Human iPSCs Generation and CRISPR/cas:
iPSC:
Mount Sinai School of Medicine
PI: Bruce Gelb
A working group was organized and a protocol was established for developing iPSC lines from blood samples obtained from CHD GENES subjects who harbor de novo mutations of interest. Additional blood samples are collected from enrolled subjects, the IPSC Core isolates peripheral erythroblasts and derives iPSCs that can be stored or transported to other sites for study. Two approaches are being used- subjects bearing mutations of interest are recruited and iPSCs derived from peripheral blood cells. Then, CRISPR technology will be used to correct the relevant mutation to provide an isogenic control. Alternatively, control iPSCs are being mutated using CRISPRs to introduce mutations of interest.
iPS CRISPR/cas:
Brigham and Women’s Hospital
PI: Christine Seidman
iPS cells generated from individual CHD patients exhibit extensive variability in background genotypes. To overcome the variability inherent in research with human iPS cells, the PCGC elected to introduce selected mutations into isogenic pluripotent human stem cells using CRISPR technology. The iPS/CRISPR Core has identified and selected an iPS cell line that demonstrates consistent robust differentiation into cardiomyocytes for mutagenesis. Multiple lines have been generated with mutation in candidate genes and are being analyzed at iPS, mesoderm, early cardiac progenitor, late cardiac progenitor, and cardiomyocyte stages of differentiation.