Figure 2: Ectopic expression of Gata6 in hiPSCs induces a heterogeneous multi population niche including CXCR4⁺ definitive endoderm.

(a) Cell line A expresses EBFP2 (blue fluorescent protein) constitutively and GATA6-2A-EGFP (GATA6 and green fluorescent protein) in a Dox-inducible fashion. Cell line B expresses mKate2 (red fluorescent colour), constitutively. (b) Cell line A and B were mixed and seeded at a 9:1 ratio and induced with Dox (1,000 ng ml⁻¹) (c). By day 5, the two cell lines proliferate and segregate to distinct high EGFP⁺(also EBFP⁺) and mKate2⁺ subpopulations. When the blue cells express high EGFP (high GATA6), they look blue and green. If they express low amounts of EGFP they look blue (very weakly green) and stay together with mKate⁺ population. (d) Undifferentiated uninduced hiPSCs at day 3 are enriched in Nanog. (e) Dox-induced ectopic expression of lentivirally delivered GATA6 leads to segregation into GATA6⁺ or Nanog⁺ subpopulations. hiPSCs were seeded as single-cell suspension(+Dox, 1,000 ng ml⁻¹). Insets show that Nanog⁺ are weakly GATA6⁺. (f) Brachyury⁺ (also known as T) cells are interspersed with FoxA2⁺ endodermal cells at day 3 for a Dox-induced experiment. (g) Immunostaining for HA, FoxA2 and Nanog. (h) Analysis of the immunostaining: GATA6-HA induces endoderm, but is suppressed in the Nanog⁺ cluster. Cells with low Nanog levels require less GATA6 (HA epitope) to differentiate into endoderm (FoxA2). Scale bars, 200 μm.