All About Differentiation – Tools and Tips for Success

By on July 24, 2014
Expert Session Summaries

We recently finished our Ask the Expert discussion on Efficient cell specific differentiation systems for iPSC. This week we had many interesting questions and helpful protocol suggestions for differentiation of iPSCs into several cell types including dopaminergic neurons, epidermal cells cardiomyocytes and hepatocytes. Answers also provided information on topics involving detecting differentiation using imaging, cell density, culturing without feed layer, single-cell sequencing, confirming and evaluating differentiation, and the use of small molecules in differentiation.

The process of directed differentiation is critical to getting the desired cell types and numbers needed for drug screening, translational cell therapy and regenerative medicine applications. Most of the existing methods of differentiation are suboptimal, involving laborious mechanical and manual steps leading to issues of reproducibility and reduced efficiency in downstream processing of functionally mature lineages. The complex developmental process of differentiation and the challenges associated need to be efficiently deciphered in order to successfully direct the hPSC differentiation to target cell types.

During this Ask the Experts session, we discussed the challenges associated with hPSC differentiation into different cell types and how these processes can be efficiently simplified with tools and cGMP cell culture media systems for robust, efficient and scalable differentiation.

This Ask the Expert session was hosted by Dr. Mohan C. Vemuri, the Director of Research and Development for Cell Biology at Thermo Fisher Scientific. In this capacity, Dr. Vemuri leads R&D activities in stem cell product development in the areas of human iPSC, adult stem cells, immune cells and cell lineage specific differentiation in GMP environment for research use and subsequently for use in cell therapy with regulatory compliance.

Prior to this role, Dr. Vemuri served on the faculty at Children’s Hospital of Philadelphia where his research efforts focused on developing improved methods for fetal transplantation of engineered hematopoietic stem cells for blood and bone marrow transplantation. Dr. Vemuri previously served on the faculty at Thomas Jefferson Medical School, where he developed cell screening assay systems for Parkinson’s disease drug discovery. Dr. Vemuri collaborates with researchers in academia and industry, striving towards the successful utilization of stem cells in regenerative cell therapies. He holds a Ph.D in Cell Biology from Sri Venkateswara University in India and performed his postdoctoral work at the National Institutes of Health.

Dr. Vemuri has authored over 50 publications and has edited or co‐edited several stem cell focused books, including Stem Cell Assays, Regulatory Networks in Stem Cells, MSC assays and applications, MSCs and Cell Therapy, Neural Development and Stem Cells and most recently, Pluripotent Stem Cell Assays by Springer Press.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – Efficient cell specific differentiation systems for iPSC.

Question:

I am looking for a system that can detect differentiation using imaging techniques that are not invasive to the culture. Do you have any recommendations?

The Answer:

There are two possible approaches you could use for a system to detect differentiation using non-invasive imaging techniques. The first is simple phase contrast microscopy. Using this approach you would look for the spike appearance of differentiating cells. Using imaging tools you can mask the undifferentiated cells and mark the differentiated cells. You may want to look in to an imaging tool hosted by the NIH which would also allow you to quantify this method. The second method I would recommend is utilizing a tool offered by Life Technologies – the Alkaline Phosphatase Live Stain. This is a non-invasive technology by which undifferentiated cells can be stained in 20 minutes or less by fluorescence. Cells that are not picked by this stain are differentiated cells. The Alkaline Phosphatase Live Stain is a quick and easy tool to use for detecting differentiated cells.

Click for more information about the Alkaline Phosphatase Live Stain – http://www.lifetechnologies.com/order/catalog/product/A14353?CID=search-product

Click to see a selection of tools for live and fixed cell staining of pluripotent stem cells – http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/pluripotent-stem-cell-detection/pluripotent-stem-cell-antibodies.html?icid=cvc-stemcelldetection-c2t1

Question:

What is the best differentiation medium for hepatogenic differentiation of iPS cells derived from human skin fibroblasts?

The Answer:

Several researchers are working on iPSC to hepatic differentiation. Commonly, the first step required is definitive endoderm differentiation. Following the definitive endoderm fate, the cells need to be taken through posterior foregut fate and then towards hepatic. There is no one culture media to accomplish these different steps available today. Currently this process involves the use of specific media systems at each of the step during the differentiation process.

The following reference may be useful to you, however we have not performed this protocol in our labs and cannot comment on reproducibility.
• Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells (PMID: 23681950) – http://stemcellstm.alphamedpress.org/content/2/6/409.full.pdf+html

Question:

Our lab is differentiating iPSCs into cardiomyocytes and we have initial success for a few times then our efficiency drops. The only thing that is changing is cell density. Do you think the cell density is affecting our efficiency and if so, what kind of passaging do you recommend for the best efficiency?

The Answer:

iPSC quality of the starter population and seeding densities are critical for cardiac differentiation depending on the protocol you are adopting for your differentiation to cardiomyocytes. If you have expanding populations in the beginning during differentiation, the stochastic ratios alter and could lead to less robust methods of generating desired cell types. Similarly if the starting quality of iPSCs is poor, it will eventually result in poor differentiation of desired lineages.

One product you could try to improve your efficiency is the new PSC Cardiomyocyte Differentiation kit offered by Life Technologies. This kit offers a simple and reproducible three-step protocol to generate cardiomyocytes. Early cardiomyocytes can be generated as early as eight days and can be matured for the length of time required. Cells differentiated using this kit are applicable to studies including cardiotox screening, cardiac development, disease modeling, etc.

Learn more about the PSC Cardiomyocyte Differentiation Kit – http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-differentiation/culture-systems-reagents-cardiomyocyte-differentiation.html

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