[Show abstract][Hide abstract] ABSTRACT: Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.
[Show abstract][Hide abstract] ABSTRACT: Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.
[Show abstract][Hide abstract] ABSTRACT: Genomic abnormalities may accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Characterization of the mechanisms enabling survival and expansion of abnormal hESCs is important due to consequences of genetic changes for the therapeutic utilization of stem cells. Furthermore, these cells provide an excellent model to study transformation in vitro. We report here that the histone deacetylase proteins, HDAC1 and HDAC2, are increased in karyotypically abnormal hESCs when compared to their normal counterparts. Importantly, similar to many cancer cell lines, we found that HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs, whereas normal cells are more resistant to the treatment. The decreased proliferation correlates with downregulation of HDAC1 and HDAC2 proteins, induction of the proliferation inhibitor, cyclin-dependent kinase inhibitor 1A (CDKN1A), and altered regulation of tumor suppressor protein Retinoblastoma 1 (RB1). Through genome-wide transcriptome analysis we have identified genes with altered expression and responsiveness to HDAC inhibition in abnormal cells. Most of these genes are linked to severe developmental and neurological diseases and cancers. Our results highlight the importance of epigenetic mechanisms in the regulation of genomic stability of hESCs, and provide valuable candidates for targeted and selective growth inhibition of karyotypically abnormal cells.
Stem Cell Research 07/2013; 11(3):1022-1036. · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation.
We investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively.
Plakoglobin and γ-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells.
Decreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/γ-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.
Breast cancer research: BCR 05/2012; 14(3):R86. · 5.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pluripotent potential of embryonic stem cells has often seen them touted as the future of regenerative medicine. The road to any therapeutic success however, must stretch back to teratocarcinoma, the tumour from which pluripotent stem cells (embryonal carcinoma cells) were first derived. This 2011 meeting in Cardiff acted as a historical perspective from which the impact of embryonal carcinoma cell research on the present pluripotent stem cell landscape could be observed, with many of the early luminaries in this field still very active. The meeting addressed the genetic and epigenetic make-up of pluripotent stem cells, the mechanisms which control their fate, and their relationship to the early embryo proper. With each speaker tasked with revisiting previous questions, this meeting demonstrated how far has been travelled, yet how far is left to go.
The International journal of developmental biology 01/2012; 56(4):197-206. · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.
[Show abstract][Hide abstract] ABSTRACT: Disentangling the complex interactions that govern stem cell fate choices of self-renewal, differentiation, or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632, HA-1077, and H-8 all strongly inhibit the kinases ROCK and PRK2, highlighting the important role of these kinases in EC cell survival. Two molecules, GF109203x and rottlerin, induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells, caused the cell cycle arrest, and repressed the expression of pluripotency-associated genes.
Journal of Biomolecular Screening 05/2011; 16(6):603-17. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Understanding the complex mechanisms that govern the fate decisions of human embryonic stem cells (hESCs) is fundamental to their use in cell replacement therapies. The progress of dissecting these mechanisms will be facilitated by the availability of robust high-throughput screening assays on hESCs. In this study, we report an image-based high-content assay for detecting compounds that affect hESC survival or pluripotency. Our assay was designed to detect changes in the phenotype of hESC colonies by quantifying multiple parameters, including the number of cells in a colony, colony area and shape, intensity of nuclear staining, and the percentage of cells in the colony that express a marker of pluripotency (TRA-1-60), as well as the number of colonies per well. We used this assay to screen 1040 compounds from two commercial compound libraries, and identified 17 that promoted differentiation, as well as 5 that promoted survival of hESCs. Among the novel small compounds we identified with activity on hESC are several steroids that promote hESC differentiation and the antihypertensive drug, pinacidil, which affects hESC survival. The analysis of overlapping targets of pinacidil and the other survival compounds revealed that activity of PRK2, ROCK, MNK1, RSK1, and MSK1 kinases may contribute to the survival of hESCs.
Stem Cell Research 09/2010; 5(2):104-19. · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.
Biochemical Society Transactions 08/2010; 38(4):1046-50. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.
[Show abstract][Hide abstract] ABSTRACT: Appropriate culture of murine embryonic stem cells is critical to enabling introduced mutations to be passed through the germ line. ES cells must be carefully cultured to ensure that pluripotency is maintained. The feeder cells and foetal calf serum used to culture the cells can significantly influence germ line transmission potential. Additionally, ES cells can be karyotypically unstable, so determining the karyotype of ES cell lines will increase your confidence in the ability of the manipulated cells to contribute to the germ line in chimaeric mice.
[Show abstract][Hide abstract] ABSTRACT: The availability of high-throughput biochemical and imaging techniques that can be used on live mice has increased the possibility of undertaking longitudinal studies to characterize skeletal changes such as bone mineral content and density. Further characterization of bone morphology, bone quality, and bone strength can also be achieved by analyzing dissected bones using techniques that provide higher resolution. Thus, the combined use of high-throughput [e.g., biochemical analysis of plasma, radiography and dual-energy X-ray absorptiometry (DEXA)] and secondary phenotyping techniques (e.g., histology, histomorphometry, Faxitron digital X-ray point projection microradiography, biomechanical testing, and micro-computed tomography) can be utilized for comprehensive characterization of bone structure and quality and to elucidate the underlying molecular mechanisms giving rise to musculoskeletal disorders.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms that regulate bone mass are important in a variety of complex diseases such as osteopenia and osteoporosis. Regulation of bone mass is a polygenic trait and is also influenced by various environmental and lifestyle factors, making analysis of the genetic basis difficult. As an effort toward identifying novel genes involved in regulation of bone mass, N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has been utilized. Here we describe a mouse mutant termed Yoda that was identified in an ENU mutagenesis screen for dominantly acting mutations. Mice heterozygous for the Yoda mutation exhibit craniofacial abnormalities: shortened snouts, wider skulls, and deformed nasal bones, underlined by altered morphology of frontonasal sutures and failure of interfrontal suture to close. A major feature of the mutant is reduced bone mineral density. Homozygosity for the mutation results in embryonic lethality. Positional cloning of the locus identified a missense mutation in a highly conserved region of the ankyrin repeat domain 11 gene (Ankrd11). This gene has not been previously associated with bone metabolism and, thus, identifies a novel genetic regulator of bone homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screens in the mouse are being used to elucidate gene function and develop disease models. Many of the earlier screens focused on identifying dominant mutations, whereas many newer mutagenesis programs have arisen that focus on identifying recessive mutations. Recessive screens require more complex breeding and phenotyping procedures, yet little information is available on the optimal breeding and phenotyping strategies for identifying recessive mutations. Optimization involves minimizing the numbers of mice that must be bred and subjected to phenotypic screens while maximizing the number of mutant phenotypes that can be identified. Analysis of expected frequencies of mutants has been used to determine which of the typically used mating and screening strategies will produce the best returns in terms of identifying recessive phenotypes. As a general guideline, to minimize the number of mice to be screened, the optimal strategy is to mate a single generation 2 (G2) female and G1 male and screen either 11 or 17 G3 offspring to obtain at least 1 or 2 homozygous mutants, respectively. When the expense of producing and housing the mice is the greatest cost factor and the phenotype is so robust that a single outlier will suffice, then the optimal strategy is to mate 2 G2 sisters with the G1 male parent and screen a single litter from each. Intercrossing of G2 brothers and sisters is not an efficient method for maximizing returns from ENU screens.
Journal of the American Association for Laboratory Animal Science: JAALAS 12/2007; 46(6):44-9. · 1.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the field of mammalian functional genomics, one of the main aims in the post-genomic era is to elucidate the function of all genes in the genome. The powerful technology of gene targeting in embryonic stem cells has enabled the simple generation of mice lacking a specific gene. However, it is evident that in a proportion of such knockout mice no deviation in phenotype could be detected. Advancements in the field of mouse phenotyping and use of extensive phenotyping tests on each knockout showed that abnormal phenotypes were sometimes detected in physiological areas where they were not initially anticipated, or only manifested under certain conditions, emphasizing the need for careful phenotypic investigation. Nevertheless, the effect of some genes became evident only upon inactivation of another gene, pointing to the phenomenon of biological robustness. Unlike in yeast, this phenomenon has not yet been analysed systematically in the mouse. In this review, we present examples of mouse knockouts that lend support to the concept of robustness, discuss the mechanisms by which it may have evolved, as well as speculate on the reasons for its evolution.
Briefings in Functional Genomics and Proteomics 07/2007; 6(2):91-103.
[Show abstract][Hide abstract] ABSTRACT: Most gene-targeted mice are produced on a mixed genetic background of C57BL/6 and substrains of 129/Sv. Mating chimeric mice containing 129/Sv-derived embryonic stem cells that are wild type at the agouti locus (A) in a nonagouti (a) donor genetic background with inbred C57BL/6 mice that are homozygous for the nonagouti allele allows the use of coat color to detect germline transmission. Agouti pups from such a cross indicate germline transmission of embryonic stem cell-derived genetic material. However, 129/Sv substrains and C57BL/6 are genetically and phenotypically quite different and, consequently, differing genetic contributions of the 2 backgrounds may influence the phenotype under investigation. To avoid this problem yet maintain the usefulness of the coat color system in detecting germline transmission, we have generated a new strain of mouse by selectively introducing the nonagouti locus into a 129/Sv inbred background. This mouse strain contains 129/Svderived genetic material almost entirely except for a small region surrounding the nonagouti allele. Germline transmission can be detected in the usual manner, but the agouti offspring will be almost identical to 129/Sv inbred mice. Thus, the system allows the generation of gene-targeted mutations on a 129/Sv genetic background.
Journal of the American Association for Laboratory Animal Science: JAALAS 06/2007; 46(3):37-40. · 1.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-ethyl-N-nitrosourea (ENU) mutagenesis in mice has become a standard tool for (i) increasing the pool of mutants in many areas of biology, (ii) identifying novel genes involved in physiological processes and disease, and (iii) in assisting in assigning functions to genes. ENU is assumed to cause random mutations throughout the mouse genome, but this presumption has never been analyzed. This is a crucial point, especially for large-scale mutagenesis, as a bias would reflect a constraint on identifying possible genetic targets. There is a significant body of published data now available from both phenotype-driven and gene-driven ENU mutagenesis screens in the mouse that can be used to reveal the effectiveness and limitations of an ENU mutagenesis approach. Analysis of the published data is presented in this paper. As expected for a randomly acting mutagen, ENU-induced mutations identified in phenotype-driven screens were in genes that had higher coding sequence length and higher exon number than the average for the mouse genome. Unexpectedly, the data showed that ENU-induced mutations were more likely to be found in genes that had a higher G + C content and neighboring base analysis revealed that the identified ENU mutations were more often directly flanked by G or C nucleotides. ENU mutations from phenotype-driven and gene-driven screens were dominantly A:T to T:A transversions or A:T to G:C transitions. Knowledge of the spectrum of mutations that ENU elicits will assist in positional cloning of ENU-induced mutations by allowing prioritization of candidate genes based on some of their inherent features.
Environmental and Molecular Mutagenesis 04/2007; 48(2):124-42. · 3.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.