A New FACS Approach Isolates hESC Derived Endoderm Using Transcription Factors
(A) A representative experiment using two-channel FACS analysis of GATA4 and SOX17. Compared with the isotype negative control (bottom panels), three distinct cellular populations: SOX17+GATA4−, SOX17+GATA4+, and SOX17−GATA4+ are emerging gradually upon differentiation: at day 1, 13% are SOX17+GATA4+, increasing to 23% by day 3. Another significant population consists of 18% SOX17−GATA4+ at day 1 and 25% at day 3. (B) After 5 days of differentiation, using three-way multichannel FACS analysis for SOX17, GATA4, and CXCR4, we found that the SOX17+GATA4+ population dominates the culture (62%) and CXCR4 is expressed in 49% of the cells, most of which are SOX17+GATA4+CXCR4+ (41%). There are also approximately 27% GATA4+CXCR4− cells, which comprises the population of SOX17+GATA4+CXCR4− cells (21%). (C) Post sorting, FACS analysis demonstrated that 97% of day 5 SOX17+GATA4+CXCR4+ cells were positive for GATA4, 88% were SOX17 positive, and 95% were CXCR4 positive. This was consistent over 5 separate experiments. (D) Expression analysis using RT-qPCR demonstrates that day 5 SOX17+GATA4+CXCR4+ and day3 SOX17+GATA4+ cells have higher level of expression of SOX17, GATA4 and CXCR4 than unsorted fixed cells or day 3 SOX17−GATA4− (d3SOX17negGATA4neg) cells.