iSCORE-PD: an isogenic stem cell collection to research Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by complex genetic and environmental factors. Genome-edited human pluripotent stem cells (hPSCs) offer the uniique potential to advance our understanding of PD etiology by providing disease-relevant cell-types carrying patient mutations along with isogenic control cells. To facilitate this experimental approach, we generated a collection of 55 cell lines genetically engineered to harbor mutations in genes associated with monogenic PD ( A53T, A30P, Ex3del, Q129X, Ex1-5del, G2019S, FS, R498X/FS, c.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side.

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides.

Stem Cells, Genome Editing, and the Path to Translational Medicine.

The derivation of human embryonic stem cells (hESCs) and the stunning discovery that somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) holds the promise to revolutionize biomedical research and regenerative medicine. In this Review, we focus on disorders of the central nervous system and explore how advances in human pluripotent stem cells (hPSCs) coincide with evolutions in genome engineering and genomic technologies to provide realistic opportunities to tackle some of the most devastating complex disorders.

S-Nitrosylation of PINK1 Attenuates PINK1/Parkin-Dependent Mitophagy in hiPSC-Based Parkinson's Disease Models.

Mutations in PARK6 (PINK1) and PARK2 (Parkin) are linked to rare familial cases of Parkinson's disease (PD). Mutations in these genes result in pathological dysregulation of mitophagy, contributing to neurodegeneration. Here, we report that environmental factors causing a specific posttranslational modification on PINK1 can mimic these genetic mutations.

Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression.

Genome-wide association studies (GWAS) have identified numerous genetic variants associated with complex diseases, but mechanistic insights are impeded by a lack of understanding of how specific risk variants functionally contribute to the underlying pathogenesis. It has been proposed that cis-acting effects of non-coding risk variants on gene expression are a major factor for phenotypic variation of complex traits and disease susceptibility. Recent genome-scale epigenetic studies have highlighted the enrichment of GWAS-identified variants in regulatory DNA elements of disease-relevant cell types.

Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders.

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.

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.

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.

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.

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.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

Parkinson's disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs).

Identification and rescue of α-synuclein toxicity in Parkinson patient-derived neurons.

The induced pluripotent stem (iPS) cell field holds promise for in vitro disease modeling. However, identifying innate cellular pathologies, particularly for age-related neurodegenerative diseases, has been challenging. Here, we exploited mutation correction of iPS cells and conserved proteotoxic mechanisms from yeast to humans to discover and reverse phenotypic responses to α-synuclein (αsyn), a key protein involved in Parkinson's disease (PD).

Toward eliminating HLA class I expression to generate universal cells from allogeneic donors.

Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression.

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.

Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations.

Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation.

Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells.

Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs.

Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs.

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway.

Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing.

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.