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While observing PDX-1 expression is a good sign, it is not sufficient to confirm successful generation of pancreatic progenitor cells. PDX-1 expression is also observed in neighbouring regions outside the developing pancreas, including the stomach and duodenum. It is critical to ensure that the PDX-1-expressing cells are of the pancreatic lineage. The best way to do this is by checking for co-expression of NKX6.1, as upregulation of both of these transcription factors is indicative of the transition to the pancreatic progenitor stage, and is observed in the human fetal pancreas at around 9 weeks of gestation. During human pluripotent stem cell differentiation to pancreatic progenitor cells, PDX-1 expression is upregulated during Stage 3, or between days 6 and 9 of the protocol. NKX6.1 expression is typically low at this stage, increasing significantly by the end of Stage 4. Additional markers that can also be used to ensure your cells are pancreatic progenitors include SOX9 and PTF1α. See Figure 3 on this page for a gene expression profile of these markers throughout the stages of human ES and iPS cell differentiation to pancreatic progenitor cells. To ensure efficient generation of PDX-1+/NKX6.1+ pancreatic progenitor cells, we recommend using our new pancreatic progenitor differentiation kit.