ABSTRACT
Transcription factors (TFs) have been introduced to drive the highly efficient differentiation of human-induced pluripotent stem cells (hiPSCs) into lineage-specific oligodendrocytes (OLs). However, effective strategies currently rely mainly on genome-integrating viruses. Here we show that a synthetic modified messenger RNA (smRNA)-based reprogramming method that leads to the generation of transgene-free OLs has been developed. An smRNA encoding a modified form of OLIG2, in which the serine 147 phosphorylation site is replaced with alanine, OLIG2S147A, is designed to reprogram hiPSCs into OLs. We demonstrate that repeated administration of the smRNA encoding OLIG2 S147A lead to higher and more stable protein expression. Using the single-mutant OLIG2 smRNA morphogen, we establish a 6-day smRNA transfection protocol, and glial induction lead to rapid NG2+ OL progenitor cell (OPC) generation (>70% purity) from hiPSC. The smRNA-induced NG2+ OPCs can mature into functional OLs in vitro and promote remyelination in vivo. Taken together, we present a safe and efficient smRNA-driven strategy for hiPSC differentiation into OLs, which may be utilized for therapeutic OPC/OL transplantation in patients with neurodegenerative disease.
Subject(s)
Induced Pluripotent Stem Cells , Neurodegenerative Diseases , Alanine , Humans , Induced Pluripotent Stem Cells/metabolism , Neurodegenerative Diseases/metabolism , Oligodendrocyte Transcription Factor 2/genetics , Oligodendrocyte Transcription Factor 2/metabolism , Oligodendrocyte Transcription Factor 2/pharmacology , Oligodendroglia , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine/metabolism , Transcription Factors/metabolismABSTRACT
It has been shown that following demyelination, Oligodendrocyte Progenitor Cells (OPCs) migrate to the lesion site and begin to proliferate, and differentiate. This study aimed to investigate the effects of Hydroxychloroquine (HCQ) on the expression of OLIG-2 and PDGFR-α markers during the myelination process. C57BL/6 mice were fed cuprizone pellets for 5 weeks to induce demyelination and return to a normal diet for 1 week to stimulate remyelination. During the Phase I all of the animals except CPZ and Vehicle groups were exposed to HCQ (2.5, 10, and 100 mg/kg) via drinking water. At the end of the study, animals were euthanized, perfused and the brain samples were assessed for myelination and immunohistochemistry evaluation. What is remarkable is the high rate of Olig2 + cells in the groups treated with 10 and 100 mg/kg HCQ in the demyelination phase and its decreasing trend in the remyelination phase. However, there was no significant difference between groups during phase I and Phase II based on the percentage of olig-2+/total cells in the corpus callosum region. The number of PDGFR-α+ cells in the group treated with 10 mg/kg HCQ was significant in the first phase (p value < 0.05). Considering that the 100 mg/kg HCQ group had the highest level of PDGFR-α as well as the highest level of myelin repair in LFB staining, it could be inferred that it was the most effective dose in inducing proliferation and migration of OPCs.
Subject(s)
Demyelinating Diseases , Multiple Sclerosis , Animals , Corpus Callosum/pathology , Cuprizone/toxicity , Demyelinating Diseases/chemically induced , Demyelinating Diseases/drug therapy , Demyelinating Diseases/metabolism , Disease Models, Animal , Hydroxychloroquine/pharmacology , Mice , Mice, Inbred C57BL , Multiple Sclerosis/chemically induced , Multiple Sclerosis/drug therapy , Multiple Sclerosis/metabolism , Myelin Sheath/metabolism , Oligodendrocyte Transcription Factor 2/metabolism , Oligodendrocyte Transcription Factor 2/pharmacology , Oligodendrocyte Transcription Factor 2/therapeutic use , Oligodendroglia/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Receptors, Platelet-Derived Growth Factor/pharmacology , Receptors, Platelet-Derived Growth Factor/therapeutic useABSTRACT
AIMS: Olig2 is one of the most critical factors during CNS development, which belongs to b-HLH transcription factor family. Previous reports have shown that Olig2 regulates the remyelination processes in CNS demyelination diseases models. However, the role of Olig2 in contusion spinal cord injury (SCI) and the possible therapeutic effects remain obscure. This study aims to investigate the effects of overexpression Olig2 by lentivirus on adult spinal cord injury rats. METHODS: Lenti-Olig2 expression and control Lenti-eGFP vectors were prepared, and virus in a total of 5 µL (108 TU/mL) was locally injected into the injured spinal cord 1.5 mm rostral and caudal near the epicenter. Immunostaining, Western blot, electron microscopy, and CatWalk analyzes were employed to investigate the effects of Olig2 on spinal cord tissue repair and functional recovery. RESULTS: Injection of Lenti-Olig2 significantly increased the number of oligodendrocytes lineage cells and enhanced myelination after SCI. More importantly, the introduction of Olig2 greatly improved hindlimb locomotor performances. Other oligodendrocyte-related transcription factors, which were downregulated or upregulated after injury, were reversed by Olig2 induction. CONCLUSIONS: Our findings provided the evidence that overexpression Olig2 promotes myelination and locomotor recovery of contusion SCI, which gives us more understanding of Olig2 on spinal cord injury treatment.
