Author: Bridget Milky
Milky, Bridget, 2023 Developing new monolayer culture conditions to increase adhesion, survival and maturation of hPSC-derived neuronal models, Flinders University, College of Medicine and Public Health
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Recent advances in stem cell research and cellular reprogramming have led to novel approaches for advancement in medical research. Induced pluripotent stem cell (iPSC) technology has transformed the field of neuroscience in the last two decades by generating otherwise inaccessible patient-derived live brain cells while meeting ethical standards. Modelling the human brain in vitro is an invaluable tool for thorough exploration of disease mechanisms and high-throughput screening for patient-specific treatment options. Neuronal cell culture models are rapidly advancing in complexity and specificity, however the core foundations of such models are in many cases not optimised before a novel method is developed. This is particularly evident in two-dimensional monolayer cultures of human PSC-derived neurons. The benefits of high-throughput capabilities, experimental diversity and high reproducibility falter over extended culture periods, which are necessary for human neurons to achieve physiological functional activity.
This thesis addresses the technical limitations surrounding monolayer models of human neurons and astrocytes, specifically supporting long-term cultures (months). Adequate cell adhesion is the foremost requirement for stability over time, where current human neuronal models detach within days during experiments that involve glass culture surfaces. Chapter 1 explores the development of a novel surface coating that supports adhesion for as long as 6 months on glass and other standard surfaces (with the appropriate extracellular matrix counterpart). Recapitulating the brain microenvironment in cell culture has made progress in the last decade with the development of physiological neuromedium BrainPhys (Bardy et al., 2015), however additional supportive factors required for complete culture medium are currently neither standardised nor optimised. Chapters 2-3 explore neuron-supportive factors and antioxidants (respectively) for their ability to accelerate functional development and sustain neuronal viability long-term, respectively. Lastly, the cellular profile of the brain comprises both neurons and glia whereby glia (particularly astrocytes) have critical roles in neuronal development and health. To both support neurons in culture and accurately recapitulate the brain, Chapter 4 explores the role of astrocytes in neuronal cultures by contributing to the development of an in-house astrocyte PSC differentiation protocol that allows the study of astrocytes in health and disease. The outcomes of this candidature have the capacity to benefit all laboratories using human PSC-derived neurons and astrocytes therefore can be applied to inform models of many neurological and psychiatric disorders.
Keywords: pluripotent stem cells, iPSC, neuronal models, human neurons, brain, long-term culture, tissue cultureware, culture media, adhesion, neuronal maturation
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Cedric Bardy