Figure 1. eTeSR™ Improves the Genetic Stability of hPSC Cultures Maintained Long-Term Using Single Cell Passaging

Individual clonal sub-lines were derived from H1 and H9 hPSC lines using single-cell deposition. Clones (represented by each rectangle) were screened for recurrent abnormalities using the hPSC Genetic Analysis Kit prior to initiating long-term single-cell passaging experiments. Clones from each line were then cultured for 20 weeks (30 passages) using an automated platform in either eTeSR™ or media developed and optimized for aggregate passaging. After 20 weeks, clones were screened again using the hPSC Genetic Analysis Kit and any 20q and 12p abnormalities detected were confirmed using FISH. hPSCs cultured with routine single-cell passaging demonstrated significantly fewer clones (4% vs. 50%) that developed common recurrent abnormalities when maintained in eTeSR™, compared to media optimized for aggregate-based passaging.

Figure 2. hPSCs Cultured As Single Cells in eTeSR™ Exhibit Improved Cell Expansion and Attachment

(A) Representative culture morphology of undifferentiated human ES (H9) and iPS cell lines (SCTi003-A and WLS-1C) maintained in eTeSR™ using single-cell passaging on Corning® Matrigel®-coated plates. hPSCs maintained in eTeSR™ display a homogeneous morphology that is consistent between hPSC lines. (B) Four hPSC lines were single-cell passaged in either mTeSR™1, mTeSR™ Plus, or eTeSR™ on Corning® Matrigel®-coated plates using TrypLE™ Express for 11 passages. Cultures were maintained using a 4-5-5 day passaging schedule (see manual) using a restricted feeding schedule for mTeSR™ Plus and eTeSR™, and daily feeding for mTeSR™1. eTeSR™-maintained cultures show improved expansion rates compared to mTeSR™1 and mTeSR™ Plus when using single-cell passaging. (C) Representative whole-well images of the STiPS-R038 hPSC line 24 hours post-plating stained with Hoechst 33342. hPSCs were seeded at 15,000 cells/well in either mTeSR™1, mTeSR™ Plus, or eTeSR™ supplemented with 10 μM Y-27632 on Matrigel®-coated 96-well plates. Plates were fixed, stained for Hoechst 33342, and imaged using the IXM Micro (Molecular Devices).

Figure 3. eTeSR™ Supports Efficient Gene-Editing in hPSCs

Using the ArciTect™ CRISPR-Cas9 system, GFP-labeled hPSC lines (H1 and WLS-1C) were electroporated in the presence of a ribonucleic protein (RNP) complex consisting of Cas9 and a guide RNA targeting the GFP sequence. A donor template was also included encoding a two base pair change, resulting in the conversion of the GFP sequence to a BFP sequence, which can be determined using flow cytometry. Data points in the graph represent the gene-editing outcomes of three independent experiments. Both knock-out (A) and knock-in (B) conditions show effective gene-editing with eTeSR™-maintained cultures, displaying a higher knock-in efficiency compared to mTeSR™ Plus-maintained cultures. The mock condition shows eTeSR™-maintained cells that have been incubated with the RNP complex and donor template but have not been electroporated. Error bars represent standard deviation of replicates.

Figure 4. High Cloning Efficiencies Are Achieved Following Single-Cell Deposition Using eTeSR™ Supplemented with CloneR™2

(A) Four hPSC lines were seeded at 1 cell/well using single-cell deposition in eTeSR™ supplemented with CloneR™2 on Vitronectin-XF™-coated 96-well plates. Each data point represents one 96-well plate over three independent experiments. The average cloning efficiency across all four cell lines in eTeSR™ was 51 ± 3%. (B) Representative 96-well plate imaged at Day 8 post-plating, showing colonies generated in eTeSR™ using single-cell deposition. Orange circles highlight wells with a colony present.

Figure 5. hPSCs Maintained in eTeSR™ Express Markers of the Undifferentiated State and Differentiate Efficiently to Three Germ Layers

(A) hPSCs maintained in eTeSR™ express markers of the undifferentiated state with > 98% of cells staining positive for OCT4 and TRA-1-60, as determined by flow cytometry at passage 12 and 20. Data points represent the percent positive at each time point with bars representing the average over the two time points for each cell line (n = 4 cell lines). (B) Four hPSC lines were maintained in eTeSR™ for at least 7 passages before being differentiated toward the three germ layers using the STEMdiff™ SMADi Neural Induction Kit, STEMdiff™ Mesoderm Induction Medium, and STEMdiff™ Definitive Endoderm Kit. All hPSC lines demonstrated efficient differentiation to all three lineages. Error bars represent the mean and standard deviation of three technical replicates.

Figure 6. Global Gene Expression Profiles Are Comparable Between eTeSR™ Single-Cell Passaged Cultures and mTeSR™ Plus Aggregate Passaged Cultures

Whole transcriptome analysis was performed using the Illumina NextSeq 500 on three hPSC lines passaged as either aggregates in mTeSR™ Plus or as single cells in eTeSR™. (A) PCA analysis shows that samples cluster by cell line with minimal effect from cell culture medium and passaging technique. (B) A heat map of global gene expression showing comparable gene expression between conditions. No gene ontology or signaling pathway enrichment was detected (n = 3 hPSC lines).