We recently finished our Ask the Expert discussion on Directed Differentiation of Pluripotent Stem Cells. This week we had several interesting questions and helpful tips on topics including challenges with reproducibility of differentiation protocols, maintaining differentiation potential after thaw, selection of cell culture surfaces and methods for confirming differentiation. Cell lines covered included neural stem cells, cardiomyocytes, hepatocytes, and blood cells.

This Ask the Expert session was sponsored by Thermo Fisher Scientific and hosted by Dr. Mohan C Vemuri. Dr. Vemuri is 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.

Scientists interested in the challenges associated with hPSC differentiation to neural, cardiac, and definitive endoderm lineages and how these processes can be efficiently simplified with tools and cGMP cell culture media systems should visit this Ask the Expert session. Below is a sneak peek, for a full transcript of the discussion, please see – Ask the Expert – Directed Differentiation of Pluripotent Stem Cells.

Question:

Why are many published differentiation protocols are not reproducible?

The Answer:

Many of these methods are suboptimal and a high percentage of methods even if published in highly reputed journals can not be reproduced. Variations come from the cell lines used, quality of the pluripotency state to begin with, use of undefined components in media and matrix used , and operator variations ( say a novice from an expert. All these contribute to reproducibility of the study. If researchers use a standard commercial kit – at least the set claims of those commercial kits can be verified clearly and get the results. The suppliers stand behind those products to support.

Question:

I am having trouble retaining differentiation potential after thawing, any recommendations?

The Answer:

Generally it depends the efficiency of your differentiation method. But if you are seeing the problem due to freezing and thawing, try ThermoFisher’s RevitaCell™ Supplement (100X), on the day the cells are thawed. Following 24hrs in culture, there is no need to use this supplement. Detailed instructions are available. Also some limitations apply, as some specific cells may not respond. http://www.lifetechnologies.com/order/catalog/product/A2644501?ICID=search-product

Question:

We are looking for the simplest most successful method for differentiation cardiomyocytes. I have investigated 4 methods, we are most familiar with embyoid bodies but I have read that guided differentiation has the best efficiency. What do you recommend? Do you have a protocol that you have seen success with?

The Answer:

Cardiomyocyte differentiation through embryoid body methods is quite variable in addition to the interline variation from iPSCs. Thermofisher has developed a monolayer method of cardiac differentiation that bypasses EB formation, and one can achieve efficient differentiation with cardiomyocyte differentiation kit (for example >90% cardiomyocytes using H9 line). The kit, supporting materials and detailed protocols are available on web site:
http://www.lifetechnologies.com/order/catalog/product/A25042SA?ICID=search-product