Fig. 3

General labelling strategy for generation 2 T2EC cells identification. On day 1, a population of mother-cells was stained using CTV. The CTV positive population was divided into six subgroups, and each subgroup was uniquely barcoded using a combination of CFSE and CTY concentrations, resulting in six distinct fluorescent barcodes. One mother-cell from each subgroup was then retrieved and combined in a well for approximately 24 hours of culture (resulting in a total of six mother cells, each with a unique fluorescent barcode). On day 2, following the first division, a fourth dye, CTFR, was introduced to label sister-cells with a different intensity in order to be able to discriminate the cells relationship after the subsequent division. On day 3, cells which underwent 2 divisions, determined by the intensity of CTV, were sorted into single-cells, and their fluorescent intensities for CTY, CFSE and CTFR signals were recorded. Finally, a dedicated script was used to infer the relationships of cells based on the fluorescent intensities (see "Methods" section)