Paul Burridge, PhD, assistant professor, Department of Pharmacology, Center for Pharmacogenomics, Northwestern University, and Chengzu Long, PhD, assistant professor, Division of Cardiology, New York University School of Medicine, talk to contributing editor Tanuja Koppal, PhD, about some of the recent innovations in culturing human-derived induced pluripotent stem cells (iPSCs). They discuss best practices when setting up a new lab for iPSC work, some of the inherent challenges, and emerging applications.
Q: What’s the difference between working with stem cells and other mammalian cells? Any advice to scientists who are looking to set up labs for stem cell work?
Paul Burridge, PhD: I started working with human embryonic stem cells back in 2003, and since then things have progressed very quickly in the field. In 2008, we made our first human iPSCs and realized how easily these cells can be generated compared with embryonic stem cells. As we are interested in studying human disease at the single-cell level, generating patient-derived iPSCs is the most logical way forward. To do this work at Northwestern University, we built our large cell culture facility dedicated solely to iPSC work to reduce potential cross-contamination. Stem cells are easy to culture when you have robust, wellthought- out protocols, yet every stem cell lab does things slightly differently. We don’t use antibiotics when culturing iPSCs, so the training needs to be a little different and the aseptic conditions need to be a little more thorough than with other types of cell culture. People who work with iPSCs are very rigid in how they handle the cells, when they passage the cells, when to begin for differentiation; all that can be very challenging. You should have the mindset that it’s going to be hard work and time-consuming, and there isn’t much flexibility in the system. We don’t anthropomorphize the cells; this is a science, not an art.
The cost of the cell culture media, matrices, and routine maintenance of the cells tends to be more expensive for iPSCs. The media for iPSCs is about five times higher than that for traditional cell culture, and the commercial iPSC media is about ten times more expensive than the media we make in-house. Many of the big labs still buy commercial media because it is a big endeavor to make it in-house. It took a number of years for us to get everything formulated and quality tested. We go through about ten liters every week, so we wouldn’t be able to do all the work we are doing from a cost perspective if we bought commercial cell culture media. Our lot-to-lot variability is also as good or better than with commercial cell culture media because we make such large batches for our own use.
Q: What have been some of the biggest changes or improvements in stem cell work?
A: Stem cells have moved from being a boutique science to a standard technique that most of the labs can use. The questions and requirements for stem cell culture have changed. The progression of culturing cells from mouse embryonic fibroblasts to using synthetic cell matrices is one of the big changes. The use of chemically defined cell culture and differentiation media is also an improvement. One of the big developments in regard to assays has been the move from six-well plates to high-throughput 384-well plates. The type of work we do also has changed. Previously, we would work on techniques for reprogramming and differentiation of stem cells, and today we are working on patient-derived disease modeling and drug testing using CRISPR-based gene editing in stem cells. The access to whole genome sequencing has also changed what we do, allowing us to progress to pharmacogenomics studies. I am a developmental biologist by training, but I now work in a pharmacology department, and that’s indicativeof what’s happened in the stem cell field.
Q: Where are the current gaps and limitations in stem cell work and what’s being done to address them?
A: I’m sure everyone will agree that the most disappointing aspect of iPSC development is the matrix for cell growth. The technology that we use right now is what we used in 2003. All we have done is use a much lower concentration of the matrix. Despite many papers covering novel synthetic matrices, we have found that these are not that much better than the traditional substrates, and are too expensive. All of us would like to use a synthetic matrix or have our plates coated synthetically, but when you use about 200 plates a week it needs to be very cost-effective. We also are very interested in automation, but right now the equipment doesn’t exist for us to automate the way we would like to in a cost-effective way. The reprograming in iPSCs can be automated well, but automating the differentiation step is difficult. The cardiac lineage has by far the most advanced stem cell differentiation protocol, and there is room for the other lineages, such as endothelial cells and hepatocytes, to catch up. There has not been enough recognition in terms of the value of improving differentiation protocols.
Paul Burridge, PhD, is an assistant professor in the Department of Pharmacology at Northwestern University Feinberg School of Medicine and founding faculty of the Center for Pharmacogenomics. Dr. Burridge began his career in genomics and bioinformatics at the Sanger Institute. He completed a PhD in human stem cell biology in human development at the University of Nottingham before pursuing postdoctoral fellowships at the Johns Hopkins University Institute for Cell Engineering in pediatric oncology and then at the Stanford University Institute for Stem Cell Biology and Regenerative Medicine in radiology/cardiology.
When you set up an induced pluripotent stem cell (iPSC) lab, it has to be very clean and you have to decontaminate everything often. Bacterial contamination is easy to see, but every few weeks you have to test for mycoplasma contamination too. When you’re working with iPSCs, you have to assign a dedicated cell culture hood, incubator, refrigerator, and water bath so you don’t contaminate the iPSCs with other cell types or with iPSCs from a different patient. When you get cells from a different lab, you must create a master tube for those cells, so if anything goes wrong you can always recover the cells. You should keep the backup tubes in your own lab, but in a different liquid nitrogen tank. This is painful, but it’s important to set this up to store your most important samples and cells.
We started doing gene editing studies in mouse models, then we realized that to do real translational work, it’s also important to do these studies in human cells. So three years ago we started working with human-derived iPSCs. Culturing iPSCs can be very time-consuming, but new technologies have made it easier to culture these cells. Previously, you had to use mouse feeder cells to culture iPSCs, but now you have feeder-free systems, where all you have to do is plate your cells on some biological matrix to culture them. While setting up a new lab, you don’t want to do too much troubleshooting and you don’t want to spend too much time making your own cell culture media. The commercial media are costlier than the homemade ones, but they are more stable and reliable. Once the lab is set up and you need a lot of culture media to keep your cells growing, then you can start making it yourself. When deciding which cell culture media to buy, do your homework and ask people what has worked for them. Ask vendors to send you samples of their media so you can run quick experiments and see what works. There are many new reagents out there that you can use to grow and enhance your cells.
A perfect match: gene editing and iPSCs
iPSCs can be obtained either from skin fibroblasts or from blood, and the reprogramming efficiency is different depending on the source. Blood samples are easier to get from the patient, but the reprogramming efficiency is lower in iPSCs derived from blood than from the skin fibroblasts. The genetic background of iPSCs is different for different patients, so when you see phenotypes in iPSCs, you are not sure whether they are due to a particular mutation that you are studying. With gene editing, you can correct a certain mutation in iPSCs and then compare it to its isogenic pair, which has the same genetic background. When it’s difficult to get the cells from a patient, you can use wild-type iPSCs and generate the disease mutation using gene editing tools. You can now generate the disease model in your lab and compare it to the wild-type iPSCs. This is a very fast, robust, and high-throughput way to study diseases. Gene editing in iPSCs is simple, but tricky. However, it’s possible to pick up all the various strategies and concepts with some prior knowledge of molecular biology and cloning. Culturing the iPSCs needs more advanced training compared with regular mammalian cell culture.
Although iPSCs are being used to generate disease models in a dish, it is still controversial because some models are not quite accurate. For instance, to study cardiovascular disease you can use iPSCs to get beating cardiomyocytes, but these cells are premature and neonatal without the organized structure that you see in adult cardiomyocytes. We need the right in vitro cell culture conditions for iPSCs to differentiate into adult cells. This is the same for neuronal cells.
Chengzu Long, PhD, a newly appointed principal investigator and assistant professor, is currently conducting research on advancing novel genome editing technology to model and treat human genetic diseases at New York University School of Medicine’s Leon H. Charney Division of Cardiology. After receiving his bachelor’s degree in bioengineering, he earned a Master of Science degree in microbiology and then worked at the National Institute of Biological Science, Beijing, where he studied pathogenhost interactions. Dr. Long then went to the University of Texas Southwestern Medical Center for his doctoral work and joined Dr. Eric Olson’s laboratory to study mechanisms of degenerative disease using mouse models with genetic modifications.