Human stem cell modeling for amyotrophic lateral sclerosis
Yang, Meimei
Yang, Meimei
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Publication Date
2022-09-28
Type
Thesis
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Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs). Most ALS cases are sporadic, with no family history or known genetic association. Therefore, an extensive collection of sporadic ALS models is required when attempting to identify common underlying mechanisms of pathology or test new therapeutic interventions. Induced pluripotent stem cells (iPSCs) are a valuable tool for disease modelling, drug screening and cell therapy, especially for sporadic cases for which there are no animal models available. But the lack of differentiation protocol to derive enriched and functional MNs from iPSCs hinders the utility of these models. In this PhD project, we recruited two healthy individuals and four sporadic ALS patients and reprogrammed their fibroblasts into iPSCs using non-integration Sendai Viruses carrying four “Yamanaka reprogramming factors” of OCT3/4, SOX2, c-MYC and KLF4. All iPSC lines were characterized by pluripotency and spontaneous differentiation capacity. Other quality control (QC) tests, including transgene integration-free assay, karyotyping, SNP fingerprinting, and mycoplasma test, were also carried out to ensure they were suitable for further applications. In the second part of this PhD project, I established a novel, rapid, simple, but efficient differentiation protocol for deriving spinal motor neurons (sMNs) on monolayer culture by modulating the timing of adding small molecule compound E based on another previously published protocol. The novel protocol can differentiate iPSCs into a highly pure population (>95%) of sMNs expressing mature MN markers in 18 days. The efficiency of producing sMNs by this novel protocol was evaluated by the expression of stage-specific MN markers during the differentiation process via ICC and qPCR. I also validated the MN differentiation protocol with TUJ1, MAP2, synaptic markers and excitatory neuronal markers. Ultimately, I investigated the subtype of MNs derived from our novel protocol and identified them with the cervical fate of spinal cord identity. Then we assessed the electrophysiological activities of our iPSC-derived sMNs using multi-electrode array (MEA) and calcium imaging. Extensive network firing was detected in 30-day iPSC-derived MNs from MEA plates, indicating that the iPSC-derived sMNs have formed functional synapses and neurotransmission. During the longitudinal MEA recording, we identified elevated excitability and abnormal firing patterns of sALS iPSC-derived sMNs during aging. These results indicated that altered excitability could be used as a target for drug screening and testing. To further investigate the functional maturity, we carried out calcium imaging on 18-day and 28-day sMNs to reveal the calcium dynamics in ALS iPSC-derived MNs. Both 18-day and 28-day sMNs showed active calcium transients, but 28-day sMNs had higher maturity. This was concluded from other calcium transient properties and their responsiveness to the TTX, glutamate receptor agonist and antagonist. In summary, a mini-iPSC biobank was derived for sporadic ALS research. A novel MN differentiation protocol was established, which is valuable for investigating disease phenotype and developing new drugs for ALS. A preliminary phenotype of hyperexcitability was identified in sALS-derived sMNs which may facilitate the drug screening and testing.
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NUI Galway
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Attribution-NonCommercial-NoDerivs 3.0 Ireland
CC BY-NC-ND 3.0 IE
CC BY-NC-ND 3.0 IE
CC BY-NC-ND 3.0 IE
CC BY-NC-ND 3.0 IE
CC BY-NC-ND 3.0 IE
CC BY-NC-ND 3.0 IE