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Cell Journal، جلد ۱۳، شماره Supplement، صفحات ۰-۰
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عنوان انگلیسی Is-12: Discovery of Potential Therapeutic Compouns for Familial Dysautonomia Using Patient-Specific and Symptom-Relevant iPSC Derived Neural Crest Precursors
چکیده انگلیسی مقاله Patient-specific human induced pluripotent stem cells (hiPSCs) offer unprecedented promise in disease modeling and drug discovery by providing unlimited number of symptom-relevant cells. Taking advantage of our novel methodology of direct differentiation and prospective isolation of neural crest (NC) from human pluripotent stem cells, we successfully modeled Familial Dysautonomia (FD) with hiPSC technology. The results from these studies offered previously unknown clues for disease pathogenesis and allowed us to validate several candidate drugs. To extend our approach beyond the validation of a few candidate compounds, we present here our data on the first high-throughput screening (HTS) drug discovery effort performed in FD-iPSC derived symptom-relevant NC precursors. FD-iPSCs differentiated into NC using our standard MS-5 differentiation protocol and purified by flow cytometry using HNK1 (CD57) antibody. Enriched NC population was further expanded for 2 weeks yielding > 0.5 x 109 cells that were could be cryo-preserved prior to performing HTS assay. To establish culture condition in 384-well-plates, we optimized seeding density, proliferation rate and coating reagents. Furthermore, by using commercially available kits, automated protocols for RNA extraction and qRT-PCR were developed to measure level of wild type (WT) and mutant (MU) IKBKAP expression. We selected qRT-PCR as the primary HTS readout given that reduction in transcriptional levels of wild-type IKBKAP is closely correlated with FD disease symptoms. Using our optimized culture conditions in 384-well-plates and automated qRT-PCR protocols, we tested the impact of chemical compound libraries (>7000 compounds, FDA-approved drugs and natural products) on WT and MU IKBKAP expression in patient-specific and symptom-relevant NCs. Expression levels were normalized to housekeeping gene and all assays were performed in three biological repeats per compound. We identified a total of 43 primary hits that were subjected to various validation studies, cytotoxicity assays and dose-response studies (12 concentrations). Finally, 8 candidate compounds were selected based on increased level of WT IKBKAP transcript, which was significantly correlated with enhanced IKAP protein. In further functional analysis, we found that more than 3 compounds showed the rescue of mis-regulation of autonomic gene expression (ASCL1 and SCG10). However, we could not find any significant increase in the migration defect in FD-NC. Our drug discovery effort using symptom-relevant and patient-specific cells is a proof of concept demonstration for HTS with disease-specific hiPSC lines. We hope that our proposed paradigm (specification/isolation of symptom-relevant cell type, disease modeling and HTS for drug discovery) will prove suitable for other iPSC based disease models and lead to novel treatments for many currently intractable diseases. In conclusion, our studies illustrate the potential of using patient-specific hiPSCs for HTS-based drug discovery.
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/1610
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