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 last week’s 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.
[/one_third]This week, Dr. Michael Riedel will be answering your questions about pancreatic cell differentiation including how to use the STEMdiff™ Pancreatic Progenitor Kit to ensure high efficiency differentiation and generate pancreatic progenitors for pancreatic research. If you have any questions regarding differentiating to the endoderm lineage, Dr. Riedel is happy to discuss that as well as he also oversees the STEMdiff™ Definitive Endoderm Kit that can be used to generate multipotent definitive endoderm (DE) cells.
Have you seen differences in differentiation potential between ES and iPS cells? What about reproducibility?
0 0 0 Total: 0 The STEMdiff™ Pancreatic Progenitor Kit was optimized with reproducibility in mind. We designed both the medium formulation as well as the protocol to maximize the efficiency of differentiation across multiple human embryonic stem (ES) and induced pluripotent stem (iPS) cell lines (see Figure 2 here for the supporting data). At STEMCELL … Continued
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?
0 0 0 Total: 0 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 … Continued
I’m using the protocol published by Rezania et al. (2014) which uses a seven-stage protocol to differentiate hESCs to insulin-producing beta cells. How does your kit’s protocol compare to the published one?
0 0 0 Total: 0 The recent publications by Rezania et al. and Pagliuca et al. describe culture methods for the generation of maturing pancreatic beta cells. These maturing beta cells express insulin and are able to secrete it in a glucose-dependent manner, although with altered secretion kinetics. The cells generated at the end of … Continued
After researching protocols for differentiation to pancreatic cells I have found a couple that use chemically defined medium. I have not tried these, but haven’t had much luck weaning cells from serum or other animal products in the past. Is your kit chemically defined? Have you seen any problems or even differences in the cells cultured with serum vs. those cultured without.
0 0 0 Total: 0 There is significant interest in many biological fields to move away from the use of serum and other undefined components in cell culture media. It is thought that removing undefined components will help reduce variability associated with lot-to-lot changes in the composition of these undefined components. In addition, stockpiling ‘good’ … Continued
0 0 0 Total: 0 The STEMdiff™ Pancreatic Progenitor Kit is designed and suitable for research use applications only. The medium is defined and serum-free but is not intended for clinical use.
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?
0 0 0 Total: 0 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 … Continued
We have been testing different protocols to generate pancreatic progenitors from human embryonic stem cells and while we get a good percentage of cells expressing PDX-1 we have much variation in expression levels of NKX6.1. Have you seen this variation too? Does your kit correct for this variation?
0 0 0 Total: 0 Achieving robust and high levels of NKX6.1 expression in human pluripotent stem cell-derived pancreatic progenitor cells has been a significant challenge in the field. As demonstrated in the 2013 publication from Dr. Tim Kieffer’s Lab at the University of British Columbia, in collaboration with BetaLogics (Rezania et al., 2013), enriching … Continued
I’m interested in generating a diseased iPS cell line. Will this kit be compatible with my new iPS cell line?
0 0 0 Total: 0 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 … Continued
Is using your kit easier than using currently published protocols? Should we expect a similar learning curve when starting to use the kit?
0 0 0 Total: 0 The STEMdiff™ Pancreatic Progenitor Kit is supplied as two basal media and six pre-mixed supplements, all of which have been rigorously performance tested in differentiation assays to assure the user receives a high performing product. The user is simply required to combine the appropriate basal medium and supplement(s) and apply … Continued
Our lab is evaluating the differentiation potential of several iPSCs generated using patient cells. What do you recommend as the most high thoughput and consistent method?
0 0 0 Total: 0 To verify that human induced pluripotent stem cells are indeed pluripotent, there are several available methods. Each have their advantages and disadvantages. For example, the teratoma assay can provide information about the trilineage differentiation potential of human iPS cell lines in a single experiment, but has the disadvantage of being … Continued
We need to generate iPSCs from Type 1 diabetes patients then differentiate. How would this fit into your kit workflow?
0 0 0 Total: 0 STEMCELL Technologies has developed a comprehensive suite of tools to generate, characterize, expand and differentiate human induced pluripotent stem (iPS) cells. The STEMdiff™ Pancreatic Progenitor Kit is the latest tool within this workflow that provides robust and reproducible differentiation across different human iPS cell lines. The kit has been validated … Continued
How important is the confluence when beginning the differentiation protocol and why do they need to be single cells and not colonies or monolayer?
0 0 0 Total: 0 Starting confluence can be a critical factor in the efficient generation of definitive endoderm. This is especially important when the differentiation procedure starts with the plating of a single cell suspension of undifferentiated pluripotent stem cells. Because confluence can be a relatively subjective measurement, we prefer to plate a specific … Continued
We are trying this kit to differentiate human pancreatic precursors from H1 cells. We found that from Stage 3 the cells became multilayers. We stained stage 4 cells on cell culture plate, but because the cells clusters are thick, we can’t get clear images using common immunofluorescence microscope . How did you prepare samples for the Stage 4 immunohistochemistry assay? Did you culture the cells on cell culture plate, or on the coverslips? Did you get the images from Confocal Microscope?
0 0 0 Total: 0 During the 14 days of differentiation, there is significant proliferation of cells. A confluent monolayer (single cell depth) is achieved very early in the protocol, certainly by day 2 when the cells are effectively definitive endoderm. Proliferation continues throughout the remaining stages such that after plating 800,000 cells to start, … Continued
It’s become more and more difficult to get glass pipettes. Do you use glass pipettes or plastic pipettes for this kit of differentiation and for cell culture without antibiotics ?
0 0 0 Total: 0 The media comprising the STEMdiff™ Pancreatic Progenitor Kit are compatible with standard plastic micropipette tips. We recommend that the user practice sterile technique and avoid the use of antibiotics.
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:
0 0 0 Total: 0 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 … Continued