Figure 6. In vitro three dimensional organotypic culture and in vivo expression of type VII collagen.

a-c 3D organotypic culture. Uncorrected or base edited fibroblasts from c.553 patient cells were layered with transformed keratinocytes on a supportive matrix and stained by hematoxylin and eosin. a Uncorrected fibroblast cell 3D culture. The red arrows show the detached epithelial layer due to a fragile dermal epidermal junction that is structurally deficient when the culture contains uncorrected fibroblasts. b ABE corrected cell 3D culture. Improved structural integrity without epithelial layer detachment (green arrows) was observed when ABE corrected cells with restored C7 were employed. c Epithelium detachment quantification. The percent of detached epithelia observed by microscopy and scored by an expert was quantified for eight and three experimental replicates for uncorrected and ABE corrected fibroblasts, respectively. Mean and standard deviation are graphed and p value from Student’s t-test are shown. d-e In vivo teratoma. iPSC were injected into immune deficient mice and representative images of d COL7A1 defective and e base edited iPSC teratoma derived skin equivalents are shown. Both RDEB null and base edited samples were stained with polyclonal anti-human type VII collagen (red) and anti-human cytokeratin (CK5; green) antibodies as well as DAPI nuclear stain. Scale bars=50 μm. At inset, lower right is the base edited nucleotide that was observed following amplification of human COL7A1 DNA from the in vivo teratoma tissue.