Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

Abstract

Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10 days in vitro. Finally, we demonstrate that zebrafish OPCs differentiate into Myelin Basic Protein (MBP)-expressing OLs when co-cultured with human motor neurons differentiated from induced pluripotent stem cells (iPSCs). This shows that the basic mechanisms of oligodendrocyte differentiation are conserved across species and that understanding the regulation of zebrafish OPCs can contribute to the development of new treatments to human diseases.

Electrophysiological Properties of Adult Zebrafish Oligodendrocyte Progenitor Cells

“…Isolation and FACS of OPCs from the adult zebrafish spinal cord was adapted from Kroehne et al (2017). Briefly, transgenic Tg( olig2 :eGFP) adult zebrafish of 6–12 months old were terminally anesthetized in 0.1% w/vol Tricaine (Sigma Aldrich) in E3 medium for zebrafish with 10 −5 % v/v methylene blue.…”

“…OPCs are subsequently purified by fluorescence activated cell sorting (FACS) and set in culture immediately after sorting. OPCs remain viable and undifferentiated for up to 10 days in vitro (DIV) (Kroehne et al, 2017). At 3 DIV, whole-cell patch-clamp is used to assess their electrical properties.…”

“…In order to support the results of the patch-clamp, a recently published RNAseq dataset of OPCs and oligodendrocytes from the adult zebrafish spinal cord from our laboratory (Kroehne et al, 2017) was investigated for genes related to ion channels. Briefly, this dataset was generated by using SMARTer Ultra Low Input RNA for Illumina Sequencing Kit—HV from Clontech, followed by a library prep using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB).…”

“…Thus, we examined the functional expression of cell-type specific ion channels in OPCs from the adult zebrafish spinal cord by whole-cell patch-clamp. To access adult zebrafish OPCs, we first developed an in vitro system for the culture of primary OPCs from the adult zebrafish spinal cord (Kroehne et al, 2017). This setup enables the robust and highly reproducible isolation of OPCs by fluorescence activated cell sorting (FACS).…”

Single cell sequencing of radial glia progeny reveals the diversity of newborn neurons in the adult zebrafish brain

“…3C) supports their generation from RG in the adult zebrafish brain. In addition, flow cytometry of double transgenic fish that combine the RG-specific her4.1:mCherry reporter with the OPC-specific reporter olig2:gfp (Shin et al, 2003;Kroehne et al, 2017;Tsata et al, 2019) [Tg(her4.1:mcherry; olig2:gfp)] revealed that 28.6±3.9% of all olig2:GFP cells were mCherry low /GFP high double positive ( Fig. S4B,C).…”