33 research outputs found
Concise Review: Exciting Cells: Modeling Genetic Epilepsies with PatientâDerived Induced Pluripotent Stem Cells
Human induced pluripotent stem cell (iPSC) models of epilepsy are becoming a revolutionary platform for mechanistic studies and drug discovery. The skyrocketing pace of epilepsy gene discovery is vastly outstripping the development of in vivo animal models. Currently, antiepileptic drug prescribing to patients with specific genetic epilepsies is based on smallâscale clinical trials and empiricism; however, rapid production of patientâderived iPSC models will allow for precision therapy. We review iPSCâbased studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSCâbased neurological disease models. We also discuss iPSCâderived cardiomyocyte studies of arrhythmiaâinducing ion channelopathies that exemplify novel drug discovery and use of multielectrode array technology that can be translated to epilepsy research. Beyond initial studies of Rett, Timothy, PhelanâMcDermid, and Dravet syndromes, the stage is set for groundbreaking iPSCâbased mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life. Stem Cells 2016;34:27â33Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134416/1/stem2203_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134416/2/stem2203.pd
Fibroblast growth factor 2 regulates activity and gene expression of human postâ mitotic excitatory neurons
Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature, postâ mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has antiâ depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogeninâ 2 (NEUROG2) to generate a homogenous population of postâ mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline, the vast majority of cells are postâ mitotic, and the gene expression profile recapitulates that of excitatory neurons within 6Ă days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f, we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2â responsive genes were determined by RNAâ Seq. FGF2â regulated genes previously identified in nonâ neuronal cell types were upâ regulated (EGR1, ETV4, SPRY4, and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuronâ specific genes were also identified that may mediate FGF2â dependent increases in synaptic efficacy including NRXN3, SYT2, and GALR1. Since several of these genes have been implicated in MDD previously, these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD.Alterations in fibroblast growth factor (FGF) signaling have been implicated in major depressive disorder (MDD). In this article, human stem cells are differentiated into glutamatergic neurons. FGF2 treatment of these neurons increases activity as determined using calcium imaging. RNAseq studies implicate a number of genes in this regulation of neuronal activity by FGF2 including SYT2, NRXN3, and GALR1.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/1/jnc14255-sup-0001-SupInfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/2/jnc14255-sup-0002-TableS1-S2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/3/jnc14255.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/4/jnc14255_am.pd
Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans
Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have
fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in
25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16
regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of
correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP,
while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in
Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium
(LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region.
Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant
enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the
refined data for existing association signals, we estimate that these loci now explain âź38.9% of the familial relative risk of PrCa,
an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of
PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent
signals within the same regio
Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received
Background
The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy.
Objective
To report outcomes according to treatment received in men in randomised and treatment choice cohorts.
Design, setting, and participants
This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy.
Intervention
Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment.
Outcome measurements and statistical analysis
Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores.
Results and limitations
According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa.
Conclusions
Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group.
Patient summary
More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common
Functional and quality of life outcomes of localised prostate cancer treatments (prostate testing for cancer and treatment [ProtecT] study)
Objective
To investigate the functional and quality of life (QoL) outcomes of treatments for localised prostate cancer and inform treatment decision-making.
Patients and Methods
Men aged 50â69âyears diagnosed with localised prostate cancer by prostate-specific antigen testing and biopsies at nine UK centres in the Prostate Testing for Cancer and Treatment (ProtecT) trial were randomised to, or chose one of, three treatments. Of 2565 participants, 1135 men received active monitoring (AM), 750 a radical prostatectomy (RP), 603 external-beam radiotherapy (EBRT) with concurrent androgen-deprivation therapy (ADT) and 77 low-dose-rate brachytherapy (BT, not a randomised treatment). Patient-reported outcome measures (PROMs) completed annually for 6âyears were analysed by initial treatment and censored for subsequent treatments. Mixed effects models were adjusted for baseline characteristics using propensity scores.
Results
Treatment-received analyses revealed different impacts of treatments over 6âyears. Men remaining on AM experienced gradual declines in sexual and urinary function with age (e.g., increases in erectile dysfunction from 35% of men at baseline to 53% at 6âyears and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6âyears. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6âyears, and after EBRT this was reported by 69% and 74%, respectively (Pâ<â0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1âpad/day, persisting for 20% at 6âyears, compared with no change in men receiving EBRT or AM (Pâ<â0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6âyears and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (Pâ<â0.001) with lesser effects after BT. No treatment affected mental or physical QoL.
Conclusion
Treatment decision-making for localised prostate cancer can be informed by these 6-year functional and QoL outcomes
Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation
Induced pluripotent stem cells (iPSCs) are becoming mainstream tools to study mechanisms of development and disease. They have a broad range of applications in understanding disease processes, in vitro testing of novel therapies, and potential utility in regenerative medicine. Although the techniques for generating iPSCs are becoming more straightforward, scientists can expend considerable resources and time to establish this technology. A major hurdle is the accurate determination of valid iPSC-like colonies that can be selected for further cloning and characterization. In this study, we describe the use of a gammaretroviral vector encoding a fluorescent marker, mRFP1, to not only monitor the efficiency of initial transduction but also to identify putative iPSC colonies through silencing of mRFP1 gene as a consequence of successful reprogramming
PARK2 patient neuroprogenitors show increased mitochondrial sensitivity to copper
Abstract: Poorly-defined interactions between environmental and genetic risk factors underlie Parkinson's disease (PD) etiology. Here we tested the hypothesis that human stem cell derived forebrain neuroprogenitors from patients with known familial risk for early onset PD will exhibit enhanced sensitivity to PD environmental risk factors compared to healthy control subjects without a family history of PD. Two male siblings (SM and PM) with biallelic loss-of-function mutations in PARK2 were identified. Human induced pluripotent stem cells(hiPSCs) from SM, PM, and four control subjects with no known family histories of PD or related neurodegenerative diseases were utilized. We tested the hypothesis that hiPSC-derived neuroprogenitors from patients with PARK2 mutations would show heightened cell death, mitochondrial dysfunction, and reactive oxygen species generation compared to control cells as a result of exposure to heavy metals (PD environmental risk factors). We report that PARK2 mutant neuroprogenitors showed increased cytotoxicity with copper (Cu) and cadmium (Cd) exposure but not manganese (Mn) or methyl mercury (MeHg) relative to control neuroprogenitors. PARK2 mutant neuroprogenitors also showed a substantial increase in mitochondrial fragmentation, initial ROS generation, and loss of mitochondrial membrane potential following Cu exposure. Our data substantiate Cu exposure as an environmental risk factor for PD. Furthermore, we report a shift in the lowest observable effect level (LOEL) for greater sensitivity to Cu-dependent mitochondrial dysfunction in patients SM and PM relative to controls, correlating with their increased genetic risk for PD
Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing
Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene, and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines, even in the absence of patient tissue
Genomic Instability Associated with p53 Knockdown in the Generation of Huntington's Disease Human Induced Pluripotent Stem Cells.
Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown