Generation of mutation-corrected induced pluripotent stem cell lines derived from adrenoleukodystrophy patient by using homology directed repair.

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. Previously, we generated an ALD patient-derived SCHi001-A iPSC model. In this study, we have performed the first genome editing of ALD patient-derived SCHi001-A iPSCs using homology-directed repair (HDR). The mutation site, c.1534G > A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal iPSC plulipotency marker expression following genome editing. Mutation-corrected iPSCs from SCHi001-A iPSC line can be used in research into the pathophysiology of and therapeutics for ALD.

Activation of dickkopf-1 and focal adhesion kinase pathway by tumour necrosis factor α induces enhanced migration of fibroblast-like synoviocytes in rheumatoid arthritis.

OBJECTIVE: The objective of this study was to investigate the roles of dickkopf-1 (DKK-1) and integrin-related focal adhesion kinase (FAK) by TNF-α on the migration of fibroblast-like synoviocytes (FLSs) in RA. METHODS: Wound scratch assays were performed to assess FLS migration. Western blotting was used to measure the levels of DKK-1, Wnt signalling molecules and FAK signalling molecules. Quantitative real-time PCR was used to measure the expression levels of DKK-1, integrin αv, laminin, fibronectin, E-cadherin, MMP-8 and MMP-13. The concentrations of DKK-1, TNF-α and GSK-3ß were measured by ELISA. Genetic silencing of TNF-α was achieved by the transfection of small interfering RNA into cells. RESULTS: Migrating RA FLSs exhibited higher levels of DKK-1 and TNF-α expression compared with those in OA FLSs and/or stationary RA FLSs. Moreover, migrating FLSs exhibited significantly higher levels of FAK, p-JNK, paxillin and cdc42 expression, whereas the level of cytosolic ß-catenin was lower. WAY-262611, Wnt pathway agonist via inhibition of DKK-1, markedly inhibited cell migration of RA FLSs through the accumulation of cytosolic ß-catenin and suppression of FAK-related signalling pathways. TNF-α treatment to RA FLSs up-regulated expression of DKK-1, integrin αv, fibronectin, laminin and MMP-13. TNF-α stimulation also suppressed cytosolic ß-catenin and E-cadherin expression in a time-dependent manner. Moreover, TNF-α small interfering RNA-transfected migrating FLSs exhibited decreased activation of integrin-related FAK, paxillin, p-JNK and cdc42 signalling pathways. CONCLUSION: This study demonstrates that the activation of DKK-1 and the integrin-related FAK signalling pathway stimulated by TNF-α induces the dissociation of ß-catenin/E-cadherin, thus promoting RA FLS migration.