Delayed platelet recovery is seen following transplantation with suboptimal numbers of CD34+ cells, particularly from heavily pretreated donors, or following umbilical cord blood (UCB) transplantation. Ex vivo expansion of CD34+ cells is being used to overcome this problem, but issues have arisen concerning the quality and quantity of stem cells (HSC) generated and their long-term engraftment potential in vivo. The PI proposes to evaluate parameters of HSC function in cytokine- stimulated cultures of UCB and mobilized PB CD34+ cells using Flk2/flt3 ligand (FL) and Tpo to enhance HSC self-renewal in comparison with co- cultures on marrow stroma or endothelial cells. HSC will then be evaluated in long term culture-initiating and cobblestone area-forming assays and NOD-SCID repopulation. If defects are seen, he shall determine whether these are due to defective marrow homing which in turn may relate to defective expression of the chemokine receptor CXCR-4 on HSCs and impaired response to marrow chemotactic factor SDF-1. He will use transmembrane chemotaxis assays and endothelial adhesion and transmigration assays to determine if there are defects in CD34, c-kit and adhesion molecule expression. If defects are found he will use adenoviral gene delivery to (a) enhance expression of the stable transmembrane isoforms of c-kit ligand and FL on stromal and endothelial cells; (b) increase flk-2, c-kit, and CXCR-4 expression on HSC to improve marrow homing and chemotaxis and counteract TGF beta inhibition. Selective expansion of CD41a+ megakaryocytes will be undertaken to evaluate their chemotactic response to SDF-1 and their ability to generate platelets in vitro and following transfer to NOD-SCID mice. Proliferative senescence related to progressive telomere shortening is seen in vitro and in vivo in hematopoietic cells and endothelium. The PI proposes to transduce the gene for the catalytic component of telomerase (hTERT) into primitive hematopoietic cells, marrow and umbilical endothelium, and CD34+Flk-l+ circulating endothelial progenitors using retroviral and adenoviral gene delivery with selectable markers. Telomerase activity will be correlated with telomere length in our ex vivo cell expansion systems with measurement of the numbers of population doublings be achieved. All of the assay systems outlined above for evaluation of stem and endothelial function will be applied to potentially """"""""immortalized"""""""" populations after prolonged passage to determine if there is retention of normal function, whether there is functional rejuvenation - i.e., adult populations acquiring features of neonatal cells, or whether there is a tendency for """"""""immortalized"""""""" cells to undergo neoplastic transformation.
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