Sponsored by: Thermo Fisher Scientific
Session ends: May 22nd, 2015, 3:00pm MST
Answers by: Dr. Mohan C Vemuri , Thermo Fisher Scientific, Inc.
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.
Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion on directed differentiation of human pluripotent stem cells.
Questions & Answers
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 If the critical growth factors that support pluripotency state, such as TGF beta and FGF2 are reduced or completely withdrawn, the cells transition to spontaneous differentiation.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 […]» Read MoreMany 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 […]» Read MoreNeural stem cells can be differentiated to either neurons or astrocytes following the protocol described at this weblink: http://www.lifetechnologies.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/differentiating-neural-stem-cells-into-neurons-and-glial-cells.html» Read MoreTo 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 […]» Read MoreAs 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 […]» Read MoreGenerally 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 […]» Read MoreFor neural, generally it is poly L Lysine coating followed by laminin coating surfaces.» Read MoreCardiomyocyte 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 […]» Read MoreAlong 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 […]» Read MoreWhich 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, […]» Read More