HB9-GFP Lentivirus

Of the several gene mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are elevated, resulting in a slightly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and why it occurs in only a subset of patients remain unclear. To model the acquisition of C9orf72 hypermethylation and investigate the potential role of 5-hydroxymethylcytosine (5hmC), researchers generated induced pluripotent stem cells (iPSCs) from ALS patients with C9orf72 promoter hypermethylation. The data showed that 5mC levels were reduced by reprogramming and then regained after neuronal specification, while 5hmC levels increased after reprogramming and were highest in iPSCs and motor neurons. The researchers confirmed the presence of 5hmC within the C9orf72 promoter in postmortem brain tissue from hypermethylated patients. These findings suggest that iPSCs are a valuable model system for examining epigenetic perturbations caused by C9orf72 mutations and reveal a potential role for cytosine demethylation.

To examine the acquisition of C9orf72 promoter hypermethylation during neural development, researchers generated neural progenitor cells (NPCs) and terminally differentiated motor neurons from iPSCs using a two-stage differentiation protocol. Expression of NPC cell markers Nestin and Sox2 was confirmed by immunodetection (Figure 1C), while motor neuron specification was confirmed by expression of cytoskeletal neuron markers Tuj1 and Isl1 (Figure 1D, E). Motor neuron specification was further confirmed using a lentiviral vector encoding a green fluorescent protein (GFP) reporter gene driven by the motor neuron-specific homeobox gene promoter HB9 (HB9-GFP lentivirus)(Figure 1F). Endogenous expression of Tuj1, Isl1, and HB9 was increased in motor neuron cultures compared to iPSCs. The C9orf72 repeat expansion was maintained throughout reprogramming and motor neuron specification, as determined by Southern blot analysis and repeat-primed PCR. Interestingly, repeat size varied in different cell types, suggesting repeat instability during motor neuron specialization, a trend that has been reported previously.

Figure 1. Characterization of patient-derived iPSCs, NPCs, and motor neurons. (Esanov R, et al., 2016)