Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Abstract

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-∆7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

“…In addition, the TUJ1-positive FB cells derived from ADAR1 KD H9 cells exhibited much shorter growing neurites at all differentiation stages ( Figure 1F). Meanwhile, although the differentiation efficiency of MNs was only about 30%-40% as reported previously [37], we found that ADAR1 knockdown also dramatically repressed the formation of MN clusters ( Figure 1H) and HB9-positive MNs ( Figure 1I). The resulting MNs also exhibited a significant reduction of neurite outgrowth ( Figure 1I).…”

“…At 10 and 17 days of neural differentiation, hESC-derived primitive and definitive neuroepthelial (NE) cells with scramble shRNA treatment presented typical columnar NE morphology and were organized into rosettes [36][37][38] (Figure 1D and 1E), similar to the structure of NE cells from WT hESC induction (as discussed below and data not shown). Noticeably, although differentiating hESCs with stable ADAR1 knockdown also formed rosette-like structures, these cells expressed low levels of PAX6, SOX1 and FOXG1 ( Figure 1D and Supplementary information, Table S1), and formed much Table S1.…”

“…Importantly, consistent with the aberrant MN formation from ADAR1 KD H9 cells ( Figure 1H and 1I), genes related with MNs and their functions were also clustered (Supplementary information, Figure S3B-S3D). Given that the differentiation efficiency of MNs is only about 30%-40% [37,41], we focused on the differentiation to FB neurons for our subsequent analyses.…”

“…As ADAR1 knockdown has little effect on pluripotency, we then proceeded to study the effect of ADAR1 deficiency on differentiation with a protocol summarized in Supplementary information, Figure S2A [36][37][38]. Briefly, hESCs were induced into the neural lineage by generating EBs followed by culturing in neural medium.…”

“…H9 cells were induced to differentiate into the region-specific neural cells according to the published protocols [37][38][39][40][41]. To infect hESCs with lentivirus, H9 cells grown on Matrigel (BD Biosciences) were digested into single cells with Accutase (Millipore).…”