Programming Human Induced Pluripotent Stem Cells to Generate Functional Oligodendrocyte Progenitors Using the Human Tfome

Scalable production of engraftable oligodendrocyte progenitor cells (OPCs) with high purity could revolutionize treatment for patients with myelin-degenerative diseases such as multiple sclerosis. To date, only a few protocols have shown OPC generation from human induced pluripotent stem cells (hiPSCs), and require 48-180 days with low efficiency. We have developed a rapid engineering method using transcription factor (TF)-mediated direct reprogramming of hiPSCs which overcomes these hurdles and significantly increases the potential clinical use. We demonstrated that our engineering method is capable of generating O4-positive OPCs (ideal stage for transplantation) with up to 70% efficiency in 4 days in stem cell media, 10x faster than most published protocols. We used droplet-based single cell RNA-sequencing to evaluate the homogeneity of our TF-based cell programming and we showed that the pluripotency genes are down-regulated in the majority of the cell population after four days of induction while oligodendrocyte-specific markers such as PLP and CNP are up-regulated as a relatively pure population. The ability of OPCs to mature into myelinating oligodendrocytes was confirmed through protein expression, microscopy and flow cytometery, demonstrating that after 8 days the OPCs can become MOG-positive and GalC-positive. To validate the ability of the generated OPCs to form myelin, we created co-cultures of OPCs with Neurogenin-induced neurons derived from hiPSCs and performed transmission electron microscopy (TEM). We showed that our method produces cells that exhibit robust myelin formation around developing neurons. Our method for hiPSC rapid cell type engineering appears to overcome many of the hurdles of current hiPSC differentiation processes and improves their potential clinical application in myelin-degenerative diseases.