Abstract
Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.
Highlights
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells, have the potential to differentiate into several types of functional cells
After culturing for 1 day in medium without supplements, the medium was replaced with medium containing retinoic acid (RA) to help determine positions along the embryonic anterior/posterior axis and Sonic Hedgehog (SHH), which served as a ventralizing factor, because oligodendrocyte progenitor cells (OPCs) arise as NE cells in the ventral ventricular zone [21]
Results from the present study showed the ability of monkey ESCs to differentiate into functional oligodendrocytes
Summary
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to differentiate into several types of functional cells. It is too complicated to consistently maintain the cells in an undifferentiated state and to differentiate the cells into desired functional cells To address these problems, previous studies have developed alternative methods, such as feeder-free and dissociated monolayer culture systems [1, 2]. Previous studies have developed alternative methods, such as feeder-free and dissociated monolayer culture systems [1, 2] In these reports, the PSCs were maintained in undifferentiated states, expressed many pluripotency markers, and had the differentiation capacity by teratoma formation. We previously reported a dissociated monolayer and feeder-free culture system for primate PSCs using cynomolgus monkey ESCs, CMK6SFF, and CMK970 [3] Using this culture system, ESCs were passaged many times and propagated at a high proliferation rate, while retaining the typical ESC properties. These cells were able to differentiate into cortical neurons
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