The differentiative capacity of primitive human hematopoietic cells can be profoundly affected by ex-vivo culture. Due to the growing number of protocols proposed for stem cell gene therapy and ex-vivo expansion, it is crucial to define methods to preserve the potentiality of human stem cells in culture while promoting self-renewal divisions. We have previously demonstrated that human CD34+ cells maintained in suspension culture for 74 hours in the presence of the cytokines IL-3, IL-6, and stem cell factor (SCF) support hematopoiesis in immune deficient (bnx) mice for up to four moonths, but fail to sustain long-term hematopoiesis. In contrast, CD34+ cells cultured on monolyers of marrow stromal cells or the COOH-terminal fragment of fibronectin sustain both short-term and long-term hematopoiesis. We hypothesize that human hematopoietic progenitors may be induced into a program of terminal differentiation if they are maintained with cytokines in suspension culture. Engagement of the integrins VLA-4 or VLA-5 by binding to the fibronectin CS-1 or RGD sequences, respectively, or increased activity of TGFbeta1 on adherent cells, may act to reduce the frequency of cell cycle progression, and the induction of programs of differentiation induced by cytokines. Human CD34+ and CD34+/38- cells will be cultured in suspension vs. on sub-fragments of fibronectin, with and without addition of TGFbeta1 or anti-TGFbeta1 neutralizing antibody, in serum-containing vs. serum-free media, and with addition of selected combinations of the cytokines IL-3, IL-6, SCF, FL, MIP-1alpha, and LIF. In some experiments, antisense oligonucleotides to transcription factors implicated in induction to differentiation (c-myb, pim-1, E2F, and MZF1, will be added during the culture period. We will measure molecular and cellular effects of each condition, alone and in combination, on progenitors (CD34+, colony-forming cells, long-term culture initiating cells) and long-lived hematopoietic stem cells (CD34+/38-, immune deficient mouse repopulatig cells). Molecular changes and alterations in the capacity for multilineage generation and short-term and long-term growth properties of cells cultured in each ex-vivo condition will be determined. Together, the results of the proposed studies will define conditions wihch promote primitive progenitor survival and self-renewal ex vivo, and will give insight into the molecular changes that precede lineage commitment in primitive human hematopoietic cells.
