Project description:Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

Project description:BACKGROUND:Oligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals. METHODS:Our protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq). RESULTS:We demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice. CONCLUSIONS:This is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.

Project description:Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients. RNA sequencing of oligodendrocyte progenitor cells treated with vehicle, miconazole or clobetasol for 0, 2, 6, or 12 hours. Cells were plated 1.5 hours prior to addition of drug.

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Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination.

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Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination.

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