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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.
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 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.
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 hormones (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.
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.
Single-cell RNA sequencing (scRNA seq) analysis of myelinoids generated in 2018, confirmed that there were distinct populations of oligodendrocytes throughout multiple stages of development in oligocortical spheroids which closely matched the single-cell transcriptome data obtained from human fetal cortex. Due to their close transcriptomic resemblance to human fetal brain data, the regulatory landscape of cells within cerebral organoids can inform on the underlying regulatory mechanisms governing human brain development. (2025). 9780128215319 ISBN 9780128215319
In 2020, researchers described an approach to obtain meaningful scRNA seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data from brain organoids. The protocol can likely translate to myelin organoids due to the similar biology between cerebral organoids and myelinoids.
Using the Orgo-Seq framework, three datasets (bRNA-seq from donor derived organoids, scRNA-seq data from cerebral organoids and fetal brains in precious studies, and bRNA-seq from the BrainSpan Project of human post-mortem brains) were used to study copy number variants in autism spectrum disorder. They leveraged several datasets to identify the types of cells present and cell specific driver genes in patient derived organoids.
Brain organoids serve as a human-derived model through which genetic variation and its impact on cell specific processes and association with neurodevelopmental and neurodegenerative disorders can be studied. Specifically, myelinoids provide a system to study the cell type specific effects in oligodendrocytes that are disrupted by genetic variants. Overall, Orgo-Seq provides a quantitative and validated framework for investigating driver genes and their role in neurological and neurological disorders. In the future, Lim et al., aim to develop a precision medicine framework to identify gene networks and effects of genetic variants in an organoid system, which would include myelinoids, that recapitulates the patient's exact genetic background.
With the absence of human brain tissue, myelinoids offer unprecedented opportunities for studying oligogenesis and myelination. While animal models are valuable for studying human diseases, they do not fully recapitulate human brain development and show many discrepancies affecting their translatability to human physiology. Considering resemblance of myelin organoids to the human brain, they have been proposed as models bridging between animal models and human physiology.
Other hPSC derived oligodendrocytes systems have been established, such as the two dimensional (2D) monolayer oligodendrocytes models. However, when compared to 2D systems, myelin organoids more faithfully recapitulate the structure and functionality of the developing human brain containing a more physiologically relevant microenvironment including their 3D cytoarchitecture, neural circuits, cell interactions and an overall more physiologically relevant microenvironment.
While cerebral organoids form the brain cytoarchitecture and composition, they generally lack oligodendrocytes, the cells responsible for myelination in the central nervous system. The myelinoid protocol pioneered in 2018, and subsequently modified by others, offer a reproducible method for generating organoids with robust OPC and oligodendrocytes populations that track the endogenous neurons forming functional neuronal networks ensheathed with myelin.
Finally, the ability to generate myelinoids from patient derived hPSCs (induced-PSCs) offer major advantages and opportunities to explore patient-specific pathogenesis over the developmental and maturation stages of oligodendrocytes. This allows for the development of personalized therapeutic approaches.
As is the case with every model system, myelinoids have their limitations. Due to the methods involved with generating the organoids, there can be a large degree of experimental variability. Additionally, due to the long duration over which myelination occurs, optimizing the dosage of molecules and treatments involved in myelin development can be difficult. The advantage of drug screening in this model comes with its own limitations. It can be difficult to scale myelinoid experiment to an appropriate scale for high throughput screening due to the long duration of protocols and limited efficiency.
Myelinoids capture a large number of cell types found in vivo, however, they fail to capture all cell types. Microglia are absent in some myelinoids as was observed in the 2021 protocol. Myelinoids also do not capture any behavioral abnormalities.
Finally, a challenge with all organoid cultures is that they rely on diffusion for nutrients to reach cells. Therefore, many organoids will develop a necrotic center due to a lack of nutrients making their way to the innermost cells. Recently, developing vascularized organoids has been of interest and may potentially alleviate this issue. However, myelinoids as described in current protocols are not vascularized.
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