Fig. 4

Effect of cell density on PDX1 and NKX6.1 co-expression in protocol 2-dissociated pancreatic progenitors. a–c Differences in morphology of pancreatic progenitors dissociated and re-plated at either half or quarter of the definitive endoderm (DE) density. Note the 3D structures that appeared at the quarter DE density re-plated culture (b, c). Generation of high PDX1⁺/NKX6.1⁺ co-positive population in half DE density (d) and a novel PDX1^–/NKX6.1⁺ population in quarter DE density re-plated culture (e, f). g Graphical summary for generation of the monolayer PDX1⁺/NKX6.1⁺ population and compact three-dimensional (3D) structures comprising of a PDX1^–/NKX6.1⁺ population at a half and a quarter of the endoderm re-plating densities. The generation of the two pancreatic progenitor populations is a result of a combined effect orchestrated by endodermal cell dissociation, cell density, and extracellular matrix on pancreatic differentiation. All data shown are representative results from at least three independent experiments. Scale bars in a, b and e = 50 μm and d, c and f = 50 μm