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Sep 26, 2011

Technique Developed to Generate Myelinating Cell Progenitors for Drug Screening and Cell Therapeutics

  • A technique for generating pure populations of mouse oligodendrocyte progenitor cells (OPCs) that can differentiate into myelin-producing cells both in vivo and in vivo is reported by scientists at Case Western Reserve University School of Medicine and Stanford University School of Medicine. The method involves directing the differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions, which, the team claims, results in the rapid and efficient production of highly expandable populations of mouse OPCs.  

    Case Western’s Paul J. Tesar, Ph.D., and colleagues, say the availability of pure populations of OPCs will facilitate high-throughput screening approaches for drug compounds that modulate the functional differentiation of OPCs and could pave the way to the development of cell-based therapies for the diseases characterized by demyelination, such as multiple sclerosis. They report on the technique in Nature Methods in a paper titled “Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells".

    Curing diseases that feature demyelination of nerve cells isn’t yet possible because there are no techniques for promoting re-myelination, the researchers report. In theory, reversing such disorders could be effected either by use of a drug that restores the myelinating capacity of endogenous cells, or the transplantation of OPCs that retain the functional capacity to respond to signals and generate myelinogenic oligodendrocytes. 

    In vivo, OPCs are found in or close to demyelinated lesions, but they generally don’t re-myelinate demyelinated axons, and identifying compounds that can trigger OPC differentiation in vivo has been hampered by the inability to derive large enough quantities of OPCs for screening, the team adds. And while previous research has demonstrated that myelogenic oligodendrocytes can be generated from neural stem cells and pluripotent stem cells, these approaches often result in a mixture of uncharacterized neural precursors rather than pure populations of functional OPCs.

    The team sought to develop a technique for efficiently generating OPCs from mouse EpiSCs, a cell type that displays features and a developmental state in common with human embryonic stem cells and human induced pluripotent stem cells (iPSCs). The first stage was to specify EpiSCs into a neuroectodermal lineage by modulating activin-nodal and bone morphogenic protein (BMP) signaling pathways using small molecule inhibitors. This led to the EpiSCs rapidly downregulating the expression of pluripotency genes and upregulating neuroectodermal genes, and undergoing morphological changes to form radially organized neural rosettes. In fact, over 99% of EpiSC colonies formed rosettes that expressed the neuroectodermal genes Pax6 and Sox1, they claim.

    The next stage was to pattern the "naive" EpiSC-derived neural rosettes in a region-specific manner using cues including sonic hedgehog (SHH) and retinoic acid. This resulted in upregulation of the OPC-relevant transcription factors Olig2 and Nkx2.2, and the expression pattern of factors in the rosettes that mimicked the nonoverlapping expression that occurs in the ventral ventricular zone of the developing mouse neural tube.

    In vivo, OPCs proliferate in response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signals, and migrate extensively to populate the central nervous system, the researchers continue. To try and replicate this they dissociated the patterned EpiSC-derived neural rosettes and plated them onto a laminin substrate in the presence of PDGF-AA, FGF2, and SHH. The primary passaging led to the EpiSC-derived neural rosettes immediately giving rise to βIII-tubulin+ neurons and presumptive OPCs that expressed Olig2 or Nkx2.2. “These results recapitulate in vivo cell-specification events at the ventral ventricular zone of the developing spinal cord that are known to give rise to both neurons and OPCs,” the team states.

    While the resulting neurons didn’t survive culture past three days, the presumptive OPCs proliferated in the presence of PDGF-AA, FGF2, and SHH, and within days produced a confluent, nearly homogeneous cell population that displayed a typical bipolar morphology and expressed transcription factors and cell surface markers of OPCs. Encouragingly, the EpiSC-derived OPCs could be expanded for at least eight passages, and yielded what the team claims was previously unobtainable numbers of pure OPCs. To demonstrate the robustness of the technique, the procedure was repeated using four different EpiSC cell lines.

    To investigate the differentiation capacity of the EpiSC-derived OPCs, the cells were treated with thyroid hormone (T3) in the absence of both FGF2 and PDGF-AA. Under these conditions the putative OPCs stopped proliferating and differentiated exclusively into oligodendrocytes that displayed the oligodendrocyte cell-surface marker O4. By day three, O4+ oligodendrocytes had upregulated classical and defining markers of bona fide oligodendrocytes, such as myelin basic protein (Mbp), proteolipid protein 1 (Plp1), myelin-associated glycoprotein (Mag), and myelin oligodendrocyte glycoprotein (Mog). By day four the vast majority of EpiSC-derived OPCs had become oligodendrocytes. Again, this procedure was replicated using OPCs derived from four independent EpiSC lines, with no evidence of any cell-line specific differences in efficiency or timing of differentiation.

    The researchers moved on to test the functional and myelinogenic properties of EpiSC-derived OPCs either by culturing them in vitro with neurons or by injecting them into the brains of congenitally hypomyelinated mice in vivo. Low density EpiSC-derived OPCs cultured with mouse cortical neurons differentiated into MBP+ cells, with much of the MBP staining showing alignment with βIII-tubulin+ axons, “which is suggestive of their myelinogenic capacity,” they report. EpiSC-derived OPCs were also evaluated in an organotypic slice culture assay using tissue from early postnatal shiverer pups, which lack Mbp and compact myelin. When the cells were injected into forebrain slices, they exclusively differentiated into oligodendrocytes, leading to substantial numbers of Mbp+ segments and compact myelin.

    For evaluation of EpiSC-OPCs in vivo, cells were delivered into the developing corpus callosum region of newborn, immunocompetent shiverer pups. Brain analysis 3–7 weeks later demonstrated numerous patches of MBP+ myelin sheaths in transplant recipient animals, but not in untransplanted control animals. Importantly, the team adds, “EpiSC-derived OPCs appeared to migrate extensively in the host central nervous system as we observed myelination at sites distant from the injection such as the contralateral striatum.” No teratomas or aberrant cellular growths were found in any of the mice transplanted with EpiSC-derived OPCs over the course of the evaluation.

    The researchers also wanted to demonstrate the feasibility of using EpiSCs as a screening platform to identify compounds or proteins that prompt OPCs to generate myelinating oligodendrocytes. To this end they established a simple proof-of-principle study in which EpiSC-derived OPCs were subjected to three different regimens that modulate either the Notch, Wnt–β-catenin, or BMP signaling pathways that have previously been implicated in oligodendrocyte development or differentiation.

    They found that treating EpiSC-derived OPCs with T3 differentiation medium in the presence of the Notch ligand Jagged1 (Jag1) had no impact on the rate or number of O4+ oligodendrocytes. However, a similar protocol involving addition of an inhibitor of GSK3β (which acts as a negative regulator of Wnt–β-catenin signaling) resulted in concentration-dependent inhibition of cell differentiation into to O4+ oligodendrocytes. “These results suggest that canonical Wnt–β-catenin signaling, activated by inhibition of GSK3β, may positively regulate the OPC state and block the transition to oligodendrocytes,” the authors state. For the third test they exposed EpiSC-derived OPCs to signaling cues that had previously been shown to trigger OPCs to develop into astrocytes. As expected, this resulted in the majority of EpiSC-derived OPCs cells undergoing morphological and gene expression changes indicative of respecification into astrocytes. “Our system is also a robust platform to perform high-throughput screens for drugs that modulate the functional differentiation of mouse OPCs,” they conclude.

    The ability to generate virtually limitless supplies of functional OPCs in the mouse could in addition have obvious implications for future cell therapies in humans, the researchers stress. “As EpiSCs have defining features and differentiation responses in common with human embryonic stem cells and iPSCs, the potential extension of our OPC differentiation regimen to human pluripotent cells is an exciting prospect for the treatment of human myelin-related disorders in the future...Studies will take advantage of the purity and scalability of this system to optimize transplantation strategies (injection site, timing, and dosing) for functional recovery from dysmyelinating conditions.”

    The researchers claim their developmental system will in addition faciliate detailed studies into the gene expression changes and signaling pathways involved in the formation of an oligodendrocyte cell lineage during development, and potentially provide new insights into the causes of relevant congenital disorders.

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