Regeneration of functional neurons for treatment of spinal cord injury and ALS
A spinal cord injury, neuron technology, applied in nervous system diseases, nervous system cells, biochemical equipment and methods, etc., can solve problems such as unmet treatment of ALS
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[0200] Materials and methods
[0201] animal use
[0202] GAD-GFP mice (Tg[Gad1-EGFP]94Agmo / J) and wild-type C57BL / 6 mice were purchased from Jackson Laboratory and bred in house. Mice (male and female) aged 2-4 months were used. Mice were housed on a 12 hour light / dark cycle and provided with adequate food and water. All animal use and research were approved by the Institutional Animal Care and Use Committee (IACUC) of the Pennsylvania State University. All procedures were performed according to protocols and guidelines approved by the National Institutes of Health (NIH).
[0203] Retrovirus and AAV production
[0204] A retroviral vector expressing GFP and NeuroD1-GFP under the CAG promoter (pCAG) was previously described (Guo et al., Cell Stem Cell, 14:188-202 (2014)). Retroviral packaging, purification and titration were performed as previously described (Guo et al., Cell Stem Cell, 14:188-202 (2014)). For AAV-mediated gene expression, the Cre-Flex system was applied...
example 1
[0213] Example 1 – NeuroD1 reprograms reactive astrocytes into neurons in the injured spinal cord
[0214] SCI has been studied for decades, but to date, therapies for treating SCI patients remain limited. In addition to axonal degeneration, neuronal loss after SCI is a major obstacle to functional recovery. It was previously demonstrated that expression of NeuroD1 in reactive astrocytes following brain injury can directly convert astrocytes into neurons (Guo et al., Cell Stem Cell, 14:188-202 (2014)). In the present study, it was investigated whether this in vivo direct transformation technique could be used to regenerate functional new neurons in the injured spinal cord. To target division-responsive astrocytes after injury, a retrovirus expressing the ectopic gene primarily in dividing cells but not in non-dividing neurons was employed. Retrovirus expressing NeuroD1 was injected 4 days after puncture injury (dpi) when many division-reactive astrocytes had been detected ...
example 2
[0216] Example 2 – NeuroD1 converts dorsal spinal cord astrocytes into Tlx3+ glutamatergic neurons
[0217] After demonstrating astrocyte-to-neuron conversion in the spinal cord, we next investigated which neuronal subtypes arise through NeuroD1-mediated conversion. The dorsal horn of the spinal cord contains two major neuronal subtypes: glutamatergic neurons and GABAergic neurons (Abraira and Ginty, Neuron, 79:618-639 (2013)). During spinal cord development, two transcription factors, Tlx3 and Pax2, appear to play a role in determining cell fate specification in the dorsal horn (Cheng et al., Nature Neurosci., 8:1510-1515 (2005); and Huang et al., Developmental Biology (Dev. Biol.), 322:394-405 (2008)). Interestingly, by examining AAV NeuroD1-GFP-infected cells in the dorsal horn at 8wpi, it was found that the majority of NeuroD1-transformed neurons were Tlx3+ (62.6±3.3%), suggesting that the majority of glutamatergic neurons are sub- type( Figure 3A ). In contrast, on...
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