Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: The focus of this proposal is on the development of T-lymphocytes from hESC in vitro. Aim 1: Seventeen non-approved hESC lines will be initially evaluated for their ability to become hematopoietic progentiors (HP) using conditions designed to generate embryoid bodies or upon co-culture with stromal cells. Aim 2: Simultaneously, HP will be isolated based on expression of cell surface markers and on stromal lines in various cytokines to induce differention of T-cell progenitors. The final aim will focus on attempting to induce mature T-cell development by engineering stromal cell lines to express integrin ligands or human MHC molecules. An attempt to reproduce the three dimensional structure of the thymus with collagen matrix will also be made. SIGNIFICANCE AND INNOVATION: The generation of mature T-cells from hESCs is a potentially important step for providing cells for specific immune functions in disease and for clarifying the ontogenetic steps of lymphocyte maturation. The proposed work may also disclose whether the few NIH-approved cell lines are so inefficient in generating T cells because they have lost the ability to differentiate despite the use of different culture conditions and stromal cell support, or for more basic biological reasons. To do this, the PI will explore 17 recently reported hESC lines from the Melton lab. A particularly difficult step is the conversion of the immature CD4+, CD8+ phenotype into CD4+, CD8- and CD4-, CD8+, of the mature T cell population. The lack of CD4+ T cells should be anticipated from the lack of class II HLA ligands in the system using murine stroma; thus, the comparison between the use of embryoid bodies and OP9-DL1 assisted cultures with growth factors and cytokines. To enhance the maturation of early CD4+, CD8+ cells Dr. Robey’s solution is to transduce the HLA class I and class II MHC genes and then monitor the phenotypic expression of informative markers using CD34+ cells from umbilical cord blood. This will shorten the identification of an optimal culture system to be used with the most promising cell lines and culture conditions. In addition, she will attempt to develop effector and regulatory T cells. These are all reasonable and innovative, though laborious approaches. The innovation in this proposal reflects the willingness to screen many (17) cell lines under many conditions in an effort to achieve the stated goals. State-of-the-art methodology will be utilized and novel, although sketchily described, strategies will be utilized in an attempt to achieve the final steps in T cell maturation. Overall, one reviewer ranks the proposal fairly high with respect to innovation. The work's significance lies in the importance of understanding the differentiation of hESCs into hematopoietic cells and the potential long term use of in vitro generated lymphocytes for therapuetic purposes. STRENGTHS: This is submitted by a highly seasoned and well funded investigator who has made many important contributions to experimental immunology and specifically to T-cell differentiation. Her colloborator, Dr. Winoto, is also an accomplished experimental immunologist who brings expertise in molecular biology to the proposal. Overall the enviroment seems very strong for conducting this research. The proposal is generally well thought out and encompasses a diversity of approaches to improve the chances of success. Both PI and Co-PI are well-known with solid track record in experimental immunology. Support staff is excellent, the approaches are elegant and innovative and some preliminary data is presented. WEAKNESSES: The technical descriptions are very skimpy, although all procedures are well within the technology used by this lab with the possible exception of achieving the T-cell selection of non-self reactive and immunologically active. The proposed strategies to recreate the enviroment to support terminal T-lymphocyte development are minimally described. For example, how will human MHC be expressed to achieve both negative and positive regulation of T-cell development. Which integrins will be expressed and what is the basis for their choice? How will collagen matrix re-create the 3 diminisional structure of the thymus microenviroment? DISCUSSION: This proposal aims to study differentiation of T cells from hESCs. The three aims of this proposal describe a three-stage protocol for generating mature T-cells that is elegant and well-constructed. Seventeen (17) cells lines will be used to test the effects of many growth conditions using FACS as a monitoring method. There was some discussion over the rationale to test 17 cell lines. 17 cell lines seems excessive but may be potentially justified based on heterogenity issues; the PI wants to screen enough to know that the conditions will work. It was noted that some of the NIH approved lines have been tested without success for differentiation to mature T lymphocytes. In the case of these lines, there is some question whether the issue is one of passage number or a more fundamental issue. The environment for completing this work is strong. From an immunological point of view, the most interesting part of this proposal is the selection of +/- (CD4,CD8)cells from hESC lines. T cell derivation has previously failed but it isn't known why. There was a certain vagueness in the proposal as to exactly what the applicant proposes to do; this aspect was not very well described in what is otherwise an elegant proposal. The proposed approach seems to be to test a lot of lines and thus figure it out.