Biology subjects

Pitstick, A.

Publications and source records attributed to Pitstick, A..

2 recordsLinked to original sources

bioRxiv2021

High-throughput functional analysis of CFTR and other apically localized channels in iPSC derived intestinal organoids

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. In these proof-of-concept studies, we show how this fluorescence-based assay of apical membrane potential can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This multiparameter phenotypic platform promises to expand CF therapy discovery to include strategies to target multiple determinants of epithelial fluid transport.

molecular biology↗

bioRxiv2017

A de novo missense mutation in TUBA1A results in reduced neural progenitor survival and differentiation

Mutations in tubulins have been implicated in numerous human neurobiological disorders collectively known as \"tubulinopathies.\" We identified a patient with severe cortical dysgenesis and a novel de novo heterozygous missense mutation in Tubulin Alpha 1a (TUBA1A, c.1225 G>T). Induced pluripotent stem cells derived from this individual were differentiated into two dimensional neural rosette clusters to identify underlying mechanisms for the severe cortical dysgenesis phenotype. Patient-derived clones showed evidence of impaired neural progenitor survival and differentiation with abnormal neural rosette formation, increases in cell death, and fewer post-mitotic neurons. These features correlate with the drastically underdeveloped cortical tissues seen in the proband. This is the first experimental evidence in human tissue suggesting a mechanism underlying the role for TUBA1A in cortical development.\n\nSUMMARY STATEMENTVariants in tubulin genes often lead to severe congenital brain malformations. Here we identify a new mutation in TUBA1A and use iPSCS to show this alters proliferation, differentiation and survival of neural progenitors.

genetics↗