Browsing by Subject "iPSC"

ASH1L is a Histone lysine methyltransferase belonging to the KMT family, which plays an important role in epigenetic gene regulation during development, and has been linked to neurodevelopmental disorders (NDDs). Mutations in ASH1L have been linked to NDDs including intellectual disability, autism spectrum disorder and Tourette’s syndrome. Induced pluripotent stem cell (iPSC) based models in combination with CRISPR/Cas9 gene editing provide powerful tools for studying the genetic causes of NDDs. The broad aim of this thesis was the creation of genetically modified iPSC lines for modelling NDDs linked to ASH1L. Patient and healthy cell lines were obtained from the Northern Finland Intellectual Disability cohort. With the long-term goal of generating a model by which to understand the impact of genetic background on reported causative mutations, CRISPR/Cas9-based genetic engineering was employed to correct the mutation in a patient cell line, and conversely, to generate a patient mutation in a healthy line. iPSC lines are known to be intrinsically variable and require thorough characterization of their genetic stability and pluripotency before use. Therefore, the secondary aim of this thesis was to subject newly reprogrammed iPSC lines to a battery of assays to first determine their suitability for downstream applications. Single-guide RNAs (sgRNAs) were designed to target a site ≤16 bp from the edit site. Single-stranded oligodeoxynucleotides (ssODNs) were used as HDR templates, incorporating the mutation of interest and 3-4 silent mutations to prevent binding by sgRNA after successful HDR. The Cas9-sgRNA complex and HDR template were introduced into the cell by nucleofection. Both mutations are frameshift mutations and are predicted to cause loss of function. Editing efficiency was evaluated with a T7E1 assay after nucleofection. Individual clones were isolated and MiSeq was used to sequence the region to a read depth of >1000reads per clone around the edit site to identify successful edits in these clones that can be used in downstream NDD modelling applications. Edit efficiencies were found to vary between sgRNAs and cell lines. In the correction attempts, guides were found to be almost entirely ineffective, producing only a single successfully edited clone among the combined 192 isolated clones. In the knock-in lines, both guides were effective at producing edited clones. The knock-in guide with the highest predicted efficiency and the shortest edit distance predictably produced the highest number of edits, but also a higher number of homozygotic knock-ins.

Schizophrenia is a debilitating psychiatric disorder associated with reduced life expectancy. The biological mechanism of schizophrenia is nebulous; however, many findings point to the central nervous system and neurons, where a reduction in dendritic spines has been indicated by previous research. The genetic findings support the involvement of synapses in the pathogenesis of schizophrenia. To study the biological properties stemming from genetics, relevant model systems and efficient methods are needed. Induced pluripotent stem cell (iPSC) technology offers a robust method for modeling the biological processes underlying schizophrenia. Somatic cells, e.g. fibroblasts, can be reprogrammed back to a pluripotent state resembling embryonic stem cells, and further differentiated into any cell type of the body, which might not be otherwise accessible. This allows establishing and characterizing neuronal cultures from patient and control cell lines, potentially revealing biological differences associated to the disease phenotype. The field of schizophrenia research has adopted iPSC technology and multiple studies have been conducted. These include assessments of synaptic density in the produced neuronal cultures, many of which reported decreased density associated with schizophrenia. In this thesis, a modified version of Nehme et al. (2018) protocol was used to differentiate iPSCs into neurons in co-cultures with human iPSC-derived astrocytes. The overarching aim was to construct an immunocytochemistry (ICC) -based assay to measure synaptic density in the produced co-cultures. First, suitable markers for characterization by ICC were tested and selected. The markers were selected to inform about neuronal identity, maturity, and synapses of the differentiated neurons. Next, the culturing conditions were optimized regarding the cell density and coating of the culturing wells. Finally, to estimate the utility of the assay, a pilot study was performed with three cell lines derived from a healthy control and a monozygotic twin pair discordant for schizophrenia. iPSCs from these cell lines were differentiated into neurons in co-cultures with astrocytes, and then characterized with ICC using selected markers and image analysis software. The synaptic density was quantified for each cell line. The performance of the assay was evaluated with analysis of variance (ANOVA) and restricted maximum likelihood model (RELM). An assay to quantify synaptic structures in mature neurons was established. The average synaptic density for all cell lines was approximately 1 synapse per 100μm of neurite. Analysis of the data produced with the assay revealed a notable batch effect and technical variation. This suggests that further optimization is needed to reduce variance from undesired sources. The pilot data suggests that the differences in synaptic density between cases and controls may be modest, further highlighting the need for minimizing noise in the assay to improve signal to noise ratio. However, indicated by power analysis, large sample sizes are needed to identify meaningful differences between cases and controls. In light of these results, more attention should be drawn to the methodology in the field of iPSC-based studies, as the principals of the assay constructed here were similar to other synaptic assays used in previous publications.

Schizophrenia, a mental disorder affecting over 1% of the world’s population, has a 41-65% chance of being acquired in monozygotic twins, and shows a complex heritable pattern. Research has shown the involvement of various neuronal and glial cell types in the disorder’s progression. Recent studies are focusing on cortical interneurons, as clinical features of schizophrenia such as working memory deficits emerge due to the abnormal activity of these cells . The advent of induced pluripotent stem cell (iPSC) technology has made it easier to study schizophrenia disease mechanisms, with studies revealing differences in morphological and physiological properties of cortical interneurons in patients with schizophrenia. In this thesis , the aim was to optimize iPSC-interneuron differentiation protocol and live-cell imaging method suitable for disease modelling. Interneurons were differentiated from iPSCs with overexpression of inducible transcription factor, Achaete-scute homolog 1 (ASCL1). The iPSCs were derived from twin pairs discordant for schizophrenia and from healthy controls. Expression of interneuron-specific markers was verified using RT-qPCR and validated at the protein level by an immunocytochemistry (ICC) assay in the control cell lines first. Additionally, to estimate the formation of neurites and differences in neurite length and branching, the differentiated interneurons from the controls were subjected to live-cell imaging by IncuCyte S3 live-cell imaging system. Imaging parameters such as cell body cluster filter was optimized to visualize the neurites. To study interneuron involvement in schizophrenia, iPSCs from one twin pair discordant for schizophrenia were successfully differentiated. Interneurons strongly expressed Gamma-aminobutyric acid (GABA) neurotransmitter related neuronal markers: glutamate decarboxylase 67 (GAD67) and GABA at protein level. The neurons were identified as somatostatin (SST) subtype GABAergic neurons by their mRNA and protein expression. While it was possible to observe differences in gene expression, there were no clear differences in the morphology of the differentiated cells as well as the localization of markers in comparison to the healthy controls. Further studies should focus on having a protracted time for differentiation where more mature interneurons can be produced by establishing co-cultures with excitatory neurons. This will help replicate the in vivo cortical machinery which in turn will aid in better understanding of disease mechanisms.