Journal: Nature Communications

Article Title: A scalable and cGMP-compatible autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa

doi: 10.1038/s41467-024-49400-z

Figure Lengend Snippet: a Uniform manifold approximation projection (UMAP) of integrated scRNA-seq of H9 ES cell differentiation towards iSCs on Day 7 reveals three lineages: ectoderm (orange), mesoderm (blue), and neuroectoderm (green). b Violin plots depicting relative expression (RPKM) of representative ectoderm, mesoderm and neuroectoderm genes across scRNA-seq clusters. c ITGA6 expression analyzed via RNA-seq in H9 ES cells during iSC differentiation. Time points as indicated (D: day; n = 2 biological replicates; NHKs used as positive control n = 1). d Flow cytometry of day 45 unsorted H9 ES cells. Cells double positive for ITGA6 HI PE (y-axis) and K14 HI FITC ( x axis) are in high gate (red) and lower ITGA6/K14 LOW expressing cells are in low gate (blue); expression of K18 (PER-CP; x axis) in subpopulations (right). n = 1. e Bright-field image (top left) of FACS sorted ITGA6 HI H9 ES cell-derived iSCs expanded and used for organotypic stratification (top right). Note normal polarization and stratification; Collagen 7 (green), Involucrin (red), DAPI (blue); additional images, K14, K10 (red). Bright-field image (bottom left) of unsorted H9 ES cell-derived iSCs used for organotypic stratification (bottom right). Note disorganized layering and stratification. n = 1, scale indicated. f iPS cell to iSC differentiation strategy employing cell enrichment via AutoMACS pro-separator, following a defined cGMP-compatible protocol. g Flow cytometry analysis of % ITGA6 positivity measured before and after AutoMACS enrichment. h % Coupling efficiency (CE) determined by the equation (5) %CE= live sorted iSCs/iPS cell input*100. g , h Data from five independent differentiated patient cell lines ( n = indicated; mean, SEM;). i UMAPs of integrated scRNA-seq data from five patient- and H9 ES-derived iSCs post-ITGA6 enrichment and in vitro expansion reveal 8 clusters (C1–C8) comprising the DEBCT product. j Individual UMAP plots from overlaid scRNA-seq datasets from ( i ) with color scheme as indicated. Four ectoderm (C1, C2, C4, C7), 3 mesoderm (C3, C8, C5), and 1 melanocyte/neuroectoderm-derived (C6) clusters were identified. C5/C8 clusters indicated by dotted outline were present at variable quantities (see text, Fig. and Supplementary Fig. ). Source data are provided as a Source Data file. Panel f created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: After wash steps, cells were fixed and permeabilized (eBioscience™ Intracellular Fixation & Permeabilization Buffer Set, Cat # 88-8824-00), then stained for antibodies of interest for 30 min at 4 °C (FITC anti-K14 (CBL197 F, Millipore), PerCP anti-K18 (NB120- 7797, Novus), ITGA6 (PE anti-CD49F Cat #555736, BD)), all at 1:100 dilution.

Techniques: Cell Differentiation, Expressing, RNA Sequencing Assay, Positive Control, Flow Cytometry, Derivative Assay, In Vitro