Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system

IntroductionOligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). Although OLs can be differentiated from human-induced pluripotent stem cells (hiPSCs), the in vitro modeling of axon myelination in human cells remains challenging. Brain microphysiological systems (bMPS, e.g. organoids) are complex three-dimensional (3D) cultures that offer an ideal system to study this process as OLs differentiate in a more in vivo-like environment; surrounded by neurons and astrocytes, which support the myelination of axons. MethodsHere, we take advantage of CRISPR/Cas9 technology to generate a hiPSC line in which proteolipid protein 1 (PLP1), an OLs marker, is tagged with super-fold GFP (sfGFP). While generating the PLP1-sfGFP reporter, we used reverse transfection and obtained higher Knock-In (KI) efficiency compared to forward transfection (61-72 vs. 46%). ResultsAfter validation of the KI and quality control of the PLP1-sfGFP line, selected clones were differentiated into bMPS, and the fidelity, specificity, and function of the tagged PLP protein were verified in this model. We tracked different stages of oligodendrogenesis in the verified lines based on PLP1-sfGFP(+) cells' morphology, and the presence of PLP1-sfGFP surrounding axons during bMPS' differentiation. Finally, we challenged the bMPS with cuprizone and quantified changes in both the percentage of PLP1-sfGFP expressing cells and the intensity of GFP expression. DiscussionThis work demonstrates an efficient method for generating hiPSC KI lines and the description of a new 3D model to study OL differentiation, migration, and maturation both during in vitro neurodevelopment as well as in response to environmental chemicals or disease-associated stressors.

Automated summary

In this study, a hiPSC line with tagged proteolipid protein 1 (PLP1) with super-fold GFP (sfGFP) was generated using CRISPR/Cas9 technology. The fidelity, specificity, and function of the tagged PLP protein in a 3D model were validated and confirmed. The differentiation stages of oligodendrogenesis were tracked based on the morphology of PLP1-sfGFP(+) cells and the presence of PLP1-sfGFP surrounding axons during bMPS differentiation. Finally, the bMPS was challenged with cuprizone, and changes in the percentage of PLP1-sfGFP expressing cells and the intensity of GFP expression were quantified.