Cytoplasmic and mitochondrial metabolism of phenotypically distinct iPSC-generated astrocytes throughout differentiation and reactivity using advanced quantitative microscopy.

Abstract

Förster Resonance Energy Transfer (FRET) is a widely used technique for monitoring cellular changes via engineered biosensors. However, its application in characterizing phenotypically and functionally distinct cellular subtypes has been somewhat limited. Astrocytes, intricate glial cells with diverse morphology and functions, display regional specificity in the brain and are critical in neurodegenerative diseases. Yet, generating distinct subtypes of astrocytes in vitro remains a challenge for deepening our understanding of neuro-glial interactions.

This PhD research focuses on elucidating the molecular metabolic processes underlying astrocyte maturation and reactivity. Through the use of cytokines BMP4 and CNTF in independent differentiation protocols, novel in vitro iPSC-derived metabolically distinct astrocyte subtypes were generated. A significant novelty of this thesis lies in the application of Fluorescence Lifetime Imaging (FLIM)-FRET as a quantitative method to assess metabolic differences between these astrocyte subtypes, offering a new lens into their metabolic profiles as they mature and transition into reactive states. A major focus of the thesis was the development of a novel two-photon Bessel dual-view selective plane illumination microscopy light sheet system for further 3D investigation of astrocyte metabolism. Despite significant developmental progress, the implementation of this advanced system faced challenges due to the COVID-19 pandemic, limiting experimental continuity.

The thesis reveals that distinct metabolic pathways emerge after day 7 (the point of comparability), despite observable phenotypic differences at this juncture. By day 14, CNTF astrocytes demonstrate lower lactate and ATP levels, whereas BMP4 astrocytes exhibit higher lactate, particularly in proximity to the nucleus, with ATP levels comparable to those of day 7; perhaps elucidating to distinct functional roles. Lactate-to-pyruvate ratios remained consistent throughout maturation and reactivity across the subtypes.

These findings, along with the two-photon Bessel light sheet system's future potential, offer promising avenues for 3D metabolic investigations. This work provides metabolically and phenotypically diverse astrocyte models that will enhance neural modelling in both developmental and disease contexts, overcoming some of the limitations associated with in vivo studies.

Date of Award	1 Dec 2024
Original language	English
Awarding Institution	King's College London
Supervisor	Simon Ameer-Beg (Supervisor) & Andrea Serio (Supervisor)