Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro

Abstract

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.

Therapeutic Aspects of Mesenchymal Stem Cell-Based Cell Therapy with a Focus on Human Amniotic Epithelial Cells in Multiple Sclerosis: A Mechanistic Review

“…Previous studies have demonstrated that autologous and allogeneic AD-MSCs can be useful in the treatment of various diseases such as MS, diabetes, Parkinson's disease, autoimmune encephalomyelitis (56)(57)(58)(59). In line with this notion, Li et al revealed that intravenously injection of AD-MSC to EAE mice contributed to the reduction in infiltrations of inflammatory cells in the CNS and significantly inhibited demyelination and axonal loss (8,59). These cells release various growth factors including anti-apoptotic, angiogenic and neurotrophic mediators which have significant roles in many cellular mechanisms such as cell differentiation, proliferation, and maturation (60).…”

Transcriptomic analysis of loss of Gli1 in neural stem cells responding to demyelination in the mouse brain

“…One NSC subset, which is enriched in the ventral SVZ, expresses the transcription factor Gli1 and generates interneurons in the olfactory bulb and astrocytes in the healthy mouse brain 26,27 . This population of NSCs is also present in the human SVZ 27 as well as in NSCs derived from human induced pluripotent stem cells and human embryonic stem cells 28 . Remarkably, and in contrast to its fate in the healthy adult brain, this Gli1 pool of NSCs is specifically recruited to the white matter in response to demyelination and go on to differentiate into remyelinating oligodendrocytes 20,27,29 .…”

“…Remarkably, and in contrast to its fate in the healthy adult brain, this Gli1 pool of NSCs is specifically recruited to the white matter in response to demyelination and go on to differentiate into remyelinating oligodendrocytes 20,27,29 . Inhibition of Gli1 in these cells further enhances their remyelination potential and is neuroprotective resulting in functional improvement in mouse models of MS 27,28 . Importantly, Gli1 expressing stem-like cells respond to injury in other organs like the liver, lung, bone marrow, kidney and heart with loss of Gli1 resulting in attenuated injury in these organs [30][31][32][33][34][35][36][37] .…”

The Role of Sonic Hedgehog Pathway in the Development of the Central Nervous System and Aging-Related Neurodegenerative Diseases

“…In vivo , blocking SHH may lead to insufficient differentiation of granular neurons and Bergmann glial cells and abnormal development of Purkinje neurons ( Wang and Almazan, 2016 ). According to numerous research studies, SHH can mediate to produce oligodendrocyte precursors ( Namchaiw et al, 2019 ; Starikov and Kottmann, 2020 ). By the chemical inhibitor or anti-SHH antibody, inhibiting SHH may inhibit the differentiation of oligodendrocytes ( Yang et al, 2020 ).…”