The Alzheimer’s disease γ-secretase generates higher 42:40 ratios for β-amyloid than for p3 peptides

Alzheimer’s disease is characterized by intracerebral deposition of β-amyloid (Aβ). While Aβ40 is the most abundant form, neurotoxicity is mainly mediated by Aβ42. Sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases gives rise to full-length Aβ (Aβ1-x) and N-terminally truncated Aβ′ (Aβ11-x) whereas cleavage by α- and γ-secretases leads to the shorter p3 peptides (Aβ17-x). We uncovered significantly higher ratios of 42- versus 40-ending variants for Aβ and Aβ′ than for p3 secreted by mouse neurons and human induced pluripotent stem cell (iPSC)-derived neurons or produced in a cell-free γ-secretase assay with recombinant APP-CTFs. The 42:40 ratio was highest for Aβ′, followed by Aβ and then p3. Mass spectrometry analysis of APP intracellular domains revealed differential processing of APP-C83, APP-C89, and APP-C99 by γ-secretase already at the ε-cleavage stage. This mechanistic insight could aid in developing substrate-targeted modulators of APP-C99 processing to specifically lower the Aβ42:Aβ40 ratio without compromising γ-secretase function

Emerging concepts in neural stem cell research: autologous repair and cell-based disease modelling

The increasing availability of human pluripotent stem cells provides new prospects for neural-replacement strategies and disease-related basic research. With almost unlimited potential for self-renewal, the use of human embryonic stem cells (ESCs) bypasses the restricted supply and expandability of primary cells that has been a major bottleneck in previous neural transplantation approaches. Translation of developmental patterning and cell-type specification techniques to human ESC cultures enables in vitro generation of various neuronal and glial cell types. The derivation of stably proliferating neural stem cells from human ESCs further facilitates standardisation and circumvents the problem of batch-to-batch variations commonly encountered in "run-through" protocols, which promote terminal differentiation of pluripotent stem cells into somatic cell types without defined intermediate precursor stages. The advent of cell reprogramming offers an opportunity to translate these advances to induced pluripotent stem cells, thereby enabling the generation of neurons and glia from individual patients. Eventually, reprogramming could provide a supply of autologous neural cells for transplantation, and could lead to the establishment of cellular model systems of neurological diseases

Blood-derived integration-free iPS cell line UKBi011-A from a diagnosed male Alzheimer's disease patient with APOE kappa 4/epsilon 4 genotype

Alzheimer's disease (AD) is most the frequent neurodegenerative disease, and the APOE epsilon 4 allele is the most prominent risk factor for late-onset AD. Here, we present an iPSC line generated from peripheral blood cells of a male AD patient employing Sendai virus vectors encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC. The characterized iPSC line expresses typical human pluripotency markers and shows differentiation into all three germ layers, complete reprogramming vector clearance, a normal SNP genotype and maintenance of the APOE epsilon 4/epsilon 4 allele

Robust Generation of Cardiomyocytes from Human iPS Cells Requires Precise Modulation of BMP and WNT Signaling

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling

Energy Metabolism Disturbances in Cell Models of PARK2 CNV Carriers with ADHD

The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification

Combining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of a-synuclein (aSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs ac-cording to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or aSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses aSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets

Regulation of stem cell therapies under attack in Europe: for whom the bell tolls

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients

Generation of a set of isogenic iPSC lines carrying all APOE genetic variants (epsilon 2/epsilon 3/epsilon 4) and knock-out for the study of APOE biology in health and disease

APOE genotype is the strongest genetic risk factor for Alzheimer's Disease (AD). The low degree of homology between mouse and human APOE is a concerning issue in preclinical models currently used to study the role of this gene in AD pathophysiology. A key objective of ADAPTED (Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was to generate in vitro models that better recapitulate human APOE biology. We describe a new set of induced pluripotent stem cells (iPSC) lines carrying common APOE variants (epsilon 2,epsilon 3, and epsilon 3/epsilon 4) and a knock-out isogenic to the parental APOE epsilon 4/epsilon 4 line (UKBi011-A)

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.close11812