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Patient-specific stem cell-derived models for Alzheimer’s disease and related neurodegenerative disorders

Final Report Summary - STEMMAD (Patient-specific stem cell-derived models for Alzheimer’s disease and related neurodegenerative disorders)

Patient-specific stem cell-derived models for Alzheimer’s disease and related neurodegenerative disorders (STEMMAD) “STEMMAD”

The focus of STEMMAD is an area of immense societal importance: Neurodegenerative diseases and their potential cure. Future treatment of incurable neurodegenerative disorders, including Alzheimer’s disease (AD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA), and Huntington’s disease (HD) has to be tailored to individual patients or cohorts of patients to obtain an optimal effect. It has been our overall aim to derive patient-specific in vitro neural cell models that in the future will allow for such customized treatment. The basic principle in STEMMAD was the collection of skin biopsies from genetically and clinically well-characterized patients and fibroblasts were isolated and reprogrammed into induced pluripotent stem cells (iPSCs), which, in turn, were differentiated into neural progenitor cells (NPCs) and specific subpopulations of neurons. We expected that these neurons would express molecular characteristics of the patient’s disease phenotype and thereby be representative as patient-specific neural cell models or patient-specific “microbrains”. The models have been characterized for functional disease parameters (disease phenotype) and used for studies of molecular pathogenesis, and indeed, we have succeeded in demonstrating the faithfulness of the patient-specific in vitro models. The project has been structured into four coherent work packages (WPs) which will be shortly presented in the following with respect to objectives and results.

WP1 entitled “Patients and establishment of iPSC” had the objectives of (1) selection and tissue sampling from patients and (2) reprogramming of patient and control fibroblasts into iPSCs. Skin biopsies were collected from well-characterized control subjects and patients suffering from AD, FTD and SCA. Instead of focusing on HD, we put emphasis on including patients with Trisomy 21 representing a special case of AD. We successfully generated iPSC lines from all control subjects and patients using an episomal plasmid approach. All of these published 19 iPSC lines are karyotypically normal, express expected pluripotency markers at mRNA and protein levels and are devoid of the episomal reprogramming plasmids, which makes these foot-print-free iPSCs the desired kind for future cell modelling and replacement strategies. They have been tested for their in vitro differentiation potential and are all capable of differentiating into endoderm, mesoderm and ectoderm using the embryonic body method. In addition, several CRISPR/Cas9 gene edited isogenic control cell lines have been generated for the disease-causing mutations. The precise repair of a single nucleotide, which is disease-causing, followed by neural differentiation of patient and isogenic control iPSC has enabled us to identify the phenotypes truly caused by the mutations due to their absence in the isogenic controls. In conclusion, we have generated a unique battery of iPSC lines from several neurodegenerative diseases as well as their isogenic controls using CRISPR/Cas9 gene editing for the use in the subsequent WPs and continued research after the project.

WP2 entitled “Differentiation of iPSC into NPCs and neurons, verification of disease phenotype and studies of potential disease modulating treatments” included the objectives of (1) establishment of NPCs and neurons from control and patient-derived iPSC and (2) verification of disease phenotype. A robust in vitro neural differentiation protocol was developed, which allowed us to generate cortical type neurons from human iPSCs, independent from the disease. Marker gene expression studies at mRNA and protein levels were investigated and cortical neuronal differentiation was clearly verified. Hence, we successfully established in vitro cellular disease models for studies of the disease-related phenotypes. Using the models, we compared neurons derived from iPSC lines of early-onset familial AD (fAD) patients, all caused by mutations in PSEN1 gene, and non-demented control individuals and isogenic control cell lines.

Relative to the controls, neurons derived from fAD patients with mutations in PSEN1 exhibited aberrant levels of extracellular amyloid β (Aβ)40 and Aβ42 as well as, in some cases, increased Aβ42/Aβ40 ratios. Likewise, increased phosphorylation of TAU protein was noted as well as elevated sensitivity to oxidative stress and amyloid oligomer solutions. Moreover, abnormal mitochondria respiration and reduced ATP production, defects in glucose metabolism, fragmented Golgi complexes and reduced numbers of synaptic vesicles were noted. Hence, our model system for fAD was capable of recapitulating known disease phenotypes all well as identifying novel phenotypic features of the condition. FTD is another leading cause of dementia and within this project we have extensively studied the cellular pathology of patients with CHMP2B mutations and their CRISPR/Cas9 gene edited isogenic controls. We were able to recapitulate the patient post-mortem brain phenotype of enlarged endo-lysosomal structures in our in vitro cell models. Moreover, we revealed that mitochondria in CHMP2B mutant neurons were mis-localized to the perinuclear domain and a substantial amount lacks proper cristae formation, which resulted in impaired mitochondrial respiration and ATP generation. Other disease phenotypes were elevated radical oxygen species levels, reduced neurite outgrowth and increased levels of intracellular iron. All of these disease phenotypes were rescued in the isogenic controls underlining that the CHMP2B mutation is indeed causative for the observed pathological features. Yet another disorder examined is SCA including both subtypes SCA2 and SCA3. Neurons derived from SCA3 patient iPSCs have been carefully examined, but relative to controls they did not show pathologies related to the protein Ataxin as expected. The in vitro cell models for SCA2 are still under development, but CRISPR/Cas9-edited isogenic control cell lines have been established.

WP3 entitled “Transdifferentiation of fibroblasts into NPCs and neurons” had the objective (1) reprogramming of fibroblasts directly into neurons. It has recently been shown that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Based on the published methods, we tested two different approaches: First the direct conversion of fibroblast cells into neurons was tested, and later a fibroblast to NPC protocol was investigated. In short, none of the methodologies were found to be efficient enough to be implemented in the derivation of our in vitro cell models. The yields of neurons were significantly lower than with the iPSC-based methodology and the outcomes were inconsistent.

WP4 entitled “Unravelling of molecular pathological pathways from patient-specific neural cell models” had the objective (1) disease pathway analysis. We have investigated the disease phenotypes and disease-relevant pathways in both fAD and FTD. For FTD we have performed RNA sequencing and proteomic studies that correlate with the cellular pathologies pointing to novel pathways relevant for development of potential therapeutic interventions including aberrant iron homeostasis. For fAD, our proteomics and RNA sequencing data are currently being integrated with the abnormal neuronal phenotypes and will give us detailed insights into altered disease-relevant pathways.

The STEMMAD project has continuously been disseminated to the public domain through a website (http://www.stemmad.eu).

In conclusion, STEMMAD has resulted in significant scientific progress with respect to establishment of unique patient-specific iPSC-based in vitro cell models as well as the use of these models for identification of novel cellular and molecular disease phenotypes. Moreover, a wealth of highly competent academic fellows has resulted from the executed recruitments and secondments.