lasted 210 days and involved the addition of various growth factors and media at specific time points. Due to the prolonged duration of the Madhavan et al. 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 by Marton et al., in 2019. This protocol was able to generate organoids with compact myelin formation by day 160. In 2019, Kim et al. demonstrated that myelinated organoid generation could be accelerated even 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 and myelinated axons were observed by day 105, effectively halving the duration of the protocol developed by Madhavan et al., the year prior. A protocol of similar duration to the 2019 protocols was established by James et al. 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. James et al. 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 which was validated in the study through generating organoids recapitulating the disease pathology observed in Nfasc 155-/- patients. In this paper, they referred to their myelinated organoids as “myelinoids” thus creating the category of organoids referred to as myelinoids. Shaker et al., 2021, recognized that the lengthy differentiation protocols renders myelinoids less practical for high throughput experimentation such as high throughput drug screening. To address this, Shaker et al. 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.