Fig. 3

Differentiation of hPSCs into different populations of MPCs using protocol 2. Immunofluorescence analysis of MPCs generated using protocol 2 showing the generation of a large number of PDX1⁺/NKX6.1⁺ cells (a). Further investigation of the PDX1⁻/NKX6.1⁺ structures, which were generated after dissociation of the differentiated cells, showed that this population rearranged in 3D rounded structures (arrowheads) enclosed and surrounded by PDX1⁺/ NKX6.1⁺ expressing cells (b, c). These centrally located cellular 3D aggregates displayed an expression of NKX2.2 (arrow) in contrast to the surrounding cells that showed little or almost no NKX2.2 expression (d). NKX2.2 was expressed in some NKX6.1 expressing cells (arrowhead) in a 3D structure (e). The square indicates cells magnified in the insets on the right. All data shown are representative results from at least three independent experiments. Scale bars = 100 μm