Dissecting Risk Haplotypes in Sporadic Alzheimer's Disease

Understanding how genetic risk variants contribute to complex diseases is crucial for predicting disease susceptibility and developing patient-tailored therapies. In this issue of Cell Stem Cell, Young et al. (2015) dissect the function of common non-coding risk haplotypes in the SORL1 locus in the pathogenesis of sporadic Alzheimer's disease using patient-derived induced pluripotent stem cells

Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution

Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.National Institutes of Health (U.S.) (Grant HD 045022

Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation

β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this

Parkinson's Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.Howard Hughes Medical Institute (Collaborative Innovation Award)Life Sciences Research Foundation (Merck Fellow)Michael Stern Parkinson's Research FoundationMorris K. Udall Center for Excellence in Parkinson’s Research (grant P50NS39793)National Institutes of Health (U.S.) (NIH grant R37-CA084198)National Institutes of Health (U.S.) (NIH grant RO1-CA087869)National Institutes of Health (U.S.) (grant NIH RO1-HD045022

Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors

The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.United States. National Institutes of HealthNew York State Stem Cell ScienceNational Institute of Neurological Disorders and Stroke (U.S.)Starr Foundation (Tri-Institutional Starr Stem Cell Scholars Fellowship

Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.Facultad de Ciencias MédicasInstituto de Investigaciones Bioquímicas de La Plat

Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells