We recently finished our Ask the Expert discussion on Differentiating Human ES and iPS Cells to Pancreatic Progenitor Cells. During this Ask the Expert session, we discussed the steps involved in differentiation of ES and iPSCs to pancreatic progenitor cells. Specific topics included generated diseased induced pluripotent stem cell lines, evaluating differentiation potential, using a kit vs. published protocols, methods for confirming differentiation, and starting confluence.

Differentiating human embryonic stem (ES) or induced pluripotent stem (iPS) cells to pancreatic progenitors and downstream differentiated cells is a challenging and labor-intensive process. With the availability of a new differentiation kit that provides a standardized and straight-forward protocol, generating functional PDX-1⁺/NKX6.1⁺ pancreatic progenitor cells with high efficiency has become much easier.

In the post, Cool Tool – New Robust Kit for Efficient Generation of Functional Pancreatic Progenitor Cells, the challenges in the field of pancreatic cell research were discussed along with how the new STEMdiff™ Pancreatic Progenitor Kit can address them. This kit efficiently differentiates multiple human ES and iPS cell lines to pancreatic progenitors that are capable of maturing into endocrine and exocrine cells. The new tool allows researchers to study pancreatic development and disease without having to worry about optimizing the cell culture conditions.

This session was sponsored by STEMCELL Technologies and hosted by Dr. Michael Riedel. Dr. Michael Riedel is a Senior Scientist and the team lead for epithelial cell products in R&D at STEMCELL Technologies. Dr. Riedel’s team develops cell culture media and reagents for the isolation, expansion and differentiation of both primary and pluripotent stem cell-derived epithelial cells from tissues including the pancreas, liver, lung and intestine. Dr. Riedel obtained his PhD from the University of Alberta and received postdoctoral training at the University of British Columbia where his work focused on the development of cell and gene therapies for diabetes.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Differentiating Human ES and iPS Cells to Pancreatic Progenitor Cells.

Question:

In my lab we are using a published protocol for generating insulin producing beta cells. We are seeing significant variability, but am not sure if it is the protocol or some other factor. What is the level of variability you find with your kit and how simple would it be to train technicians to use this method instead? Is it easier than currently published protocols?

The Answer:

The STEMdiff™ Pancreatic Progenitor Kit is comprised of 2 basal media and 6 supplements that are used in specific combinations during the 2 week differentiation. Accompanying the kit is a detailed step-by-step protocol, including a schematic of the differentiation process. Because the medium is supplied in this modular format, there is no need for complex medium manufacturing on the part of the user. Simply mix the appropriate stage-specific supplement and basal medium and add it to your cells each day to achieve robust and efficient differentiation. With our own in house testing, we find that variability is quite low between experiments and across cell lines. For example, we measured the percent PDX-1⁺/NKX6.1⁺ cells at the end of Stage 4 across 10 different experiments using the H1 cell line. The data show an average differentiation efficiency of 69.3 ± 11.9% (mean ± SD) with a range of 46.9 – 83.3%. Within a given experiment, duplicate wells showed an average difference of 1.9 ± 1.2% (mean ± SD). Finally, we show that the average efficiency of differentiation is similar in human embryonic stem cell lines and induced pluripotent stem cell lines. For these data, please refer to Figure 2B here. For the kit protocol, please view this product information sheet.

Question:

I’m interested in generating a diseased iPS cell line. Will this kit be compatible with my new iPS cell line?

The Answer:

Reproducible differentiation across both human embryonic stem (ES) and induced pluripotent stem (iPS) cell lines is something we strive for when developing our STEMdiff™ kits. Demonstrating such reproducible differentiation across cell lines and across experiments is especially critical when studying human iPS cell lines from diseased patients or in studies where the genome of the cell line has been modified to mimic or correct a disease state. In these studies, changes in differentiation efficiency can be better attributed to the disease state or genetic modification, rather than the culture protocol, when a robust and reproducible system is used during the differentiation. We have presented data indicating similar efficiencies of differentiation in two human ES cell lines and two human iPS cell lines (see Figure 2 here) and we continue to add data for additional human iPS cell lines. Therefore, we have confidence that the STEMdiff™ Pancreatic Progenitor Kit is as likely to be compatible with newly derived human iPS cell lines as those protocols detailed in the literature.

Question:

My cells show good PDX1 expression – can I use this marker to confirm successful pancreatic progenitor differentiation? Can I use these cells for downstream differentiation to endocrine cells?

The Answer:

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.