We propose to study factors that regulate self-renewal of candidate human stem cell populations capable of myeloid and lymphoid differentiation. We have developed two novel long-term bone marrow culture systems termed """"""""stroma-non-contact"""""""" culture and long-term natural killer cell culture (LTNK) that allow: a) expansion of primitive, multilineage hematopoietic progenitors; and b) their differentiation to the myeloid or lymphoid lineage. The use of these culture systems makes it therefore possible to assess both multilineage and self-renewal capacity of candidate human stem cells. We hypothesize that multi-lineage stem cells can be induced to self-renew by a combination of cytokines and stromal factors, both diffusible and stromal bound, allowing (1) high efficiency retroviral gene transfer and (2) long-term expansion of this population ex vivo.
In Specific Aim 1, we will evaluate the role of stroma, stromal components, diffusible factors secreted by stromal feeders and defined cytokines on the short term proliferation and hence retroviral gene transfer into multi-lineage progenitors from human marrow. In additional studies we will examine the mechanisms through which stromal ligands, stromal soluble factors or cytokines increase retroviral transduction. These studies will include evaluation of progenitor proliferation as a result of contact between progenitors and stromal components or incubation with specific cytokines, and the effect of these culture conditions on expression of retroviral receptors on progenitors.
In Specific Aim 2, we will examine the culture conditions that will result in long-term expansion of marrow derived primitive progenitors capable of both myeloid and lymphoid differentiation. We will examine the role of growth inhibitory cytokines (MIP-1a, platelet factor 4, and AcSDKP), growth promoting cytokines (IL3 and IL7) and diffusible stroma-derived factors on the long-term expansion of retrovirally marked Lin-34+DR-, multilineage progenitors to demonstrate """"""""proliferation,"""""""" the expanded progenitors will be subcultured in 4 secondary cultures and self-renewal of a retrovirally marked stem cell I determined by PCR analysis of progeny present in the different secondary cultures. To demonstrate """"""""multilineage potential"""""""" expanded progenitors will be subcultured in both myeloid (stroma-non-contact) and lymphoid (LTNK) differentiation cultures and outgrowth of common retrovirally marked clonogenic cells and NK cells demonstrated by PCR. We will use a combination of inverse PCR and/or PCR using a partially degenerate upstream primer.
In Specific Aim 3, we will use the same methodologies as described in SAl and SA2 to compare the self-renewal capacity of primitive multilineage progenitors from umbilical cord blood and fetal liver, thought to have significantly greater proliferative potential and to require different """"""""growth factors"""""""" for proliferation than their counterparts in adult marrow. These studies are intended to define further the proliferation and multilineage differentiation capacity of human hematopoietic stem cells of various ontogeny. Methods for improved retroviral transduction of hematophil tic stem cells will be defined. Finally, further development of the """"""""stroma-non-contact"""""""" and LTNK culture systems should provide us with an assay system amenable for serial evaluation of proliferation and pluripotency of human hematopoietic stem cells.
