Concise Review: Exciting Cells: Modeling Genetic Epilepsies with Patient‐Derived 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 small‐scale clinical trials and empiricism; however, rapid production of patient‐derived iPSC models will allow for precision therapy. We review iPSC‐based studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSC‐based neurological disease models. We also discuss iPSC‐derived cardiomyocyte studies of arrhythmia‐inducing 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, Phelan‐McDermid, and Dravet syndromes, the stage is set for groundbreaking iPSC‐based mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life. Stem Cells 2016;34:27–33Peer 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 6 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 ∼38.9% 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 50–69 years 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 6 years 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 6 years. 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 6 years and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6 years. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6 years, and after EBRT this was reported by 69% and 74%, respectively (P < 0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1 pad/day, persisting for 20% at 6 years, compared with no change in men receiving EBRT or AM (P < 0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6 years and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (P < 0.001) 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