| 研究課題/領域番号 |
20K07458
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
TERENZIO Marco 沖縄科学技術大学院大学, 分子神経科学ユニット, 准教授 (60867513)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | ALS / protein translation / biomarkers / human IPSC / motor neurons |
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| 研究実績の概要 |
We are looking at what drives the rate of progression of neurodegeneration in Amyotrophic Lateral Sclerosis (ALS) by directly deriving motor neurons (MNs) from ALS patient’s human pluripotent stem cells (iPSC) to assess how local mechanisms of protein translation is changed in motor cells from ALS patients.
1) We successfully established a differentiation protocol for iPSC into MNs using commercially available hiPSC lines. To this extent, we purchased the HPS1005 hiPSC line from the Riken BRC Cell Bank and adapted the protocol described in Bossolasco et al., 2018, Stem Cell Res. 30: 61-68 to induce MN differentiation. Successful generation of MNs was confirmed with established MN markers such as ChAT and the transcription factor HB9.
2) We have developed a microfluidic chamber (MFC) device suitable for the culture of MNs and adult sensory neurons. These MFCs allow for the study of local translation via incorporation of puromycin to follow newly translated proteins via imaging as well as by biochemical retrieval and Mass Spectrometry analysis (Forester et al, 2018; Terenzio et al, 2018). We have used said MFCs to successfully grow MNs and tested effective fluidic isolation by monitoring axonal transport of Lysosomes and mitochondria.
3) we have started to work on the establishment of co-cultures between MNs and myoblasts to recreate a functional neuromuscular junction in vitro.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We have optimized the protocols and constructed the tools, i.e. MFCs, to be able to conduct the proteomic analysis planned for the FY2021. We are now ready to produce the samples for the proteomic analysis that was planned for FY2021. Candidates from this analysis will be validate in 2021 and 2022.
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| 今後の研究の推進方策 |
1. We will finalize the protocol to obtain co-cultures of human myoblasts and MNs.
2. MN-muscle co-cultures in MFCs will be used to generate axonal preparations for identification of axonally synthesized proteins, which will be labeled by the incorporation of puromycin followed by biochemical retrieval and Mass Spectrometry analysis. We will compare healthy MN cultures with cultures of MNs from ALS patients. Candidates of interest emerging from this analysis will then be tested to verify axonal local translation and impact on known physiological hallmark of ALS (i.e. axonal transport, MN survival) and compared it to MNs generated from ALS patients.
3. We will collect samples from conditioned media of human MNs cultures to perform proteomic analysis with sub-picomolar immunodetection by single molecular array with the aim of identifying secreted proteins in the medium, which could be used as biomarkers for ALS in the clinic.
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| 次年度使用額が生じた理由 |
1. We experienced some delays in obtaining human IPSCs to use for the proteomic screen. Thus, the money for the purchase of the cell lines and the proteomic analysis has been carried over to FY2021.
2. We will use the carryover money to acquire ALS-patient’s derived human iPSCs as planned. The lysates and the media from cultures of healthy and patient-derived MNs will be collected and we will outsource the proteomic analysis for axonally translated proteins and disease progression biomarkers as described before.
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