ShRNA Knockdown of ID genes in Human Embryonic Stem Cells as a Possible Path Towards B and T Cell Development
2009-04-08
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
ShRNA Knockdown of ID genes in Human Embryonic Stem Cells as a Possible Path Towards B and T Cell Development
Authors
Published Date
2009-04-08
Publisher
Type
Presentation
Abstract
Human Embryonic Stem Cells (hESCs) are pluripotent, self-renewing cells capable of becoming any cell in the human body. Previously, our lab has derived multiple cell types from hESCs, with a particular interest in hematopoietic (blood cell) development. We have been able to successfully derive natural killer (NK) cells, a type of lymphocyte with potent anti-tumor activity. However, to date we have been unable to derive other lymphocytes (T and B cells) from hESCs. Using Umbilical Cord Blood (UCB) as a comparison for early hematopoietic development, we deduced that one possible factor for this difference could be a relatively high expression of the Inhibitor of Differentiation (IDs) transcription factors in hESCs compared to UCB. ID proteins bind to and negatively regulate basic Helix-Loop-Helix (bHLH) proteins responsible for many differentiation programs within the cell. In particular, ID2 and ID3 are known to promote NK cell development and inhibit B and T cell development. My project has been to introduce shRNA constructs into hESCs to inhibit expression of ID2 and ID3 as a means to better promote T and B cell development from hESCs. Two shRNA systems have been designed. The first uses a lentiviral vector in which the shRNA construct is constitutively expressed. The second uses a lentiviral vector containing a CRE-conditional shRNA expression system. We have introduced both of these vectors into Ntera2 embryonal carcinoma cells and hESCs. For the constitutive shRNAs, we demonstrate partial knockdown of ID2 and ID3 via qRTPCR. These constitutive knockdown ID hESCs become more difficult to culture in an undifferentiated state, and over time may have lost some of their potency. This potential problem with constitutive knockdown cells highlights the need for the inducible system, which is in progress. We anticipate that the ability to inhibit ID2/3 expression at specific timepoints of hESCs differentiation into hematopoietic cells could solve this problem and facilitate development of B and T cells from hESCs.
Description
Additional contributor: Dan S. Kaufman, Department of Medicine and Stem Cell Institute, Academic Health Center
Related to
Replaces
License
Series/Report Number
Funding information
This research was supported by funding provided to the Kaufman lab from NIH/NHLBI, The Stem Cell Institute Research Foundation, and The Leukemia Research Fund of the University of Minnesota Masonic Cancer Center.
Isbn identifier
Doi identifier
Previously Published Citation
Suggested citation
Taylor, Russ. (2009). ShRNA Knockdown of ID genes in Human Embryonic Stem Cells as a Possible Path Towards B and T Cell Development. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/49078.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.