DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) differentiate into all of the blood cell lineages. Ex vivo expansion of HSCs would greatly facilitate cell and gene therapies for viral diseases (e.g., HIV) and genetic blood disorders, and would improve the prospects for umbilical cord blood transplants in adults. However, HSC division in culture is associated with differentiation into cells with decreased potential. This contrasts with sustained HSC expansion in vivo, and led to the hypothesis that a stem cell "niche" supports self-renewal in the bone marrow. The niche includes matrix- and cell-associated cytokines, as well as direct contact with stromal cells. A culture surface will be developed for controlled presentation of cell adhesion molecule (CAM) ligands and cytokines that are expressed by stromal cells in order to mimic the niche. HSCs express many CAMs and cytokine receptors, so it is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. Presenting multiple CAM ligands and cytokines in the proper distribution is problematic on a static surface. A dynamic membrane-mimetic surface will be used to allow CAM ligands and cytokines to reorient themselves to match the location of CAMs and receptors on HSCs, while remaining localized to the culture surface. Lipid-linked peptide mimics will be synthesized for the 3 binding domains offibronectin (Fn), which has been reported to enhance HSC expansion. Different combinations of the peptide-lipids will be used to evaluate individual and synergistic effects of the Fn domains on: (1) cell adhesion via a centrifugation assay, (2) integrin-modulated signal transduction via immunoblots with phospho-specific antibodies, (3) CAM clustering via confocal microscopy, (4) HSC selfrenewal via flow cytometry for cell division tracking and cell-surface expression of the HSC markers CD34 and Thy-1, and (5) differentiation into the major hematopoietic cell lineages via progenitor cell assays and flow cytometry for lineage-specific antigen expression, Lipid-linked mimics of heparan sulfate, which plays an essential role in the support of HSC expansion by stromal cell lines, and a peptide mimic of thrombopoietin, which is a key cytokine for HSC survival and expansion, will also be synthesized. These compounds will be evaluated, alone and in combination with each other and the Fn domain mimics, in order to further enhance HSC expansion.
|Effective start/end date||7/1/03 → 12/31/08|
- National Heart, Lung, and Blood Institute (5 R01 HL074151-04)