stemdiff definitive endoderm kit tesr-e8 optimized  (STEMCELL Technologies Inc)

Journal: Stem Cell Reports

Article Title: High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs

doi: 10.1016/j.stemcr.2019.04.014

Figure Lengend Snippet: Generation of CFTR dTomato Reporter iPSCs and Differentiation into Intestinal Epithelia (A and B) PCA analysis. Global comparison of different hiPSC lines (healthy donor 1 [MHHi001-A, non-edited, shown in black], donor 2 [MHHi002-A-derived cell lines; CF parental line shown in green and seamless corrected line shown in blue], healthy donor 6 [MHHi006-A-derived line, genetically edited, shown in gray]) by PCA show a clear clustering of the two isogenic lines based on donor 2 and more divergence from the two other hiPSC lines (donor 1 and donor 6), both in the undifferentiated state on day 0 (A) and on day 15 of directed differentiation (B). n = 3 independent differentiations for each line. For further analyses, also see Figure S1 . (C) Schematic illustration of the targeting strategy applied for TALEN-mediated integration of the neomycin-2A-dTomato nuc reporter cassette. The CFTR gene is targeted at exon 1 in-frame with the ATG start codon. Gray arrows indicate primers used to confirm targeted integration of the reporter construct. NeoR , neomycin resistance gene; T2A, self-cleaving peptide sequence; dTomato nuc , dimeric variant of dsRed fluorescent protein coupled to nuclear membrane location signal; frt, flippase recognition target site; PGK, phosphoglycerate kinase promoter; HygroR , hygromycin resistance gene. (D) Schematic illustration of the protocol for iPSC differentiation toward CFTR dTomato -expressing intestinal epithelial cells. iPSCs cultured as monolayer were induced to DE via the STEMdiff Definitive Endoderm Kit. Following the application of dorsomorphin and IWP-2, the intestinal specification was promoted using BMP4, CHIR, and FGF10. (E) Representative fluorescence microscopy showing emerging dTomato nuc -positive cells during the time course of directed differentiation of CFTR KO/Tom cells toward epithelial cells. BF, bright field. Scale bars represent 100 μm. (F) Quantification of dTomato nuc -positive cells on day 15 of directed differentiation of four CFTR -dTomato nuc reporter iPSC lines toward intestinal epithelial cells (mean ± SEM from three independent differentiations). (G) Fold change of dTomato nuc and CFTR expression during the time course of directed differentiation of four CFTR -dTomato nuc reporter iPSC lines (mean ± SEM from three independent differentiations). The knockout allele in the CFTR KO/Tom line was generated via deletion of the Kozak sequence, which will result in mRNA transcription but not in CFTR protein translation (see also Figure S1 E).

Article Snippet: In a stepwise differentiation protocol, iPSCs from monolayers in essential 8 (E8) medium were first differentiated to DE via a STEMdiff Definitive Endoderm Kit (TeSR-E8 Optimized) from STEMCELL Technologies.

Techniques: Comparison, Derivative Assay, Construct, Sequencing, Variant Assay, Membrane, Expressing, Cell Culture, Fluorescence, Microscopy, Knock-Out, Generated