From clinical specimens to human cancer preclinical models—a journey the NCI-cell line database—25 years later

The intramural the National Cancer Institute (NCI) and more recently the University of Texas Southwestern Medical Center with many different collaborators comprised a complex, multi-disciplinary team that collaborated to generated large, comprehensively annotated, cell-line related research resources which includes associated clinical, and molecular characterization data. This material has been shared in an anonymized fashion to accelerate progress in overcoming lung cancer, the leading cause of cancer death across the world. However, this cell line collection also includes a range of other cancers derived from patient-donated specimens that have been remarkably valuable for other types of cancer and disease research. A comprehensive analysis conducted by the NCI Center for Research Strategy of the 278 cell lines reported in the original Journal of Cellular Biochemistry Supplement, documents that these cell lines and related products have since been used in more than 14 000 grants, and 33 207 published scientific reports. This has resulted in over 1.2 million citations using at least one cell line. Many publications involve the use of more than one cell line, to understand the value of the resource collectively rather than individually; this method has resulted in 2.9 million citations. In addition, these cell lines have been linked to 422 clinical trials and cited by 4700 patents through publications. For lung cancer alone, the cell lines have been used in the research cited in the development of over 70 National Comprehensive Cancer Network clinical guidelines. Finally, it must be underscored again, that patient altruism enabled the availability of this invaluable research resource.     

Neural lineage differentiation of human pluripotent stem cells: Advances in disease modeling

Brain diseases affect 1 in 6 people worldwide. These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease. Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models, tissue culture models, and epidemiologic patient data that are commonly used to study brain disease. One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells (hPSCs) to neural lineages including neurons, astrocytes, and oligodendrocytes. Three-dimensional models such as brain organoids have also been derived from hPSCs, offering more physiological relevance due to their incorporation of various cell types. As such, brain organoids can better model the pathophysiology of neural diseases observed in patients. In this review, we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation

Objectives:  Defects of the mitochondrial genome (mtDNA) cause a series of rare, mainly neurological disorders. In addition, they have been implicated in more common forms of movement disorders, dementia and the ageing process. In order to try to model neuronal dysfunction associated with mitochondrial disease, we have attempted to establish a series of trans mitochondrial mouse embryonic stem cells harbouring pathogenic mtDNA mutations.
Materials and methods:  Trans mitochondrial embryonic stem cell cybrids were generated by fusion of cytoplasts carrying a variety of mtDNA mutations, into embryonic stem cells that had been pretreated with rhodamine 6G, to prevent transmission of endogenous mtDNA. Cybrids were differentiated into neurons and assessed for efficiency of differentiation and electrophysiological function.
Results:  Neuronal differentiation could occur, as indicated by expression of neuronal markers. Differentiation was impaired in embryonic stem cells carrying mtDNA mutations that caused severe biochemical deficiency. Electrophysiological tests showed evidence of synaptic activity in differentiated neurons carrying non-pathogenic mtDNA mutations or in those that caused a mild defect of respiratory activity. Again, however, neurons carrying mtDNA mutations that resulted in severe biochemical deficiency had marked reduction in post-synaptic events.
Conclusions:  Differentiated neurons carrying severely pathogenic mtDNA defects can provide a useful model for understanding how such mutations can cause neuronal dysfunction.     

Application of reprogrammed patient cells to investigate the etiology of neurological and psychiatric disorders

Cellular reprogramming allows for the de novo generation of human neurons and glial cells from patients with neurological and psychiatric disorders.Crucially,this technology preserves the genome of the...     

Primary Skin Fibroblasts as a Model of Parkinson's Disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues.     

Synaptopathies: diseases of the synaptome

The human synapse proteome is a highly complex collection of proteins that is disrupted by hundreds of gene mutations causing over 100 brain diseases. These synaptic diseases, or synaptopathies, cause major psychiatric, neurological and childhood developmental disorders through mendelian and complex genetic mechanisms. The human postsynaptic proteome and its core signaling complexes built by the assembly of receptors and enzymes around Membrane Associated Guanylate Kinase (MAGUK) scaffold proteins are a paradigm for systematic analysis of synaptic diseases. In humans, the MAGUK Associated Signaling Complexes are mutated in Autism, Schizophrenia, Intellectual Disability and many other diseases, and mice carrying orthologous mutations show relevant cognitive, social, motoric and other phenotypes. Diseases with similar phenotypes and symptom spectrums arise from disruption of complexes and interacting proteins within the synapse proteome. Classifying synaptic disease phenotypes with genetic and proteome data provides a new brain disease classification system based on molecular etiology and pathogenesis.     

Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC) lines from Rett syndrome patients as in vitro disease model

Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5-6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.     

No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.     

Induced pluripotent stem cells for modelling human diseases

Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation.     

Analysis of the molecular pathophysiology of sleep disorders relevant to a disturbed biological clock

Genetic studies have revealed several clock gene variations/mutations involved in the manifestation of sleep disorders or interindividual differences in sleep–wake patterns, but only part of the genetic risk can be explained by the gene variations/mutations identified to date. Recent progress in research into circadian rhythm generation has provided efficient tools for eliciting the molecular basis of clock-relevant sleep disorders, complementing traditional genetic analysis. While the human master clock resides in the suprachiasmatic nucleus of the hypothalamus (central clock), peripheral tissue cells also generate self-sustained circadian oscillations of clock gene expression (peripheral clock), enabling estimation of individual human clock properties through a single collection of skin fibroblasts or venous blood cells. Some of the established cell lines exhibit autonomous circadian oscillations of clock gene expression, and introduction of clock gene variations into these cell lines by gene targeting makes it possible to investigate changes in the circadian phenotype induced by these variations/mutations without the need for generating transgenic animals. Estimation of human clock properties using peripheral tissue cells, in addition to genetic analysis, will facilitate comprehensive explication of the genetic risk of a variety of disorders relevant to biological clock disturbances, including sleep disorders, mood disorders, and metabolic diseases.     

Human embryonic stem cells carrying mutations for severe genetic disorders

Human embryonic stem cells (HESCs) carrying specific mutations potentially provide a valuable tool for studying genetic disorders in humans. One preferable approach for obtaining these cell lines is by deriving them from affected preimplantation genetically diagnosed embryos. These unique cells are especially important for modeling human genetic disorders for which there are no adequate research models. They can be further used to gain new insights into developmentally regulated events that occur during human embryo development and that are responsible for the manifestation of genetically inherited disorders. They also have great value for the exploration of new therapeutic protocols, including gene-therapy-based treatments and disease-oriented drug screening and discovery. Here, we report the establishment of 15 different mutant human embryonic stem cell lines derived from genetically affected embryos, all donated by couples undergoing preimplantation genetic diagnosis in our in vitro fertilization unit. For further information regarding access to HESC lines from our repository, for research purposes, please email dalitb@tasmc.health.gov.il.     

Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants

The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.     

Drosophila as a Model Organism for Investigating Molecular and Cellular Etiologies Underlying Complex Neurological Disorders in Humans

The fruit fly Drosophila has been utilized as a powerful biological system to address fundamental questions concerning neurological disorders in humans, since the related basic molecular components and signal transduction pathways in humans are mostly conserved in Drosophila. In addition, Drosophila offers great experimental advantages in genetics, behavioral analysis and cell and molecular biology. Pathogenesis and etiologies underlying several monogenic neurological disorders including familial Parkinson disease, Alzheimer's disease, or ataxia have been faithfully replicated in Drosophila system when causative mutations of those disorders were transgenically introduced or loss of function mutations of endogenous homologues were made. However, more than 90% of reported cases of neurological disorders are complex forms whose inheritance patterns do not follow a monogenic inheritance. Nevertheless, complex disorders are more often observed among the families or relatives of affected patients, strongly suggesting that they are most likely to be caused by interaction of multiple genetic mutations or combinations of genetic and environmental risk factors. Complex neurological disorders are include sporadic forms of Parkinson disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dystonia, epilepsy, and mental retardation, etc. Our understanding of the genetic defects and environmental risk factors involved in the onset and progression of complex neurological disorders are, however, still rudimentary. Thus identifying unknown factors involved in the onset and progression of complex neurological disorders in humans is one of the major challenges in medical sciences.     

Can cellular models revolutionize drug discovery in Parkinson's disease?

The study of mechanisms that underlie Parkinson's disease (PD), as well as translational drug development, has been hindered by the lack of appropriate models. Both cell culture systems and animal models have limitations, and to date none faithfully recapitulate all of the clinical and pathological phenotypes of the disease. In this review we examine the various cell culture model systems of PD, with a focus on different stem cell models that can be used for investigating disease mechanisms as well as drug discovery for PD. We conclude with a discussion of recent discoveries in the field of stem cell biology that have led to the ability to reprogram somatic cells to a pluripotent state via the use of a combination of genetic factors; these reprogrammed cells are termed “induced pluripotent stem cells” (iPSCs). This groundbreaking technique allows for the derivation of patient-specific cell lines from individuals with sporadic forms of PD and also those with known disease-causing mutations. Such cell lines have the potential to serve as a human cellular model of neurodegeneration and PD when differentiated into dopaminergic neurons. The hope is that these iPSC-derived dopaminergic neurons can be used to replicate the key molecular aspects of neural degeneration associated with PD. If so, this approach could lead to transformative new tools for the study of disease mechanisms. In addition, such cell lines can be potentially used for high-throughput drug screening. While not the focus of this review, ultimately it is envisioned that techniques for reprogramming of somatic cells may be optimized to a point sufficient to provide potential new avenues for stem cell-based restorative therapies.     

DNA repair and neurological disease: From molecular understanding to the development of diagnostics and model organisms

In both replicating and non-replicating cells, the maintenance of genomic stability is of utmost importance. Dividing cells can repair DNA damage during cell division, tolerate the damage by employing potentially mutagenic DNA polymerases or die via apoptosis. However, the options for accurate DNA repair are more limited in non-replicating neuronal cells. If DNA damage is left unresolved, neuronal cells die causing neurodegenerative disorders. A number of pathogenic variants of DNA repair proteins have been linked to multiple neurological diseases. The current challenge is to harness our knowledge of fundamental properties of DNA repair to improve diagnosis, prognosis and treatment of such debilitating disorders. In this perspective, we will focus on recent efforts in identifying novel DNA repair biomarkers for the diagnosis of neurological disorders and their use in monitoring the patient response to therapy. These efforts are greatly facilitated by the development of model organisms such as zebrafish, which will also be summarised.     

Voltage-gated calcium channels and disease

Voltage-gated calcium channels are a family of integral membrane calcium-selective proteins found in all excitable and many nonexcitable cells. Calcium influx affects membrane electrical properties by depolarizing cells and generally increasing excitability. Calcium entry further regulates multiple intracellular signaling pathways as well as the biochemical factors that mediate physiological functions such as neurotransmitter release and muscle contraction. Small changes in the biophysical properties or expression of calcium channels can result in pathophysiological changes leading to serious chronic disorders. In humans, mutations in calcium channel genes have been linked to a number of serious neurological, retinal, cardiac, and muscular disorders.