Methods to induce human hematopoietic stem cells (HSC) into cycle in vitro without induction of programs of differentiation and a loss of engraftment potential are central goals of ex rive stem cell expansion and gene therapy. Currently, culture leads to a progressive loss of pluripotent stem cells, so engineered HSC populations cannot yet be returned to an ablated donor with the confidence that they will sustain long-term multilineage blood cell production. In the initial period of funding, we developed methods to recruit quiescent stem cells into cycle using cyclin-dependent kinase inhibitor reduction strategies, and we also examined the response of primary human HSC to culture in suspension vs. on the COOH domain of fibronectin (FN). We determined that human HSC lose the capacity to sustain long-term engraftment in immune deficient mice if cultured for 72 hours in suspension culture with cytokines. Binding to FN in the same conditions preserved the engraftment capacity. We initially hypothesized that engagement of the integrins alpha4beta1 and alpha5beta1 to FN maintains the primitive phenotype of HSC by sustained activation and nuclear localization of MAPK proteins, resulting in phosphorylation of GATA-2, induction of e-ets-1 and maintenance of expression of c-myb. In suspension, we predicted that MAPK would remain in the cytoplasm, c-ets-1 would not be induced, and c-myb levels would drop, causing a loss of expression of CD34, fit3, and e-kit. We have verified a portion of these hypotheses at the molecular level. Recruitment of primary human CD34+ cells into cycle occurs more rapidly in suspension culture than in cells on FN, through increased cyclin E and cyclin B-associated kinase activities. Cells cultured on FN have higher levels of nuclear ERK2 (MAPK2) and maintain higher levels of c-myb, GATA-2, and CD34 proteins than cells cultured in suspension. The molecular profile of the cells cultured on FN correlates with the maintenance of long term engraftment capacity in immune deficient beige/nude/xid mice. These molecular and in vivo data indicate that HSC cultured in suspension may commit to differentiation more rapidly than HSC cultured on FN. In the proposed studies, we will determine cause and effect, rather than correlation. We hypothesize that maintaining expression of c-myb and GATA-2 from non-integrating lentiviral vectors during culture of human stem cells in suspension, with CDK inhibitor reduction, will sustain the pluripotency of the engrafting human stem cell pool while permitting more rapid cycling to allow expansion. The HSC will be marked with integrating retroviral vectors in the different culture conditions, then studied at the molecular level, in vitro, and in vivo. An increase in the numbers of T cell and myeloid progeny bearing the same proviral integration site, recovered from bnx mice, will indicate that HSC with lymphomyeloid potential were transduced to higher levels in our optimized cultures than in control conditions. The proposed studies will define in vitro culture conditions which induce stem cell division without forcing lineage commitment during retroviral-mediated transduction or ex-vivo stem cell expansion_ and will give insight into the molecular changes that precede lineage commitment vs. maintenance of pluripotentiality in primary human hematopoietic stem cells.
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