Investigating myelination in human bioengineered neuronal organoids (BENOs)

by Marie-Kristin Schreiber
Doctoral thesis
Date of Examination:2024-03-13
Date of issue:2024-05-21
Advisor:Dr. Maria-Patapia Zafeiriou
Referee:Dr. Maria-Patapia Zafeiriou
Referee:Prof. Dr. André Fischer
Persistent Address: http://dx.doi.org/10.53846/goediss-10500

 

 

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Abstract

English

Myelination is a critical process for the optimal functioning of both the central and peripheral nervous systems. It involves wrapping nerve fibers with a myelin sheath to ensure the swift transmission of electrical signals, thereby facilitating rapid neuron-neuron communication. While in vivo models have significantly advanced our understanding of myelination, there remains a gap in our ability to observe human oligodendrocyte lineage cells’ responses to various stimuli or genetic alterations that lead to dysmyelination or demyelination. Demyelination is the damaging loss of myelin in nerves, while dysmyelination is the faulty myelin formation. This study aimed to investigate myelination using a human brain organoid model, bioengineered neuronal organoids (BENOs), and to model Pelizaeus-Merzbacher disease (PMD), a disorder characterized by dysmyelination. We employed genome engineering and human induced pluripotent stem cell (iPSC) technologies in combination with machine learning-based analysis to trace oligodendrocytes in living tissues. Through temporal bulk RNA sequencing analysis of BENOs between days 60 and 150, we delineated gene expression patterns, shedding light on neurogenesis and gliogenesis. Further gene expression analysis revealed the cell-line-independent presence of oligodendrocytes start ing from day 90. To better understand and model diseases like PMD, we introduced two novel reporter systems, bioluminescent (lumi-PLP1) and fluorescent (f -PLP1), to monitor PLP1 expression and myelination in real-time. These systems effectively identified oligodendrocytes and their myelination activity. Furthermore, we developed a new 3D Oligodendrocyte Morphometrics (3DOM) analysis tool designed to quantify morphological alterations in oligo dendrocytes resulting from mutations or therapeutic interventions. 3DOM measures soma size, the extent of cellular processes, and signs of apoptosis. Leveraging our previously published BENO model, we refined myelination protocols, incorporated detailed 3D analysis tools and reporter systems for evaluating oligodendrocyte characteristics, and effectively modeled the dysmyelinating disorder PMD. In conclusion, this study developed a comprehensive human brain organoid model for examining myelination processes and the potential for dysmyelination and remyelination studies. The outcomes of this study highlight the critical need for ongoing research in this domain and showcase the potential of BENOs as a versatile platform for advancing future therapies.
Keywords: Neuronal Organoids; Myelin; Pelizaeus-Merzbacher Disease; Disease Modeling; Induced Pluripotent Stem Cells; Fluroescent Reporter; Bioluminescent Reporter; Proteolipid Protein 1 (PLP1)