Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9hi, SSEA-1− Cells in Embryonic Stem Cell-Derived Embryoid Bodies
Figure 5
Derivation of E-RoSHL1 cell line from CD9hi, SSEA1lo cell population in EB culture.
a) One week after plating 5 day old EBs on gelatinized culture plates, the cells were harvested, labelled with anti-CD9 antibody conjugated with PE and anti-SSE4-1 antibody conjugated with FITC, and sorted.
CD9hi, SSEA1lo cells in the P1 quadrant were selected as putative RoSH cells and plated on gelationized feeder plate;
b) Morphology of semi-confluent E-RoSHL1 and E-RoSH2.1.
Bar represent 15 µm; c) Pairwise comparison of global gene expression between E-RoSHL1 and E-RoSH cells.
Global gene expression analysis were performed by hybridizing total RNA from two biological samples each of E-RoSHL1, E-RoSH2.1 and E-RoSH3.2 with Illumina BeadArray containing about 24,000 unique features;
d) Flow cytometry analysis of E-RoSHL1 cells for endothelial markers before (righ panels) and 60 hours after (left panels) induction of differentiation by plating cells on matrigel.
Nonspecific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies or with secondary antibody alone;
e) Morphology of E-RoSHL1 culture one week after induction of differentiation by plating on matrigel. Bar represents 50 µm.