Figure 1.

Single-cell selection using the STRIPPER Micropipetter results in homogenous hPSC clones

(A) Phase and brightfield image panels show a single cell picked by the micropipetter tool. Top Panel: sparse single cell suspension. Scale bar: 200 μm. Bottom panel: a single cell (red arrow) being picked by the micropipetter. White arrows show the cells not being picked. Scale bar: 400 μm.

(B) Phase image panels show the establishment of healthy hPSC clones. hPSC clones with a differentiated cell population (top left of dotted line) were first dissociated and then single-cell selected using micropipetter. A suboptimal colony (red) or a colony with healthy cell morphology (green) forms 10 days after single cell selection. Scale bar: 200 μm.

(C) Fluorescence image panel shows the whole process of a GFP reporter hPSC line formed from a single cell. A ubiquitous GFP reporter was knocked in the AAVS1 (safe harbor) locus in a male control iPSC line (CON2E)⁵ using transcription activator-like effector nucleases (TALEN) gene editing. Top panel shows a colony containing both GFP positive and negative cells before single cell picking. Bottom panel shows colonies 1-, 2-, 5- and 8-days post picking. Images were taken using an EVOS FL Auto Imaging System (Thermo Fisher Scientific). Scale bar: 200 μm.

(D and E) The percentages of surviving clones of multiple hPSC lines including hESCs (in circles) and hiPSCs (in squares) after single-cell cloning. Female lines: WA-09, and SCN1B 6b/4A (unpublished cell line); Male lines: WA-01, and UM-139-2/4–6⁶). (D) On average, 51.2% of single cells of reporter hPSC clones resulted in viable clones (n = 19 experiments, SEM = 3.5%). (E) On average, 53.9% of single cells resulted in viable clones after CRISPR/CAS9 genome editing. Gene targets included POGZ,¹FMR1,⁶KCNH2,⁷RAI1 (unpublished cell line), KDM5C (unpublished cell line), ANK3 (unpublished cell line), STXBP1 (unpublished cell line) and SYNGAP1 (unpublished cell line) (n = 18 experiments; SEM = 3.8%).