Human iPSC genetically encoded fluorescent biosensor models for toxicity imaging
Creators: name: Stellon, David
Publisher: name: University of Tasmania
Year: 2024

description: The brain is the most complex organ in the human body, consisting primarily of various types of neurons and non-neuronal cells, known as neuroglia. In disease, these distinct cell populations may pathologically interplay between each other, and genetic perturbations influence cell/non-cell autonomous pathomechanisms and/or be driven by environmental agents. This presents a significant challenge for pre-clinical biomedical research modelling which must closely reflect the disease as it occurs in the patient. Neurodegenerative diseases are characterised by the progressive degeneration and/or death of nerve cells within the central or peripheral nervous system, that leads to variable clinical manifestations and can affect individuals at any age. Gathering data on pathological events leading to neurodegeneration are of critical importance to understanding aetiology, disease progression, interventional therapeutics and environmental regulations preventing neurodegeneration. Nevertheless, visualising these pathological events at the single cell resolution is currently a difficult challenge facing researchers. Neurodegenerative research is hindered by several obstacles: i) the ability to qualitatively visualise and quantitatively measure various processes; ii) obtain high throughput data for genetic/drug/toxicity screens and iii) pathway mapping - ascertaining timestamps for pathological events that may occur in parallel or as a result of the other. Genetically encoded fluorescent biosensors (GEFBs) offer a solution to all three of these challenges. Whilst their synthetic designs can be simple or complex, generally they consist of a reporter, i.e., fluorescent protein, and sensor, e.g., enzyme or cleavage site, that is sensitive to sensing specific cellular biomolecules or processes. Subsequently, the reporter's change in fluorescence intensity upon biosensing enables them to be valuable tools for real-time imaging, enabling high-throughput data to be qualitatively and quantitatively reviewed between control and test groups. In addition, GEFBs may contain localisation signals that target them to different subcellular compartments, allowing them to report activity with high spatiotemporal resolution in pathologically significant and discrete locations. The intelligent and synthetic design of GEFBs allows researchers to develop, expand upon and utilise novel sensors, for their own areas of interest and experimental pipelines. The advent of GEFB technology supersedes more traditional methods of cell lysis/fixation, animal sacrifice and biopsies that are low-throughput and time consuming. Furthermore, stable expression of GEFBs into induced pluripotent stem cells (iPSCs) allows researchers to generate genotypically and phenotypically relevant human disease models for preclinical testing. Nonetheless, utilising GEFBs for neurodegenerative disease research faces several challenges. This includes identifying disease relevant biosensors, stable cell expression for long-term imaging experiments, incorporating GEFBs into different experimental designs and multiplexing more than one GEFB to avoid bleed-through whilst preserving cell line identity. In this way, fluorescent imaging time-lapse experiments would enable researchers to observe and analyse multiple analytes or processes that would characterise a particular toxin-induced phenotype, disease model genotype and drug mode of action. In Chapter 1 of this thesis, a published review of GEFB applications in neurodegenerative disease modelling using iPSCs is presented. This includes the advantages and disadvantages of GEFBs against other current methods of measuring cellular activities; a list of relevant GEFBs for neurodegenerative research; discussion of GEFB applications in toxicity, genetic and drug screening as well as overcoming multiplexing/pooling issues currently hindering GEFB experimental design. Following this, Chapter 2 details a published method for generating stably expressed GEFB cell lines in iPSCs, presenting the protocol for CRISPR/Cas-mediated knock-in using a crRNA (GUCACCAAUCCUGUCCCUAG) designed to target the adeno-associated virus integration site 1 safe harbour site of the human genome and subsequent quality control. This includes Sanger sequencing of eight loci that are potential crRNA off-target sites for indel mutations. Chapter 3 applies the aforementioned methodology to generate multiple GEFB-iPSC lines that are relevant to measuring pathological alterations in disease. Of the seven GEFB-iPSC lines sequenced, no indels were detected. Furthermore, pluripotency testing using cell surface (SSEA4, TRA-1-60, TRA-1-81) and transcription factor markers (NANOG, OCT4, SOX2), in addition to embryoid body assays, demonstrated sufficient differentiation capacity of GEFB-iPSCs. Functional analysis of GEFB reporting using various small molecules demonstrated the relevant cellular biomolecule/process activation or inhibition in cells. In Chapter 4, multiple receptor antagonists were used to provide insight into the limited data on functional phenotyping of in vitro iPSC-derived neuronal models using multielectrode array, with results showing appropriate electrophysiological responses to dAP5 (25 µM), CNQX (25 µM), NBQX (10 µM), bicuculline (10 µM) and picrotoxin (100 µM). This included the absence of synchronised burst fire (SBF) when exposed to dAP5, CNQX and NBQX, in comparison to increased SBF activity with bicuculline and picrotoxin. These findings would help to resolve a potential limitation of using GEFB-iPSC-derived neurons, by forming a basis for comparing the genetically engineered cells to the parental cell line; expression of exogenous GEFB protein may interfere with cell homeostasis and subsequently neuronal activity. Finally, Chapter 5 explores how various neurotoxins (BMAA, it’s isomers and chlorpyrifos) could be tested on GEFB iPSC-derived brain cells, to interrogate pathomechanisms of human environmental exposure to cyanotoxins and pesticides. To conclude, this thesis has contributed knowledge for the growing need to utilise novel tools for advancing neurodegenerative research, highlighting GEFB and iPSC technology for deciphering complex pathomechanisms and as valuable toxicity, genetic and drug screens. The benefits and challenges of GEFBs discussed help researchers discern traditional and current methods from GEFBs, allowing them to better understand and maximize utility in their own experimental designs. In addition, the CRISPR/Cas knock-in protocol established provides researchers a fast-track guide for developing their own GEFB-iPSC lines for custom high-throughput experimental disease models, that is not limited to neurodegenerative research, due to the differentiation potential of iPSCs, allowing broad application and fostering of this technology to other areas of investigation.