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
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