RESUMO
Oncogenic KRAS mutations in hematopoietic stem cells cause RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). KRAS plays essential roles in stemness maintenance in some types of stem cells. However, its roles in pluripotent stem cells (PSCs) are poorly understood. Here, we investigated the roles of KRAS on stemness in the context of induced PSCs (iPSCs). We used KRAS mutant (G13C/WT) and wild-type isogenic (WT/WT) iPSCs from the same RALD patients, as well as wild-type (WTed/WT) and heterozygous knockout (Δed/WT) iPSCs, both obtained by genome editing from the same G13C/WT clone. Compared with WT iPSCs, G13C/WT iPSCs displayed enforced retention of self-renewal and suppressed capacity for neuronal differentiation, while Δed/WT iPSCs showed normalized cellular characteristics similar to those of isogenic WTed/WT cells. The KRAS-ERK pathway, but not the KRAS-PI3K pathway, was shown to govern these G13C/WT-specific phenotypes, indicating the strong impact of the KRAS-ERK signaling upon self-renewal and differentiation propensity in human iPSCs.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Alelos , Síndrome Linfoproliferativa Autoimune , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células Cultivadas , Aberrações Cromossômicas , Análise Mutacional de DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Edição de Genes , Perfilação da Expressão Gênica , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Cariótipo , Imagem Molecular , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.