Stepwise Chemically Defined Differentiation of Human iPSCs into Oligodendrocytes with Harvestable Developmental Intermediates

This article describes a chemically defined stepwise protocol to differentiate human induced pluripotent stem cells (hiPSCs) into mature oligodendrocytes via harvestable developmental intermediates. The workflow recapitulates in vitro neural lineage specification through embryoid body formation, neuroectoderm induction, the generation of glial-restricted progenitors, and the transition through oligodendrocyte progenitor cells (OPCs) to terminally differentiated, myelinating oligodendrocytes. Lineage progression is driven exclusively by small molecules and growth factors selected based on developmental and pharmacological evidence, without the use of exogenous transcription factor overexpression. The protocol enables isolation and expansion of physiologically relevant intermediate populations, making it suitable for mechanistic studies, disease modeling, and drug screening. Differentiation efficiency and purity can be validated by immunocytochemistry, flow cytometry, and co-culture-based myelination assays. The method reliably yields high-purity oligodendroglial cultures within approximately 90 days and is readily adaptable across hiPSC lines. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Thawing and plating of human iPSCs on Geltrex-coated plates Support Protocol 1: Passaging of human iPSCs using ReLeSR Basic Protocol 2: Dissociation of iPSC colonies into clusters for suspension embryoid body formation and early basal differentiation Basic Protocol 3: Embryoid body differentiation for enhancing ectoderm by suppressing mesoderm and endoderm using specific growth factor cocktail Basic Protocol 4: Selection of embryoid bodies and plating for neuroectoderm differentiation Basic Protocol 5: Differentiation of neuroectodermal cells to glial-restricted progenitors Basic Protocol 6: Magnetic-activated cell sorting of A2B5⁺ cells for differentiation to oligodendrocyte progenitor cells Basic Protocol 7: Optimization of culture protocol for differentiation of oligodendrocyte progenitor cells to pre-oligodendrocytes Basic Protocol 8: Optimization of culture protocol for differentiation of pre-oligodendrocytes to immature oligodendrocytes Basic Protocol 9: Optimization of culture protocol for differentiation of immature oligodendrocytes to mature oligodendrocytes Basic Protocol 10: Co-culture of iPSC-derived neurons with iPSC-derived oligodendrocytes for myelination assay Support Protocol 2: Differentiation of iPSCs to neurons Support Protocol 3: Immunocytochemistry analysis Support Protocol 4: Flow cytometry analysis.