A myelinoid or myelin organoid is a three dimensional in vitro cultured model derived from human pluripotent stem cells (hPSCs) that represents various brain regions, the spinal cord or the peripheral nervous system in early fetal human development. Myelinoids have the capacity to recapitulate aspects of brain developmental processes, microenvironments, cell to cell interaction, structural organization and cellular composition. The differentiating aspect dictating whether an organoid is deemed a cerebral organoid/brain organoid or myelinoid is the presence of myelination and compact myelin formation that is a defining feature of myelinoids. Due to the complex nature of the human brain, there is a need for model systems which can closely mimic complicated biological processes. Myelinoids provide a unique in vitro model through which myelin pathology, neurodegenerative diseases, developmental processes and therapeutic screening can be accomplished.
History
In vitro models have been a critical component of many biological studies. Monolayers, or 2D cultures, have been widely used in the past, however, they are limited by their lack of complexity and fail to recapitulate tissue architecture involved in biological processes occurring in vivo. Model organisms, such as Mus musculus, Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae, recapitulate biological complexity better than 2D monolayer cultures. However, these model organisms do not perfectly capture human biology. Specifically, there are stark differences in brain development between mice and humans. Major developmental differences include variability in division patterns of neural stem cells and localization and types of glial cells that occur at specific stages in development.
Leveraging pluripotent stem cell technologies, brain organoids and cerebral organoids were developed to fill the gap in model systems to study human specific brain development and pathology in vitro. The first cerebral organoid was established in 2013. Since then, various protocols have emerged for generating organoids for different brain regions such as cerebellar, hippocampal, midbrain, forebrain, and hypothalamic organoids. Cerebral organoids provide a neurological model through which diseases, development and therapeutics can be studied. However, a major constraint of cerebral organoids is that they lack robust myelin formation and are therefore not well suited to studies investigating white matter.
This limitation of cerebral organoids was addressed in 2018 when brain organoids containing a robust population of myelinating oligodendrocytes were generated. The process of generating these myelinated brain organoids lasted 210 days and involved the addition of various growth factors and media at specific time points. Due to the prolonged duration of the 2018 protocol, there were efforts to speed up and streamline the differentiation and generation of these myelinated organoids. A similar protocol which differed slightly in growth factors added and timing of media changes was described in 2019. This protocol was able to generate organoids with compact myelin formation by day 160.
Another protocol developed in 2019 demonstrated that myelinated organoid generation could be accelerated further. Using a novel protocol, myelin basic protein (MBP), a marker for oligodendrocyte differentiation and myelination in the CNS, was detectable as early as day 63 (9 weeks) and myelinated axons were observed by day 105 (15 weeks), effectively halving the duration of the protocol.
A protocol of similar duration was established in 2021, however, the resulting organoids differ slightly in their biological context. This protocol leveraged the fact that spinal cord myelination is observed prior to cortical myelination. This protocol generated organoids with robust myelination with a ventral caudal cell fate. These organoids, although not technically brain organoids, can also be used to study myelin disease pathology, validated in the study through generating organoids recapitulating the disease pathology observed in Nfasc 155-/- patients. In this protocol, they referred to their myelinated organoids as "myelinoids" thus creating the category of organoids referred to as myelinoids.
In 2021, a group of researchers aimed to address the fact that the lengthy differentiation protocols renders myelinoids less practical for high throughput experimentation such as drug screening. To do this, scientists developed a human induced pluripotent stem cell (hiPSC) line that relies on early expression of an oligodendroglial gene which enabled the accelerated generation of myelinated organoids in just 42 days. To date, this is the fastest protocol for generating mature oligodendrocytes in a brain organoid.
Culturing methods
To generate organoids, human pluripotent stem cells (hPSCs) are allowed to aggregate into embryoid bodies (EBs) in low attachment plates (in suspension), which are then cultivated in a rotating bioreactor with lineage specific factors to promote cell amplification, growth and differentiation. EBs have the capacity to differentiate into all embryonic germ layers, mesoderm, endoderm and ectoderm. In vivo, the nervous system, including myelin, is generated from the ectoderm. To recapitulate this in vitro and generate myelin organoids, the EBs are cultured in media with specific growth factors and supplements that lead to ectodermal differentiation specifically, followed by subsequent neural induction. More specifically, neural induction factors are added to induce the formation of neural progenitor cells which give rise to neurons and glial cells, including oligodendrocytes, in vivo.
A well established method used to efficiently differentiate hPSC into neural cells is by dual inhibition of SMAD signaling using dorsomorphin (also known as compound C) and SB431542. To promote further proliferation of neural precursor cells specific growth factors are added to the media such as epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Before neural and glial induction, the spheroids are generally embedded in an extracellular matrix, such as Matrigel, and transferred to a rotating bioreactor where different small molecules and growth factors are continuously supplemented to promote the differentiation of cells into specific structures and cell types.
In vivo, neuronal induction precedes oligodendrocyte formation. Therefore, in culture, neuronal induction factors are added first to induce neuro-cortical patterning of the spheroids, followed by factors that induce oligodendrocyte precursor cell (OPC) formation and differentiation into oligodendroglia. To promote formation of neurons from neural precursor cells, brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) can be added to the media. Subsequently, factors such as platelet-derived growth factor AA (PDGF-AA) and insulin-like growth factor 1 (IGF-1) are added to the media to result in an expansion of the OPC populations present within the organoid by promoting OPC proliferation and survival.
Finally, factors that induce OPC differentiation into oligodendrocytes, and ultimately myelinating oligodendrocytes, are added. This includes thyroid hormone (T3), which has been shown to induce oligodendrocyte generation from OPCs in vivo. The organoids are maintained in suspension where they grow and mature until required for analysis. The fundamentals of this workflow are generally used to obtain myelin organoids; however, various protocols that rely on it have introduced multiple modifications for different purposes. Madhavan et al. was the first to establish a reproducible protocol that allowed for generating organoids with robust OPC and oligodendrocytes populations, and therefore myelination; they are referred to as myelin organoids, or myelinoids.
Properties and components The generation of myelin organoids generally relies on neurocortical patterning factors that establish the structural and cellular framework necessary for the induction of oligodendrogenesis later on in the differentiation protocol. Therefore, the properties and components of myelin organoids in the early stages of differentiation are very similar to that observed in cerebral organoids where populations of neural progenitor cells, precursors of neurons and glial cells, start to emerge and self-organize into distinct layers that recapitulate features of the cortex during early embryogenesis.
At such early stages, myelin organoids start to form large continuous neuroepithelial that encompass a fluid filled cavity representative of a brain ventricle. The progenitor cells surrounding the putative ventricle organize into distinct layers defined by specific neural markers that become more defined as the organoid matures. The layers include a ventricular zone surrounding the cavity with cells expressing PAX6, SOX2 and Ki67, followed by the outer subventricular zone and intermediate zone with cells expressing Ki-67 and TBR2, and finally cortical plate layer with cells expressing CTIP2, MAP2 and TBR1.
Following neurocortical patterning, the oligodendrocyte lineage growth factors drive the expansion of native populations of OPCs distributed causing a substantial increase in their numbers which express SOX6, SOX10 and OLIG2, markers of glial induction and OPC specification. As the myelin organoid matures, the OPC cells differentiate into oligodendrocytes that express proteolipid protein 1 (PLP1), the predominant component of myelin, and MYRF25, an oligodendrocyte specific transcription factor. The oligodendrocytes are distributed throughout the neuronal layers, where upon maturation, their processes express MBP and CNP (an early myelination marker), begin extending to wrap and myelinate the axons surrounding them. The myelin undergoes maturation, refinement and compaction eventually leading to the formation of functional neuronal networks with compactly wrapped myelin lamellae. Further myelin maturation leads to distinct axonal subdomains with a paranodal axo-glial junction (PNJ) and node of Ranvier. The observation of paranodal and nodal assembly is protocol dependent, some observe paranodal and nodal assembly, some do not. Overall, the oligodendrocytes in myelin organoids demonstrate the ability to form compact myelin that wraps and organizes around neuronal axons recapitulating the three dimensional architecture of myelinated axonal networks in humans.
Applications Disease modelling
Myelinoids recapitulate various fundamental aspects of brain development and myelination, and therefore related disease and pathology. Given that, they can be used to model various diseases and understand disease mechanisms associated with myelin defects including neurodegenerative diseases, CNS injury, PMD, and NFASC.
Pelizaeus-Merzbacher disease (PMD)
PMD is a rare monogenic disease caused by various mutations of the X-linked proteolipid protein 1 gene (PLP1). PLP1 is a critical protein for myelin formation. PMD is classified as a leukodystrophy, meaning that it is a disease affecting the white matter of the brain. Madhavan et al. tested how well their myelinoid system could recapitulate the established cellular pathology of PMD. Organoids were derived from three patients with varying disease severity where the subject with a deletion, a duplication, and a point mutation had mild, moderate and severe phenotypes respectively. Their results demonstrated that the myelinating oligocortical spheroids generated recapitulated the degrees of cellular pathology associated with the genetic variants, therefore can serve as models for understanding the relationships between PMD genotypes and phenotypes, which have not been fully characterized yet, therefore can serve as models for understanding the relationships between PMD genotypes and phenotypes, which have not been fully characterized yet.