A Micropatterned Human-Specific Neuroepithelial Tissue for Modeling Gene and Drug-Induced Neurodevelopmental Defects.

The generation of structurally standardized human pluripotent stem cell (hPSC)-derived neural embryonic tissues has the potential to model genetic and environmental mediators of early neurodevelopmental defects. Current neural patterning systems have so far focused on directing cell fate specification spatio-temporally but not morphogenetic processes. Here, the formation of a structurally reproducible and highly-organized neuroepithelium (NE) tissue is directed from hPSCs, which recapitulates morphogenetic cellular processes relevant to early neurulation. These include having a continuous, polarized epithelium and a distinct invagination-like folding, where primitive ectodermal cells undergo E-to-N-cadherin switching and apical constriction as they acquire a NE fate. This is accomplished by spatio-temporal patterning of the mesoendoderm, which guides the development and self-organization of the adjacent primitive ectoderm into the NE. It is uncovered that TGFß signaling emanating from endodermal cells support tissue folding of the prospective NE. Evaluation of NE tissue structural dysmorphia, which is uniquely achievable in the model, enables the detection of apical constriction and cell adhesion dysfunctions in patient-derived hPSCs as well as differentiating between different classes of neural tube defect-inducing drugs.

Correction: EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR.

[This corrects the article DOI: 10.18632/oncotarget.26446.].

EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by six-color crystal digital PCR.

Background: Detection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated. Methods: We developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and evaluated this assay on 82 tumor and plasma samples from NSLC patients. Results: Limits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wild-type DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease. Conclusion: This 6-color Crystal digital PCR assay could represent a robust solution using digital PCR for the monitoring of EGFR mutations.