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

Abstract

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.

A novel approach to increase glial cell populations in brain microphysiological systems

“…An advantage of GEM over commercial media is that the key factors driving and enhancing the glial populations are known and can be easily modified to tailor culture needs. We previously showed that Neurobasal Plus medium in combination with B27 plus supplement also increased the glial populations in comparison to the Electrophysiology kit (23), however an advantage of GEM is that cultures can be modulated in a more flexible manner than when proprietary formulations are used.…”

“…Briefly, hiPSCs and hESCs were cultured in mTESR-Plus medium (StemCell Technologies at 5% O 2 , 5% CO 2 and 37 °C). The cells were characterized based on expression of stemness markers (Oct4, Nanog and TRA-1-60) by flow cytometry (iPSC characterization see Romero et al, 2023(23), ESC characterization is summarized in Supplemental Figures 1 and 2). Stem cells were then differentiated in a monolayer to neuroprogenitor cells (NPCs) using a serum-free, chemically defined neural induction medium (Gibco, Thermo Fisher Scientific).…”

“…Stem cells were then differentiated in a monolayer to neuroprogenitor cells (NPCs) using a serum-free, chemically defined neural induction medium (Gibco, Thermo Fisher Scientific). NPCs were expanded and quality controlled for expression of NPS markers, Nestin, Sox2 (Romero et al, 2023 (23) and Supplemental Figures 3 and 4). To initiate bMPS differentiation, a single cell suspension of 2 × 10 6 NPCs was distributed per well into uncoated 6-well plates.…”

“…Brain MPS were differentiated from two induced Pluripotent Stem Cell (iPSC) lines; NIBSC-8 (female origin, UK National Institute for Biological Standards and Control (NIBSC)), and CS2PFYiCTR-nx.x (here CS2, male origin from Cedars-Sinai Medical Center Stem Cell repository), and from two embryonic stem cell (ESC) lines, H9 and Reus1, which were previously genetically modified (22). All cell lines are mycoplasma-free, with normal karyotypes, and were differentiated following our previously published two-step protocol with modifications to reflect glia enrichment conditions (Romero et al, 2023). Briefly, hiPSCs and hESCs were cultured in mTESR-Plus medium (StemCell Technologies at 5% O2, 5% CO2 and 37 °C).…”

First Organoid Intelligence (OI) workshop to form an OI community

“…Smirnova showed how, oligodendrogenesis can be quantified in response to different stimuli using a CRISPR-Cas9-engineered GFP-PLP1 reporter line (Romero et al, 2023). Smirnova's group is currently working on the implementation of a microfluidic system to increase perfusion and biomass (for energy efficiency in biocomputing), as well as biocomplexity.…”