Journal: Blood Advances

Article Title: Gene-edited pseudogene resurrection corrects p47 phox -deficient chronic granulomatous disease

doi: 10.1182/bloodadvances.2016001214

Figure Lengend Snippet: Functional analysis of gene-targeting correction of p47-CGD patient HPCs. HPCs from p47-CGD subject 3 (homozygous exon 2 ΔGT NCF1) were cultured in Stemspan SFEMII supplemented with stem cell factor, Flt3 ligand, and thrombopoietin and gene targeted with ZFN mRNA and donor B or donor D rAAV6 at day 2 of culture and analyzed at day 10 of culture under conditions inducing myeloid differentiation with 50 ng/mL granulocyte colony-stimulating factor. Shown first on the left are uncorrected myeloid differentiating HPCs from the patient in whom no oxidase-positive cells were detected. Shown last on the right are myeloid differentiating HPCs from a healthy control, where 33% of cells fall into the oxidase-positive gate. Shown on the middle-left and on the middle-right panels, respectively, are myeloid differentiating minigene (donor B) or exon 2 replacement (donor D) corrected HPCs from the patient where significant numbers of oxidase-positive cells are detected with MFI approaching that of a healthy control subject.

Article Snippet: The harvested HPCs were further expanded in stage 3 media or stem cell media (StemSpan SFEMII medium, STEMCELL Technologies) containing 50 ng/mL stem cell factor, FLT-3L, thrombopoietin, 5 ng/mL interleukin 3 (IL-3), and 25 ng/mL IL-6.

Techniques: Functional Assay, Cell Culture