Taylor, Russ2009-04-172009-04-172009-04-08https://hdl.handle.net/11299/49078Additional contributor: Dan S. Kaufman, Department of Medicine and Stem Cell Institute, Academic Health CenterHuman 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.en-USGenetics, Cell Biology and DevelopmentCollege of Biological SciencesDepartment of Genetics, Cell Biology and DevelopmentAcademic Health CenterDepartment of MedicineStem Cell InstitutePresentation