Hematopoietic reconstitution following stem cell transplantation is often slow and clinically must be supported by the administration of growth factors, antibiotics and/or platelet transfusions. Unfortunately, these approaches do not completely abrogate the nadir in blood cell counts or usefully accelerate hematologic recovery. To address this problem, we recently demonstrated that the rate of engraftment by highly enriched murine hematopoietic stem cells (HSCs) could be accelerated by 1- 2 weeks when they were partially differentiated into functional progenitors by cytokine-mediated expansion in vitro. Significantly, engraftment of expanded hematopoietic cells was farther accelerated by treating transplant recipients with the tetrapeptide cell cycle inhibitor AcSDKP. However, when we evaluated the ability of ex vivo expanded progenitors to """"""""home"""""""" to the bone marrow or spleen using a novel assay developed to track their localization in vivo, we observed a 10-fold reduction in engraftment efficiency which correlates with a downregulation of the integrin beta1 subunit common to 6 adhesion molecules. This proposal aims to test the hypothesis that the homing defect of expanded hematopoietic cells can be reversed and their clinical utility amplified through modulation of integrin expression and cell cycle status.
Aim l will define the phenotype of the early-engrafting cell in mice by flow cytometric sorting of normal and expanded bone marrow cells into populations differing in their expression of adhesion molecules and cycling status. The functional capacity of these fractions to rapidly regenerate circulating blood cells in vivo will be assayed by transplantation into myeloablated hosts.
Aim 2 will compare the homing of normal and expanded cells differing in these properties and test the hypothesis that AcSDKP accelerates engraftment of expanded HSCs by arresting cell cycle progression and improving homing. The relationship between cycling status, integrin expression and homing will be farther examined through studies of cytokine-mobilized and neonatal HSCs with rapid versus delayed engraftment kinetics, respectively.
The final aim of this proposal will examine the possibility that homing and hematopoietic reconstitution kinetics of ex vivo expanded cells can be improved by using adenoviral vectors to transiently enforce expression of adhesion molecules that are down-regulated during culture. These studies should identify transplantable cells which facilitate earlier recovery from cancer therapy and begin to define both intrinsic and extrinsic approaches toward maximizing their numbers and engraftment potential.
