Generation of Functional Oligodendrocyte Progenitor Cells Through Serial Replating of iPSC-Derived NPC Spheres

Oligodendrocytes (OLs) are essential for myelin formation in the central nervous system, and their loss or dysfunction is a hallmark of various demyelinating and neurodegenerative disorders. Although oligodendrocyte precursor cells (OPCs) represent a promising cell source for remyelination therapies, existing protocols for generating OPCs from human-induced pluripotent stem cells (iPSCs) are often limited by prolonged culture duration, low efficiency, and cellular heterogeneity. Here, we report an efficient and reproducible platform for generating OPCs from iPSC-derived neural progenitor cells (iNPCs) through stage-specific modulation of developmental signaling pathways. Directed differentiation of iNPCs recapitulated key developmental transitions, progressing through OLIG2⁺/NKX2.2⁺ progenitors to CD140a⁺/O4⁺ OPCs within a significantly shortened timeframe compared to conventional approaches. Notably, iNPC-derived spheres functioned as a progenitor-like niche, enabling sustained OPC production through serial replating. Purified OPCs could differentiate into MBP⁺ oligodendrocytes and demonstrated myelination capacity both in vitro, via nanofiber ensheathment and in vivo following transplantation into shiverer (shi/shi) mice, where they formed myelin sheaths around host axons. Despite these advances, OPC differentiation and maturation efficiencies remained suboptimal, highlighting the need for further optimization. Collectively, our findings establish a scalable and time-efficient strategy for iPSC-derived OPC generation and underscore their potential for disease modeling and cell-based remyelination therapies.