Directed Differentiation of Pluripotent Stem Cells
Company: Thermo Fisher Scientific, Inc.
Job Title: Director of Research and Development
Introduction
The promise of human pluripotent stem cells will be realized only when these cells are successfully coaxed into different cell types found in the human body, through the process of directed differentiation. This 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 will be discussing the challenges associated with hPSC differentiation to neural, cardiac, and definitive endoderm lineages, how Thermo Fisher Scientific can address how these processes can be efficiently simplified with tools and cGMP cell culture media systems for robust, efficient and scalable differentiation of these two critical cell lineages. Use of these reagent systems will enable researchers to precisely control and direct the differentiation to terminal lineages in a relatively easy manner, and speedily with high efficiency.
This Ask the Expert session is 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.
Question 1
I am currently using monolayer culture for differentiation of blood cells. I am looking at embryoid bodies and was wondering if there is a benefit to this method and also challenges with the method.
Which cell types are you starting with as monolayer is an important consideration for differentiation.
If pluripotent stem cells (PSC)are started as monolayer and driving the differentiation towards Hematopoietic stem cells (HSC), starting as adherent monolayer of PSC and harvesting HSC as suspension culture in the same dish is an ideal set up.
In contrast, if HSC are plated for differentiation towards blood lineages, generally HSC are floating cells and the differentiated blood lineages are adherent cell types.
In either case embryoid body generation can be bypassed and does not confer any special advantage.
Question 2
What is your preferred method for confirming differentiation?
Along the path of differentiation, ensuring early, middle and late stage specific marker expression for different lineages and finally testing the cells for their physiological function – for example, if the cells are being differentiated to dopaminergic neurons, analyzing the dopamine release in these cultures and finally testing the cells in in vivo animal transplants to confirm full differentiation.
Question 3
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?
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
Question 4
What cell culture surface do you use for neural cell differentiation?
For neural, generally it is poly L Lysine coating followed by laminin coating surfaces.
Question 5
I am having trouble retaining differentiation potential after thawing, any recommendations? Thank you!
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 6
It seems we are getting very poor doubling times from our recently differentiated cardiomyocytes. What should we be expecting?
As the pluripotent stem cells start to differentiate ( cardiac in this case), the expansion potential of PSC gets declined. The differentiated cells do not divide further, unless they are still in some progenitor state. Considering this dynamics in differentiating cell cultures, it is quite natural that doubling times tend to get poor and poor and eventually stop doubling. It is normal in differentiating cell cultures.
Question 7
What growth factor or factors are key to hepatocyte differentiation from iPSCs?
To begin with as endoderm, one would critically need high levels of Activin, FGF & BMP, followed by hepatocyte growth factor ( HGF) and Oncostatin in later stages of hepatic differentiation.
For detailed steps and concentration of specific growth factors used in different steps, I recommend the protocol described : Ma et al : Stem Cells Translational Medicine: 2 : 409-419, 2013
Question 8
Do you have recommendation for getting neural stem cells to differentiate into astrocytes, not neurons?
Neural stem cells can be differentiated to either neurons or astrocytes following the protocol described at this weblink:
Question 9
Why are many published differentiation protocols are not reproducible?
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 10
How many passages can I get before I see spontaneous differentiation in my iPSCs?
Passage number is not a limitation to see spontaneous differentiation for iPSCs. If the critical growth factors that support pluripotency state, such as TGF beta and FGF2 are reduced or completely withdrawn, the cells transition to spontaneous differentiation. Those are the actual limiting factors that determine whether an iPSC cell has to be maintained in PSC state or move to differentiated state.
This post is sponsored by Thermo Fisher Scientific, Inc..