We have already used this kit and we can confirm the reproducibility of the kit in the final stage 4. However we would like to ask you 3 main points of interest:

Answer

1. We have observed that the cells between stage 3 and stage 4 show a low adherence efficiency in coverslips compared to the plate surface (both of them are treated with hES-qualified matrigel). Could you suggest how this could be improved?

In general, culturing cells on coverslips provides a significant advantage for imaging. We have found that placing coverslips in larger well formats increases the risk that the coverslip will move during medium changes, which in turn can cause cell loss. We find that when culturing on a coverslip is preferred, reducing the size of the culture well to restrict the possible movement of the coverslip improves the ability to perform successful differentiations. In addition, extra care should be taken to avoid damaging the cell layer during aspiration and medium exchanging. Damaging the cell layer by accidental pipette contact can lead to loss of the monolayer.

2. Are the cells at the end of stage 4 are comparable to the pancreatic endoderm (PE) or pancreatic endocrine precursors (PEP) cells, according to the characterization of the Rezania et al, 2014 paper?

The cells generated using the STEMdiff™ Pancreatic Progenitor Kit are best compared to the Pancreatic Endoderm (PE) as described in Rezania et al., 2014. As highlighted in this publication, a key difference between the PE stage (Stage 4) and the Pancreatic Endocrine Precursors (PEPs; Stage 5) is the expression of NEUROD1 and the restriction of the cells towards the endocrine lineage. While we observe an upregulation of NEUROD1 in Stage 4 compared to earlier stages of differentiation, this level of upregulation is not to the level demonstrated in Stage 5 cells as shown in Rezania et al., 2014. Furthermore, through in vivo maturation studies that were performed in collaboration with Dr. Timothy Kieffer at The University of British Columbia, we found that Stage 4 cells generated using the STEMdiff™ Pancreatic Progenitor Kit were capable of maturing towards both endocrine and exocrine (including ductal) lineages, suggesting that they had not yet been restricted solely to the endocrine pancreatic lineage.

3. Could you suggest a protocol for the in vitro differentiation of the progenitor cells produced by the kit into mature pancreatic endocrine β- cells?

There are three publications in the field that have thus far demonstrated in vitro protocols to generate maturing insulin-producing beta cells from pancreatic progenitor cells. These are Rezania et al., 2014; Pagliuca et al., 2014; and Russ et al., 2015. To test whether pancreatic progenitor cells generated using the STEMdiff™ Pancreatic Progenitor Kit are capable of maturation towards functional beta cells, we cultured Stage 4 cells in the Stage 5 and Stage 6 media as described in both the Rezania et al. and Pagluica et al. publications. In both experiments, we observed increases in insulin and glucagon gene expression by qPCR (see Figure 6 here). These data suggest that either protocol is compatible with pancreatic progenitor cells generated using this kit.