Experimentally Induced Sepsis Causes Extensive Hypomyelination in the Prefrontal Cortex and Hippocampus in Neonatal Rats.

Neonatal sepsis is associated with cognitive deficit in the later life. Axonal myelination plays a pivotal role in neurotransmission and formation of learning and memory. This study aimed to explore if systemic lipopolysaccharide (LPS) injection would induce hypomyelination in the prefrontal cortex and hippocampus in developing septic neonatal rats. Sprague-Dawley rats (1-day old) were injected with LPS (1 mg/kg) intraperitoneally. By electron microscopy, axonal hypomyelination was evident in the subcortical white matter and hippocampus. The expression of myelin proteins including CNPase, MBP, PLP and MAG was downregulated in both areas of the brain at 7, 14 and 28 days after LPS injection. The frequency of MBP and PLP-positive oligodendrocyte was significantly reduced using in situ hybridization in the cerebral cortex and hippocampus at the corresponding time points after LPS injection, whereas the expression of NG2 and PDGFRα was noticeably increased. In tandem with this was reduction of Olig1 and Olig2 expressions which are involved in differentiation/maturation of OPCs. Expression of NFL, NFM, and NFH was significantly downregulated, indicating that axon development was disrupted after LPS injection. Morris Water Maze behavioral test, Open field test, Rotarod test, and Pole test were used to evaluate neurological behaviors of 28 days rats. The rats in the LPS group showed the impairment of motor coordination, balance, memory, and learning ability and represented bradykinesia and anxiety-like behavior. The present results suggest that following systemic LPS injection, differentiation/maturation of OPCs was affected which may be attributed to the inhibition of transcription factors Olig1 and Olig2 expression resulting in impairment to axonal development. It is suggested that this would ultimately lead to axonal hypomyelination in the prefrontal cortex and hippocampus, which may be associated with neurological deficits in later life.

Continuous tamoxifen delivery improves locomotor recovery 6h after spinal cord injury by neuronal and glial mechanisms in male rats.

No treatment is available for patients with spinal cord injury (SCI). Patients often arrive to the hospital hours after SCI suggesting the need of a therapy that can be used on a clinically relevant window. Previous studies showed that Tamoxifen (TAM) treatment 24h after SCI benefits locomotor recovery in female rats. Tamoxifen exerts beneficial effects in male and female rodents but a gap of knowledge exists on: the therapeutic window of TAM, the spatio-temporal mechanisms activated and if this response is sexually dimorphic. We hypothesized that TAM will favor locomotor recovery when administered up-to 24h after SCI in male Sprague-Dawley rats. Rats received a thoracic (T10) contusion using the MACSIS impactor followed by placebo or TAM (15mg/21days) pellets in a therapeutic window of 0, 6, 12, or 24h. Animals were sacrificed at 2, 7, 14, 28 or 35days post injury (DPI) to study the molecular and cellular changes in the acute and chronic stages. Immediate or delayed therapy (t=6h) improved locomotor function, increased white matter spared tissue, and neuronal survival. TAM reduced reactive gliosis during chronic stages and increased the expression of Olig-2. A significant difference was observed in estrogen receptor alpha between male and female rodents from 2 to 28 DPI suggesting a sexually dimorphic characteristic that could be related to the behavioral differences observed in the therapeutic window of TAM. This study supports the use of TAM in the SCI setting due to its neuroprotective effects but with a significant sexually dimorphic therapeutic window.

Derivation of telencephalic oligodendrocyte progenitors from human pluripotent stem cells.

Oligodendrocytes are the main myelinating cell of the adult CNS and are vulnerable to injury in diverse disorders such as spinal cord injury, stroke, trauma, pharmacological and radiation toxicity, as well as neuroinflammation. Human pluripotent stem cells are attractive sources of oligodendrocyte lineage cells and provide a promising treatment strategy for exogenous myelin repair through transplantation. This unit describes a protocol for the step-wise differentiation of forebrain late oligodendrocyte progenitor cells (OPCs) from human pluripotent stem cells in defined chemical in vitro culture conditions. It involves a stepwise progression of oligodendrocyte progenitors through their known developmental phases, starting with the expression of appropriate transcription factors (Olig2, Nkx2.2), the upregulation of PDGFRA, followed by the appearance of O4-expressing cells, then O1 expression and finally mature myelin-binding protein (MBP) expressing cells. Validation of cell fate is performed by extensive transcriptomal profiling, as well in vitro myelination essays with hESCs derived neuronal cells. Recapitulating forebrain oligodendrocyte development may generate cells more suitable for transplantation strategies for disorders primarily involving the telencephalon.

Enhancing oligodendrocyte differentiation by transient transcription activation via DNA nanoparticle-mediated transfection.

Current approaches to derive oligodendrocytes from human pluripotent stem cells (hPSCs) need extended exposure of hPSCs to growth factors and small molecules, which limits their clinical application because of the lengthy culture time required and low generation efficiency of myelinating oligodendrocytes. Compared to extrinsic growth factors and molecules, oligodendrocyte differentiation and maturation can be more effectively modulated by regulation of the cell transcription network. In the developing central nervous system (CNS), two basic helix-loop-helix transcription factors, Olig1 and Olig2, are decisive in oligodendrocyte differentiation and maturation. Olig2 plays a critical role in the specification of oligodendrocytes and Olig1 is crucial in promoting oligodendrocyte maturation. Recently viral vectors have been used to overexpress Olig2 and Olig1 in neural stem/progenitor cells (NSCs) to induce the maturation of oligodendrocytes and enhance the remyelination activity in vivo. Because of the safety issues with viral vectors, including the insertional mutagenesis and potential tumor formation, non-viral transfection methods are preferred for clinical translation. Here we report a poly(ß-amino ester) (PBAE)-based nanoparticle transfection method to deliver Olig1 and Olig2 into human fetal tissue-derived NSCs and demonstrate efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. This approach is potentially translatable for engineering stem cells to treat injured or diseased CNS tissues. STATEMENT OF SIGNIFICANCE: Current protocols to derive oligodendrocytes from human pluripotent stem cells (hPSCs) require lengthy culture time with low generation efficiencies of mature oligodendrocytes. We described a new approach to enhance oligodendrocyte differentiation through nanoparticle-mediated transcription modulation. We tested an effective transfection method using cell-compatible poly (ß-amino ester) (PBAE)/DNA nanoparticles as gene carrier to deliver transcription factor Olig1 and Olig2 into human fetal tissue-derived neural stem/progenitor cells, and showed efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. We believe that this translatable approach can be applied to many other cell-based regenerative therapies as well.