2 research outputs found
Development of a human pluripotent stem cell-derived in vitro model of myelination
Myelination is essential for central nervous system (CNS) formation, health, and
function. Its development is an adaptive and regulated process that, when perturbed,
leads to disease. However, our understanding of myelin formation in health and
disease is limited by the paucity of human models of myelination. I sought to develop
a human stem cell-derived in vitro model of myelination that would enable studies of
myelin development in the context of pharmacological, physiological, or genetic
perturbations.
Three-dimensional human induced pluripotent stem cell-derived, spinal cord
organoids were generated containing NF-H+ neurons, GFAP+ astrocytes, PDGFRα+
oligodendrocyte progenitor cells, and MBP+ oligodendrocytes. With prolonged culture,
myelin formation was evident, demonstrated by (i) thick MBP+ tubular structures co-localising with NF-H+ axons; (ii) organisation of myelinated axon domains, as
determined by appropriate clustering of paranodal and nodal proteins; and (iii)
compact myelin lamellae visualised by transmission electron microscopy. iPSC
‘myelinoids’ demonstrate temporal development of myelination, with both myelin
sheath length and compaction increasing over time. The morphology of individual
myelinating oligodendrocytes could also be analysed, and exposure to a
pharmacological cytoskeletal modulator potentiated myelin sheath number per cell,
as expected. Furthermore, a novel automated pipeline to quantify myelin volume
across entire myelinoids was developed, which showed that myelin volume—specific
to myelinated axons—increased over time. Automated analysis of
oligodendrogenesis and myelin formation was demonstrated as an alternative method
for investigating the effects of small molecules or trophic factor signalling on
oligodendrogenesis and myelin development.
Neuronal activity can regulate myelination by oligodendrocytes in model organisms.
However, whether myelination by human oligodendrocytes is responsive to neuronal
activity has not previously been investigated. I found that blocking synaptic vesicle
release via tetanus toxin (TeNT) impaired myelin sheath generation by individual
oligodendrocytes and led to a reduction in total myelin volume across myelinoids.These results demonstrate that human myelinating oligodendrocytes respond to
changes in neuronal activity.
An advantage of iPSC-based models is the generation of patient-derived cultures that
enable aspects of human disease to be modelled in vitro. In order to demonstrate that
myelinoids could be used to model disorders of myelin, myelinoids were generated
from a patient with a homozygous recessive mutation in the gene for Neurofascin
(NFASC). This nonsense mutation is predicted to specifically affect the glial-expressed form of Neurofascin, Nfasc155 (a component of the paranodal axoglial
junction assembly expressed by glia), the deficiency of which results in a
neurodevelopmental disorder characterised by hypotonia, amimia, and areflexia.
Patient-derived myelinoids demonstrated impaired formation of paranodal axoglial
junctions, while nodal Neurofascin remained intact, recapitulating the major pathology
of this disease.
Finally, oligodendrocyte pathology is prevalent in amyotrophic lateral sclerosis (ALS),
a rapidly progressing neurodegenerative disorder of both upper and lower motor
neurons. However, the mechanism by which oligodendrocytes contribute to disease
is not yet understood. TDP-43 is a critical DNA/RNA binding protein involved in RNA
metabolism whose cytoplasmic misaccumulation and aggregation in neurons and glia
is a pathological hallmark of ALS. Disease causing mutations in the encoding gene,
TARDBP, account for 5-10% of familial forms of ALS. To determine whether mutant
TDP-43 affects myelin development, myelinoids were generated from both patient-derived and CRISPR/Cas9 gene-corrected iPSCs. I found that myelinoids derived
from mutant and gene-corrected iPSCs showed no difference in oligodendrogenesis
or myelin formation.
Collectively, this work shows that iPSC myelinoids provide a robust platform for
investigating human myelin development in both health and disease
Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration
Although microglial activation is widely found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the underlying mechanism(s) are poorly understood. Here, using human-induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harboring the most common ALS/FTD mutation (C9orf72, mC9-MG), gene-corrected isogenic controls (isoC9-MG), and C9orf72 knockout hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated immune response upon stimulation with lipopolysaccharide. Analysis of the C9ORF72 interactome revealed that C9ORF72 interacts with regulators of autophagy and functional studies showed impaired initiation of autophagy in mC9-MG and C9KO-MG. Coculture studies with motor neurons (MNs) demonstrated that the autophagy deficit in mC9-MG drives increased vulnerability of mC9-MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated both cell-autonomous functional deficits in hiPSC-MG and MN death in MG-MN coculture. Together, these findings reveal an important role for C9ORF72 in regulating immune homeostasis and identify dysregulation in myeloid cells as a contributor to neurodegeneration in ALS/FTD.</p