioxidant response element-dependent genes. Oxidative-stress-inducing reagent led to an increase in the intracellular accumulation of ROS and cell proliferation in Jdp2 knockout mouse embryonic fibroblasts. In Jdp2-Cre mice mated with reporter mice, the expression of JDP2 was restricted to granule cells in the brain cerebellum. The induced pluripotent stem cells (iPSC)-like cells were generated from DAOY medulloblastoma cell by introduction of JDP2, and the defined factor OCT4. iPSC-like cells expressed stem cell-like characteristics including alkaline phosphatase activity and some stem cell markers. However, such iPSC-like cells also proliferated rapidly, became neoplastic, and potentiated cell malignancy at a later stage in SCID mice. This study suggests that medulloblastoma cells can be reprogrammed successfully by JDP2 and OCT4 to become iPSC-like cells. These cells will be helpful for studying the generation of cancer stem cells and ROS homeostasis.
Mechanism of Induction: Induced Pluripotent Stem Cells (iPSCs)
Induced Pluripotent Stem Cells (iPSCs) are self renewable and can differentiate to different types of adult cells, which has shown great promises in the field of regenerative medicine. iPSCs are reprogrammed from human somatic cells through ectopic expression of various transcription factors viz. Oct4, Sox2, Klf4, and c-Myc (OSKM). This novel technology enables derivation of patient specific cells, which possess a potential cure for many diseases. During the last decade, significant progresses have been achieved in enhancing the reprogramming efficiency, safety of iPSCs derivation, development of different delivery techniques by various research groups. Nevertheless, it is important to resolve and define the mechanism underlying the pluripotent stem cells. Major bottleneck which arises during iPSCs generation is the availability of source material (cells/tissues), difficulty to deliver transcription factors with no aberrant genetic modifications and limited reprogramming efficiency. Reprogramming may be achieved by employing different cocktails with number of different transcription factors, application of miRNA and some small molecules such as (Valproic acid, CHiR99021, Sodium butyrate, Vitamin C, Parnate etc). Similarly, various starting source materials have been demonstrated for iPSC based therapies including fibroblasts, cord blood, peripheral blood, keritinocytes, urine, etc., with their specific uses and limitations. Moreover, with the advent of many new reprogramming techniques, various direct delivery methods have been introduced such as using synthetic mRNA expressing pluripotent gene network has been shown to be an appropriate technique to deliver transcription factors and a dozen of small molecules which can replace transcription factors or enhance reprogramming efficiency. This article addresses the iPSCs technology mechanisms, progresses and current perspectives in the field.
Genotoxic Effects of Culture Media on Human Pluripotent Stem Cells
Scientific Reports
Culture conditions play an important role in regulating the genomic integrity of Human Pluripotent Stem Cells (HPSCs). We report that HPSCs cultured in Essential 8 (E8) and mTeSR, two widely used media for feeder-free culturing of HPSCs, had many fold higher levels of ROS and higher mitochondrial potential than cells cultured in Knockout Serum Replacement containing media (KSR). HPSCs also exhibited increased levels of 8-hydroxyguanosine, phospho-histone-H2a.X and p53, as well as increased sensitivity to γ-irradiation in these two media. HPSCs in E8 and mTeSR had increased incidence of changes in their DNA sequence, indicating genotoxic stress, in addition to changes in nucleolar morphology and number. Addition of antioxidants to E8 and mTeSR provided only partial rescue. Our results suggest that it is essential to determine cellular ROS levels in addition to currently used criteria i.e. pluripotency markers, differentiation into all three germ layers and normal karyotype through multiple passages, in designing culture media.