Figure 6

Growth dynamics and karyotypic stability of hPSC cultures maintained on different culture surfaces. (A) (i) The number of days taken for cultures of H9, hiPS-NHF1.3 (NHF) and Genea-02 (GEN) hPSCs to reach passaging density on surfaces coated with Geltrex™ (GX), cRGDfK-PAPA (RGD), StemAdhere™ (SA) or Synthemax™ (SX) is presented alongside (ii) the mean number of cells harvested at the end of each passage and the (iii) viability of harvested cells, all grouped by surface type. Mean +/− SD, n = 30 (10 passages for each of 3 cell lines). *p \(\le \) 0.05, **p ≤ 0.01. (iv) The number of days taken for each passage to reach harvesting confluence is also presented for every passage of each cell line on each surface. (B) hPSC cultures were harvested with EDTA for continuing culture, and occasionally harvested with TrypLE™ Express for flow cytometric analysis. After treatment with a dissociation agent and pipetting with media, cells that remained adherent were physically removed with a cell scraper. The proportion of cells that required scraping was estimated by visual assessment at each harvest and is presented for cultures treated with (A) EDTA (n = 30 harvests) or with (B) TrypLE™ Express (n = 8 harvests for Geltrex™ and n = 6 harvests for each of cRGDfK-PAPA, StemAdhere™ and Synthemax™). Means are presented with scale bars representing standard deviations. *p \(\le \) 0.05, **p ≤ 0.01, ***p ≤ 0.001 by unpaired two-tailed t-tests. (C) G-banding karyotyping assessment of hPSCs maintained on defined culture surfaces is summarised. The type and frequency of karyotypic abnormalities observed are plotted and arranged. Karyotypes are represented by the following colours: black: partial duplication of chromosome 20 [add(20)], black stripes on grey: loss of one sex chromosome (45, X), dark grey: includes additional unidentifiable chromosome (47, + mar), grey crosshatch on pale grey: trisomy of chromosome 12 (+12), light grey: normal karyotype (i) by surface type and (ii) by surface type and then by cell line. Full karyotypes are shown in Supplementary Figures 11 and 12 (D) The PluriTest™ assay was applied to passage 10 cultures and generated a transcriptional karyotype, which compared the expression levels of groups of genes in sample data sets to homologous genes in the pluripotent reference data sets. A heat map is formed displaying cytobands in which overall gene expression levels are higher (darker red indicates higher expression) or lower (blue, with darker blue indicating lower expression) in samples than is predicted by the pluripotency model. Horizontal red lines (arrow) indicate upregulation of genes in each cytoband of an entire chromosome (Chr); vertical red lines (arrowheads) indicate examples of cytobands that are upregulated in all samples compared to the reference data set.