Proper epithelial development and homeostasis depends on strict control of oriented cell division. Current evidence shows that this process is regulated by intrinsic polarity factors and external spacial cues. Due to the lack of appropriate model system that can recapitulate skin's architecture, deregulation of spindle orientation in human epithelial carcinoma has never been investigated. Using an inducible model of human squamous cell carcinoma (SCC), we demonstrate that RAS-dependent suppression of PAR3 accelerates epithelial disorganization during early tumorigenesis. Diminished PAR3 led to loss of E-cadherin mediated cell adhesion, which in turn contributed to misoriented cell division. Pharmacological inhibition of the MAPK pathway downstream of RAS activation reversed the defects in PAR3 expression, E-cadherin mediated cell adhesion and mitotic spindle orientation. Thus, temporal analysis of human neoplasia provides a powerful approach to study cellular and molecular transformations during early oncogenesis, which allowed identification of PAR3 as a critical regulator of tissue architecture during initial human SCC development.
CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.
This protocol describes the immunocytochemistry for staining motor neurons derived from induced pluripotent stem cells (iPSCs) using the hNIL transgenic factors in a CLYBL safe harbor site. For the protocol on this differentiation, refer to the Clelland Lab’s Differentiation of iPSCs with the hNIL construct into motor neurons protocol.
This protocol describes the differentiation of induced pluripotent stem cells (iPSCs) into motor neurons using the hNIL transgenic factors in a CLYBL safe harbor site.
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